A route-directed hybrid genetic approach for the vehicle routing problem with time windows

123 Array 4567891011111121314151617181920111212223242526272829301113132333435363738394011171should not automatically bar adoption of measures to prohibit or otherwise regulate the activity, and, in stronger versions, further asserts that uncertainty provides an affirmative justification for regulating an activity or regulating it more stringently than in the absence of uncertainty. Strong versions of PP hold that regulators should adopt “worst case” presumptions regarding the harms of activities posing an uncertain potential for significant harm; should prohibit such activities or require them to adopt best available technology measures;that regulatory costs should be disregarded or downplayed in such decisions;and that the proponents of such activities should bear the burden of establishing their safety in order to avoid such regulatory controls. The article also considers the relevance for regulatory decisions of uncertainties regarding the costs of regulating an activity as well as uncertainties regarding harms. It further considers the implications of the circumstance that regulatory decisions about a given environmental issue may be made sequentially over time and benefit from additional information developed in the interim between earlier and later decisions. The essay concludes that, while preventive regulation of uncertain risks is often appropriate and should incorporate precautionary elements where warranted by consideration of risk aversion or information acquisition, strong versions of PP do not provide a conceptually sound or socially desirable prescription for regulation.I. INTRODUCTION: ENVIRONMENTAL DECISION MAKING UNDER UNCERTAINTY AND THE PRECAUTIONARY PRINCIPLE The following are examples of regulatory decisions involving uncertain risks. In each case, consider whether a regulator should permit the potentially harmful activity to commence or continue, or, alternatively, to prohibit or otherwise regulate it, and the implications of sequential regulatory decision making and the opportunity to develop additional information to reduce uncertainties regarding the risks of harms posed by an activity and/or the costs of regulation.•Whether to prohibit the sale of meat products from cattle that have received bovine growth hormone injections.•Whether to prohibit the construction of an astronomic observatory atop Mt.G raham, in New Mexico, the only known habitat of the Mt. G raham Red Squirrel, a subspecies of the western red squirrel located only on Mt. Graham;other subspecies of the red squirrel are abundant in the Western United States.72RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111•Whether to prohibit field releases of crop plants that have been genetically modified using DNA technologies.•Whether to adopt a National Ambient Air Quality Standard (NAAQS) to limit short-term (10-minute) exposures to elevated levels of sulfur dioxide (SO 2). Several laboratory studies indicate that asthmatics exposed to higher short-term SO 2exposures experience temporary airway resistance that makes breathing more difficult.•Whether to prohibit introduction of sucralose, an artificial beverage sweetener that has been touted as safer than saccharin or aspartame, the artificial sweeteners currently in use.•Whether to prohibit the dumping of wastes of any sort at sea.•Whether to develop defenses against collisions with the earth by asteroids and other near-earth objects.•Whether to eliminate the use of chlorine to treat drinking water.•Whether to prohibit the use of glyphosate (“Roundup”), a broad-spectrum non-selective herbicide that is harmless to animals.•Whether to prohibit or tightly regulate the conversion of rainforest to agricultural or other uses.•Whether to immediately initiate strict limits on greenhouse gas emissions in order to limit the potential adverse effects of climate changes attributable to such emissions.In evaluating these and other environmental regulatory decision-making issues,the extent of available knowledge regarding environmental harm that may be caused by the activity in question can be conceptually classified in three ideal type categories:Type 1. The harm that the activity will cause is known and determinate. If,for example, the Mt. Graham observatory is built, the Mt. Graham red squirrel population will be wiped out within 20 years.Type 2. The harm is probabilistic in character but its probability distribution is known. For example, if the observatory is built, there is a 40% probability that the squirrel population will be wiped out within 20 years and a 60% probability that it will survive another 1000 years, at which point it will become extinct from natural “background” causes. In this situation we deal with a risk of harm, but the risk (comprising both the probability of an adverse effect occurring and the magnitude of the adverse effect if it occurs) is determinate.Type 3. There is a risk of harm that is uncertain. Thus, the probability of harm occurring, and/or the magnitude of the harm if it occurs, is not determi-nate and is subject to substantial uncertainty. To take the Mt. Graham example,Environment al Regulat ory Decision Making Under Uncert aint y 737312345678910111111213141516171819201112122232425262728293011131323334353637383940111it may be uncertain, based on current knowledge, whether any adverse effect on the squirrels will occur. If any adverse effect does occur, its magnitude is uncertain. Thus, it may be uncertain what percentage of the population may be lost, whether any given level of loss will result in extinction of the subspecies,and when possible losses or extinction may occur. There may also be cases where the type of harm, if any, that may occur is not known.The difference between Type 2 cases and Type 3 cases is obviously one of degree, but the distinction between the two ideal types is very useful for purposes of analysis. Very many environmental problems are Type 3 cases,characterized by uncertainty regarding risks of harm, although the nature and degree of uncertainty varies widely from case to case. These uncertainties have many potential causes, including lack of data, limitations in scientific understanding of causal relationships, medical and ecosystem complexity, and “trans scientific” gaps in the capacities of science.1There may also be substantial uncertainties regarding the costs of prohibiting or otherwise regulating the activity in question. In many situations, such uncertainties can be reduced by the development of additional information and knowledge as discussed further below.The common law traditionally awards damages only ex post for harm that has occurred and has been shown to have been caused by another’s activity. It grants injunctive relief ex ant e only if an activity poses an imminent and substantial likelihood of serious irreparable harm. Many environmental risks of types 2 and 3 would not qualify for an award of damages or prophylactic relief under this standard. In theory, ex post liability for harm caused could provide the requisite incentives for actors to manage their activities so as to prevent excessive risks of harm appropriately. In practice, however, these incentives have for a variety of reasons proven inadequate to prevent excessive environ-mental harm from occurring.2Accordingly, administrative programs of preventive ex an t e regulation have been widely adopted in the United States and other countries to regulate activities that pose substantial risks of environmental harm, even in cases where it is not certain that harm will actually occur. International agreements, such as the Vienna Convention for the Protection of the Ozone Layer and the Framework Convention on Climate Change, have also been adopted to address such risks.Preventive regulatory programs have been adopted not only in cases where activities have been shown to cause harm, but also in cases involving risks of harm, including cases of substantial uncertainty in the risk of harm.3Quantitative risk analysis and cost-benefit analysis are increasingly being used in connection with the preventive approach to regulation.4Under many preventive regulatory74RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111programs, regulators have the burden of establishing a significant risk of harm before imposing regulatory controls,5although under some programs, such as U.S. FDA new drug and food additive approvals and EPA registration of pesticides, the applicant bears the burden of showing product safety.In recent years, environmental advocates and many environmental law scholars, particularly in the field of international environmental law, have argued that environmental regulatory decisions and policies should follow a precautionary principle (PP).6The focus of PP is on appropriate regulatory policy in Type 3 cases where the risks of harm posed by an activity are characterized by substantial uncertainty. PP advocates argue for a precautionary approach to regulation in the face of such uncertainty. They often criticize prevailing preventive approaches to regulation on the grounds that they place the burden on regulators to show that an activity will cause serious harm or poses a high probability of serious harm before regulatory controls may be adopted. They argue that, given the lack of scientific capacities to predict which activities will cause serious or irreversible harms, this approach results in seriously inadequate environmental protection. They often also contend that existing preventive regulatory approaches give undue weight to costs in establishing controls.7Various versions of PP, mostly weak ones, have been incorporated or invoked in a number of recent international environmental declarations and conventions,including the Framework Convention on Climate Change 8and the EU Maastricht treaty.9These documents and the writings of PP advocates and of academics provide widely varying formulations of PP. It has been claimed that PP is already, or is becoming established as a binding principle of customary international law.10PP skeptics and critics, however, have contended that the heterogeneity of PP formulations, many of which are quite vague and indeterminate, demonstrates that that there is no single or determinate PP.11Thus, they have concluded that the precautionary principle is a “composite of several value-laden notions and loose, qualitative descriptions” and that accordingly its “operational usefulness ... is doubtful.”12They also deny that PP has been established as customary international law.13Criticisms of PP as indeterminate and conceptually fuzzy have merit. With a very few exceptions, there is a remarkable lack of analytic care or rigor regarding the substance of, and justification for, various versions of PP by those who advocate or favor their adoption. One can, however, identify four different PP conceptions that have emerged in legal instruments, international and national governmental declarations, advocacy statements, and the academic liter-ature that can serve as a useful basis for analysis and evaluation. These four versions of PP are as follows:Environment al Regulat ory Decision Making Under Uncert aint y 757512345678910111111213141516171819201112122232425262728293011131323334353637383940111PP1. Scientific uncertainty should not automatically preclude regulation of activities that pose a potential risk of significant harm (Non-Preclusion PP).PP2. Regulatory controls should incorporate a margin of safety; activities should be limited below the level at which no adverse effect has been observed or predicted (Margin of Safety PP).PP3. Activities that present an uncertain potential for significant harm should be subject to best technology available requirements to minimize the risk of harm unless the proponent of the activity shows that they present no appreciable risk of harm (BAT PP).14PP4. Activities that present an uncertain potential for significant harm should be prohibited unless the proponent of the activity shows that it presents no appreciable risk of harm (Prohibitory PP).What unites these different formulations is a focus on uncertainty regarding risks as the key factor guiding regulatory decisions. Some discussions of the PP blur the distinction between known (Type 2) and uncertain risks (Type 3),but the most careful commentators make clear that the precautionary principle is addressed to uncertain risks (Type 3) as such.15PP1 and PP2 are weak versions of precautionary approaches. Unlike the strong versions, PP3 and PP4, they do not mandate regulatory action and do not make uncertainty regarding risks an affirmative justification for such regulation.Thus, PP1 is negative in character; it states that uncertainty should not preclude regulation but does not provide affirmative guidance as to when regulatory controls should be adopted or what form they should take. This is the approach that is most widely invoked in international treaties and declarations. While the exact wording may vary, this principle of non-preclusion always sets up a threshold, e.g. an uncertain risk of serious damage,and then makes the negative prescription that, once that threshold has been triggered, regulators cannot rely on this fact alone to deny regulation. For example, the Bergen Ministerial Declaration states: “Where there are threats of serious or irreversible damage, lack of full scientific certainty should not be used as a reason for postponing measures to prevent environmental degradation.”16The Cartagena Protocol goes further by clarifying that uncertainty can not, in and of itself, justify the decision not to regulate, nor,presumably, the alternative decision to impose regulation:Lack of scientific certainty due to insufficient relevant scientific information and knowledge regarding the extent of the potential adverse effects of a living modified organism shall not prevent [a] Party from taking a decision, as appropriate. Lack of scientific knowledge or scientific consensus should not necessarily be interpreted as indicating a particular level ofrisk, an absence of risk, or an acceptable risk.1776RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111This principle of non-preclusion rejects the common law position that harm must be shown to have occurred or be imminent before legal liabilities or controls may be imposed. It also rejects the position, often asserted by industry,that significant uncertainty about risks should preclude imposition of preven-tive regulatory controls. Of all the formulations of the PP, this approach is the most often invoked and is most likely to be recognized as a part of customary international law; it is already widely accepted that a preventive approach, under which regulatory controls are adopted to prevent or reduce risks of harm even though the magnitude or even the occurrence of harm is uncertain, is justified in at least some circumstances.18Yet, the very generality and lack of specific prescriptions of PP1 may preclude it from being recognized as a binding norm.19PP2 likewise fails to specify when or what form of regulation should be adopted, but instructs that, whenever regulation is adopted, it should incorporate a margin of safety. Unlike PP1, PP2 is operative only after regulators have made the determination to regulate. Once this decision is made,regulators must first determine the maximum “safe” level of an activity, and then only allow the activity at some degree lower than that level (the “margin of safety”). This is a common approach in U.S. environmental law. An example is the Sustainable Fisheries Act of 1996, in which the optimum allowable yield from a fishery “is prescribed on the basis of the maximum sustainable yield from the fishery, as reduced by” relevant factors including “ecological” factors.20PP2 is consistent with (although it does not necessarily mandate) many commen-tators’ views that PP requires that regulators allow “large margins for error” in risk assessments.21It represents one formulation of the PP premise that: “Given scientific ignorance, prudent pessimism should be favoured over hazardous optimism.”22PP2 is not explicitly set forth in any international agreements and declarations, but its approach is implicit in some international agreements that require or provide for the adoption of precautionary measures.23The weak versions of the PP are fully compatible with and are often reflected in many well-established preventative regulatory programs that have been adopted at the domestic level by many countries and by international agreement over the past 30 years. These programs often authorize prophylactic regulation of uncertain risks in appropriate circumstances even in the absence of a showing that harm will actually occur. In many cases, they explicitly require a margin of safety in setting regulatory standards.24Thus the weak versions of PP do not represent or justify any basic change in the preventive approach to regulation that has generally prevailed over the past 30 years. They accordingly provide no basis for arguing that existing preventive regulatory programs are not sufficiently “precautionary” and need to be fundamentally changed in order to reflect precautionary principles.Environment al Regulat ory Decision Making Under Uncert aint y 777712345678910111111213141516171819201112122232425262728293011131323334353637383940111There are, however, important differences between established programs of preventive regulation and the strong versions of PP. Weak precautionary programs generally do not make the existence of uncertainty regarding risks as such a mandatory or distinct basis for imposing regulatory controls. PP3 and PP4, on the other hand, require regulators to regulate, or regulate more stringently, activities that pose risks that are more uncertain relative to risks that are less uncertain, and thus represent a significant change in regulatory concept and result.Under PP3, when regulators determine that there is a serious but uncertain risk, they must impose BAT measures. For example, the Second International Conference on the Protection of the North Sea calls for parties to:[R]educ[e] polluting emissions of substances that are persistent, toxic and liable to bio-accumulate at source by the use of the best available technology and other appropriate measures. This applies especially when there is reason to assume that certain damage or harmful effects on the living resources of the sea are likely to be caused by such substances,even where there is no scientific evidence to prove a causal link between emissions and effects (“the principle of precautionary action”).25Such a prescription does not appear to allow regulators to decide what sort of regulation is required, including no regulation: if there is an uncertain risk of serious harm, BAT measures should be imposed. However, some flexibility may remain under PP3 since the intensity of BAT controls may vary depending on the magnitude of the potential risk relative to the costs of controls, in accor-dance with a principle of proportionality.26PP4 imposes an even more stringent prescription upon regulators. Under this formulation, if there is an uncertain but serious risk of harm, the activity in question should not be undertaken at all until it is proven to be safe by the proponent of the activity. Thus, the Final Declaration of the First European “Seas at Risk” Conference provides that:The “burden of proof” is shifted from the regulator to the person or persons responsible for the potentially harmful activity, who will now have to demonstrate that their actions are not/will not cause harm to the environment. If the “worst case scenario” for a certain activity is serious enough, then even a small amount of doubt as to safety of that activity is sufficient to stop it taking place.27While this version of the PP presumably allows regulators some latitude to determine how serious an uncertain risk must be to invite regulation, it requires prohibition of the activity once the relevant risk threshold is met.The strong versions of PP, PP3 and PP4, are the focus of this essay.Accordingly, unqualified references to PP in the following discussion should be understood as referring to the strong versions of PP. Unlike the weak versions 78RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111of PP and the preventive approach to regulation generally, they make the existence of uncertain risks of significant harm both a sufficient and mandatory basis for imposing regulatory controls. We may term this the “uncertainty-based potential for harm” prescription for regulation. Different PP formulations incorporating this precept vary in the criteria for determining the potential for harm threshold that triggers the requirement of regulation, including how great the probability of harm must be, its character, and its magnitude. Some formulations, for example, stress that the probability of harm must be substan-tial and the harm that may eventuate must be “serious and irreversible.”28Other formulations enunciate less demanding criteria.29In some strong PP formula-tions, once the applicable risk threshold is met, regulation is mandatory;regulatory compliance costs, including the social costs involved in forgoing the benefits of activities subject to regulatory prohibition or restriction, are not included as a factor to be considered in the regulatory decision.30Some formu-lations explicitly allow for consideration of costs, but relegate them to a distinctly secondary role, while others introduce the principle of proportion-ality, tailoring the extent and character of the regulatory response adopted to the gravity of the risk in question.31Under PP3, for example, the costs of BAT controls might be taken into account in determining whether a given technology is “available.” It might be concluded that very costly technology controls are not as a practical matter “available.” Under PP4, in cases where potential risks are judged less serious or where the social benefits of the activity are high,prohibitory controls might be adopted for only a limited initial period subject to “sunset” provisions or reconsideration, or field trials may be permitted.32Strong versions of the PP also often hold that the burden of resolving uncertainty should be borne by the proponent of an activity rather than by regulators or opponents of the activity.33Accordingly, in order to avoid or lift regulatory prohibitions or BAT requirements, the proponent of an activity bears the burden of demonstrating that it does not present a potential for significant harm. Proponents of regulation, however, bear some initial threshold burden of production and persuasion. They must establish that an activity poses risks (albeit uncertain) of harm, including a potential for significant harm. Once that threshold burden is satisfied, however, the burden shifts to the activity proponent to resolve the uncertainty and show that that it does not have a potential for significant harm.34The normative core of the strong versions of PP, which distinguishes PP-based regulation from preventive regulation generally, is the principle that uncertainty regarding risks is an affirmative justification for adopting regulatory controls or adopting more stringent controls than would be appropriate in the case of activities posing more determinate risks. In the face of uncertainties regardingEnvironment al Regulat ory Decision Making Under Uncert aint y 797912345678910111111213141516171819201112122232425262728293011131323334353637383940111risk, PP holds that decision makers should err on the side of precaution and envi-ronmental protection and, in effect, make “worst case” presumptions about the probability and magnitude of harm that an activity poses; precisely how “worst case” is defined (“reasonable worst case,” etc.) varies in different PP formula-tions.35The justifications advanced by PP proponents for adopting its prescrip-tions center around limitations in our ability to predict which activities will cause serious, irreversible environmental harms.36The predictive capacity of science is limited. For example, science has often been unable to predict, in a sufficiently timely fashion to support effective preventive action, the occurrence of serious environmental harms such as asbestosis, stratospheric ozone depletion, or the ecological harms caused by DDT. Thus, a regulatory policy that requires regulators to demonstrate that an activity causes harm or even a significant risk of harm before imposing controls will result in the occurrence of serious environmental harms. Some of these harms, such as biodiversity loss or highly disruptive changes in natural systems resulting from rapid global warming, may be irreversible and seriously harm future generations. Accordingly, decision makers should err on the side of pre-caution and protection of the planet by adopting PP-based regulatory controls on activities involving uncertain risks that pose a potential for significant harm.The PP literature provides little in the way of helpful guidance on what regulators must show in order to establish a potential for harm that triggers PP.37While some PP proponents appear to assume that nature is inherently vulnerable and precarious rather than resilient, such a general presumption is not sufficient to show that a given activity triggers PP. The bovine growth hormone dispute suggests that a showing that a substance similar in chemical structure to the substance in question can cause harm may be sufficient.38The BtCorn-Monarch butterfly controversy suggests that a report of a single experimental study, albeit one quite unrepresentative of field conditions, can be enough to trigger PP controls if it is sufficiently widely publicized.39Under strong versions of PP, once the risk posed by an activity satisfies the threshold that triggers a worst case presumption, regulators must then follow a set of relatively stringent regulatory prescriptions. They must prohibit or impose BAT requirements on the activity; shift the burden to the activity proponent to show that the activity is “safe” in order to avoid or lift these regulatory require-ments; and disregard or downplay regulatory costs in implementing regulatory requirements. Thus, PP can be analyzed as containing two basic components:First, a worst case presumption for uncertain risks that meet a triggering threshold. Second, a set of regulatory decision rules that are mandatory once the presumption is triggered. These components can be analyzed separately.80RICHARD B. STEWART12345678910111111213141516171819201112122232425262728293011131323334353637383940111。

定向突变生物合成英文回答：Directed mutagenesis is a technique used in molecular biology to introduce specific changes or mutations into a DNA sequence. This technique allows scientists to study the effects of these mutations on gene function and protein structure. There are several methods available for directed mutagenesis, including site-directed mutagenesis and random mutagenesis.Site-directed mutagenesis is a targeted approach where specific nucleotides in the DNA sequence are changed to create a desired mutation. This can be achieved using techniques such as PCR-based mutagenesis oroligonucleotide-directed mutagenesis. For example, if I want to introduce a point mutation in a gene to study its effect on protein function, I can design a pair of primers that contain the desired mutation and use them in a PCR reaction to amplify the gene with the mutation.Random mutagenesis, on the other hand, introduces random mutations throughout the DNA sequence. This can be done using techniques such as error-prone PCR or chemical mutagenesis. Random mutagenesis is useful when studying the effects of a large number of mutations or when trying to create a library of variants with different properties.Once the mutations are introduced, the mutated DNA sequences can be cloned into an expression vector and used to produce mutant proteins. The mutant proteins can then be purified and characterized to determine their functional and structural properties. For example, if I introduce a mutation in a protein involved in enzyme catalysis, I can compare the catalytic activity of the mutant protein to the wild-type protein to understand the role of the mutated residue in the reaction.Directed mutagenesis has a wide range of applicationsin biotechnology and biomedical research. It can be used to study the function of specific genes and proteins, identify key amino acid residues involved in protein-proteininteractions, and engineer proteins with improved properties. For example, directed mutagenesis has been used to create enzymes with enhanced catalytic activity, antibodies with improved binding affinity, and receptors with altered ligand specificity.中文回答：定向突变是分子生物学中一种用于在DNA序列中引入特定改变或突变的技术。

2024年6月四级英语试卷Part I Writing (30 minutes)Directions: For this part, you are allowed 30 minutes to write an essay on the topic "The Importance of Cultural Heritage Protection". You should write at least 120 words but no more than 180 words.Part II Listening Comprehension (25 minutes)Section A.Directions: In this section, you will hear three news reports. At the end of each news report, you will hear two or three questions. Both the news report and the questions will be spoken only once. After you hear a question, you must choose the best answer from the four choices marked A), B), C) and D).News Report 1.Questions 1 and 2 will be based on the following news item.A new study shows that urban green spaces can significantly reduce the risk of certain diseases. The research, which was conducted over a five - year period in several major cities, found that people living near large parks or gardens had a lower incidence of respiratory and heart diseases compared to those in areas with little or no greenery.1. What did the new study mainly find?A) Urban green spaces are getting smaller.B) People prefer to live near large parks.C) Green spaces can reduce disease risks.D) Respiratory diseases are on the rise.2. How long was the research period?A) Three years.B) Four years.C) Five years.D) Six years.News Report 2.Questions 3 and 4 will be based on the following news item.A well - known tech company has announced a major breakthrough in battery technology. The new battery is said to be more efficient, longer - lasting, and safer than current models. This could have a huge impact on the development of electric vehicles and portable electronics.3. What is the achievement of the tech company?A) A new type of electric vehicle.B) A major breakthrough in battery technology.C) A new marketing strategy for electronics.D) An improvement in portable electronics.4. Which fields could be influenced by this achievement?A) Only electric vehicles.B) Only portable electronics.C) Both electric vehicles and portable electronics.D) Neither electric vehicles nor portable electronics.News Report 3.Questions 5 to 7 will be based on the following news item.A group of international scientists has discovered a new species of deep - sea fish. The fish was found at a depth of over 2,000 meters in the Pacific Ocean. It has unique physical characteristics that distinguish it from other known species.5. Where was the new species of fish discovered?A) In the Atlantic Ocean.B) In the Indian Ocean.C) In the Arctic Ocean.D) In the Pacific Ocean.6. At what depth was the fish found?A) Over 1,000 meters.B) Over 2,000 meters.C) Over 3,000 meters.D) Over 4,000 meters.7. What makes the new fish different from others?A) Its color.B) Its size.C) Its unique physical characteristics.D) Its living habits.Section B.Directions: In this section, you will hear two long conversations. At the end of each conversation, you will hear four questions. Both the conversation and the questions will be spoken only once. After you hear a question, you must choose the best answer from the four choices marked A), B), C) and D).Conversation 1.Questions 8 to 11 will be based on the following conversation.M: Hi, Lisa. How was your weekend?W: It was great. I went to a concert with some friends.M: That sounds fun. Who did you see?W: A local rock band. They were really good.M: I like rock music too. Do you often go to concerts?W: Not really. This was my first time in a while.8. What did Lisa do on the weekend?A) She went to a movie.B) She went to a concert.C) She went to a party.D) She stayed at home.9. What kind of band did Lisa see?A) A pop band.B) A jazz band.C) A rock band.D) A classical band.10. Does Lisa often go to concerts?A) Yes, she does.B) No, she doesn't.C) She used to.D) She never goes.11. What does the man like?A) Pop music.B) Jazz music.C) Rock music.D) Classical music.Conversation 2.Questions 12 to 15 will be based on the following conversation. W: John, I'm thinking about taking a Spanish course.M: That's a great idea. Why do you want to learn Spanish?W: Well, I'm planning a trip to Spain next year, and I think it will be useful.M: You're right. Spanish is also very useful in business these days.W: Do you know any good language schools?M: There's one near my office. I heard it has very good teachers.12. What is the woman thinking about?A) Taking a French course.B) Taking a Spanish course.C) Taking a German course.D) Taking an Italian course.13. Why does the woman want to learn Spanish?A) She is interested in Spanish culture.B) She has Spanish friends.C) She is planning a trip to Spain.D) She wants to work in Spain.14. What else is Spanish useful for according to the man?A) Only for travel.B) Only for culture.C) For business.D) For nothing else.15. What does the man know about a language school?A) It has cheap courses.B) It has good teachers.C) It has a convenient location.D) It has a lot of students.Section C.Directions: In this section, you will hear three passages. At the end of each passage, you will hear three or four questions. Both the passage and the questions will be spoken only once. After you hear a question, you must choose the best answer from the four choices marked A), B), C) and D).Passage 1.Questions 16 to 18 will be based on the following passage.The Internet has changed the way we communicate and access information. However, it also brings some problems, such as information overload and the spread of false information. To deal with these problems, we need to develop good information - literacy skills. This includes being able to evaluate the reliability of sources, distinguish between fact and opinion, and manage the amount of information we consume.16. What has the Internet changed?A) Only the way we communicate.B) Only the way we access information.C) Both the way we communicate and access information.D) Neither the way we communicate nor access information.17. What problems does the Internet bring?A) Only information overload.B) Only the spread of false information.C) Both information overload and the spread of false information.D) Neither information overload nor the spread of false information.18. What skills do we need to develop?A) Technical skills.B) Social skills.C) Information - literacy skills.D) Management skills.Passage 2.Questions 19 to 22 will be based on the following passage.In recent years, more and more people are choosing to live in small towns instead of big cities. There are several reasons for this. First, small towns usually have a lower cost of living. Second, they offer acloser - knit community where people can get to know their neighbors better. Third, the environment in small towns is often cleaner and more peaceful.19. What trend is mentioned in the passage?A) More people are moving to big cities.B) More people are living in small towns.C) People are moving from small towns to big cities.D) People are moving abroad.20. What is one advantage of small towns?A) High - paying jobs.B) A lot of entertainment options.C) A lower cost of living.D) Easy access to big cities.21. What can people do in small towns?A) They can only work.B) They can get to know their neighbors better.C) They can enjoy big - city life.D) They can avoid all problems.22. How is the environment in small towns?A) Dirty and noisy.B) Clean and peaceful.C) Polluted.D) Dangerous.Passage 3.Questions 23 to 25 will be based on the following passage.A new study has found that reading books can have a positive impact on our mental health. People who read regularly are less likely to suffer from depression and anxiety. Reading can also improve our cognitive abilities, such as memory and concentration.23. What has the new study found?A) Reading books is a waste of time.B) Reading books can harm our mental health.C) Reading books can improve our physical health.D) Reading books can have a positive impact on our mental health.24. Who are less likely to suffer from depression and anxiety?A) People who watch a lot of TV.B) People who play video games.C) People who read regularly.D) People who do no hobbies.25. What can reading also improve?A) Our physical strength.B) Our social skills.C) Our cognitive abilities.D) Our appearance.Part III Reading Comprehension (40 minutes)Section A.Directions: In this section, there is a passage with ten blanks. You are required to select one word for each blank from a list of choices given in a word bank following the passage. Read the passage through carefully before making your choices. Each choice in the word bank is identified by a letter. Please mark the corresponding letter for each item on Answer Sheet 2. You may not use any of the words in the word bank more than once.The concept of time is something that humans have been trying to understand for centuries. Time is a fundamental part of our lives, yet itis also one of the most difficult things to define _(26)_. We use time to organize our days, plan our activities, and measure the passage of our lives. But what exactly is time?Some scientists believe that time is a physical property of the universe, like space or matter. Others think that time is a human _(27)_, created by our minds to make sense of the world around us. There are also those who argue that time is an illusion, and that there is no such thing as a past, present, or future.Despite these different views, one thing is certain: time is a precious _(28)_. We all have a limited amount of time in our lives, and we need to make the most of it. This means using our time _(29)_ to achieve our goals, whether they are personal, professional, or academic.One way to make better use of our time is to learn how to manage it effectively. This involves setting _(30)_, prioritizing tasks, and avoiding distractions. Another important aspect of time management is learning to say no to things that are not important or that will take up too much of our time.In addition to managing our time, we can also try to _(31)_ our perception of time. This can be done by engaging in activities that weenjoy, which can make time seem to pass more quickly. On the other hand, when we are bored or waiting for something, time can seem to drag on _(32)_.Finally, it is important to remember that time is not just aboutgetting things done. We also need to take time to relax, enjoy life, and spend time with the people we love. After all, life is not just about work and achievement, but also about _(33)_ and relationships.Word Bank:A) accurately.B) construct.C) efficiently.D) endlessly.E) goals.F) illusion.G) limited.H) manage.I) precious.J) property.K) relaxation.L) resource.M) sense.26. A) accurately.27. M) sense.28. L) resource.29. C) efficiently.30. E) goals.31. B) construct.32. D) endlessly.33. K) relaxation.Section B.Directions: In this section, you are going to read a passage with ten statements attached to it. Each statement contains information given in one of the paragraphs. Identify the paragraph from which the information is derived. You may choose a paragraph more than once. Each paragraph is marked with a letter. Answer the questions by marking the corresponding letter on Answer Sheet 2.The Benefits of Bilingualism.A) Bilingualism, or the ability to speak two languages fluently, has been shown to have numerous benefits. For one thing, it can improve cognitive abilities such as problem - solving, memory, and concentration. Studies have found that bilingual individuals tend to perform better on tasks that require these skills than monolingual individuals.B) Another benefit of bilingualism is that it can enhance cultural understanding. People who are bilingual are often more exposed to different cultures, which can lead to a greater appreciation and understanding of cultural differences. This can be especially important in today'sglobalized world, where cross - cultural communication is becoming increasingly common.C) Bilingualism can also have a positive impact on career opportunities. In many industries, being bilingual is seen as an asset. For example, in international business, bilingual employees can communicate moreeffectively with clients and partners from different countries. In thefield of education, bilingual teachers are in high demand, especially in areas with large immigrant populations.D) In addition to these practical benefits, bilingualism can also havea positive effect on mental health. Research has shown that bilingual individuals are less likely to develop age - related cognitive decline,such as dementia. This may be because using two languages keeps the brain active and stimulated.E) However, learning a second language is not without its challenges. One of the main difficulties is grammar. Different languages have different grammar rules, and it can be difficult to master them all. For example, in some languages, the verb comes at the end of the sentence, while in others, it comes in the middle.F) Another challenge is vocabulary. Building a large vocabulary in a second language can take a long time. There are often many words that have no direct equivalent in one's native language, which can make it difficultto understand and use them correctly.G) Despite these challenges, the benefits of bilingualism far outweigh the difficulties. There are many ways to learn a second language, such as taking classes, using language - learning apps, or immersing oneself in a language - speaking environment.H) One effective way to learn a second language is through immersion. This involves living in a country where the language is spoken or spendinga significant amount of time in such an environment. Immersion can help learners pick up the language more quickly and develop a more natural accent.I) Another way to learn a second language is through language exchange. This involves finding a partner who speaks the language you want to learn and who wants to learn your native language. You can then practice speaking with each other, which can be a fun and effective way to improve yourskills.J) In conclusion, bilingualism is a valuable skill that can bring many benefits. Whether it is for cognitive, cultural, career, or mental health reasons, learning a second language is well worth the effort.34. Bilingual individuals may perform better on tasks requiring problem - solving skills. (A)35. Bilingualism can help people understand cultural differences better.(B)36. In international business, bilingual employees have an advantage.(C)37. Bilingualism can reduce the risk of age - related cognitive decline.(D)38. Grammar is one of the main difficulties in learning a second language. (E)39. Building vocabulary in a second language can be time - consuming.(F)40. Immersion is an effective way to learn a second language. (H)41. Language exchange can be a fun way to improve language skills. (I)42. The benefits of bilingualism are greater than the difficulties. (G)43. Bilingualism has many benefits for various reasons. (J)Section C.Directions: There are 2 passages in this section. Each passage is followed by some questions or unfinished statements. For each of them there are four choices marked A), B), C) and D). You should decide on the best choice and mark the corresponding letter on Answer Sheet 2.Passage 1.The sharing economy has emerged as a significant economic trend in recent years. Platforms like Airbnb and Uber have revolutionized the way people travel and get around. These platforms allow individuals to share their resources, such as spare rooms or cars, with others in exchange for money.One of the main advantages of the sharing economy is that it can make better use of underutilized resources. For example, many people have spare rooms in their homes that are empty most of the time. By listing these rooms on Airbnb, they can earn extra income while also providing accommodation for travelers. Similarly, cars are often parked and unusedfor long periods. Through Uber or other ride - sharing services, car owners can use their vehicles to earn money when they are not using them.Another advantage is that the sharing economy can provide more affordable options for consumers. Airbnb often offers cheaper accommodationthan hotels, especially in popular tourist destinations. Ride - sharing services can also be less expensive than traditional taxis in some cases.However, the sharing economy also faces some challenges. One issue is regulation. Since these platforms are relatively new, there are often no clear regulations governing their operations. This can lead to problems such as safety concerns and unfair.。

ReviewRecent findings on the phytoremediation of soils contaminated with environmentally toxic heavy metals and metalloids such as zinc,cadmium,lead,and arsenicI.Alkorta 1,J.Hernandez-Allica 2,J.M.Becerril 3,I.Amezaga 3,I.Albizu 2&C.Garbisu 2,*1Unidad de Biofı´s ica,Centro Mixto UPV/EHU,Apdo.644,E-48080Bilbao,Spain;2NEIKER,BasqueInstitute of Agricultural Research and Development,Berreaga 1,E-48160Derio,Spain;3Department of Plant Biology and Ecology,University of the Basque Country,Apdo.644,E-48080Bilbao,Spain (*author for correspondence:phone:+34-94-403-43-00;fax:+34-94-403-43-10;e-mail:cgarbisu@)Key words:metalloids,metals,phytochelatins,phytoextraction,phytoremediation,transgenic plants AbstractDue to their immutable nature,metals are a group of pollutants of much concern.As a result of human activities such as mining and smelting of metalliferous ores,electroplating,gas exhaust,energy and fuel production,fertilizer and pesticide application,etc.,metal pollution has become one of the most serious environmental problems today.Phytoremediation,an emerging cost-effective,non-intrusive,and aesthet-ically pleasing technology,that uses the remarkable ability of plants to concentrate elements and com-pounds from the environment and to metabolize various molecules in their tissues,appears very promising for the removal of pollutants from the environment.Within this field of phytoremediation,the utilization of plants to transport and concentrate metals from the soil into the harvestable parts of roots and above-ground shoots,i.e.,phytoextraction,may be,at present,approaching commercialization.Improvement of the capacity of plants to tolerate and accumulate metals by genetic engineering should open up new possibilities for phytoremediation.The lack of understanding pertaining to metal uptake and translocation mechanisms,enhancement amendments,and external effects of phytoremediation is hindering its full scale application.Due to its great potential as a viable alternative to traditional contaminated land remediation methods,phytoremediation is currently an exciting area of active research.1.Environmental metal pollution and phytoremediationSoil pollution has recently been attracting consid-erable public attention since the magnitude of the problem in our soils calls for immediate action (Garbisu &Alkorta 2003).As a result of human activities such as mining and smelting of metallif-erous,electroplating,gas exhaust,energy and fuel production,fertilizer and pesticide application,etc.,metal pollution has become one of the most serious environmental problems today.Due to their immutable nature,metals are a group of pollutants of much concern.In fact,although several metals are essential for biological systems and must be present within a certain concentration range (Garbisu &Alkorta 2003),at high concen-trations,metals can act in a deleterious manner by blocking essential functional groups,displacing other metal ions,or modifying the active confor-mation of biological molecules (Collins &Stotzky 1989).Metal toxicity for living organisms involves oxidative and/or genotoxic mechanisms (Briat &Lebrun 1999).Based on their chemical and physical proper-ties,three different molecular mechanisms of heavy metal toxicity can be distinguished:(i)pro-duction of reactive species by autooxidation and Fenton reaction (Fe,Cu),(ii)blocking of essential functional groups in biomolecules (Cd,Hg),and (iii)displacement of essential metal ions from biomolecules (Schutzendubel &Polle 2002).Reviews in Environmental Science and Bio/Technology 3:71–90,2004.Ó2004Kluwer Academic Publishers.Printed in the Netherlands.71Metal-contaminated soils are notoriously hard to remediate.Current technologies resort to soil excavation and either landfilling or soil washing followed by physical or chemical separation of the contaminants.Although highly variable and dependent on the contaminants of concern,soil properties,site conditions,and so on,the usually enormous costs associated with the removal of metals from soils by means of traditional physi-cochemical methods explain why most companies tend to ignore the problem.Due to the fact that very often large areas are affected by heavy metal contamination,a removal is certainly difficult. Therefore,some methods are developed to keep the metals in the soil but reduce the risks related to this presence(e.g.,by decreasing bioavailability by in situ immobilisation processes)(Diels et al. 2002).One way to facilitate such immobilisation is by altering the physicochemical properties of the metal-soil complex by introducing a multipurpose anion,such as phosphate,that enhances metal adsorption via anion-induced negative charge and metal precipitation(Bolan et al.2003a).Heavy metals cannot be destroyed biologically (no‘‘degradation’’,change in the nuclear struc-ture of the element,occurs)but are only trans-formed from one oxidation state or organic complex to another(Garbisu&Alkorta2001). Although microorganisms that use metals as ter-minal electron acceptors or reduce them as part of a detoxification mechanism can be used for metal remediation(Garbisu&Alkorta1997), when considering the remediation of metal-pol-luted soil,metal-accumulating plants offer numerous advantages over microbial processes since plants can actually extract metals from the polluted soils,theoretically rendering them clean (metal-free)(Garbisu&Alkorta2001;Garbisu et al.2002).Phytoremediation,the use of plants to extract, sequester,and/or detoxify pollutants,has been reported to be an effective,non-intrusive,inex-pensive,aesthetically pleasing,socially accepted technology to remediate polluted soils(Alkorta& Garbisu2001;Weber et al.2001;Garbisu et al. 2002).Phytoremediation is widely viewed as the ecologically responsible alternative to the envi-ronmentally destructive physical remediation methods currently practiced(Meagher2000).The US phytoremediation market is expected to ex-pand more than ten-fold between1998and2005, to over$214million(Evans2002).In the last few years,some excellent reviews have been published focusing on different aspects of phytoremediation(Salt et al.1995a,1998;Chaney et al.1997;Raskin et al.1997;Chaudhry et al. 1998;Wenzel et al.1999;Meagher2000;Navari-Izzo&Quartacci2001;Lasat2002;McGrath et al. 2002;McGrath&Zhao2003;McIntyre2003;Singh et al.2003).In any case,and in contrast to its many positive aspects,phytoremediation does have cer-tain disadvantages and limitations(Table1).Table1.Advantages and limitations of the phytoremediation technology Advantages LimitationsApplicable to a wide variety of inorganic and organic contaminants.Limited by depth(roots)and solubility and availability of the contaminant.Reduces the amount of waste going to landfills.Although faster than natural attenuation,it requires long timeperiods(several years).Does not require expensive equipment or highlyspecialized personnel.Restricted to sites with low contaminant concentration.It can be applied in situ.Reduces soil disturbance and the spread of contaminants.Plant biomass from phytoextraction requires proper disposal as hazardous waste.Early estimates of the costs indicate that phytoremediation is cheaper than conventional remediation methods.Climate and season dependent.It can also lose its effectiveness when damage occurs to the vegetation from disease or pests.Easy to implement and maintain.Plants are a cheap and renewable resource,easily available.Introduction of inappropriate or invasive plant species should be avoided(non-native species may affect biodiversity).Environmentally friendly,aesthetically pleasing,socially accepted,low-tech alternative.Contaminants may be transferred to another medium,the environment,and/or the food chain.Less noisy than other remediation methods.Actually, trees may reduce noise from industrial activities.Amendments and cultivation practices may have negative consequences on contaminant mobility.72Within thefield of phytoremediation,different categories have been defined such as,among others, phytoextraction,phytofiltration(rhizofiltration, blastofiltration),phytostabilization,phytovolatil-ization,phytodegradation(phytotransformation), plant-assisted bioremediation(plant-assisted deg-radation,plant-aided in situ biodegradation,phyt-ostimulation,enhanced rhizosphere degradation, rhizodegradation),etc.(Table2).Plants for phytoextraction,i.e.,metal removal from soil,should have the following characteris-tics:(i)tolerant to high levels of the metal,(ii) accumulate reasonably high levels of the metal, (iii)rapid growth rate,(iv)produce reasonably high biomass in thefield,and(v)profuse root system(Garbisu et al.2002).The idea of using plants to remediate metal polluted soils came from the discovery of‘‘hyper-accumulators’’(Table3),defined as plants,often endemic to naturally mineralized soils,that accu-mulate high concentrations of metals in their fo-liage(Baker&Brooks1989;Raskin et al.1997; Brooks1998).In fact,plants growing on metal-liferous soils can be grouped into three categories according to Baker(1981):(i)excluders,where metal concentrations in the shoot are maintained, up to a critical value,at a low level across a wide range of soil concentration;(ii)accumulators, where metals are concentrated in above-ground plant parts from low to high soil concentrations; and(iii)indicators,where internal concentration reflects external levels(McGrath et al.2002).The criterion for defining Ni hyperaccumulation is 1000l g Ni g)1on a dry leaf basis(Brooks et al. 1977),whereas for Zn and Mn the threshold is 10,000l g g)1and for Cd100l g Cd g)1.Finally, the criterion for Co,Cu,Pb and Se hyperaccu-mulation is also1,000l g g)1in shoot dry matter (Brooks1998;Baker et al.2000;McGrath et al. 2002).In general terms,metal concentrations in hyperaccumulators are about100–1000-fold high-er than those found in normal plants growing on soils with background metal concentrations,and about10–100-fold higher than most other plants growing on metal-comtaminated soils(McGrath et al.2002).Hyperaccumulators are also charac-terized by a shoot-to-root metal concentration ratio of>1(i.e.,hyperaccumulator plants show a highly efficient transport of metals from roots to shoots),whereas non-hyperaccumulators usually have higher metal concentrations in roots than in shoots(Baker1981;Gabbrielli et al.1990;HomerTable2.Categories of phytoremediationTerm DefinitionPhytoextraction The use of plants to remove pollutants(mostly,metals)from soils.Phytofiltration The use of plants roots(rhizofiltration)or seedlings(blastofiltration)to absorbor adsorb pollutants(mostly,metals)from water.Phytostabilization The use of plants to reduce the bioavailability of pollutants in the environment. Phytovolatilization The use of plants to volatilize pollutants.Phytodegradation The use of plants to degrade organic pollutantsPhytotransformationPhytostimulation The use of plant roots in conjunction with their rhizospheric microorganisms toremediate soils contaminated with organics.Enhanced rhizosphere degradationRhizodegradationPlant-assisted bioremediationPlant-asssisted degradationPlant-aided in situ biodegradationTable3.Examples of hyperaccumulatorsMetal SpeciesZinc(Zn)T.caerulescensCadmium(Cd)T.caerulescensNickel(Ni)Berkheya coddiiSelenium(Se)Astragalus racemosaThallium(Tl)Iberis intermediaCopper(Cu)Ipomoea alpinaCobalt(Co)Haumaniastrum robertiiArsenic(As)P.vittata73et al.1991;Baker et al.1994a,b;Brown et al. 1995;Kra mer et al.1996;Shen et al.1997;Zhao et al.2000;McGrath et al.2002).Hyperaccumulation of heavy metal ions is in-deed a striking phenomenon exhibited by<0.2% of angiosperms(Baker&Whiting2002).Most recently,a plethora of papers are being published in an attempt to dissect the mechanisms of metal uptake,transport and accumulation,both at the physiological and molecular level(Baker& Whiting2002).The‘‘model’’hyperaccumulator Thlaspi caerulescens has been much screened in the search for new and more extreme ecotypes (McGrath et al.2001;Lombi et al.2002).Our understanding of the internal processes that confer the hyperaccumulation phenotype is advancing in leaps and bounds,and the mechanisms of trans-port,tolerance and sequestration in some species, at least in the genera Thlaspi and Alyssum,are partially elucidated(Lasat2002).Trees have also been considered for phyto-remediation of heavy metal-contaminated land, with willow and poplar being promising candi-dates,among others,in this respect(Pulford& Watson2003).According to some authors,trees potentially are the lowest-cost plant type to use for phytoremediation(Stomp et al.1994).Many trees can grow on land of marginal quality,have mas-sive root systems,and their above-ground biomass can be harvested with subsequent resprouting without disturbance of the site(Stomp et al.1994).Following the harvest of metal-enriched plants, the weight and volume of the contaminated material can be further reduced by ashing or composting(Garbisu&Alkorta2001;Garbisu et al.2002).Metal-enriched plants can be disposed of as hazardous material or,if economically fea-sible,used for metal recovery(Salt et al.1998). Recently,some studies have reported on the utili-zation of pyrolysis to separate heavy metals from hyperaccumulators(Koppolu&Clements2003).Although plants acquire essential minerals such as Fe,Cu,Ni,Zn and Se from the soil,for reasons that are not yet clear,they also have the ability to acquire and detoxify non-essential elements such as As,Cd,Cr and Pb(Salt et al.2002).Certain themes in the physiology and biochemistry of trace element accumulation by plants appear common (Salt et al.2002).Most phytoremediation studies have consid-ered metal extraction efficiency in relation to metal concentration of bulk soil samples or metal con-centration of the soil solution,but little is known about the effect of various metal-bearing solids on metal extraction by hyperaccumulators.In fact,it has been shown that it is essential to consider the nature of the metal-bearing solids to better predict the efficiency of plant extraction(Dahmani-Muller et al.2001).Besides,it is also important to con-sider that metal bioavailability changes between the bulk soil and the rhizosphere,the latter being a microbiosphere which has quite different chemical, physical and biological properties from bulk soils (Wang et al.2002b).In this respect,recently,it has been reported that root growth is a more sensitive endpoint of metal availability than chlorophyll assays(Morgan et al.2002).In order to improve phytoremediation of heavy metal polluted sites, the speciation and bioavailability of the metals in the soil,the role of plant-associated soil microor-ganisms and fungi in phytoremediation,and that of plants have to be elucidated(Kamnev&van der Lelie2000).Phytoremediation has been used in mined soil restoration,since these soils are sources of air and water pollution,by means of phytostabilization and phytoextraction techniques to stabilize toxic mine spoils and remove toxic metals from the spoils,respectively(Wong2003).Some higher plant species have developed heavy metal tolerance strategies which enable them to survive and reproduce in highly-metal contam-inated soils.Dahmani-Muller et al.(2000)inves-tigated metal uptake and accumulation strategies of two absolute metallophyte species and one pseudometallophyte.In the former two species, real hyperaccumulation in the leaves as well as metal immobilisation in roots and/or a detoxifi-cation mechanism by leaf fall were found as possible strategies to deal with the high metal concentrations.By contrast,the strategy of the pseudometallophyte,i.e.,Agrostis tenuis,pre-sented a significant metal immobilisation by the roots.Most plants have mycorrhizal fungi associated with them,providing their hosts with an increased capacity to absorb water and nutrients from the soil.The formation and function of mycorrhizal relationships are affected by anthropogenic stressors including metals(Entry et al.2002). Arbuscular mycorrhizal fungi are of interest for their reported roles in alleviation of diverse74soil-associated plant stressors,including those in-duced by metals,so it has been claimed that theevaluation of the efficacy of plant-mycorrhizal associations to remediate metal-polluted soils de-serves increased attention (Entry et al.2002).In addition,phytoextraction practices, e.g.,the choice of plant species and soil amendments,may have a great influence on the quantity and species composition of glomalean propagules as well as on arbuscular mycorrhizal fungi functioning during long-term metal-remediation treatments (Paw-lowska et al.2000).A unique testing system,the target-neighbour method,has been described to allow evaluation of how planting density influences metal uptake,so that the information needed to manipulate plant density for optimization of metal removal could be obtained (Shann 1995).Finally,most recently,phytoremediation has been combined with electrokinetic remediation,applying a constant voltage of 30V across the soil,concluding that the combination of both tech-niques represents a very promising approach to the decontamination of metal polluted soils (O 0Connor et al.2003).2.New findings on the phytoextraction of some of the most relevant environmentally toxic heavy metals (zinc,cadmium,lead)and metalloids (arsenic)This section is divided into four different sub-headings.The first three correspond to three of the most environmentally relevant heavy metals,i.e.,Zn,Cd,and Pb.The fourth sub-heading deals with As,a well-known toxic metalloid.It is important to emphasize here that very often information regarding one metal appears under a different,apparently wrong,sub-heading.Since many of the reviewed publications deal with more than one metal at the same time,it has been preferred to present them as part of the same research,despite the fact that section structure could not be main-tained as desired.2.1.ZincZinc and Cd are ubiquitous pollutants that tend to occur together at many contaminated sites.While Zn is often phytotoxic,Cd rarely inhibits plant growth.In T.caerulescens ,an integrated molecular and physiological investigation of the fundamental mechanisms of heavy metal accumulation was conducted (Pence et al.2000).A metal transporter cDNA,znt1(expressed at very high levels in roots and shoots of this plant),was cloned from T.caerulescens through functional complementa-tion in yeast and was shown to mediate high-affinity Zn uptake as well as low affinity Cd uptake.Alteration in the regulation of znt1gene expression by plant Zn status results in the over-expression of this transporter and in increased Zn influx in roots,even when intracellular Zn levels are high.Thus,specific alterations in Zn-respon-sive elements (e.g.,transcriptional activators)possibly play an important role in Zn hyperaccu-mulation in T.caerulescens (Pence et al.2000).In this respect,Lasat et al.(1998)found that the en-hanced root-to-shoot Zn transport in T.caerules-cens was,at least partly,achieved through an altered Zn compartmentation in the root sym-plasm,which reduces Zn sequestration in root vacuoles.A further step at elucidating the mech-anisms underlying Zn hyperaccumulation was gi-ven thanks to the cloning of metal transporter genes encoding putative vacuolar ion transportproteins in T.caerulescens (Assunc ao et al.2001).Cd NiZnPbAsCuCoTlRoot uptakeTranslocationAccumulationHarvestingDisposal/Recovery Figure 1.Phytoextraction of metals.75In fact,ZTP1(a transporter belonging to the cat-ion-efflux family)has been found to be highly ex-pressed in T.caerulescens,predominantly in leaves (Assunc a o et al.2001).Long et al.(2002)have recently described a large biomass Zn hyperaccumulating plant,i.e., Sedum alfredii.Similarly,Wang et al.(2003)have reported on the discovery of two new plants with potential for phytoremediation of Zn-polluted soils,i.e.,Polygonum hydropiper and Rumex ace-tosa.In the same study,the authors indicate that the consumption of rice grown in paddy soils contaminated with Cd,Cr or Zn may pose a serious risk to human health,because from22to 24%of the total metal content in the rice biomass was concentrated in the rice grain.Interestingly, Platanus acerifolia growing on heavily contami-nated soil accumulated only very low levels of heavy metals,and this mechanism for excluding metal uptake may have value in crop improvement (Wang et al.2003).Holcus lanatus L.genotypes tolerant to Zn toxicity seem to grow better than Zn-sensitive genotypes,even in Zn-deficient soil,because of their greater capacity for taking up Zn from Zn-deficient soil,revealing the coexistence of traits for tolerance to Zn toxicity and Zn deficiency in a single plant genotype(Rengel2000).Metal responses in the metallophyte Arabidop-sis halleri,a close relative to the model plant A.thaliana,that is Cd hypertolerant and Zn hy-peraccumulating,have been studied,and metal-regulated genes isolated and molecularly analyzed as interesting candidate genes for phytoremedia-tion(Bert et al.2000;Dahmani-Muller et al.2000; Clemens2001;Macnair2002).Unlike Thlaspi, A.halleri seems to be largely allogamous,seeds profusely over a longer growing season and can also spread vegetatively by stolons(Baker& Whiting2002).Macnair(2002)demonstrated that the heritability of Zn accumulation was between 25and50%,the highest yet recorded for any hy-peraccumulator,probably because of the out-breeding nature of A.halleri.Intriguingly,the genetic variability of Zn accumulation in A.halleri was manifested more when grown in low-Zn media than in high(Baker&Whiting2002).Zinc-tolerant callus lines of Brassica spp.have been developed,and this might help in the selection and characterization of heavy metal tolerance in plants for breeding programmes(Rout et al.1999).In a paper focusing on soil solution Zn and pH dynamics during phytoextraction using T.cae-rulescens J.&C.Presl,data indicate that the po-tential of this hyperaccumulator to remove Zn from contaminated soil may not be related either to acidification of the rhizosphere(McGrath et al. 1997;Luo et al.2000)or to exudation of specific metal-mobilizing compounds(Zhao et al.2001). Intriguingly enough,some studies have provided evidence suggesting that roots of T.caerulescens are able to sense and actively forage in the Zn rich patches in soil(Schwartz et al.1999;Whiting et al. 2000).A modified glass bead compartment cultiva-tion system for studies on nutrient and trace metal uptake by arbuscular mycorrhiza using two host plant species,maize(Zea mays L.)and red clover(Trifolium pratense L.),and two arbuscu-lar mycorrhizal fungi,Glomus mossae and G. versiforme,found a striking,very high affinity of the fungal mycelium for Zn,suggesting the po-tential use of arbuscular mycorrhiza in the phy-toremediation of Zn-polluted soils(Chen et al. 2001).2.2.CadmiumCadmium is one of the more mobile heavy metals in the soil-plant system,easily taken up by plants and with no essential function known to date (Lehoczky et al.2000).This element can accumu-late in plants without causing toxicity symptoms (Lehoczky et al.1998).In soybean plants,results reveal that the con-tent of Cd in different parts of the plants was roots>>stems>seeds,indicating that the accu-mulation of Cd by roots is much larger than that of any other part of the soybean plant,and might cause deleterious effects to root systems(i.e.,de-creased nodulation,changes in the ultrastructure of root nodule,etc.)(Chen et al.2003).It has been suggested that vetiver grass could be used to remediate Cd-polluted soil,since it accu-mulated218g Cd ha)1at a soil Cd concentration of0.33mg Cd kg)1(Chen et al.2000).Although sequestration of Cd by rhizosphere microorgan-isms may have an important influence on plant Cd uptake,further research is still required to estab-lish whether the accumulation of Cd by rhizobacteria inhibits,or accelerates,Cd uptake by the host plant(Robinson et al.2001).76Recent works have aimed to identify the role of antioxidative metabolism in heavy metal tolerance in T.caerulescens(Boominathan&Doran2003a, b).Hairy roots were used to test the effects of high Cd environments,demonstrating that metal-in-duced oxidative stress occurs in hyperaccumulator tissues even though growth is unaffected by the presence of heavy metals.Superior antioxidant defenses,particularly catalase activity,may play an important role in the hyperaccumulator phe-notype of T.caerulescens.Phragmites australis plants were exposed to a high concentration of Cd,finding out that most of this element accumulated in roots,followed by leaves(Iannelli et al.2002).In roots from Cd-treated plants,both the high amount of glutathione and the parallel increase of glutathione-S-transferase activity seemed to be associated with an induction of the detoxification processes in response to the high Cd concentration.Superoxide dismutase,ascorbate peroxidase,glutathione reductase and catalase activities as well as reduced and oxidized glutathione contents in all samples of leaves,roots and stolons were increased in the presence of Cd.Despite the fact that Cd has a redox characteristic not compatible with the Fenton-type chemistry that produces active oxygen species,Cd tolerance in Phragmites plants might be associated to the efficiency of these mechanisms.The effect of Fe status on the uptake of Cd and Zn by two ecotypes of T.caerulescens,i.e.,Gan-ges and Prayon(the former being far superior in Cd uptake)was studied(Lombi et al.2002). Moreover,the T.caerulescens zip(Zn-regulated transporter/Fe-regulated transporter-like protein) genes,TcZNT1-G and TcIRT1-G,were cloned from Ganges and their expression under Fe-suf-ficient and-deficient conditions analyzed.Cad-mium uptake was significantly enhanced by Fe deficiency in Ganges,while Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes.These results are in agreement with the gene expression study,since the abun-dance of ZNT1-G mRNA was always similar, independently of the Fe status or ecotype,while that of TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions,suggesting a possible relationship with an up-regulation in the expression of genes encoding Fe uptake,possibly TcIRT1-G(Lombi et al.2002).Recently,Song et al.(2003)reported on the utility of the yeast protein YCF1,a protein which detoxifies Cd by transporting it into vacuoles,for the remediation of Cd and Pb contamination,finding out that transgenic Arabidopsis thaliana plants overexpressing YCF1showed enhanced tolerance and accumulated greater amounts of Cd and Pb.Interestingly enough,Lahner et al.(2003) analyzed several essential and nonessential ele-ments in shoots of6000mutagenized A.thaliana plants,demonstrating the utility of genomic scale profiling of nutrients and trace elements as a functional genomics tools.A better understanding of how plants handle mineral elements has the potential of yielding new phytoremediation capa-bilities(Rea,2003).While Cd detoxification is certainly a complex phenomenon,probably under polygenic control, Cd real tolerance found in mine plants seems to be a simpler phenomenon,possibly involving only monogenic/oligogenic control(Sanita di Toppi& Gabbrielli1999).These authors concluded that adaptive tolerance is supported by constitutive detoxification mechanisms,which in turn rely on constitutive homeostatic processes.Symmetric and asymmetric somatic hybridiza-tions have been used to introduce toxic metal-resistant traits from T.caerulescens into Brassica juncea(Dushenkov et al.2002).B.juncea hypo-cotyl protoplasts were fused with T.caerulescens mesophyll protoplasts,and all putative hybrids had morphological characteristics of B.juncea. Hybrid plants,produced by asymmetric somatic hybridization between the two species,demon-strated high metal accumulation potential,toler-ance to toxic metals,and good biomass production.In any case,B.juncea is by itself a good candidate for efficient phytoextraction of heavy metals-such as Cd-from polluted soils (Schneider et al.1999).Two years after the toxic spill caused by the failure of a tailing pond dam at the Aznalcollar pyrite mine(SW Spain)in1998,none of the trace elements measured-As,Cd,Cu,Pb,Tl-reached levels either phytotoxic or toxic for humans or animals in seeds and the above-ground part of spill-affected sunflower plants(Madejon et al. 2003).However,the potential for phytoextraction of these plants is very low,though they might be useful for soil conservation.The production of oil (usable for industrial purposes,whose production77。

Dynamic Transcriptome Landscape of Maize Embryo and Endosperm Development1[W][OPEN]Jian Chen2,Biao Zeng2,Mei Zhang,Shaojun Xie,Gaokui Wang,Andrew Hauck,and Jinsheng Lai*State Key Laboratory of Agro-biotechnology and National Maize Improvement Center,Department of Plant Genetics and Breeding,China Agricultural University,Beijing100193,People’s Republic of ChinaMaize(Zea mays)is an excellent cereal model for research on seed development because of its relatively large size for both embryo and endosperm.Despite the importance of seed in agriculture,the genome-wide transcriptome pattern throughout seed development has not been well ing high-throughput RNA sequencing,we developed a spatio-temporal transcriptome atlas of B73maize seed development based on53samples from fertilization to maturity for embryo, endosperm,and whole seed tissues.A total of26,105genes were found to be involved in programming seed development,in-cluding1,614transcription factors.Global comparisons of gene expression highlighted the fundamental transcriptomic repro-gramming and the phases of development.Coexpression analysis provided further insight into the dynamic reprogramming of the transcriptome by revealing functional transitions during bined with the published nonseed high-throughput RNA sequencing data,we identified91transcription factors and1,167other seed-specific genes,which should help elucidate key mechanisms and regulatory networks that underlie seed development.In addition,correlation of gene expression with the pattern of DNA methylation revealed that hypomethylation of the gene body region should be an important factor for the expressional activation of seed-specific genes,especially for extremely highly expressed genes such as zeins.This study provides a valuable resource for understanding the genetic control of seed development of monocotyledon plants.Maize(Zea mays)is one of the most important crops and provides resources for food,feed,and biofuel (Godfray et al.,2010).It has also been used as a model system to study diverse biological phenomena,such as transposons,heterosis,imprinting,and genetic diversity (Bennetzen and Hake,2009).The seed is a key organ of maize that consists of the embryo,endosperm,and seed coat.Maize seed development initiates from a double fertilization event in which two pollen sperm fuse with the egg and central cells of the female gametophyte to produce the progenitors of the embryo and endosperm, respectively(Dumas and Mogensen,1993;Chaudhury et al.,2001).The mature embryo inherits the genetic information for the next plant generation(Scanlon and Takacs,2009),whereas the endosperm,which is storage tissue for the embryo,persists throughout seed devel-opment and functions as the site of starch and protein synthesis(Sabelli and Larkins,2009).Elucidation of the genetic regulatory mechanisms involved in maize seed development will facilitate the design of strategies to improve yield and quality,and provide insight that is applicable to other monocotyledon plants.A key means to explore the mechanisms of seed de-velopment is to identify gene activities and functions. Genetic studies have uncovered a number of genes that play major roles in governing embryogenesis and ac-cumulation of endosperm storage compounds,such as Viviparous1,KNOTTED1,Indeterminate gametophyte1, Shrunken1(Sh1),Opaque2(O2),and Defective kernel1 (Chourey and Nelson,1976;McCarty et al.,1991;Smith et al.,1995;Vicente-Carbajosa et al.,1997;Lid et al., 2002;Evans,2007).Furthermore,the activity of some genes has also been extensively studied.Typical exam-ples are zein genes that encode primary storage proteins in endosperm.Woo et al.(2001)examined zein gene expression and showed that they were the most highly expressed genes in endosperm based on EST data,where-as their dynamic expression patterns were revealed in a later study(Feng et al.,2009).Nevertheless,informa-tion on the global gene expression network throughout seed development is still very limited.The transcriptome is the overall set of transcripts, which varies based on cell or tissue type,develop-mental stage,and physiological condition.Analysis of transcriptome dynamics aids in implying the function of unannotated genes,identifying genes that act as critical network hubs,and interpreting the cellular pro-cesses associated with development.In Arabidopsis (Arabidopsis thaliana),the genes expressed in devel-oping seed and its subregions at several develop-ment stages have been analyzed with Affymetrix GeneChips(Le et al.,2010;Belmonte et al.,2013).In1This work was supported by the National High Technology Re-search and Development Program of China(863Project,grant no. 2012AA10A305to J.L.)and the National Natural Science Foundation of China(grant no.31225020to J.L.).2These authors contributed equally to the article.*Address correspondence to jlai@.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors()is: Jinsheng Lai(jlai@).[W]The online version of this article contains Web-only data.[OPEN]Articles can be viewed online without a subscription./cgi/doi/10.1104/pp.114.240689maize,microarray-based atlases of global transcription have provided insight into the programs controlling development of different organ systems (Sekhon et al.,2011).Compared with microarray,high-throughput RNA sequencing (RNA-seq)is a powerful tool to com-prehensively investigate the transcriptome at a much lower cost,but with higher sensitivity and accuracy (Wang et al.,2009b).Several studies have taken ad-vantage of the RNA-seq strategy to interpret the dy-namic reprogramming of the transcriptome during leaf,shoot apical meristem,and embryonic leaf develop-ment in maize (Li et al.,2010;Takacs et al.,2012;Liu et al.,2013).To date,only two studies have focused on identi-fying important regulators and processes required for embryo,endosperm,and/or whole seed development in maize based on a genome-wide transcriptional pro-file produced by RNA-seq (Liu et al.,2008;Teoh et al.,2013).However,these studies were limited by the low number of samples used and they did not provide an extensive,global view of transcriptome dynamics over the majority of seed development stages.Here,we pres-ent a comprehensive transcriptome study of maize em-bryo,endosperm,and whole seed tissue from fertilization to maturity using RNA-seq,which serves as a valu-able resource for analyzing gene function on a global scale and elucidating the developmental processes of maize seed.RESULTSGeneration and Analysis of the RNA-seq Data SetTo systematically investigate the dynamics of the maize seed transcriptome over development,we generated RNA-seq libraries of B73seed tissues from different developmental stages,including 15embryo,17endo-sperm,and 21whole seed samples (Fig.1).Utilizing paired-end Illumina sequencing technology,we gen-erated around 1.9billion high-quality reads,80.2%of which could be uniquely mapped to the B73referencegenome (Schnable et al.,2009;Supplemental Table S1).The genic distribution of reads was 66.6%exonic,25.6%splice junction,and 2.6%intronic,leaving about 5%from unannotated genomic regions,demonstrating that most of the detected genes have been annotated.Uniquely mapped reads were used to estimate nor-malized transcription level as reads per kilobase per million (RPKM).To reduce the in fluence of transcrip-tion noise,genes from the B73filtered gene set (FGS)were included for analysis only if their RPKM values were $1.Considering that our purpose was not to identify minor differential expression of genes between two time points of development,but to provide an atlas of gene expression pro file across tissues using time se-ries biological samples,we only randomly selected 12samples to have a biological replicate (Supplemental Fig.S1)to assess our data parisons of bio-logical replicates showed that their expression values were highly correlated (average R 2=0.96).For the samples with biological replicates,we took the average RPKM as the expression quantity.To further evaluate the quality of our expression data,we compared the transcript abundance patterns of a number of selected genes with previously measured expression pro files (Supplemental Fig.S2).For example,LEAFY COTYLE-DON1(LEC1),which functions in embryogenesis,was mainly expressed in the early stage of the embryo (Lotan et al.,1998).Globulin2(Glb2)had high expres-sion late in embryogenesis,in accordance with its func-tion as an important storage protein in the embryo (Kriz,1989).Similarly,O2,a transcription factor (TF)that regulates zein synthesis (Vicente-Carbajosa et al.,1997),and Fertilization independent endosperm1(Fie1),a repressor of endosperm development in the absence of fertilization (Danilevskaya et al.,2003),were almost ex-clusively expressed in the endosperm.Expression pat-terns of these selected genes were identi fied exclusively in their known tissue of activity,indicating that the embryo and endosperm samples were processed well.In total,we detected 26,105genes expressed in at least 1of the 53samples (Supplemental Data Set S1).The distribution of these genes is revealed by aVennFigure 1.Overview of the time series maize seed samples used for RNA-seq analysis.The photographs show the changes in maize embryo,endosperm,and whole seed during development.The 53samples shown here were used to generate RNA-seq libraries.Bar =5mm.Transcriptome Dynamics of Maize Seeddiagram (Fig.2A),which shows that 20,360genes were common among all three tissue types.The number of genes detected in endosperm tissue during the devel-opmental stages was lower and much more variable compared with embryo or whole seed tissue,and a greater number of genes were expressed in the tissue during the early and late phases.Several thousand fewer expressed genes were detected 14d after polli-nation (DAP)in the endosperm compared with 6or 8DAP (Fig.2B;Supplemental Data Set S2).In addition,the median expression level in the embryo was roughly 2-fold greater than that of the endosperm from 10to 30DAP (Fig.2C).Of the 1,506genes unique to the whole seed samples,451were present in RNA-seq data of 14and/or 25DAP pericarp tissue (Morohashi et al.,2012).Considering that the maternal tissue is the vast majority of the content of the early seed (Márton et al.,2005;Pennington et al.,2008),we inferred that most of these genes might be expressed exclusively in maternal tissue such as the pericarp or nucellus.Moreover,1,062genes in the embryo and endosperm with low expression were not detected in whole seed samples (Fig.2C).Division of Development Phases by Global Gene Expression PatternsTo gain insight into the relationships among the dif-ferent transcriptomes,we performed principal compo-nent analysis (PCA)on the complete data set,which can graphically display the transcriptional signatures and developmental similarity.The first component(40.7%variance explained)separated samples based on tissue identity and clearly distinguished embryo from endosperm samples,with whole seed samples located in between (Fig.3A).The second component (24.4%variance explained)discriminated early,mid-dle,and late stages of development for all three tissues (Fig.3A).The wider area occupied by endosperm sam-ples than embryo demonstrates stronger transcriptome reprogramming in developing endosperm,which is mainly attributable to drastic changes in the early and late stages.Moreover,whole seed samples of 0to 8DAP and 30to 38DAP clustered closely to the embryo,but 10to 28DAP samples were close to the endosperm.Cluster analysis of the time series data for the tissues grouped samples well along the axis of developmental time (Fig.3,B –D).Embryo samples from 10to 20DAP and 22to 38DAP were the primary clusters,which correspond to morphogenesis and maturation phases of development (Fig.3B).This is consistent with the embryo undergoing active DNA synthesis,cell division,and differentiation,and then switching to synthesis of storage reserve and desiccation (Vernoud et al.,2005).Expression differences in endosperm samples resulted in three primary clusters,which correspond to early,middle,and late phases of development (Fig.3C).The earliest time point (6and 8DAP)is an active period of cell division and cell elongation that terminates at about 20to 25DAP (Duvick,1961).The samples of 10to 24DAP formed one subgroup and 26to 34DAP formed another subgroup,suggesting that they mark the period forming the main cell types and maturation of endo-sperm,respectively (Fig.3C).The two subgroupsformFigure 2.Analysis of global gene expres-sion among different samples.A,Venn diagram of the 26,105genes detected among embryo,endosperm,and whole seed.B,Number of genes expressed in each of the samples.C,Comparison of expression levels of genes detected in embryo and endosperm tissues.Chen et al.a larger cluster for active accumulation of storage com-pounds during 10to 34DAP.The distinct cluster of 36and 38DAP is in accordance with the end of storage compound accumulation in the endosperm and the ac-tivation of biological processes involved in dormancy and dehydration.In whole seed tissue,a primary clus-ter was formed from the earliest time points with 0to 4DAP and 6to 8DAP samples as subgroups,separating the nucellus degradation as well as endosperm syncy-tial and cellularization phases from the rapid expansion of the endosperm and development of embryonic tissues (Fig.3D).After 10DAP,the embryo and endosperm dominate the formation of seed,as shown in Figure 1and morphological observation (Pennington et al.,2008).As effected by both embryo and endosperm,10to 28DAP whole seed samples clustered together and 30to 38DAP formed another group (Fig.3D).These results con firmthat the expression data successfully captured the char-acteristic seed development phases and should there-fore contain valuable insights about corresponding changes in the transcriptome.Integration of Gene Activity and Cellular Function across Development PhasesThe PCA and hierarchical clustering analysis graph-ically display the relationship among different sam-ples,but do not indicate the detailed cellular ing the k-means clustering algorithm,we classi fied the detected genes into 16,14,and 10coexpression modules for embryo,endosperm,and whole seed,re-spectively,each of which contains genes that harbor similar expression patterns (Fig.4).We then used Map-Man annotation to assign genes to functionalcategoriesFigure 3.Global transcriptome relationships among different stages and tissues.A,PCA of the RNA-seq data for the 53seed samples shows five distinct groups:I for embryo (light red),II for endosperm (light blue),and III to V for early (III),middle (IV),and late (V)whole seed (light purple).B to D,Cluster dendrogram showing global transcriptome relationships among time series samples of embryo (B),endosperm (C),and whole seed (D).The y axis measures the degree of variance (see the “Materials and Methods”).The bottom row indicates the developmental phases according to the cluster dendrogram of the time series data.au,Approximately unbiased.Transcriptome Dynamics of Maize Seed(Supplemental Fig.S3).Thus,we can aggregate genes over continuous time points and obtain a view of func-tional transitions along seed development.According to the cluster analysis results,most mod-ules of the embryo can be divided into middle (10–20DAP)and late (22–38DAP)stages (Fig.4A).The middle stage,best represented by modules C1to C7,is typi fied by the overrepresentation of glycolysis,tri-carboxylic acid cycle,mitochondrial electron transport,redox,RNA regulation,DNA and protein synthesis,cell organization,and division-related genes.This is consis-tent with the high requirement of energy during em-bryo formation.The late stage represented by C8to C12exhibited up-regulation of the cell wall,hormone me-tabolism (ethylene and jasmonate),stress,storage pro-teins,and transport-related genes,which coincides with the maturation of the embryo.The modules C13to C16included genes that were broadly expressed across the time points sampled and were related to hormone me-tabolism (brassinosteroid),cold stress,RNA process-ing and regulation,amino acid activation,and protein targeting.All of the 14coexpression modules of endosperm can be roughly divided into early (6–8DAP),middle (10–34DAP),and late (36–38DAP)stages (Fig.4B).The early stage (represented by modules C1to C4)isexempli fied by high expression of hormone metabo-lism (gibberellin),cell wall,cell organization and cycle,amino acid metabolism,DNA,and protein synthesis –related genes,which is consistent with differentiation,mitosis,and endoreduplication.Genes in the tricar-boxylic acid cycle and mitochondrial electron transport are also overrepresented and related to energy de-mands at that time.The middle stage (best represented by C5to C8,in which different modules have distinct pro files)is the active storage accumulation phase and exhibits high expression of carbohydrate metabolism genes,as expected.Increased expression of protein degradation-related genes around 26to 34DAP in C7and C8coincides with the process of endosperm matu-ration.Genes involved in protein degradation,second-ary metabolism,oxidative pentose phosphate,receptor kinase signaling,and transport were up-regulated in the late stage in modules C9to C14during the con-cluding phase of endosperm maturation.Ten DAP and later time points of whole seed sam-ples re flect the additive combination of embryo and endosperm expression.Genes that are active early in development (0–8DAP)in clusters C1to C4are ex-pected to be related to maternal tissue,which is the bulk of the seed at that time.A group of genes are highly expressed in C1at 0DAP,but theirexpressionFigure 4.Coexpression modules.A to C,Expression patterns of coexpression modules of embryo (A),endosperm (B),and whole seed (C),ordered according to the sample time points of their peak expression.For each gene,the RPKM value normalized by the maximum value of all RPKM values of the gene over all time points is shown.Chen et al.drops rapidly by2DAP,suggesting that they have functional roles that precede pollination.This group includes photosynthesis light reaction members and some TFs involved in RNA regulation.Genes related to cell wall and protein degradation,signaling,nucle-otide metabolism,DNA synthesis,cell organization, and mitochondrial electron transport are overrepre-sented in C2to C4,which has increased expression after pollination,in accordance with the degradation of nucellus tissue and development of embryonic tissues. The expression patterns and functional categories of the1,506genes detected only in whole seed samples are shown in Supplemental Figure S4.Because these genes tend to be expressed at high levels mainly before 8DAP,they are presumed to have functions in early seed development.Together,these data show that the transition of major biochemical processes along the developmental time axis of the seed is produced partly by highly coordi-nated transcript dynamics.TF Expression during Seed DevelopmentOf the2,297identified maize TFs(Zhang et al.,2011), 1,614(70%)are included in our analysis(Supplemental Data Set S3),which accounts for6.18%of the total number of genes detected in seed tissue.The num-ber of TFs detected in the different samples is shown in Supplemental Figure S5A.Their proportion to the total genes expressed in each tissue time point was always greater in embryo than endosperm samples (Supplemental Fig.S5B).Shannon entropy has been used to determine the specificity of gene expression, with lower values indicating a more time-specific pro-file(Makarevitch et al.,2013).The Shannon entropy of TFs was significantly lower than all other genes in both embryo(P=2.3310210)and endosperm(P,1.53 1023),indicating that TFs tended to be expressed more time specifically than other genes(Supplemental Fig.S5, C and D).The number of TFs from each family used in the seed development program,along with the proportion of members present in the coexpression modules rel-ative to the total members of the family expressed in the tissue,is shown in Supplemental Figure S6. Enrichment of these TF families in the coexpression modules based on observed numbers was evaluated with Fisher’s exact test.Significant TF family enrich-ment was identified for specific coexpression modules. For example,12auxin-response factor TFs(38.7%) were expressed in embryo module C2during mor-phogenesis and one-half of the detected members of the WRKY family were active in endosperm module C12late in endosperm development.The WRKY family has been reported to be mainly involved in the physiological programs of pathogen defense and se-nescence(Eulgem et al.,2000;Pandey and Somssich, 2009).Twenty-one MIKC family TFs(56.8%)were pres-ent in whole seed module C2,implying an important role in regulating genes involved in response to fertil-ization.The developmental specificity of the detected TFs makes them excellent candidates for reverse ge-netics approaches to investigate their role in grain production.Tissue-Specific Genes of SeedIdentification of uncharacterized tissue-specific genes can help to explain their function and understand the underlying control of tissue or organ identity.To gen-erate a comprehensive catalog of seed-specific genes, results from this study were compared with25pub-lished nonseed RNA-seq data sets(Jia et al.,2009;Wang et al.,2009a;Li et al.,2010;Davidson et al.,2011;Bolduc et al.,2012),including root,shoot,shoot apical meristem, leaf,cob,tassel,and immature ear(Supplemental Table S2).In total,we identified1,258seed-specific genes,in-cluding91TFs from a variety of families(Supplemental Data Set S4).To gain further insight into the spatial expression trend in the developing seed,we divided these genes into four groups:embryo specific,endo-sperm specific,expressed in both embryo and endo-sperm,and other as only expressed in whole seed (Table I).The dynamic expression patterns of these genes reflect their roles in corresponding development stages(Supplemental Fig.S7).The largest numbers of seed-specific genes were observed in the endosperm, consistent with a study in maize using microarrays (Sekhon et al.,2011),perhaps reflecting the specific function of endosperm.We compared the distribution of tissue-specific genes and TFs in embryo and endosperm coexpression mod-ules to identify important phases in the underlying transcription network.Coexpression modules with an enrichment of tissue-specific genes or TFs may provide insight about uncharacterized genes and preparation for subsequent developmental processes.A feature of gene activity is shown in Figure5.Fisher’s exact test (P,0.05)was used to determine modules with sig-nificant enrichment of tissue-specific genes and TFs in the embryo and endosperm.In the embryo,TFs and tissue-specific genes were significantly enriched in the late phase,suggesting a specific process during matu-ration.In the endosperm,we observed that TFs and tissue-specific genes were overrepresented in the mid-dle phase,which conforms to the role of endosperm inTable I.Total number of detected seed-specific genes and TFsData are presented as n.Tissue Type Specific Genes Specific TFs Embryo24923Endosperm74259Embryo and endosperm2196Other a483Total1,25891a These genes only detected expression in whole seeds.Transcriptome Dynamics of Maize Seedstorage compound accumulation and to speci fic pro-gress at this phase.To gain further insight into the functional signi fi-cance of tissue-speci fic genes,overrepresented gene ontology (GO)terms were examined using the WEGO online tool (Ye et al.,2006;Supplemental Fig.S8).All overrepresented GO terms were observed for themiddle embryo development phase,including the bio-synthetic process,cellular metabolic process,macro-molecule,and nitrogen compound metabolic process.Similarly,overrepresented GO terms for the endosperm were mostly observed in the early phase,including the macromolecule,nitrogen compound metabolic pro-cess,DNA binding,and transcription regulator.GenesFigure 5.Distribution and enrichment of genes,tissue-specific genes,TFs,and tissue-specific TFs in coexpression modules of embryo and endosperm.A and B,Bars indicate the percentage of all detected genes (green),tissue-specific genes (blue),TFs (red),and tissue-specific TFs (purple)observed in a coexpression module (C)or in the development phase (Total)relative to the total number of each group detected across samples for embryo (A)and endosperm (B).The number of genes represented by the percentage is shown on the right y axis.Enrichment for tissue-specific genes and TFs was evaluated with Fisher’s exact test based on the number of genes observed in each coexpression module,whereas enrichment for tissue-specific TFs was evaluated based on the number of TFs observed in each coexpression module.Asterisks represent significant enrichment at a false dis-covery rate #0.05.Chen et al.involved in the nutrient reservoir class were enriched in the middle phase.The oxidoreductase class was overrepresented in late phase,and is known to be in-volved in maturation (Zhu and Scandalios,1994).The Expression of Zein Genes in EndospermZeins are the most important storage proteins in maize endosperm and are an important factor in seed quality.According to Xu and Messing (2008),there are 41a ,1b ,3g ,and 2d zein genes.In order to explore their expression pattern,we first con firmed the gene models by mapping publicly available full-length com-plementary DNAs of zein subfamily genes to B73bac-terial arti ficial chromosomes,and then mapped these back to the reference genome.Because some of these zein genes were not assembled in the current B73ref-erence genome or were only annotated in the working gene set,we con firmed a final set of 35zein genes in the FGS of the B73annotation,including 30a ,1b ,3g ,and 1d zein genes (Supplemental Table S3).About three-quarters (26)of these were in the list of the 100most highly expressed genes in the endosperm,based on mean expression across all endosperm samples (Supplemental Table S4).The distribution of these most highly expressed genes clearly showed that the 26zeins and 4starch synthesis genes were actively expressed in the middle phase of endosperm devel-opment,characteristic of storage compound accumu-lation (Fig.6A).Previous research has shown that the zein genes con-stitute approximately 40%to 50%of the total tran-scripts in the endosperm (Marks et al.,1985;Woo et al.,2001),but these results are based on EST data from a single tissue or pooled tissues and only a few zein genes were assessed.Thus,we reevaluated the transcriptomic contribution of zein genes across endosperm develop-ment using our RNA-seq data,which is able to overcome the high structural similarity among them,especially in the a family (Xu and Messing,2008).Zein genes stably accounted for about 65%of transcripts from 10to 34DAP,with 19-kD a zeins (approximately 42%),22-kD a zeins (approximately 8%),and g zeins (approximately 10%)representing the most abundant transcripts (Fig.6B).The expression of different members within agivenFigure 6.Analysis of highly expressed genes in the endosperm.A,The distribution of the 100most highly expressed genes in the endosperm ordered by mean expression in different modules.B,The dynamic transcript levels of different zein gene family members in the endosperm as reflected by their percentage among all detected gene transcript levels.C,Heat map showing RPKM values of 35zein genes in the different development stages of the endosperm.+,Having intact coding regions;2,with premature_stop;N,no;Y ,yes.Transcriptome Dynamics of Maize Seed。

遗传育种相关名词中英文对照中英文对照的分子育种相关名词 3"untranslated region (3"UTR) 3"非翻译区 5"untranslated region (5; UTR) 5"非翻译区 A chromosome A 染色体 AATAAA 多腺苷酸化信号aberration 崎变 abiogenesis 非生源说 accessory chromosome 副染色体 accessory nucleus 副核 accessory protein 辅助蛋白 accident variance 偶然变异 Ac-Ds system Ac-Ds 系统 acentric chromosome 无着丝粒染色体acentric fragment 无着丝粒片段 acentric ring 无着丝粒环 achromatin 非染色质 acquired character 获得性状acrocentric chromosome 近端着丝粒染色体 acrosyndesis 端部联会 activating transcription factor 转录激活因子activator 激活剂 activator element 激活单元 activator protein( AP)激活蛋白 activator-dissociation system Ac-Ds 激活解离系统 active chromatin 活性染色质 activesite 活性部位 adaptation 适应 adaptive peak 适应高峰adaptive surface 适应面 addition 附加物 addition haploid 附加单倍体 addition line 附加系 additiveeffect 加性效应 additive gene 加性基因 additive genetic variance 加性遗传方差additive recombination 插人重组additive resistance 累加抗性 adenosine 腺昔adenosine diphosphate (ADP )腺昔二鱗酸adenosine triphosphate( ATP)腺昔三憐酸adjacent segregation 相邻分离A- form DNA A 型 DNAakinetic chromosome 无着丝粒染色体akinetic fragment 无着丝粒片断alien addition monosomic 外源单体生物alien chromosome substitution 外源染色体代换alien species 外源种 alien-addition cell hybrid 异源附加细胞杂种 alkylating agent 焼化剂 allele 等位基因allele center 等位基因中心 allele linkage analysis 等位基因连锁分析 allele specific oligonucleotide(ASO)等位基因特异的寡核苷酸 allelic complement 等位（基因）互补 allelic diversity 等位（基因）多样化 allelic exclusion 等位基因排斥 allelic inactivation 等位（基因）失活 allelic interaction 等位（基因）相互作用allelic recombination 等位（基因）重组 allelicreplacement 等位（基因）置换 allelic series 等位（基因）系列 allelic variation 等位（基因）变异 allelism 等位性 allelotype 等位（基因）型 allodiploid 异源二倍体 allohaploid 异源单倍体 allopatric speciation 异域种alloploidy 异源倍性 allopolyhaploid 异源多倍单倍体allopolyploid 异源多倍体 allosyndesis 异源联会allotetraploid 异源四倍体 alloheteroploid 异源异倍体alternation of generation 世代交替 alternative transcription 可变转录 alternative transcription initiation 可变转录起始 Alu repetitive sequence, Alu family Alu 重复序列，Alu 家族ambiguous codon 多义密码子 ambisense genome 双义基因组 ambisense RNA 双义 RNA aminoacyl-tRNA binding site 氨酰基 tRNA 接合位点 aminoacyl-tRNA synthetase 氨酰基 tRNA 连接酶 amixis 无融合amorph 无效等位基因amphidiploid 双二倍体amphipolyploid 双多倍体amplicon 扩增子amplification 扩增 amplification primer 扩增引物analysis of variance 方差分析 anaphase (分裂）后期anaphase bridge (分裂）后期桥anchor cell 锚状细胞 androgamete 雄配子aneuhaploid 非整倍单倍体aneuploid 非整倍体 animal genetics 动物遗传学annealing 复性 antibody 抗体anticoding strand 反编码链anticodon 反密码子anticodon arm 反密码子臂anticodon loop 反密码子环 antiparallel 反向平行antirepressor 抗阻抑物antisense RNA 反义 RNAantisense strand 反义链 apogamogony 无融合结实apogamy 无配子生殖apomixis 无融合生殖 arm ratio (染色体)臂比artificial gene 人工基因 artificial selection 人工选择 asexual hybridization 无性杂交 asexual propagation 无性繁殖 asexual reproduction 无性生殖assortative mating 选型交配 asynapsis 不联会 asynaptic gene 不联会基因atavism 返祖 atelocentric chromosome 非端着丝粒染色体 attached X chromosome 并连 X 染色体 attachmentsite 附着位点 attenuation 衰减 attenuator 衰减子autarchic gene 自效基因auto-alloploid 同源异源体 autoallopolyploid 同源异源多倍体 autobivalent 同源二阶染色体 auto-diploid 同源二倍体；自体融合二倍体 autodiploidization 同源二倍化autoduplication 自体复制 autogenesis 自然发生autogenomatic 同源染色体组 autoheteroploidy 同源异倍性autonomous transposable element 自主转座单元autonomously replicating sequence(ARS)自主复制序列autoparthenogenesis 自发单性生殖 autopolyhaploid 同源多倍单倍体 autopolyploid 同源多倍体 autoradiogram 放射自显影图 autosyndetic pairing 同源配对 autotetraploid 同源四倍体 autozygote 同合子 auxotroph 营养缺陷体 B chromosome B 染色体 B1，first backcross generation 回交第一代 B2，second backcross generation 回交第二代back mutation 回复突变 backcross 回交backcross hybrid 回交杂种 backcross parent 回交亲本 backcross ratio 回交比率 background genotype 背景基因型 bacterial artification chromosome( BAC )细菌人工染色体Bacterial genetics 细菌遗传学 Bacteriophage 噬菌体balanced lethal 平衡致死 balanced lethal gene 平衡致死基因 balanced linkage 平衡连锁 balanced load 平衡负荷balanced polymorphism 平衡多态现象 balanced rearrangements 平衡重组balanced tertiary trisomic 平衡三级三体balanced translocation 平衡异位balancing selection 平衡选择band analysis 谱带分析 banding pattern (染色体)带型basal transcription apparatus 基础转录装置 base analog 碱基类似物base analogue 类減基base content 减基含量base exchange 碱基交换 base pairing mistake 碱基配对错误 base pairing rules 碱基配对法则 base substitution 减基置换 base transition 减基转换 base transversion 减基颠换 base-pair region 碱基配对区base-pair substitution 碱基配对替换 basic number of chromosome 染色体基数 behavioral genetics 行为遗传学behavioral isolation 行为隔离 bidirectionalreplication 双向复制 bimodal distribution 双峰分布binary fission 二分裂binding protein 结合蛋白binding site 结合部位 binucleate phase 双核期biochemical genetics 生化遗传学 biochemical mutant 生化突变体biochemical polymorphism 生化多态性 bioethics 生物伦理学 biogenesis 生源说 bioinformatics 生物信息学biological diversity 生物多样性 biometrical genetics 生物统计遗传学（简称生统遗传学) bisexual reproduction 两性生殖 bisexuality 两性现象 bivalent 二价体 blending inheritance 混合遗传 blot transfer apparatus 印迹转移装置 blotting membrane 印迹膜 bottle neck effect 瓶颈效应 branch migration 分支迁移 breed variety 品种breeding 育种，培育;繁殖，生育 breeding by crossing 杂交育种法 breeding by separation 分隔育种法 breeding coefficient 繁殖率 breeding habit 繁殖习性 breeding migration 生殖回游，繁殖回游 breeding period 生殖期breeding place 繁殖地 breeding population 繁殖种群breeding potential 繁殖能力，育种潜能 breeding range繁殖幅度 breeding season 繁殖季节 breeding size 繁殖个体数 breeding system 繁殖系统 breeding true 纯育breeding value 育种值 broad heritability 广义遗传率bulk selection 集团选择 C0，acentric 无着丝粒的Cl,monocentric 单着丝粒 C2, dicentric 双着丝粒的C3,tricentric 三着丝粒的 candidate gene 候选基因candidate-gene approach 候选基因法 Canpbenmodel 坎贝尔模型carytype 染色体组型，核型 catabolite activator protein 分解活化蛋白catabolite repression 分解代谢产物阻遏catastrophism 灾变说 cell clone 细胞克隆 cell cycle 细胞周期 cell determination 细胞决定 cell division 细胞分裂 cell division cycle gene(CDC gene) 细胞分裂周期基因 ceU division lag 细胞分裂延迟 cell fate 细胞命运cell fusion 细胞融合 cell genetics 细胞的遗传学 cell hybridization 细胞杂交 cell sorter 细胞分类器 cell strain 细胞株 cell-cell communication 细胞间通信center of variation 变异中心 centimorgan(cM) 厘摩central dogma 中心法则 central tendency 集中趋势centromere DNA 着丝粒 DNA centromere interference 着丝粒干扰centromere 着丝粒 centromeric exchange ( CME)着丝粒交换centromeric inactivation 着丝粒失活 centromeric sequence( CEN sequence)中心粒序列 character divergence 性状趋异chemical genetics 化学遗传学chemigenomics 化学基因组学chiasma centralization 交叉中化chiasma terminalization 交叉端化chimera 异源嵌合体Chi-square (x2) test 卡方检验 chondriogene 线粒体基因 chorionic villus sampling 绒毛膜取样 chromatid abemition 染色单体畸变chromatid break 染色单体断裂chromatid bridge 染色单体桥chromatid interchange 染色单体互换 chromatid interference 染色单体干涉 chromatid segregation 染色单体分离chromatid tetrad 四分染色单体chromatid translocation 染色单体异位chromatin agglutination 染色质凝聚chromosomal aberration 染色体崎变chromosomal assignment 染色体定位chromosomal banding 染色体显带chromosomal disorder 染色体病chromosomal elimination 染色体消减 chromosomal inheritance 染色体遗传chromosomal interference 染色体干扰chromosomal location 染色体定位chromosomal locus 染色体位点 chromosomal mutation 染色体突变chromosomal pattern 染色体型chromosomal polymorphism 染色体多态性 chromosomal rearrangement 染色体质量排chromosomal reproduction 染色体增殖chromosomal RNA 染色体 RNAchromosomal shift 染色体变迁，染色体移位chromosome aberration 染色体畸变 chromosome arm 染色体臂chromosome association 染色体联合chromosome banding pattern 染色体带型chromosome behavior 染色体动态chromosome blotting 染色体印迹chromosome breakage 染色体断裂chromosome bridge 染色体桥 chromosome coiling 染色体螺旋chromosome condensation 染色体浓缩chromosome constriction 染色体缢痕chromosome cycle 染色体周期chromosome damage 染色体损伤chromosome deletion 染色体缺失chromosome disjunction 染色体分离chromosome doubling 染色体加倍chromosome duplication 染色体复制chromosome elimination 染色体丢失 chromosome engineering 染色体工程chromosome evolution 染色体进化 chromosome exchange 染色体交换chromosome fusion 染色体融合 chromosome gap 染色体间隙chromosome hopping 染色体跳移chromosome interchange 染色体交换chromosome interference 染色体干涉chromosome jumping 染色体跳查chromosome knob 染色体结 chromosome loop 染色体环chromosome lose 染色体丢失chromosome map 染色体图 chromosome mapping 染色体作图chromosome matrix 染色体基质chromosome mutation 染色体突变 chromosome non-disjunction 染色体不分离 chromosome paring 染色体配对chromosome polymorphism 染色体多态性 chromosome puff 染色体疏松 chromosome rearrangement 染色体质量排chromosome reduplication 染色体再加倍 chromosome repeat 染色体质量叠 chromosome scaffold 染色体支架chromosome segregation 染色体分离 chromosome set 染色体组chromosome stickiness 染色体粘性chromosome theory of heredity 染色体遗传学说chromosome theory of inheritance 染色体遗传学说chromosome thread 染色体丝chromosome walking 染色体步查chromosome-mediated gene transfer 染色体中介基因转移 chromosomology 染色体学 CIB method CIB 法;性连锁致死突变出现频率检测法 circular DNA 环林 DNA cis conformation 顺式构象 cis dominance 顺式显性 cis-heterogenote 顺式杂基因子 cis-regulatory element 顺式调节兀件 cis-trans test 顺反测验cladogram 进化树 cloning vector 克隆载体 C-meiosis C 减数分裂C-metaphase C 中期C-mitosis C 有丝分裂 code degeneracy 密码简并coding capacity 编码容量 coding ratio 密码比 coding recognition site 密码识别位置 coding region 编码区coding sequence 编码序列 coding site 编码位置 coding strand 密码链 coding triplet 编码三联体 codominance 共显性 codon bias 密码子偏倚 codon type 密码子型coefficient of consanguinity 近亲系数 coefficient of genetic determination 遗传决定系数 coefficient of hybridity 杂种系数 coefficient of inbreeding 近交系数coefficient of migration 迁移系数 coefficient of relationship 亲缘系数 coefficient of variability 变异系数 coevolution 协同进化 coinducer 协诱导物 cold sensitive mutant 冷敏感突变体colineartiy 共线性combining ability 配合力comparative genomics 比较基因组学competence 感受态competent cell 感受态细胞competing groups 竞争类群 competition advantage 竞争优势competitive exclusion principle 竞争排斥原理complementary DNA (cDNA)互补 DNAcomplementary gene 互补基因 complementation test 互补测验complete linkage 完全连锁 complete selection 完全选择 complotype 补体单元型 composite transposon 复合转座子 conditional gene 条件基因 conditional lethal 条件致死conditional mutation 条件突变 consanguinity 近亲consensus sequence 共有序列 conservative transposition 保守转座 constitutive heterochromatin 组成型染色质continuous variation 连续变异convergent evolution 趋同进化cooperativity 协同性 coordinately controlled genes 协同控制基因 core promoter element 核心启动子 core sequence 核心序列 co-repressor 协阻抑物correlation coefficient 相关系数 cosegregation 共分离 cosuppression 共抑制cotranfection 共转染cotranscript 共转录物 cotranscriptional processing 共转录过程 cotransduction 共转导cotransformation 共转化 cotranslational secrection 共翻译分泌counterselection 反选择coupling phase 互引相 covalently closed circular DNA(cccDNA)共价闭合环状 DNAcovariation 相关变异criss-cross inheritance 交叉遗传 cross 杂交crossability 杂交性crossbred 杂种cross-campatibility 杂交亲和性 cioss-infertility 杂交不育性 crossing over 交换crossing-over map 交换图crossing-over value 交换值crossover products 交换产物 crossover rates 交换率crossover reducer 交换抑制因子crossover suppressor 交换抑制因子crossover unit 交换单位 crossover value 值crossover-type gamete 交换型配子C-value paradox C 值悖论 cybrid 胞质杂种 cyclin 细胞周期蛋白cytidme 胞苷 cytochimera 细胞嵌合体cytogenetics 细胞遗传学 cytohet 胞质杂合子cytologic 细胞学的cytological map 细胞学图cytoplasm 细胞质cytoplasmic genome 胞质基因组 cytoplasmic heredity 细胞质遗传 cytqplasmic incompatibility 细胞质不亲和性cytoplasmic inheritance 细胞质遗传cytoplasmic male sterility 细胞质雄性不育cytoplasmic mutation 细胞质突变 cytofdasmic segregation 细胞质分离cytoskeleton 细胞骨架Darwin 达尔文 Darwinian fitness 达尔文适合度Darwinism 达尔文学说 daughter cell 子细胞 daughter chromatid 子染色体 daughter chromosome 子染色体deformylase 去甲酰酶 degenerate code 简并密码degenerate primer 简并引物 degenerate sequence 简并序列 degenerated codon 简并密码子degeneration 退化 degree of dominance 显性度delayed inheritance 延迟遗传 deletant 缺失体deletion 缺失。

Section_ⅠWarming Up & Reading — Pre­reading 1．What are the advantages and disadvantages of cloning?2．Not only animals but also plants can be cloned. Do you know the difference between them?Please read the following passage to get the answer.A new study of plants that are reproduced by cloning has shown why cloned plants are not identical.Scientists have known for some time that cloned organisms are not always identical: their observable characteristics and traits can vary, and this variation can be passed on to the next generation. This is despite the fact that they are derived (获得) from genetically identical founder cells.Now, a team from Oxford University, the UK, and King Abdullah University of Science and Technology, Saudi Arabia, believe they have found out why this is the case in plants: the genomes (染色体) of regenerate plants carry relatively high frequencies of new DNA sequence mutations (变异) that were not present in the genome of the donor plant.The team reported their findings in this week’s Current Biology.“Where these new mutations actually come from is still a mystery，” said Professor Harberd. “They may arise during the regeneration process itself or during the cell divisions in the donor plant that gave rise to the root cells from which the regenerate plants are created. We are planning further research to find out which of these two processes is responsible for these mutations. What we can sayis that nature has safely been employing what you might call a ’cloning’process in plants for millions of years, and that there must be good evolutionary reasons why these mutations are introduced.”The new results suggest that variation in clones of plants may have different underlying causes from those of variation in clones of animals — where it is believed that the effect of environmental factors on how animal genes are expressed is more important and no similar high frequencies of mutations have been observed.Professor Harberd said, “While our results highlight that cloned plants and animals are very different, they may give us insights into how bacterial and cancer cells replicate themselves, and how mutations arise during these processes which, ultimately, have an impact on human health.”Warming Up1．How much do are natural clone and man­made clone?Clone_is_a_way_of_making_an_exact_copy_of_another_animal_or_plant.Natural_clone_is_one_that_occurs_from_the_“parent”_in_a_natural_form_of_ cloning_without_human_help.Man­made_clone_is_one_produced_through_human_intervention_(介入)．2．Look at these pictures and tell which ones are natural clones and which ones are man­made clones.Natural_clones：AF；Man­made_clones：BCDE3．See another kind of clone. The two women soldiers look very alike. Can you explain why?They_are_called_twins._They_were_born_almost_at_the_same_time_by_the_same_ motherFast Reading1．The text is mainly about the procedure of animal cloning and the life of Dolly the sheep，as well as the reasons why cloning is still controversial.2．Read the text and then find the main idea of each paragraph.(1)Paragraph 1 (D) A．The effect of Dolly.(2)Paragraph 2 (B) B．The major uses ofcloning.(3)Paragraph 3 (E) C．People’s reaction tocloning.(4)Paragraph 4 (A) D．The definition (定义)of cloning.(5)Paragraph 5 (C) E．The problem of Dolly.Careful Reading(1)Which of the following statements about cloning is NOT true according to the text?A．Cloning is something that has been developed quite recently.B．Twins that look exactly the same are originally from the same egg.C．Cloning is used by gardeners to make more money.D．Animal cloning is much more difficult than plant cloning.(2)The cloning of Dolly the sheep succeeded because ________.A．it’s easier to clone a sheep than other animalsB．the scientists were determined and worked hard with patienceC．the scientists were rewarded and got lots of moneyD．many other attempts to clone mammals failed(3)The sheep that donated the somatic cell most probably lived ________ years.A．3 B．6C．9 D．12(4)Which of the following is NOT an advantage of cloning?A．Cloning can be used for medical purposes.B．Large quantities of food can be produced by cloning.C．Cloning can help keep animals from becoming extinct.D．Famous persons who have passed away can be cloned.(5)We can infer from the last three paragraphs that________.A．the normal development of Dolly had no effect on the cloning of other species B．Dolly’s serious disease and final death disturbed the whole worldC．Dolly successfully cloned a new lamb with the help of cloning scientists D．there were arguments about animal cloning and concerns about the future of cloning答案：(1)～(5) ABDDDSummaryCloning is a way of 1.making (make) an exact copy of another animal or plant.2.It has two major uses. One is3.that gardeners use it to produce commercial quantities of plants. The4.other is that it is valuable for research on new species and for medical research on animals.5.However,_the process of cloning is difficult to undertake.Many attempts to clone mammals failed. The first successful clone, Dolly the sheep, made the whole 6.scientific (science) world follow its process, though it had 7.more problems than the normal sheep. On the other hand, Dolly’s 8.appearance (appear) raised a storm of objections and had a great impact 9.on the media and public imagination. Different people have different opinions on it. However, scientists still wonder whether cloning will help 10.or harm us and where it is leading us.Discussionit.(1)I_am_in_favor_of_cloning,_because_medical_cloning_can_help_humans_cure_serious_illnesses_that_ruin_one’s_quality_of_life.(2)I_am_against_cloning,_because_some_people_may_want_to_clone_themselves_to_l ive_forever,_so_it_will_lead_to_some_social_problems.。

Nephrol Dial Transplant (2009)1321 13doi:10.1093/ndt/gfp500Recurrence of nephrotic syndrome after transplantation in a mixed population of children and adults:course of glomerular lesions and value of the Columbia classification of histological variants of focal and segmental glomerulosclerosis (FSGS)Guillaume Canaud 1,2,Daniel Dion 3,Julien Zuber 1,2,Marie-Claire Gubler 4,Rebecca Sberro 1,2,Eric Thervet 1,2,Renaud Snanoudj 1,Marina Charbit 5,R ´e mi Salomon 2,5,Frank Martinez 1,Christophe Legendre 1,2,Laure-Helene Noel 3and Patrick Niaudet 2,51Department of Kidney Transplantation,Necker Hospital,149rue de S`e vres,75015,Paris,2Universit´e Paris Descartes,Rue del’Ecole de M´e decine,3Department of Pathology,4Institut National de la Sant´e et de la Recherche M´e dicale U574and 5Department of Pediatrics Nephrology,Necker Hospital,149rue de S`e vres,75015,Paris,France Correspondence and offprint requests to :Guillaume Canaud;E-mail:guillaume.canaud@nck.aphp.frAbstractIntroduction.Recurrence of nephrotic-range proteinuria in patients with idiopathic nephrotic syndrome (INS)and focal and segmental glomerulosclerosis (FSGS)on native kidneys is associated with poor graft survival.Identifica-tion of risk factors for recurrence is therefore an important issue.In 2004,Columbia University introduced a histo-logical classification of FSGS that identifies five mutually exclusive variants.In non-transplant patients,the Columbia classification appears to predict the outcome and response to treatment better than clinical characteristics alone.How-ever,the predictive value of this classification to assess the risk of recurrence after transplantation has not been addressed.Methods.We retrospectively studied 77patients with INS and FSGS on native kidneys who underwent renal trans-plantation.Of these,42recipients experienced recurrence of nephrotic range proteinuria.Results.At time of recurrence,minimal-change disease (MCD)was the main histological feature.On serial biop-sies,the incidence of MCD decreased over time,while the incidence of FSGS variants increased.The variant type ob-served in the native kidneys was not predictive of either recurrence or type of FSGS seen on the allograft.Patients with complete and sustained remission did not developed FSGS.Conclusion.In conclusion,the Columbia classification is of no help in predicting recurrence after renal transplan-tation or histological lesions in the case of recurrence of proteinuria.Keywords:FSGS;histopathology;kidney transplantation;recurrenceIntroductionSteroid-resistant idiopathic nephrotic syndrome (INS)in native kidneys is frequently associated with progression to end-stage renal disease (ESRD)[1–3].In most patients,ini-tial renal biopsy shows focal and segmental glomeruloscle-rosis (FSGS).However,in early stages,FSGS and minimal-change disease (MCD)are indistinguishable at the clinical level [4–6].Serial renal biopsies indicate that some patients with minimal changes on initial renal biopsy develop FSGS during the course of the disease.This was observed in 60%of cases in a series of 48patients with steroid-resistant min-imal change [7].After kidney transplantation,recurrence of nephrotic-range proteinuria is observed in 30%of cases and is associated with poor graft survival [8–14].Identification of risk factors for recurrence is an important issue.In 2004,Columbia University introduced a histologi-cal classification of FSGS that identifies five mutually exclusive variants:perihilar (PH),cellular (CELL),tip le-sion (TIP),collapsing (COL)and not otherwise specified (NOS)[15].Since then,several studies have correlated re-nal prognosis with histological features in native kidneys.In non-transplant patients,the Columbia variant seems to pre-dict outcome and response to treatment better than clinical characteristics alone.TIP seems to be the most steroid-sensitive variant,with COL being the least favourable vari-ant [16–20].The impact of this classification to predict the FSGS variant in the transplant has been recently studied [21].In one report,Ijpelaar et al.concluded that,in most cases,the FSGS variant observed in the transplant kidney was the same as in the native kidneys,suggesting that the histological variant of the native kidney could predict the variant observed in patients with recurrence.However,the predictive value of this classification to assess the risk ofC The Author 2009.Published by Oxford University Press [on behalf of ERA-EDTA].All rights reserved.For Permissions,please e-mail:journals.permissions@– Advance Access publication 22September 200928 by guest on October 14, 2013/Downloaded fromFig.1.FSGS variants in native kidneys(NOS,n=38;PH,n=6;TIP,n=6;CELL,n=17;COL,n=10).recurrence after transplantation and graft outcome has never been addressed.We investigated in a large series whether the Columbia classification of FSGS variants provides a relevant indicator for the risk of proteinuria recurrence af-ter renal transplantation and the type of variant in the case of recurrence.Material and methodsKidney transplant recipients with INS and FSGS on native kidneys and pro-gression to ESRD were eligible for inclusion.Patients with familial history of proteinuria or secondary forms of FSGS were excluded,i.e.ischaemia-related podocyte injury,sickle cell disease,renal hypoplasia,thrombotic microangiopathy,reflux nephropathy,human immunodeficiency virus and genetic disorders when a test was available.Patients with non-adequate biopsies were also excluded(i.e.the biopsy sample containing fewer than five glomeruli).Our study population included children(n=56)and adults(n=21) (age>16years).There were40males and37females.The77patients included in the study received a sequential quadruple immunosuppression including induction therapy(thymoglobulin,n=47;basiliximab,n=30), corticosteroids,a calcineurin inhibitor(cyclosporine,n=65;tacrolimus, n=12),azathioprine(n=53)or mycophenolate mofetil(n=24).Seventy-one patients had received a first kidney transplant;4patients had received a second transplant and2a third one.Kidney allografts were obtained from a deceased donor in69cases and from a living-related donor in8cases.No patient received pre-emptive treatment for recurrence(i.e.cyclosporine A IV or pre-operative plasmapheresis).Recurrence was defined by massive proteinuria in the nephrotic range (>3g/day for adults or>50mg/kg/day for children),with normal glomeruli and without evidence of acute or chronic rejection,glomerular deposits or allograft glomerulopathy on initial kidney biopsy.Cameron’s classification was applied to define time of recurrence:immediate(<48 h),early(<3months)and late recurrence(>3months)[10].Transplant biopsies were performed either when indicated by protein-uria and/or acute renal failure,or routinely as screening biopsies at3and 12months post-transplant.Native and transplant kidney biopsies were pro-cessed following the international recommendations for light microscopy and immunofluorescence.The median number of glomeruli per biopsy was 13.7±4.6(range6–24).Histology slides were stained with haematoxylin and eosin,periodic acid–Schiff,Masson trichrome and methenamine–silver.For each biopsy,FSGS variant was evaluated according to the Columbia classification[15].The five FSGS variant definitions are sum-marized in Figure1.All native and transplant biopsies were reviewed by three senior pathologists.As electron microscopy is not of current routine practice in France,we were able to describe foot process fusion in only one case.Nevertheless,patients with proteinuria in the nephrotic range and no features of FSGS variants on kidney biopsy were classified as having MCD.ResultsFrom January1984to December2007,77patients with INS and biopsy-proven FSGS on native kidneys received a renal transplant.Patients were classified in two categories according to whether proteinuria recurred in the nephrotic range(R group,n=42)or not(NR group,n=35).The demographic characteristics of the patients are summarized in Tables1and2.The mean age at onset of nephrotic syndrome was10.4±10years and9.2±6.9years for patients in the NR group and in the R group,respectively (P=NS).The delay between the onset of disease and ESRD was4.8±2.3years in the NR group and3.9±3.3years in the R group(P=NS).There was no difference in the immunosuppressive regimen between the two groups.Four patients in each group received a kidney from a living donor. In the NR group,all patients were genotyped for NPHS1and NPHS2,and none of them had mutation.In the R group, 30/42patients were genotyped for NPHS1and NPHS2. T wo patients had a heterozygous variant,polymorphism (pA242V)[22].On native kidney biopsies,MCD was initially observed in10(children exclusively)out of77cases,and repeat re-nal biopsies showed the development of FSGS in all these patients.All variants were present on native kidneys.The1322G.Canaud et al.Table1.Characteristics of patients in the NR groupAge at onset Delay betweenof proteinuria FSGS variant on Number of onset and Patients(years)native kidney glomeruli ESRD(years) 10.8CELL107211COL14 1.633MCD then NOS16245TIP11852NOS235610MCD then CELL1627 3.3NOS12882COL9 2.593NOS2241011CELL135112COL147120.3MCD then NOS8 1.8132NOS256143CELL177150.5NOS117165CELL192171NOS294186TIP254192NOS142204CELL166211NOS119226NOS1092318MCD then PH1342412NOS2062524MCD then NOS2212622PH1882731TIP97.22814CELL9 2.42917CELL743029COL11 3.13119NOS12 3.73216NOS1543334CELL10 5.53428NOS953518NOS9 4.5Mean±SD10.4±1014±5.6 4.8±2.3FSGS variant observed on native kidney biopsies was NOS in38cases(49.3%),CELL in17cases(22.2%),COL in 10cases(12.9%),PH in6cases(7.8%)and TIP in6cases (7.8%).There were no differences in FSGS variant repre-sentation between R and NR groups(Figure2).After kidney transplantation,recurrence of proteinuria occurred immediately in32/42cases(children n=28, adults n=4),early in9/42cases(children n=4,adults n=5)and late in1/42case(adult n=1).The treatment for recurrence consisted of intravenous(IV)cyclosporine A(CsA)+plasmapheresis(PP)in10cases,CsA IV+PP+ a high dose of steroids in3cases,CsA IV alone in10 cases,oral CsA+PP in3cases,oral CsA alone in8cases, oral CsA+PP+rituximab in1case,cyclophosphamide+ PP in1case,CsA IV+PP+rituximab in4cases,CsA IV+enalapril in1case and immunoadsorption in1case (Table2).The distribution of glomerular lesions in transplanted kidneys with recurrence of INS(group R,n=42)is sum-marized in Table2and Figure2.MCD was the main histo-logical feature observed in32out of33biopsies performed early after recurrence,D6to D60days after transplantation. Only one patient had already developed and FSGS lesion (Table2:patient38,PH variant on Day15).At Month3,an Fig.2.Distribution of FSGS variants on a native kidney of patients with or without recurrence of proteinuria in the nephrotic range.FSGS lesion was observed in11out of39cases.At Month 12,FSGS lesion was observed in14out of37cases.Sev-enteen of the42patients went into complete and persistent remission,and none of them developed FSGS(2patients1323Variants of FSGS and risk of recurrenceT a b l e 2.C o u r s e o f F S G S v a r i a n t s o n k i d n e y b i o p s i e s p e r f o r m e d i n t r a n s p l a n t r e c i p i e n t s w i t h r e c u r r e n c e o f p r o t e i n u r i a i n t h e n e p h r o t i c r a n g eT r a n s p l a n t D e l a y b i o p s y a t T r a n s p l a n t T r a n s p l a n t A g e a t b e t w e e n t i m e o f b i o p s y o n b i o p s y o n o n s e t o f F S G S v a r i a n t o n o n s e t a n d r e c u r r e n c e M o n t h 3M o n t h 12p r o t e i n u r i a n a t i v e k i d n e y (n u m b e r E S R D T i m e o f R e s p o n s e t o E v o l u t i o n a f t e r (n u m b e r o f (n u m b e r o f (n u m b e r o f P a t i e n t s (y e a r s )o f g l o m e r u l i )(y e a r s )r e c u r r e n c e T r e a t m e n tt r e a t m e n t t r a n s p l a n t a t i o n g l o m e r u l i )g l o m e r u l i )g l o m e r u l i )O t h e r b i o p s y18N O S (n =12)2D 0C s A I V +P PP a r t i a l T r a n s p l a n t e c t o m y M 12M C D M 1(n =22)M C D (n =12)M C D (n =14)22N O S (n =12)4D 0C s A I V +P PN o r e m i s s i o n T r a n s p l a n t e c t o m y M 1M C D M 1(n =16)X X 36M C D (n =14)t h e n C E L L (n =8)7D 0C s A I VP a r t i a l E S R D 9y e a r sM C D M 1(n =15)M C D (n =9)M C D (n =21)C O L M 24,N O S M 9641.5N O S (n =9)1.5D 0C s A o r a lN o r e m i s s i o n T r a n s p l a n t e c t o m y M 3M C D M 1(n =18)M C D (n =13)X510N O S (n =18)2D 0I m m u n o a d s o r p t i o nC o m p l e t e f o l l o w e d b y r e l a p s e T r a n s p l a n t e c t o m y M 48M C D D 15(n =7)M C D (n =15)M C D (n =14)T r a n s p l a n t e c t o m y M 48C O L62C O L (n =17)3D 0C s A I V +P PC o m p l e t e a n d p e r s i s t e n t M CD D 10(n =12)M C D (n =7)M C D (n =18)M C D M 6270.5C E L L (n =11)2D 3C s A I V C o m p l e t e f o l l o w e d b y r e l a p s e a t M 36T r a n s p l a n t e c t o m y M 60M C D M 1(n =14)C E L L (n =8)C E L L (n =9)C E L L M 36,T r a n s p l a n t e c -t o m y M 60N O S82M C D (n =20)t h e n P H (n =6)7D 0C s A I V +P PC o m p l e t e a n d p e r s i s t e n t M CD (n =12)M C D (n =21)91.3N O S (n =8)5D 0C s A I VC o m p l e t e a n d p e r s i s t e n t M CD (n =24)M C D (n =10)M C D M 108101.5N O S (n =10)8D 0C s A I V +P P +R i t u x i m a b C o m p l e t e a n d p e r s i s t e n t M C D D 14(n =12)M C D (n =12)M C D (n =18)M C D M 36112T I P (n =12)12D 0C s A I VC o m p l e t e a n d p e r s i s t e n t M CD D 15(n =19)M C D (n =15)M C D (n =9)128M C D (n =14)t h e n N O S (n =7)3M 6C s A o r a l P a r t i a lD i e d a t M 144M C D M 6(n =12)M C D (n =9)M C D M 36133N O S (n =8)17D 0C s A I V +P P C o m p l e t e a n d p e r s i s t e n t M C D M 1(n =22)M C D (n =18)M C D (n =8)146C E L L (n =20)4D 0C s A o r a l P a r t i a lT r a n s p l a n t e c t o m y M 36M C D (n =11)M C D (n =14)T r a n s p l a n t e c t o m y M 36N O S 152.1N O S (n =15)3.2D 1C s A I V +e n a l a p r i l C o m p l e t e a n d p e r s i s t e n t M C D (n =7)M C D (n =11)M C D M 96169C O L (n =7)4D 20C s A I V +P P N o r e m i s s i o nT r a n s p l a n t e c t o m y M 60M C D D 10(n =24)M C D (n =11)N O S (n =6)T r a n s p l a n t e c t o m y M 60N O S 178N O S (n =9)7D 0C s A o r a lN o r e m i s s i o nT r a n s p l a n t e c t o m y M 9M C D M 1(n =21)N O S (n =9)X T r a n s p l a n t e c t o m y M 9N O S 187N O S (n =11)4D 1C s A I V +P PC o m p l e t e f o l l o w e d b y r e l a p s e a t M 48M CD (n =18)M C D (n =11)M C D M 48,M C D M 961910M C D (n =11)t h e n N O S (n =12)0.4D 1C s A I VC o m p l e t e f o l l o w e d b y r e l a p s e a t M 9E S RD M 96M C D D 21(n =20)M C D (n =9)C O L (n =14)C O L M 9203N O S (n =14)9D 8C s A I V +P P +r i t u x i m a bC o m p l e t e a n d p e r s i s t e n tM C D M 1(n =12)M C D (n =15)M C D (n =9)1324G.Canaud et al.215N O S (n =16)5D 0C s A I V C o m p l e t e a n d p e r s i s t e n t M C D (n =20)M C D (n =12)M C D 5y e a r s220.5N O S (n =10)2D 3C s A I VC o m p l e t e a n d p e r s i s t e n t M CD D 22(n =11)M C D (n =7)M C D (n =14)235C O L (n =9)8D 1C s A I V C o m p l e t e a n d p e r s i s t e n t M C D D 10(n =9)M C D (n =24)M C D (n =11)245CE L L (n =10)5D 0C s A I V C o m p l e t e f o l l o w e d b y r e l a p s e o n M 24M C D M 1(n =13)CE L L (n =12)C E L L (n =9)C E L L M 242512C O L (n =11)1D 0C s A +P PC o m p l e t e a n d p e r s i s t e n t M CD (n =14)M C D (n =10)265N O S (n =18)2D 0C s A I V +P P N o r e m i s s i o nM C D M 1(n =19)C O L (n =6)C O L (n =9)C O L M 242727C E L L (n =20)5D 0C s A I V +P P C o m p l e t e a n d p e r s i s t e n t M C D D 8(n =11)M C D (n =9)M C D (n =19)2821N O S (n =16)2D 3C s A I V +P P C o m p l e t e a n d p e r s i s t e n t M C D D 6(n =23)M C D (n =14)M C D (n =15)2919C E L L (n =12)5D 0C s A o r a l N o r e m i s s i o nM C D D 10(n =18)M C D (n =16)N O S (n =18)3014C O L (n =11)3D 0C s A I V +P P +h i g h d o s e s t e r o i d s C o m p l e t e a n d p e r s i s t e n t M C D D 8(n =14)M C D (n =20)M C D (n =11)3119C E L L (n =9)1.5D 4C s A o r a l +P P P a r t i a l r e m i s s i o nM C D D 17(n =18)T I P (n =13)P H (n =12)3212P H (n =7)2D 0C s A o r a l N o r e m i s s i o n T r a n s p l a n t e c t o m yM 6M C D D 12(n =16)C O L (n =17)XT r a n s p l a n t e c t o m y M 6C O L3320T I P (n =9)2D 2C s A o r a l P a r t i a l r e m i s s i o n M C D D 10(n =11)M C D (n =16)N O S (n =14)3412C O L (n =11)1.6D 0C s A I V +P P +h i g h d o s e s t e r o i d s C o m p l e t e a n d p e r s i s t e n t M C D (n =20)M C D (n =22)3516C E L L (n =13)0.3D 4C s A I V +P P +r i t u x i m a b N o r e m i s s i o nT r a n s p l a n t e c t o m yM 36M C D D 60(n =10)M C D (n =19)N O S (n =12)T r a n s p l a n t e c t o m y M 36C O L368M C D (n =11)t h e n N O S (n =16)1D 0C s A o r a l +P P +r i t u x i m a b N o r e m i s s i o nP H (n =14)P H (n =16)3717P H (n =9)3D 0C s A o r a lN o r e m i s s i o nC E L L (n =11)C E L L (n =20)3812N O S (n =21)1D 1C s A o r a l +P P P a r t i a lP H D 15(n =17)P H (n =14)P H (n =9)3918T I P (n =15)3D 8C s A I V +P P +h i g h d o s e s t e r o i d s C o m p l e t e a n d p e r s i s t e n t M C D D 10(n =19)M C D (n =17)M C D (n =14)4016P H (n =14)1.5D 1C s A I VN o r e m i s s i o nT r a n s p l a n t e c t o m y M 6M C D D 11(n =21)C O L (n =10)XT r a n s p l a n t e c t o m y M 6C O L4122N O S (n =17)4D 10C s A o r a l +P PN o r e m i s s i o nM C D D 12(n =18)C O L (n =11)C O L (n =9)428N O S (n =16)1.2D 0C y c l o p h o s p h a m i d e +P PN o r e m i s s i o n T r a n s p l a n t e c t o m y M 24M C D D 10(n =16)M C D (n =20)C E L L (n =12)T r a n s p l a n t e c t o m y M 24C E L LM e a n ±S D 9.2±6.93.9±3.319.5±11.6D ,d a y ;M ,m o n t h ;C s A ,c y c l o s p o r i n e ;I V ,i n t r a v e n o u s ;P P ,p l a s m a p h e r e s i s ,CE L L ,c e l l u l a r ;C O L ,c o l l a p s i n g ;N O S ,m o t o t h e r w i s e s p e c i f i e d ;T I P ,t i p l e s i o n ,P H ,p e r i h i l a r ;M C D ,m i n i m a l c h a n g e d i s e a s e ;X ,r e t u r n i n d i a l y s i s .1325Variants of FSGS and risk of recurrenceTable3.Course of FSGS variants on kidney biopsies performed in transplant recipients with recurrence of proteinuria on successive kidney transplant Second allograft Third allograftBiopsy at time Biopsy at timeof recurrence Month3Biopsy Month12of recurrence Month3Month12 Native First(number of(number biopsy(number(number of(number biopsy(number Patients kidney allograft glomeruli)of glomeruli)of glomeruli)glomeruli)of glomeruli)of glomeruli) 3CELL COL MCD(n=12)NOS(n=10)NOS(n=7)MCD(n=9)MCD(n=18)MCD(n=16) 7CELL NOS MCD(n=23)MCD(n=14)X19NOS COL MCD(n=16)MCD(n=15)NOS(n=8)32PH COL MCD(n=15)MCD(n=12)TIP(n=12)36NOS PH MCD(n=9)MCD(n=20)MCD(n=14)42NOS CELL MCD(n=7)MCD(n=18)MCD(n=20)MCD(n=17)MCD(n=12)MCD(n=9) CELL,cellular;COL,collapsing;NOS,not otherwise specified;TIP,tip lesion;PH,perihilar;MCD,minimal change disease;X,return in dialysis.are on long-term permanent therapy with PP).Conversely, patients who never achieved complete and sustained re-mission developed FSGS lesions.Histological findings on native kidneys were not predictive of either recurrence or category of FSGS variant on3-month or12-month biop-sies,and only3patients(Table2:patients7,16and24) had the same variants on native kidney biopsies and on 12-month transplant biopsies.In four cases(Table2:pa-tients3,7,31and35),the FSGS variant changed during the post-transplant course.Six patients experienced a recurrence on a second allo-graft and two on a third one.Histological findings were different from native kidney on the first,second or even the third allograft(Table3).In the recurrence group,6/42patients lost their graft during the first year and12/42patients during the first5 years.The five-year graft survival in this group was71.5%. In the group exempt of recurrence,the5-year graft survival was85.5%Only one patient had an electron microscopy study(Ta-ble2:patient6)at the time of nephrotic-range proteinuria recurrence with the observation of foot process effacement. DiscussionAn early transplant biopsy in patients with recurrence of nephrotic-range proteinuria without signs of rejection shows normal glomeruli by light microscopy and no de-posit by immunofluorescence.Electron microscopy,when performed,shows widespread foot process effacement.Le-sions of FSGS develop only after several weeks as a con-sequence of persistent proteinuria.Therefore,the term of FSGS recurrence is confusing,and in this paper the re-currence was considered when massive proteinuria occurs soon after transplantation even if renal biopsies did not show histological lesion of FSGS.In the absence of glomerular immune deposits and in the absence of histological sign of rejection,the diagnosis of recurrence is almost certain. Moreover,those patients with recurrence of massive pro-teinuria who respond to therapy with complete and sus-tained remission of proteinuria do not develop FSGS as it was the case in17/42patients in our series.In native kidney,we found a distribution of FSGS vari-ants different from the adult American population,but sim-ilar to the only paediatric report available[23–25].NOS and CELL were the most frequent variants observed,with a greater representation of NOS,supporting the idea that it is the commonest variant even in a paediatric popula-tion.Interestingly,we observed TIP(7.8%)and PH(7.8%) variants in children.The fact that all FSGS variants were present in the native kidney,even the TIP lesions that are often steroid sensitive and have a more favourable outcome [15],demonstrates that all FSGS variants can lead to ESRD. Interestingly,perihilar variant,usually observed in the sec-ondary form[26],was present in native kidneys of four patients who experienced recurrence.Furthermore,after transplantation,two patients suffering from recurrence had a perihilar variant.At the time of proteinuria recurrence,MCD was the most frequently histological pattern.Only one patient(patient38) had already developed FSGS(PH variant)on Day15,on the transplant kidney.One might hypothesize that this le-sion was present in the donor kidney.But,this donor had no past history of proteinuria,and pre-transplant biopsy did not show any glomerular abnormality.Furthermore, the following course of this recipient was concordant with recurrence.Thus,we interpreted this as the result of a prob-ably uncontrolled and more aggressive disease leading to the rapid constitution of FSGS.Interestingly,early FSGS was also observed in two patients in a recent study(COL on Day7and NOS on Day10)[21].At Month3and subsequently,the incidence of MCD decreased progressively while FSGS was increasingly ob-served in those patients with persistent proteinuria.Con-versely,at1year,there was a strong association between sustained remission and the absence of FSGS.So,on se-rial biopsies,the incidence of MCD decreased over time and the incidence of FSGS variants increased.As we previously observed,patients who achieved complete remission under intensive and prolonged treatment of recurrence did not develop FSGS lesion[27].These serial biopsies indicated a correlation between the clinical status and histological findings.During this period(1984–2007),many treatments were used to treat INS recurrence with good efficiency(5-year graft survival is71.5%in our study population).Concor-dant studies had demonstrated the relative efficiency of the association of intravenous cyclosporine with plasma ex-changes to obtain and sustain complete remission[27,28].1326G.Canaud et al.Fig.3.Summary of the different patterns of FSGS variant observed on a native kidney of patients with recurrence of proteinuria in the nephrotic range,and variant observed on a transplant kidney.This series confirmed that rituximab did not dramatically change the course of recurrence,and did not consistently reach remission[29,30].The aims of this study were to determine if the Columbia classification of FSGS(1)could predict the risk of re-currence of INS,and(2)the type of FSGS variant after transplantation.Here,we showed that all FSGS variants, even the PH variant,were present in the native kidney of patients with idiopathic NS progressing to ESRD,and that no difference in their distribution was observed between recurrent and non-recurrent patients.In other words,re-currence of proteinuria/NS may be observed whatever the type of FSGS variant and conversely patients with an FSGS variant(COLL,CELL)were usually associated with a se-vere clinical course.On the other hand,all types of FSGS variants were observed in the transplant kidney and we did not find any correlation between the variant present in the native kidney and those occurring in transplanted kidney (Figure3).It should be stressed that only3/21had the same FSGS variant in native and transplanted kidneys.In-terestingly,all FSGS variants were observed in transplanted kidneys with recurrence.The discrepancy between variant native and transplant kidneys may have several explanations.Several factors may play a role in the development of FSGS in nephrotic pa-tients.First of all,the probable immunological course of the disorders may lead to the production of the pathogenic circulating factor.This factor is the same before and after transplantation,and its consequences could be modified by the immunosuppressive regimen.The long-term use of im-munosuppressive drugs,especially calcineurin inhibitors, promotes obliterative vascular lesions and ischaemic ter-ritories that can lead to the development of FSGS.The form associated with ischaemia is usually the collapsing variant[31,32].Secondly,the genetic background of the transplanted kidney,different from the recipient,may be also in question.Supporting this hypothesis,recent data have highlighted that multiple MYH9SNPs and haplotypes were recessively associated with FSGS[33].In addition,the haemodynamic condition associated with a solitary kidney could favour the development of FSGS,or more specifi-cally,the PH variant.T wo other variables render it difficult to classify the variant:firstly biopsy sampling,and sec-ondly the delay between onset of proteinuria and biopsy. In this series,biopsy specimens were serially studied and a mean of13.7glomeruli per biopsy was examined allowing a precise evaluation of FSGS variants.Indeed,late biopsies after the beginning of the disease are more difficult to in-terpret because all FSGS variants found at early stages of the disease might progress towards NOS variant[15].Following transplantation,the rate of recurrence of pro-teinuria varies between30%and50%and is associated with a poor graft outcome[8–14].Several risk factors for recurrence have been identified including older age at on-set on disease,rapid progression to ESRD and recurrence on a previous graft but none of them can clearly distin-guish between patients who will and those who will not be affected by recurrence[34].On native kidneys,the recent Columbia University classification is interesting since it combines epidemiological data on native kidneys with in-formation on the prognosis and response to treatment.The two main variants are NOS and CELL.The COL variant is associated with the worse prognosis i.e.poor or no response to treatment.In contrast,patients with the TIP variant of-ten respond to corticosteroids and rarely progress to ESRD. NOS,PH and CELL variants are associated with an inter-mediate prognosis[23,25,35].The aim of our study was to evaluate the potential value of the Columbia classification of FSGS to predict risk and the type of recurrence,ques-tions that had never been addressed.Ijpelaar et al.recently identified in21cases three distinct patterns of recurrence of FSGS in renal transplant:firstly,recurrence of the same variant of FSGS;secondly,recurrence of the same variant of FSGS after an intermediate state of MCD and a third pattern of recurrence with a different FSGS variant in the allograft[21].The authors found a high rate of concordance of FSGS variant between native and transplanted kidneys. The discrepancy with our results may be explained by sev-eral differences.On the native kidney,diagnosis of FSGS and the type of variant in our series were all performed on renal biopsies with a mean of13.7±4.6(range6–24) glomeruli,whereas it was performed on the nephrectomy specimen in6/19native kidney in the Ijpelaar study.In-deed,the predominant pattern of NOS variant observed in their study is certainly explained by the course of disease progression as it was suggested in the Columbia Classifica-tion[15].In the same way,after kidney transplantation,in the Ijpelaar study,transplant biopsies were performed late, at a mean of783.4days(range10–2555),suggesting that most biopsies at the time of recurrence,which occurred classically early after transplantation,were not available.In our series,renal biopsies were performed within the first3 months of recurrence,an explanation for the diverse type of FSGS lesion observed.In conclusion,our series is the first to report serial biopsy data in kidney transplant recipients with recurrent nephrotic syndrome.Our results suggest that the Columbia classifi-cation of FSGS is not helpful in predicting neither recur-rence nor the histological variant of FSGS when recurrence develops.Conflict of interest statement.None declared.References1.Barisoni L,Schnaper HW,Kopp JB.A proposed taxonomy for thepodocytopathies:a reassessment of the primary nephrotic diseases.Clin J Am Soc Nephrol2007;2:529–5422.Habib R,Levy M,Gubler MC.Clinicopathologic correlations in thenephrotic syndrome.Paediatrician1979;8:325–3481327Variants of FSGS and risk of recurrence。

新视野大学英语视听说教程第二版4（答案）Unit 1 Enjoy your feelings!I Lead-in1. out of sorts (sadness)2. over the moon (happiness)3. feel a shiver run down one’s spine (fear)4. hit the roof (anger)II Basic listening practice1. C2. B3. D4. A5. DIII Listening inTask 11. A2. C3. D4. C5. BTask 2(1) families (2) chemicals (3) information (4) certain (5) self-esteem (6) thinking patterns (7) mood (8) divorce (9) physical abuse (10) financial difficulties (11) stress (12) anxiety Task 31. B2. D3. A4. A5. CIV Speaking outModel 1(1) No wonder you’re livid(2) I’d be mad too if someone ripped me off like that(3) they didn’t have to be so blunt(4) I almost hit the roof(5) to keep my coolModel 2(1) you’ve been down in the dumps(2) I’ve come to cheer you up(3) I’m too depressed(4) Tell me what’s on your mind(5) I’ll help you solve the biggest problem(6) Forget it. You’re on your ownModel 3(1) You seem to be on top of the world tonight(2) I’m so happy I’m about to burst(3) qualify for a bachelor’s degree(4) No wonder you're beaming(5) The multinational I was doing my field project at offered me a job at a good starting salary(6) I feel like celebratingV Let’s talk(1) shy (2) crying (3)scared(4) came down (5) fun (6) nice(7) two step (8) argue (9) touch(10) bad time (11) speak (12) comfortable(13) brother (14) adults (15) children(16) secondary (17) growing (18) learnVI Furthering listening and speakingListeningTask 1(S1) owner (S2) running (S3) drop (S4) run(S5) local (S6) yelling (S7) lives(S8) As he’s picking himself up, he sees a large man, almost seven feet tall(S9) The bartender nervously hands the big man a beer, hands shaking(S10) I got to get out of town! Do n’t you hear Big John is comingTask 21. A2. B3. C4. C5. DTask 31. T2. F3. F4. T5. FViewing and speaking(1) Seven (2) 150 (3) favourite (4) bridge (5) 111 (6) fast (7) Simple (8) trusted (9) stupid (10) did (11) No way (12) ultimate (13) limits (14) skywards (15) ￡60 (16) cheap新视野大学英语视听说教程第二版4（答案）Unit 2 Beauty can be bought.I Lead-in6 4 1 578 2 3 9II Basic listening practice1. B2. A3. C4. D5. BIII Listening inTask 11. C2. A3. A4. D5. BTask 2(1) the end (2) discounts (3) T-shirts(4) big-name brands (5) Interchangeable pieces (6) black trousers(7) several times (8) simplest (9) stylish and fashionable Task 31. A2. B3. A4. C5. DIV Speaking outModel 1(1) I’m floored(2) She’s having her nose fixed(3) I could understand getting surgery(4) She’s also having her teeth straightened(5) a better physical appearance will improve her chances of getting a good job(6) We won’t even recognize her when she gets backModel 2(1) That’s not the way a woman should stay attractive(2) I might look gorgeous, but it wouldn’t be the real me(3) people should be happy with the way they are(4) I’m worried about the wrinkles around my eyes(5) I need a facelift(6) I don’t expect my skin to be baby-smooth, but I should at least be young-looking Model 3(1) How did you find the plastic surgery there?(2) It was all pretty scary at first(3) If I hadn’t recognized your clothes, I wouldn’t have known it was you(4) underneath this surgery, it’s still the same old me(5) If I want to be competitive in the job market, I think this is indispensable(6) I’m skipping lunch, I want to stay in good shapeV Let’s talk(1) wealthy (2) clothing styles (3) figure (4) slimmer(5) construction (6) light (7) larger (8) formal(9) brightly colored (10) the rich and the poor (11) one class (12) occasions (13) Poorer peopleVI Furthering listening and speakingListeningTask 11. C2. A3. A4. B5. ATask 21. F2. T3. F4. T5. FTask 31. She chose two colors, then built her wardrobe around them.2. She has a pair of black dress slacks, with black shoes to match. If she wears that with her turquoise silk blouse and a matching necklace and earrings, she will look dressed up.3. If she wears a T-shirt with the black dress slacks, she is more casual.4. If she brings a pashmina, or another dress scarf, she can dress up the T-shirt into casual chic.5. Her suitcase will be nearly empty, with lots of room for shopping.Viewing and speaking(1) combining clothing with new technology(2) brand-new style(3) new industrial design solutions(4) electronics and fashion(5) into your collar(6) went into partnership with(7) bring them together(8) the modern-day worker(9) 600 pounds(10) to what we might expect新视野大学英语视听说教程第二版4（答案）Unit 3 Watch out when nature strikes back.I Lead-in3 1 24 8 65 7 9II Basic listening practice1. D2. A3. C4. B5. CIII Listening inTask 11. B2. C3. A4. D5. CTask 21. T2. F3. T4. F5. TTask 3(1) drought (2) tsunami (3) undersea(4) result in (5) dry spell (6) flood(7) watre supplies (8) famine (9) agriculture(10) heavy rainfall (11) melting (12) swell(13) man-made damsIV Speaking outModel 1(1) I’m floored(2) She’s having her nose fixed(3) I could understand getting surgery(4) She’s also having her teeth straightened(5) a better physical appearance will improve her chances of getting a good job(6) We won’t even recognize her when she gets backModel 2(1) That’s not the way a woman should stay attractive(2) I might look gorgeous, but it wouldn’t be the real me(3) people should be happy with the way they are(4) I’m worried about the wrinkles around my eyes(5) I need a facelift(6) I don’t expect my skin to be baby-smooth, but I should at least be young-looking Model 3(1) How did you find the plastic surgery there?(2) It was all pretty scary at first(3) If I hadn’t recognized your clothes, I wouldn’t have known it was you(4) underneath this surgery, it’s still the same old me(5) If I want to be competitive in the job market, I think this is indispensable(6) I’m skipping lunch, I want to stay in good shapeV Let’s talk(1) erupted (2) burying (3) few(4) overseas (5) rescue (6) injured(7) damaged (8) islanders (9) seven(10) biggest (11) close (12) aftershocks(13) trap (14) 20 million people (15) diseaseVI Furthering listening and speakingListeningTask 11. B2. A3. D4. A5. CTask 21. Since the company was composed entirely of men over 65, there was doubt that they would be of any assistance. But the farmer called the company anyway because the fire proved to be more than the small town fire department could handle, and there was no other help available.2. The truck drove straight toward the fire and, instead of stopping in front of the fire, drove right into the middle of the flames.3. After an hour of intense fighting, they had extinguished the fire.4. He presented the volunteer fire company with a check for $10,000.5. The captain said, "The first thing we're going to do is to get the brakes fixed on that stupid fire truck." That suggests that they drove right into the middle of the flames because of useless brakes rather than bravery.Task 31. A2. B3. C4. D5. CViewing and speaking(1) rescued (2) worst flooding (3) surprise(4) flooded (5) my son’s (6) crews(7) burst (8) hit (9) save(10) high tide (11) furniture (12) normal (13) computer system (14) drugs (15) heavy新视野大学英语视听说教程第二版4（答案）Unit 4 Is work just another four-letter word?I Lead-in3 4 1 5 8 6 9 7 2II Basic listening practice1. C2. B3. A4. B5. DIII Listening inTask 11. C2. B3. D4. A5. ATask 2(1) Relationships (2) marketing manager (3) in conflict with (4) expenses (5) ended in vain (6) training adminstrator (7) visited (8) establish closer relationships(9) long-term (10) picked upTask 31. C2. D3. D4. C5. AIV Speaking outModel 1(1) Susan speaking. How may I help you(2) Would you please transfer me to Helen Parker, extension 513(3) Mrs. Parker is out of the office(4) May I take a message(5) She can get in touch with me any time tomorrow at 613-2775-2940(6) She’ll get back to you as soon as she canModel 2(1) She was hoping to get together with you this week(2) I’m free as the breeze on Friday(3) Why not meet at about 10 in the morning at your office(4) I’ll look forward to seeing her at 10 on Friday(5) I’m afraid we have to cancel the meeting with you on Friday(6) Shall we meet some other time(7) postpone the meeting until Monday afternoon(8) Would that be convient for youModel 3(1) you are doing excellent work during this internship(2) you went out of your way to bring in new customers(3) the board of directors is unanimous in deciding to offer you a job(4) I’ll work heart and soul to promote its business(5) to take on some new responsibilities(6) I’m promoting you to assistant marketing manager(7) I’m honored that you have so much confidence in me(8) I look forward to the new challenges of the jobV Let’s talk(1) 16 (2) features (3) brief(4) eight (5) paper (6) guidelines(7) interviews (8) treatment (9) come back(10) Deadlines (11) meet (12) guidance(13) scaryVI Furthering listening and speakingListeningTask 11. D2. B3. C4. A5. CTask 21. F2. T3. F4. T5. FTask 31. They gathered for lunch to welcome the new Chief Executive Officer, Carl Martin, and say goodbye to the departing CEO, Dick Jakson.2. The departing CEO left three numbered envelopes for the new CEO.3. The message read, “Blame your predecessor.”So, the new CEO held a press conference and tactfully laid the blame at the feet of the previous CEO.4. The message read, “Reorganize.” The new CEO did it, and the company quickly rebounded.5. The message said, “Prepare three envelopes.” It simplied that it was time for Carl to leave and give three similar envelopes to the next CEO.Viewing and speaking(1) earning (2) live on (3) frustrating(4) head (5) chasing (6) television(7) editor (8) launching (9) basis(10) privileged新视野大学英语视听说教程第二版4（答案）Unit 5 Distant pastures are always greener.I Lead-in1. B2. C3. D4. CII Basic listening practice1. B2. C3. A4. C5. DIII Listening inTask 11. B2. A3. C4. C5. DTask 31. F2. T3. F4. T5. FTask 31. D2. C3. B4. C5. AIV Speaking outModel 1(1) I’m sad that you’ll be leaving us(2) We’re going to miss you(3) Assistants like you are one in a million(4) I’ll put out notices today and screen the applicants myself(5) things certainly won’t be the same without you around here(6) please feel free to call meModel 2(1) Are you sure I haven’t been doing a good job(2) you’ve been reprimanded a number of times for being late(3) I promise to do better in the future(4) this simply can’t be allowed(5) we have to let you go(6) I’m sorry it had to turn out this wayModel 3(1) It’s been operating at a loss(2) the only way out was to lay off redundant employees(3) Why do you think our corporation makes a good career move for you(4) your corporation is a well-known multinational(5) that would be a real boost to your career(6) That will help me grow professionallyV Let’s talk(1) supermarkets (2) a factory (3) capacity(4) kitchen (5) commercial (6) six(7) against (8) my own (9) appeal(10) larger (11) high-risk (12) gamble(13) one-man (14) supportVI Furthering listening and speakingListeningTask 11. A2. B3. C4. D5. ATask 21. T2. T3. F4. F5. FTask 31. She had experience as a career counselor who had counseled hundreds of people about career changes. But when her husband lost his job, they were both caught unprepared.2. They were unprepared to tell their children, unprepared for the mix of emotions, and most dramatically, unprepared for the sudden loss of routine in their lives.3. She saw her husband sitting in their home office patting his forehead and muttering to himself. “Now what am I supposed to do?”4. In the bookstore they found lots of books about job search, but not a single book on the day-to-day chanllenges people face when they get laid-off.5. They had gathered enough interesting material for a book on career trasition-thebook they needed but couldn’t find last July.Viewing and speaking(1) turn things around (2) suggestions (3) expectations (4) how many (5) three million (6) target (7) salespeople (8) market (9) brand (10) problems (11) challenges (12) money (13) hard task (14) salesmen (15) investment (16) experienced新视野大学英语视听说教程第二版4（答案）Unit 6 The truth can be stranger than fiction.I Lead-in1. UFO2. crop circle3. Great Pyramid4. Maya civilization5. Stonehenge6. statues on Easter IslandII Basic listening practice1. C2. C3. A4. D5. BIII Listening inTask 11. C2. B3. A4. D5. CTask 2(S1) terrrible (S2) misfortune (S3) reflection(S4) damaged (S5) heaven (S6) pick(S7) washed(S8) Some people claim that the number is bad luch because 13 people sat down for the Last Supper before Jesus was crucified(S9) Friday the 13th of any month is considered especially bad or unlucky, and Friday the 13th of March is the worst of them all(S10) when people talk about the “seven-year itch” they mean that every seven years a person undergoes a complete change in personalityTask 3(1) falling stock markets (2) even more superstitious (3) much less (4) constructive action (5) less lucky (6) worked(7) more satisfied (8) exams (9) trust(10) revisionIV Speaking outModel 1(1) their belief seems to make some sense(2) knocking on wood was a way to brag without being punished(3) might be frightened away(4) how the custom of kissing under the mistletoe originated(5) I haven’t the slightest idea(6) there is a custom that dates back to the ancient CeltsModel 2(1) so most people are superstitious(2) researched the relationship between superstition and luck(3) people who believe themselves to be lucky tend to go for positive superstitions(4) They worry a lot about a broken mirror(5) our fate is linked to our attitude rather than to our superstitions(6) I wouldn’t be superstitiousModel 3(1) I was visited by aliens(2) ET have visited the Earth before(3) Projects like the pyramids are difficult to explain away(4) some highly advanced civilization assisted humans in their construction(5) You’d go nuts(6) making up a story to get attentionV Let’s talk(1) mystery (2) inside out (3) largest(4) three million (5) outside (6) inner(7) his theory (8) used up (9) the base(10) 137-meter (11) purpose (12) tall, narrow(13) 100,000 or so (14) radarVI Furthering listening and speakingListeningTask 11. A2. B3. D4. D5. BTask 21. T2. T3. F4. F5. TTask 31. The magician did the same tricks over and over again.2. The captain's parrot watched every show and began to understand what the magician did in each trick. Once he understood that, he started shouting in the middle of the show.3. Each time the parrot revealed one of his secrets, the audience roared with laughter. The performance he intended to be dark and mysterious turned into a comedy.4. The ship collided with an enormous iceberg and sank. The magician found himself on a piece of wood, in the middle of the ocean, and the parrot was by his side.5. He said, "OK, I give up. But I hope you'll tell me what trick you are going to do with the boat."Viewing and speaking(1) feet (2) shapes (3) theory(4) man-made (5) look after (6) experts(7) a million (8) outer space (9) extraterrestrial (10) conceivable (11) circle maker (12) image(13) explain (14) genuine (15) lifting out of (16) rubbish (17) mystery (18) mysterious(19) farmers (20) catch (21) appear(22) landscape新视野大学英语视听说教程第二版4（答案）Unit 7 What shall we do when there’s nothing to do?II Basic listening practice1. A2. A3. D4. B5. CIII Listening inTask 11. D2. C3. A4. A5. BTask 21. D2. C3. A4. A5. BTask 3(1) rest (2) Sunday (3) industrial revolution (4) God (5) six-day work week (6) late 19th and early 20th (7) relaxation (8) great boom (9) consumer spending (9) Second World War (10) two days (12) do not work (13) religious activitiesIV Speaking outModel 1(1) Would you like to see a movie(2) Thanks for asking, but(3) I don’t really like to(4) let’s go to see a country singer tonight(5) I’m not really in the mood(6) Is there anything worth watching tonight(7) Do you mind if we watch it(8) I really wanted to watch the Rocket’s gameModel 2(1) Planning holiday trip somewhere(2) I get itchy feet(3) Will this be a trip abroad or some excursion closer to home(4) Are these whirlwind tours(5) That’s more like it. You can look around and not feel rushed(6) You didn’t let me finish(7) variety is the spice of lifeModel 3(1) don’t bug me(2) I was born tired, and I’ve been resting ever since(3) don’t you feel like exercising vigorously to stay in good shape(4) an exercise like walking benefits the mind and body(5) have it your own way(6) if you stay fat, heart trouble or high blood pressure could hopitalize you(7) you may have a point thereV Let’s talk(1) held back (2) transport (3) highest(4) subsistence (5) lower (6) 2,000-kilometer (7) minerals (8) potential (9) development (10) number one (11) tourism (12) only two (13) landscapes (14) fascinating (15) commuting (16) tourist (17) ruined (18) history(19) traditional (20) mistakesVI Furthering listening and speakingListeningTask 11. T2. T3. F4. F5. FTask 21. C2. A3. B4. B5. DTask 31. A2. B3. C4. D5. CViewing and speaking(1) book (2) search engines (3) real customers (4) judge (5) 250 pounds (6) two days (7) partying (8) click (9) targeting新视野大学英语视听说教程第二版4（答案）Unit 8 Is biotechnology our friend or enemy?I Lead-in1. 3 1 6 4 7 2 52. (1) GM crops (2) stem cell (3) genetic map (4) cloningII Basic listening practice1. B2. C3. D4. A5. CIII Listening inTask 11. B2. A3. A4. A5. DTask 2(S1) Research (S2) genes (S3) journal(S4) warn (S5) productivity (S6) grow(S7) survival(S8) Plant breeders have already used preliminary information from the rice genome to create experimental strains of rice that better resist cold and pests(S9) The new map could better explain more than just rice. Rice shares a common ancestor with other cereal crops(S10) While significant progress has been made in the analysis of the rice genome, the mapping of the human genes is also making headwayTask 31. C2. B3. D4. A5. DIV Speaking outModel 1(1) processed foods containing these ingredients aren’t labeled(2) biotech foods have the same composition as organic foods(3) Many people are dying of starvation(4) a genetically modified fish that grew to be ten times its normal size(5) that fish could feed ten times as many people(6) genetically modified foods might help solve world hungerModel 2(1) I have some reservations about it(2) they’ve cloned humans(3) How risky it is(4) Cloned animals grow abnormally large and age unnaturally fast(5) No one knows if cloning damages the mind(6) the cloning of humans should be forbidden by law(7) the cloning of human organs shouldn’t be prohibited(8) I see your pointModel 3(1) it's interfering with nature(2) nature can be pretty hard-nosed(3) A better understanding of the genetic code that controls our body would be a great help in curing many diseases(4) identify and correct genetically-caused diseases(5) Some diseases like cancer are also believed to have a genetic switch(6) it served you right(7) I’m honored that you have so much confidence in me(8) I look forward to the new challenges of the jobV Let’s talk(1) kitchens (2) opposed (3) labeled(4) completely (5) law (6) no way(7) GM ingredients (8) choice (9) tougher(10) any content (11) the public (12) fear(13) safer (14) naturally (15) cheap(16) decideVI Furthering listening and speakingListeningTask 1(1) biotechnology (2) rejected (3) special labels (4) 73 percent to 27 percent (5) a new type of tomato (6) against (7) save lives (8) improve the human condition(9) medicines (10) conventional foodsTask 21. T2. T3. F4. F5. TTask 31. D2. B3. A4. C5. CViewing and speaking(1) trees (2) native genes (3) black(4) onion (5) taste (6) poisonous (7) tested (8) toxic (9) fish (10) popular (11) bigger (12) food (13) normally (14) larger (15) threefold (16) genes (17) originally (18) different (19) risks (20) laboratory (21) environment新视野大学英语视听说教程第二版4（答案）Unit 9 You can learn how to ride the business cycle.I Lead-in4 6 1 8 7 3 2 5II Basic listening practice1. A2. C3. D4. B5. AIII Listening inTask 11. A2. C3. D4. C5. BTask 21. C2. D3. C4. A5. BTask 3(S1) downtowh (S2) loan (S3) security(S4) agreed (S5) against (S6) underground(S7) settle(S8) That will be $5,000 in principal, and $20.30 in interest(S9) We’re very happy to have had your business, and this transaction has worked out very nicely, but we are a little puzzled(S10) Where else in Manhattan can I park my car for two weeks for only $20.30 and expect it to be there when I returnIV Speaking outModel 1(1) the bubble’s going to burst(2) consumer consumption is too high at 70 percent of the GDP(3) What’s behind this big rush to buy things in America(4) Apparently the search for status symbols(5) they buy on credit(6) And too much debt may burst the bubbleModel 2(1) a 37.1 percent jump over the previous year(2) foreign trade reached $523 billion(3) Booming consumption has created opportunities for China’s exports(4) Exporters can get rebates from the customs department(5) She has gradually been decreasing tariffs and strengthening intellectual property rights(6) Some of her state-owned industries have only a blunt competitive edge(7) Some industries still depend on trade protectionismModel 3(1) the technology gap between rich and poor countries is closing(2) the gap between the North and the South was widening(3) struggling below the poverty line(4) the technological advantages of the developed countries are getting smaller(5) Does a narrower technology gap necessarily mean a narrower economic gap(6) information technology is the most powerful engine for economic growthV Let’s talk(1) trouble (2) sales (3) save(4) backed (5) salary (6) private medical(7) 100,000 pounds (8) another (9) money(10) hire (11) debt (12) face the facts(13) securing (14) difficult (15) contacts(16) schoolboy excuseVI Furthering listening and speakingListeningTask 1(1) environment (2) economy (3) minus(4) enterprise’s (5) benefits (6) demands(7) balanced, sustainable (8) generationsTask 21. C2. D3. D4. A5. BTask 31. F2. T3. F4. T5. TViewing and speaking(1) marketing campaign (2) hugh challenge (3) market(4) football (5) eight new teams (6) time-consuming (7) the marketing and the organisation新视野大学英语视听说教程第二版4（答案）Unit 10 Learn how to manage your wealth.II Basic listening practice1. D2. C3. A4. B5. CIII Listening inTask 1(1) future needs (2) shares (3) risk(4) banks (5) 30 (6) a half(7) rate (8) captical loss (9) interchangeably(10) savingsTask 21. D2. B3. C4. C5. ATask 31. T2. F3. F4. T5. TIV Speaking outModel 1(1) It’s been a bear market for several years now(2) The declining market has investors depressed(3) A bear market will eventually be followed by a bull market(4) investors are coming back to the market, and that will drive up the value of stocks(5) the market is picking up(6) shake their confidenceModel 2(1) think long term and take proactive measures(2) you’re a little short of funds at the moment(3) You simply can’t afford to upgrade your facilities without considerable outside investment(4) the interest rate for that much capital would be more than we could afford(5) explain the registration procedure for an IPO(6) you’re financially qualified to be listed on the stock marketModel 3(1) The stock market has dropped again(2) How many points did it lose this time(3) look at the stock listings for today(4) some stocks actually gained(5) My stock went up five percent overnight(6) cash in my stocks(7) I’m honored that you have so much confidence in me(8) I look forward to the new challenges of the jobV Let’s talk(1) b (2) a (3) c (4) e (5) h(6) d (7) i (8) g (9) f (10) jVI Furthering listening and speakingListeningTask 11. A2. B3. D4. B5. CTask 21. C2. B3. D4. A5. CTask 31. He has been following the roller coaster ride of his stock portfolio and it's driving him mad.2. Stocks are pretty hard to predict. So she has put her money into hedge funds.3. A hedge is a way of reducing risk. A hedge fund is a company that creates a stock portfolio that tries to balance the market activity.4. Analysts examine stocks to assess which ones are likely to go up, and which will likely go down.5. He says that if he leaves his money with a fund manager, perhaps the manager can trade his stocks in a more profitable way.Viewing and speaking(1) cash (2) easy (3) results (4) pay (5) bills (6) head (7) 26 percent (8) close the deal (9) figures (10) idea (11) plan (12) fine (13) business guide (14) voting rights (15) say (16) strong point (17) investment (18) 150,000 (19) 26 percent。

专利名称：具有杀线虫活性的几丁质-蛋白质复合物
专利类型：发明专利
发明人：罗伯特·奥斯汀·米尔殊,拉塞尔·J·麦坎德利什,巴巴拉·J·伊斯特伍德
申请号：CN85104021
申请日：19850522
公开号：CN85104021A
公开日：
19870114
专利内容由知识产权出版社提供
摘要：由甲壳纲类的含几丁质生物废料制造几丁质-蛋白质复合物。

这种复合物不同于几丁质和脱乙酰壳多糖，具有对农业和园艺有用的干扰和杀线虫活性。

应用方法是将杀线虫有效量的复合物与植物生长培养基混合。

此复合物还提供一种慢性释放氮源，使得它特别适宜于与肥料和土壤调节剂联合使用。

申请人：IGI生物科技公司
地址：美国马里兰州哥伦比亚
国籍：US
代理机构：中国专利代理有限公司
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《野生黄花苜蓿SKIP同源基因cDNA的克隆与分析》篇一一、引言随着生物技术的不断发展，基因克隆与分析技术已经成为研究植物遗传特性的重要手段。

其中，野生黄花苜蓿作为一种重要的植物资源，其SKIP同源基因的cDNA克隆与分析具有重要的研究价值。

本文旨在通过对野生黄花苜蓿SKIP同源基因cDNA 的克隆与分析，探讨其在植物生长与发育过程中的作用及调控机制。

二、材料与方法1. 材料本实验选用野生黄花苜蓿为研究对象，采用生物信息学手段筛选出其SKIP同源基因序列。

2. 方法（1）提取野生黄花苜蓿的RNA，进行反转录合成cDNA。

（2）根据已知的SKIP同源基因序列设计引物，进行PCR扩增。

（3）将PCR产物进行克隆，筛选阳性克隆并进行测序。

（4）对测序结果进行生物信息学分析，包括序列比对、开放阅读框预测、蛋白质结构预测等。

三、结果与分析1. cDNA克隆结果通过PCR扩增及克隆筛选，成功获得了野生黄花苜蓿SKIP 同源基因的cDNA序列。

序列长度为XXX bp，与已知SKIP同源基因序列具有较高的相似性。

2. 生物信息学分析（1）序列比对将获得的cDNA序列与已知的SKIP同源基因序列进行比对，发现两者具有较高的相似性，表明该序列为野生黄花苜蓿SKIP 同源基因的cDNA序列。

（2）开放阅读框预测通过生物信息学软件预测，该cDNA序列具有一个开放阅读框，编码一个蛋白质。

该蛋白质具有SKIP同源基因的典型结构域，表明其具有SKIP同源基因的功能。

（3）蛋白质结构预测通过蛋白质结构预测软件，预测了该蛋白质的三维结构。

结果显示，该蛋白质具有典型的蛋白质折叠方式，具有一定的稳定性。

同时，该蛋白质具有多个潜在的磷酸化位点，可能参与多种生物学过程。

四、讨论本研究成功克隆了野生黄花苜蓿SKIP同源基因的cDNA序列，并通过生物信息学分析，预测了其编码的蛋白质的结构与功能。

结果表明，该蛋白质具有SKIP同源基因的典型结构域和功能，可能参与植物生长与发育的调控。

高中生物选修2同步练习♦ •同步测控1. （原创题）下列说法不.正确的是（ ）A .生物技术药物一般是指利用 DNA 重组技术或其他生物技术生产的药物B .生物技术通常包括基因工程、细胞工程、发酵工程和酶工程等内容C .生物技术就是将活的生物体、生命体系或生命过程进行产业化的过程D .一种生物技术药物一般只涉及一种生物技术的应用解析：选D 。

本题考查生物技术（生物工程）、生物技术药物的概念，同时要求理清两者的关 系。

在生产生物技术药物时，要应用生物技术，并且往往要应用多种生物技术，如生产生物 技术疫苗常常应用基因工程技术和发酵技术等。

2•降钙素是一种多肽类激素，临床上用于治疗骨质疏松症等。

人的降钙素活性很低，半衰 期较短。

某科学机构为了研发一种活性高、 半衰期长的新型降钙素， 从预期新型降钙素的功 能出发，推测相应的脱氧核苷酸序列，并人工合成了两条 72个碱基的DNA 单链，两条链通过18个碱基对形成部分双链 DNA 片段，再利用 Klenow 酶补平，获得双链 DNA ，过程 如下图：ii 川 DNA 经EcoR I （识别序列和切割位点 一G AATTC —）和BamH I （识别序列和 切割位点一G GATCC —）双酶切后插入大肠杆菌质粒中。

下列有关分析错误的是 （ ）A. Klenow 酶是一种 DNA 聚合酶B .合成的双链 DNA 有72个碱基对C . EcoR I 和BamH I 双酶切的目的是保证目的基因和运载体的定向连接D •筛选重组质粒需要大肠杆菌质粒中含有标记基因解析：选B 。

合成的单链 DNA 有72个碱基，从题图看出两条单链并未左右两端对齐配对，只通过18个碱基对形成部分双链 DNA 片段，因此获得的双链 DNA 有碱基对72 + 72 - 18 =126个碱基对。

F 图是科学家新研发的制备单克隆抗体的过程，相关叙述错误的是体外堆养 A .该过程涉及基因工程和动物细胞融合技术B. 经酶A 处理后的I 与质粒具有相同的黏性末端C.若供体DNA 提供了 prG 基因（无限增殖调控基因），则该过程中的H 是已免疫的B 细胞D .通过检测和筛选得到的川细胞既能无限增殖又能分泌特异性抗体解析：选A 。

2Molecular Markers in EcologyUnderstanding Molecular MarkersIn Chapter1we started to look at the extraordinary wealth of genetic information that is present in every individual,and we started to explore how some of this information can be accessed and used in ecological studies.We will now build on this foundation by looking in more detail at some of the properties of the genetic markers that are used in molecular ecology.We will start with an overview of the different genomes,because the use and interpretation of all markers will be influenced by the way in which they are inherited.The second half of the chapter will be an overview of those molecular markers that are most commonly used in ecology.After reading this chapter you should understand enough about different molecular markers to suggest which would be most appropriately applied to general research questions. Modes of InheritanceGenetic material is transmitted from parents to offspring in a predictable manner, and this is why molecular markers allow us to infer the genetic relationships of individuals.This does not simply mean that we can use genetic data to determine whether two individuals are siblings orfirst cousins.In molecular ecology,the calculation of genetic relationships often takes into account the transmission of particular alleles through hundreds,thousands or even millions of generations.In later chapters we will look at some of the ways in which both recent and historical genealogical relationships can be unravelled,but we mustfirst understand how different genomic regions are passed down from one generation to the next.Not all DNA is inherited in the same way,and understanding different modes of inheritance is crucial before we can predict how different regions of DNA might behave under various ecological and evolutionary scenarios.Molecular Ecology, Second Edition Joanna R. Freeland and Heather Kirk and Stephen Petersen© 2011 John Wiley & Sons, Ltd. Published 2011 by John Wiley & Sons, Ltd. ISBN: 978-0-470-74834-336MOLECULAR MARKERS IN ECOLOGYNuclear versus organelleThe offspring of sexually reproducing organisms inherit approximately half of their DNA from each parent.In diploid,sexually reproducing organisms such as humans, this means that within the nuclear genome one allele at each locus came from the mother and the other allele came from the father.This is known as biparental inheritance.However,even in sexually reproducing species,not all DNA is inherited from both parents.Two important exceptions are the uniparentally inherited organelle genomes of mitochondria(mtDNA)and plastids,with the latter including chloroplasts(cpDNA).These are both located outside the cell nucleus.Mitochon-dria are found in both plants and animals,whereas plastids are found only in plants. Organelle DNA typically occurs in the form of super-coiled circles of double-stranded DNA,and these genomes are much smaller than the nuclear genome.For example at between15000and17000bp,the mammalian mitochondrial genome is approximately1/10000the size of the smallest animal nuclear genome,but what they lack in size they partially make up for in number–a single human cell normally contains anywhere from1000to10000mitochondria.Molecular markers from organelle genomes,particularly animal mtDNA,have been exceedingly popular in ecological studies because,as we shall see below,they have a number of useful attributes that are not found in nuclear genomes.Animal mitochondrial DNAMitochondrial DNA(mtDNA)is involved primarily in cellular respiration,the process by which energy is extracted from food.Animal mtDNA contains13 protein-coding genes,22transfer RNAs and two ribosomal RNAs.There is also a control region that contains sites for replication and transcription initiation.Most of the sequences are unique,in other words they are non-repetitive,and there is little evidence of either spacer sequences between genes,or intervening sequences within transcribed genes.Although some rearrangement of mitochondrial genes has been found in different animal species,the overall structure,size and arrange-ment of genes are relatively conserved(Figure2.1).In most animals,mitochondrial DNA is maternally inherited,in other words it is passed down from mothers to their offspring.There are several reasons why mtDNA markers have been used extensively in studies of animal population genetics.First of all,mtDNA is relatively easy to work with.Its small size,coupled with the conserved arrangement of genes,means that many pairs of universal primers will amplify regions of the mitochondria in a wide variety of vertebrates and invertebrates.This means that data can often be obtained without any a priori knowledge about a particular species’mitochondrial DNA sequence.Second,although the arrangement of genes is conserved,the mutation rate is relatively high.The rate of synonymous substitutions in mammalian mtDNAhas been estimated at 5.7Â10À8substitutions per site per year (Brown et al .,1982b),which is around ten times the average rate of synonymous substitutions in protein-coding nuclear genes.The non-coding control region,which includes the displacement (D)loop ,evolves particularly rapidly in many taxa.The high mutation rate in mtDNA may be partly due to the by-products of metabolic respiration,and also to less stringent repair mechanisms compared to those acting on nuclear DNA (Wilson et al .,1985).Regardless of the cause,these high mutation rates mean that mtDNA generally shows relatively high levels of polymorphism and therefore will often detect multiple genetic lineages both within and among populations.The third relevant property of mtDNA is its general lack of recombination,which means that an offspring will usually have (barring mutation)exactly the same mitochondrial genome as its mother.As a result,mtDNA is effectively a single haplotype that is transmitted from mothers to their offspring.This means that mitochondrial lineages can be identified in a much more straightforward manner than nuclear lineages which,in sexually reproducing species,are continuously pooling genes from two individuals and undergoing recombination.The effectively clonal inheritance of mtDNA means that individual lineages can be tracked over time and space with relative ease,and this is why,as we will see in Chapter 6,mtDNA sequences are commonly used in studies of phylogeography (but see also Box 2.1).The fourth reason for the ubiquity of mtDNA genetic analysis comes from the fact that mtDNA is haploid and uniparentally inherited,and is therefore effectively one-quarter the population size of nuclear DNA.Because there are fewer copies of mtDNA to start with,it is relatively sensitive to demographic events such asND2ase8ATP-ase6COIII ND5Figure 2.1Typical gene organization of vertebrate mtDNA.Unlabelled dark bands represent 22transfer RNAs (tRNAs).Gene abbreviations starting with ND are subunits of NADH dehydrogenase and those starting with CO are subunits of cytochrome c .MODES OF INHERITANCE3738MOLECULAR MARKERS IN ECOLOGYMODES OF INHERITANCE39 bottlenecks.These occur when the size of a population is temporarily reduced,for example following a disease outbreak or a catastrophic event.Even if the population recovers quickly,it will have relatively few surviving mitochondrial haplotypes compared to nuclear genotypes.As we will see in the next chapter,inferring past bottlenecks can make an important contribution towards understanding the current genetic make-up of populations.Thefifth reason why mtDNA has been the most popular marker to study the genetic diversity of animals throughout the past three decades is perhaps also the most controversial:there has been a longstanding assumption of neutrality in mtDNA evolution.This does not mean that mitochondrial DNA is functionally neutral:as noted above,it is comprised of a number of very important genes,for example some of the genes that are involved in cellular respiration.However, beneficial mutational changes are thought to be very rare,in which case the vast majority of mutations should be either deleterious(and hence selected against)or neutral(and hence maintained).A growing number of studies have questioned the neutrality of mtDNA evolution.One study identified protein-coding changes that strongly suggest convergent evolution,and hence positive selection,in the mtDNA genomes of snakes and agamid lizards.This study reported that evidence from morphology,nuclear genes and most sites in the mitochondrial genome consistently identify snakes and lizards as evolutionarily distinct lineages,but a subset of substitutions(mostly substitutions at thefirst and second codon positions which alter the encoded amino acids)in the mitochondrial genome of snakes and agamid lizards provides strong evidence that their mitochondrial sequence similarity in this genetic region is a result of convergent evolution,which would have been driven by natural selection(Castoe et al.,2009)(Figure2.2).A more general investigation of the neutrality of mtDNA was undertaken in a study by Bazin et al.(2006)in which a meta-analysis of more than1600animal species revealed that the average level of mtDNA diversity within species was remarkably similar across the board,whereas different taxonomic groups showed much more variable levels of nuclear genetic diversity.The authors of this study argue that the relative invariance of mtDNA diversity is evidence for recurrent selective sweeps which have led to adaptive evolutionary change.Possible selective mechanisms that could account for such change are reviewed in Galtier et al.(2009).Plant mitochondrial DNAAs with animals,mtDNA in most higher plants is maternally inherited,although heteroplasmy is considered to be more common in plants than in animals.For example mtDNA is paternally transmitted in the redwood tree Sequoia sempervirens and biparentally inherited in some plants in the genus Pelargonium(Metzlaff et al., 1981).The overall function of plant and animal mitochondria is similar,but their structures differ markedly.Unlike animal mtDNA,plant mitochondrial genomesregularly undergo recombination and therefore evolve rapidly with respect to gene rearrangements and duplications.As a result,their sizes vary considerably (40000to 2500000bp).This variability makes it difficult to generalize;to take one example,the mitochondrial genome of the liverwort Marchantia polymorpha is around 186608bp long and appears to include three ribosomal RNA genes,29transfer RNA genes,30protein-coding genes with known functions and around 32genes of unknown function (Palmer,1991).Although the organization of plant mitochon-dria regularly changes,evolution is slow with respect to nucleotide substitutions.In fact,in most plant species the mitochondria is the slowest evolving genome (Wolfe et al .,1987).The overall rates of nucleotide substitutions in plant mtDNA are up to 100times slower than those found in animal mitochondria (Palmer and Herbon,1988),and this low mutation rate combined with an elevated recombination rate mean that plant mitochondrial genomes have not featured prominently in studies of molecularecology.Figure 2.2An anagamid lizard and a snake,two species in which evidence of convergent,and hence adaptive,evolution has been found in the mitochondrial genomes (Castoe et al .,2009).Photos supplied by Todd Castoe.40MOLECULAR MARKERS IN ECOLOGYMODES OF INHERITANCE41 That is not to say that there are no useful applications of mtDNA data in plant studies.For many plant species,dispersal is possible through either seeds or pollen, which often vary markedly in the distances over which they can travel.For example,if seeds are eaten by small mammals they may travel relatively short distances before being deposited.In contrast,pollen may be dispersed by the wind,in which case it could conceivably travel a long way from its natal site.Even if seeds are wind dispersed they are heavier than pollen,and therefore are still likely to travel shorter distances.Nevertheless,it is also possible that the opposite scenario could occur,for example seeds that are ingested by migratory birds may travel much further than wind-blown pollen.Tracking seeds and pollen is extremely difficult,but the different dispersal abilities of the two can sometimes be inferred by comparing the distribu-tions of mitochondrial and nuclear genes.Because mtDNA is usually maternally inherited,its distribution will reflect the patterns of seed dispersal but will not be influenced by the spread of pollen,which contains only the paternal genotype. Canadian populations of the black spruce(Picea mariana)grow in areas that were covered in ice until approximately6000years ago,so we know that the populations must have been established since that time.Researchers found that current populations share the same mtDNA haplotypes,but not the same nuclear alleles(Gamache et al.,2003).This difference was attributed to the widespread dispersal of nuclear genes that were carried by wind-blown pollen,coupled with a much more restricted dispersal of mitochondrial genes which can be transported only in seeds.Because seeds do not usually travel very far,it is likely that only a few were involved in establishing populations once the ice had retreated,hence the lack of variability in mtDNA.On the other hand,the pollen that blew to these sites likely originated in multiple populations,and this has led to a much higher diversity in nuclear genotypes.A similar study in Europe used mitochondrial haplotypes to determine that Norway spruce(Picea abies)colonized Europe by following two different routes from Russia to northern and southern Scandinavia once the ice sheets retreated.Mitochondrial data revealed higher geneflow in the north and lower geneflow in the south,whereas nuclear data revealed higher genetic diversity in the south.This suggests that in southern Scandinavia the relatively low diversity of mtDNA(which resulted from limited seedflow)was augmented by pollenflow,whereas reduced pollen production is likely responsible for the lower levels of nuclear genetic diversity that are found in northern Scandinavia(Tollefsrud et al.,2009).If this study had been based solely on data from either nuclear or mitochondrial DNA we would have an incomplete,and possibly misleading,picture about the dispersal of this coniferous species. Plastids,including chloroplast DNAThe relatively low variability of plant mtDNA means that when haploid markers are desirable in plant studies,researchers more commonly turn to plastid42MOLECULAR MARKERS IN ECOLOGYgenomes including chloroplast DNA(cpDNA).Like mtDNA,cpDNA is maternally inherited in most angiosperms(flowering plants),although in most gymnos-perms(conifers and cycads)it is paternally inherited.Chloroplast DNA genomes, which in most plants are key to the process of photosynthesis,typically range from 120000bp to220000bp(the average size is around150000bp).Although recombination sometimes occurs,chloroplasts are for the most part structurally stable,and most of the size variation can be attributed to differences in the lengths of sequence repeats,as opposed to the gene rearrangement and duplication that is found in plant mtDNA.In tobacco(Nicotiana tabacum),the cpDNA genome contains approximately 113genes which include21ribosomal proteins,four ribosomal RNAs,30transfer RNAs,29genes that are necessary for functions associated with photosynthesis, and11genes that are involved with chlororespiration(Sugiura,1992).A partial arrangement of chloroplast genes in the liverwort(Marchantia polymorpha)is shown in Figure2.3.The average rate of synonymous substitutions in the chloroplast genome,at least in higher plants,is estimated as nearly three times higher than that in plant mtDNA(Wolfe et al.,1987),although this is still four to five times slower than the estimated overall rate of synonymous substitutions in plant nuclear genomes(Wolfe et al.,1989).However,this is an average mutation rate,and the use of cpDNA markers in plant population genetic studies has escalated in recent years following the discovery of highly variable microsatellite regions within the chloroplast genome.Chloroplast microsatellites are most commonly repeats of A/T nucleotides,e.g.AAAAAAAA within a sequence(or TTTTTTTT if you are reading the complementary strand).As we will see later in this chapter,the repetitive nature of microsatellites often results in a relatively high mutation rate.Figure2.3The genome of the chloroplasts found in the liverwort Marchantia polymorpha contains 121024base pairs(Ohyama et al.,1986).These make up an estimated128genes,and the approximate locations of some of these are shown on thisfigure.The dark lines mark the locations of12of the37tRNAs.Genetic data from chloroplasts play an important role in plant ecological studies,in part because cpDNA is uniparentally inherited.In one study,Silvertown et al .(2005)compared the genetic similarity of populations of sweet vernal grass (Anthoxanthum odoratum )(Figure 2.4)on the Park Grass Experiment by using both chloroplast (maternally inherited via seeds)and nuclear (biparentally inherited via seeds and pollen)DNA markers.The Park Grass Experiment is the oldest running ecological experiment in the world,and comprises multiple adjacent plots that have each received different types and amounts of nutrients over the years.These nutrient additions have caused populations to locally adapt to different plots.Despite the proximity of these plots to one another,and the fact that sweet vernal grass is wind-pollinated and should therefore experience much more pollen flow than seed flow,populations from different plots were found to be genetically more similar to one another on the basis of cpDNA compared to nrDNA.These results strongly suggest that seed flow is much higher than pollen flow.This,in turn,supports the hypothesis that it is differences in flowering times that have caused populations to differentiate from one another,because two adjacent plots with different flowering times cannot exchange pollen,but there is no reason why they cannot exchange seeds (Silvertown et al .,2005).This type of reproductive barrier among sympatric populations that has arisen as a result of adaptive evolutionary change is known asreinforcement.Figure 2.4Sweet vernal grass (Anthoxanthum odoratum ),a wind-pollinated species.Photo attributed to James Lindsey.MODES OF INHERITANCE 4344MOLECULAR MARKERS IN ECOLOGYHaploid chromosomesWhen discussing the inheritance of nuclear and organelle markers we usually refer to nuclear genes as being biparentally inherited.For the most part this is true,but sex chromosomes(chromosomes that have a role in the determination of sex) provide an exception to this rule.Not all species have sex chromosomes,for example we saw in Chapter1that crocodiles and many turtles and lizards follow environmental sex determination,which means that the sex of an individual is determined by the temperature that it is exposed to during early development. Many other species follow genetic sex determination,which occurs when an individual’s sex is genetically determined by sex chromosomes.This can happen in a number of different ways.In most mammals,and some dioecious plants, females are homogametic(have two copies of the same sex chromosome:XX), whereas males are heterogametic(have one copy of each sex chromosome:XY). The opposite is true in birds and lepidopterans,which have heterogametic females(ZW)and homogametic males(ZZ).In some other species such as the nematode Caenorhabditis elegans,the male is XO,meaning that it has only a single X chromosome.Monoecious plant species typically lack discrete sex chromosomes.In mammals,each female gives one of her X chromosomes to all of her children, male and female alike.It is the male parent’s contribution that determines the sex of the offspring;if he donates an X chromosome the offspring will be female,and if he donates a Y chromosome then the offspring will be male.The Y chromosome therefore follows a pattern of patrilineal descent meaning that it is passed down only through the male lineage,from fathers to sons(Table2.1).Because there is never more than one copy of a Y chromosome in the same individual(barring Table2.1Usual mode of inheritance of different genomic regions in sexuallyreproducing taxaGenomic region Typical mode of inheritanceAnimals with XY chromosomesAutosomal chromosomes BiparentalMitochondrial DNA Maternal in most animalsBiparental in some bivalvesY chromosome PaternalHigher plantsAutosomal chromosomes BiparentalMitochondrial DNA Usually maternalPlastid DNA(including chloroplast DNA)Maternal in most angiospermsPaternal in most gymnospermsBiparental in some plantsY chromosome Paternal in some dioecious plantsgenetic abnormalities),Y chromosomes are the only mammalian chromosomes that are effectively haploid.In addition,like mtDNA,Y chromosomes for the most part do not undergo recombination.There are two small pseudoautosomal regions at the tips of the chromosome that recombine with the X chromosome,but in between these are approximately 60Mb of non-recombining sequence (Figure 2.5).The mutation rate of Y chromosomes is higher than that of autosomes;in higher primates,for example,the ratio of male-to-female mutation rates has been estimated from DNA sequences to be about 4–6(see Makova and Li,2002,and references therein).However,despite these higher mutation rates,Y chromosome sequences tend to be relatively invariant:one study found that the variability of three genes on the Y chromosome was approximately five times lower than that of the corresponding regions of autosomal genes (Shen et al .,2000).This is likely to be at least partly attributable to selective sweeps,which are facilitated by the relatively small Y chromosome population size:the total number of Y chromosomes in any given species is one-quarter that of autosomes,and one-third that of X chromosomes.Despite relatively low levels of variability,the Y chromosome still has the potential to be a significant source of information because it is much larger than mtDNA and,unlike mitochondria,contains substantial amounts of non-coding DNA.One important property of Y chromosomes is that they allow biologists to follow the transmission of paternal genotypes in animals in much the same way that chloroplast markers can be used for gymnosperms.The majority of studies to date PseudoautosomalregionPseudoautosomalregionp arm q armSRY gene Figure 2.5Mammalian Y-chromosome.The SRY gene (sex-determining region Y)effectively converts an embryo into a male.MODES OF INHERITANCE4546MOLECULAR MARKERS IN ECOLOGYthat have included Y chromosome data have been on humans.A recent paper by Chiaroni et al.(2009)summarizes a lot of the work that has been done in this area.A total of20human Y haplogroups(groups of closely related Y chromosome haplotypes)have now been identified and mapped around the world.The oldest haplogroups are found primarily in Africa,which supports the idea of Africa as the place where humansfirst evolved.Other haplogroups are more or less restricted to other parts of the world such as Europe and the Middle East,or south-east Asia,and by determining the evolutionary relationships of these groups,inferences can be made about human evolution and dispersal around the globe.Despite the tremen-dous potential for obtaining information about reproduction and dispersal from Y chromosomes,there remain relatively few species(apart from humans)for which Y chromosome markers have been developed,although this situation may change in the near future(Greminger et al.,2010).Identifying hybridsIt should be apparent from the examples in the previous section that there is often an advantage to using multiple molecular markers that have contrasting modes of inheritance.These potential benefits are further illustrated by studies of hybrid-ization.Hybrids can be identified from their genotypes because hybridization results in introgression,theflow of alleles from one species(or population)to another.As a result,hybrids will typically contain a mixture of alleles from both parental species,for example a comparison of grass species in the genus Miscanthus revealed that M.giganteus was a hybrid of M.sinensis and M.sacchariflorus,because it had one ITS allele from each parental species,and a plastid sequence that identified the maternal lineage as M.sacchariflorus(Hodkinson et al.,2002).This is known as cytonuclear disequilibrium,which occurs in hybrids that have cyto-plasmic markers(mitochondrial or chloroplast DNA)from one species or pop-ulation,and nuclear markers from another.Some examples of cytonuclear disequilibrium are given in Table2.2.The lack of recombination and mode of inheritance of cytoplasmic genomes mean that one species can potentially retain another species’mitochondrial genome for a very long period of time following introgression;conversely,nuclear introgression will be eroded over time if hybrids continually back-cross to their parental species.As a result,a comparison of phylogenetic trees(trees that show evolutionary relationships among lineages and species;see Chapter6)that are based on nuclear versus organelle DNA can be used to infer past hybridization events.This was the approach taken in a study of canyon tree frogs(Hyla arenicolor)(Figure2.6)and Arizona tree frogs(H.wrightorum),in which some mtDNA haplotypes of H.arenicolor were more similar to haplotypes of H. wrightorum than they were to other haplotypes of H.arenicolor,whereas nuclear genes were consistently more similar within than between species.This wasinterpreted as evidence for historical hybridization which led to the introgression of mtDNA from H.wrightorum into H.arenicolor ,but not vice versa (Klymus et al .,2010).Finally,although in this section we have been primarily concerned with how genomes with different patterns of inheritance can be used to identify hybrids,it is worth noting that hybrids can also be identified using only biparentally inherited markers;see Box 2.2.Table 2.2Some examples of cytonuclear disequilibrium in hybridsHybridNuclear DNA mtDNA or cpDNA Reference Chipmunks (Tamias amoenus ÂT.ruficaudus )T.amoenus canicaudus T.ruficaudus Good et al .(2008)Collared lizards (Crotaphytuscollaris ÂC.reticulatus )C.reticulatus C.collaris McGuire et al .(2007)Water frogs (Rana ridibunda ÂR.lessonae )R.ridibunda R.lessonae Pl €o tner et al.(2008)Subspecies of the common reed(Phragmites australis australis ÂP.a.americanus )P.a.americanus P.a.australis Paul et al .(2010)Figure 2.6A canyon tree frog (Hyla arenicolor )is camouflaged in its rocky habitat.Historical genetic introgression from Arizona tree frogs (H.wrightorum )is evident in mitochondrial,but not nuclear,DNA (Klymus et al .,2010).Photo reproduced with permission of Katy Klymus.MODES OF INHERITANCE 47Uniparental markers:A cautionary noteWhile uniparental markers have many useful applications in molecular ecology,it is important to keep in mind that they also have some drawbacks.We have already referred to the high recombination rates of plant mtDNA and the low mutation rates of cpDNA.While neither of these considerations are particularly relevant to animal mtDNA,there are other limitations that are common to all mtDNA and cpDNA markers.For one thing,organelles behave as single inherited units and are therefore effectively single locus markers.As we will see throughout this text,data from a single locus allow us to retrace the history of only a single genetic unit (gene or genome),which may or may not be concordant with the history of the species in question.This is particularly true of mtDNA,cpDNA and Y chromosomes,because their reduced effective population sizes relative to autosomal DNA means that theirhaplotypes48MOLECULAR MARKERS IN ECOLOGY。

NFATc1Regulates PD-1Expression upon T Cell Activation1Kenneth J.Oestreich,*Hyesuk Yoon,*RafiAhmed,*†and Jeremy M.Boss2*PD-1is a transmembrane protein involved in the regulation of immunological tolerance.Multiple studies have reported an association between high levels of PD-1expressed on T cell surfaces and exhaustion in lymphocyte populations when challenged by chronic viral infections,such as HIV.By using model systems consisting of murine EL4cells,which constitutively express PD-1, and primary murine CD8T cells that express PD-1upon T cell stimulation,we have identified two tissue-specific hypersensitive sites at the5؅CR of the PD-1locus.Gene reporter assays in CD8T cells have shown that one of these sites has robust transcrip-tional activity in response to cell stimulation.Cell treatment with the calcineurin inhibitor cyclosporine A or a NFAT-specific inhibitor led to a sharp reduction in PD-1expression in the constitutive and inducible systems.Furthermore,analysis of this region by chromatin immunoprecipitation assay revealed NFATc1binding associated with gene activation in EL4and primary CD8T cells.Mutation of the NFATc1binding site in PD-1reporter constructs resulted in a complete loss of promoter activity.Together, these results demonstrate that PD-1gene regulation occurs in part via the recruitment of NFATc1to a novel regulatory element at the pdcd1locus and provides the molecular mechanism responsible for the induction of PD-1in response to T cell stimulation.The Journal of Immunology,2008,181:4832–4839.P rogrammed death-1(CD279)is a transmembrane protein that shares homology with the B7/CD28family of T cellsignaling molecules(1).PD-1interactions with its ligands PD-L1and/or PD-L2provides a negative regulatory signal to CD4 and CD8T cells that results ultimately in a phenotype termed T cell exhaustion(2,3).Exhausted T cells have lost the ability to combat invading pathogens,synthesize cytokines,and proliferate(4–6).Spe-cific examples include T cell responses toward chronic viral infections caused by HIV and lymphocytic choriomeningitis virus(LCMV)(7–11).Molecular blockade of the interaction between PD-1and its li-gands results in the reinvigoration of the exhausted T cell’s effector functions,enabling cytotoxic action and cytokine release,suggesting that the PD-1pathway is reversible(11,12).Depending on the model used,genetic studies showed that deletion of the PD-1gene(pdcd1)in mice resulted in the manifestation of spontaneous autoimmune diseases,including arthritis,gastritis,and cardiomyopathy(13–15).Other studies have concluded that the PD-1/PD-L1pathway also plays a role in the onset of diabetes and en-cephalomyelitis in mice(16,17).In humans,polymorphisms within pdcd1have been linked to multiple sclerosis,rheumatoid arthritis, type1diabetes,and systemic lupus erythematosus(18–21).Addi-tionally,aberrant PD-1function and/or misregulation have been im-plicated in aiding tumor evasion from the immune system and playing a role in graft-vs-host disease(18–26).These studies suggest that PD-1is likely to be critically involved in the development and main-tenance of peripheral tolerance.PD-1expression is restricted to cells of the immune system. Early studies demonstrated that PD-1was up-regulated in thymo-cyte populations exposed to apoptotic stimuli(27).PD-1is ex-pressed on thymocytes at the early double negative and double positive stages;however,the full significance of PD-1expression at these stages is not known.In addition to appearing on the sur-faces of exhausted T cells,PD-1is transiently expressed following T cell activation.Upon T cell activation,family members of the NFAT are de-phosphorylated and translocate to the nucleus where they can ac-tivate target genes(28).Nuclear translocation of NFAT is abro-gated by inhibition of the phosphatase activity of calcineurin with immunophilin-interacting compounds such as cyclosporine A(29–31).Examination of cDNA microarray data comparing exhausted, effector,and memory CD8T cells showed an increase in NFATc1 (also called NFAT2)expression,suggesting a possibility for its role in regulating PD-1transcription(32).Despite PD-1’s role in immune responses and its association with disease,little is known about the regulation of this critical gene.In this study,an unbiased approach was taken to determine the mechanism of PD-1regulation during T cell activation as afirst step in understanding the regulatory pathway that controls this gene during complex immune responses.The results have identi-fied two5Јelements that are hypersensitive to DNase I,function together to regulate expression following T cell activation,and, when activated,bear marks of transcriptionally active chromatin. We also show that one of these elements binds and uses NFATc1 as its critical factor in regulating PD-1expression following acti-vation.These studies,therefore,describe key elements contribut-ing to the transcriptional regulatory mechanism that initiates and defines the PD-1pathway.Materials and MethodsCells and cultureThe murine B and T cell lymphoma cell lines A20and EL4were obtained from the American Type Culture Collection.Cells were maintained in RPMI1640supplemented with5%FBS(HyClone),5%bovine calf serum (HyClone),100U/ml penicillin/streptomycin,4.5g/L glucose,1.0mM sodium pyruvate,and10mM HEPES.Primary splenocytes were obtained from C57BL/6mice,and CD8T cells were isolated by negative selection using a magnetic bead purification kit from Miltenyi Biotec.This process*Department of Microbiology and Immunology and†Emory Vaccine Center,EmoryUniversity School of Medicine,Atlanta,GA30322Received for publication June13,2008.Accepted for publication July25,2008.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18U.S.C.Section1734solely to indicate this fact.1This work was supported by a grant from the Concerned Parents for AIDS Research.K.J.O.was supported by a Ruth Kirschstein Institutional Postdoctoral Training Awardfrom the National Institutes of health(T32AI007610).2Address correspondence and reprint requests to Dr.Jeremy M.Boss,Department ofMicrobiology and Immunology,Emory University,1510Clifton Road,Atlanta,GA30322.E-mail address:boss@Copyright©2008by The American Association of Immunologists,Inc.0022-1767/08/$2.00The Journal of Immunology results in CD8T lymphocyte populations of ϳ95%purity as determined by flow cytometry.Where indicated,cells were treated with the following:50ng/ml PMA (Sigma-Aldrich),2mM ionomycin (Sigma-Aldrich),1mg/ml cyclosporine A (CsA)3(Sigma-Aldrich),and 2mM cell-permeable VIVIT (EMD Biosciences).MiceC57BL/6mice were used for these studies and treated and cared for ac-cording to approved protocols.Real-time RT-PCR analysisRNA was prepared using the RNeasy kit (Qiagen).Two micrograms of total RNA were used for each reverse transcriptase reaction conducted using the GeneAmp RNA PCR kit (Applied Biosystems).Real-time PCR was used to quantify the amount of PD-1mRNA,and results were nor-malized to that of 18S rRNA.Each experiment was performed at least three times,and SE bars were calculated.A list of the primers used in this manuscript can be found in Supplemental Table I.4DNase I hypersensitivity assaysDNase I hypersensitivity assays were conducted as described by Lu et al.(33).In brief,nuclei were isolated from 2ϫ107cells and treated with increasing concentrations of DNase I enzyme (Worthington Biochemical).Following digestion,genomic DNA was isolated and digested with the appropriate restriction enzymes (NEB).DNA was subjected to gel elec-trophoresis and subsequent blotting to a nitrocellulose membrane over-night.Following blotting,membranes were hybridized with a gene-specific radiolabeled probe,and the results were visualized by autoradiography.The protocol for the PCR-based hypersensitivity assay was adapted from Schoenborn et al.(34).In brief,nuclei were harvested as above,treated with increasing concentrations of DNase I,and genomic DNA was isolated following phenol/chloroform extraction and ethanol precipitation.Real-time PCR was conducted using PCR amplicons spanning portions of the PD-1locus and relative hypersensitivity was determined by quantifying the level of retained DNA following DNase I treatment.Relative fold change was normalized to samples that received no DNase I treatment and a control fragment that remained refractory to DNase I cleavage.These assays were performed on three individually prepared sets of DNA.The data were averaged and plotted with SE bars.Gene reporter assaysThe pGL3basic plasmid (Promega)containing the firefly reporter gene was used in transient transfection transcriptional reporter assays.PCR frag-ments containing the PD-1promoter region with CR B or both B and C were cloned into the vector using an Xho I restriction site.In all assays,the results were normalized to the signal obtained for an expression vector constitutively expressing the renilla luciferase gene that was cotransfected.Transfections were conducted by electroporation in the case of EL4cells and by nucleofection (Amaxa)for primary CD8T cells.At least three independent experiments were performed,and SE bars were calculated based on the combined errors from the data obtained in each experiment.Chromatin immunoprecipitation (ChIP)assaysChIP assays were performed as previously described (35).In brief,chro-matin was isolated from 4ϫ107cells.Chromatin was precleared using protein A beads,and IP with appropriate Abs was conducted overnight.Anti-H3K9Ac ,anti-H3K18Ac ,and anti-H3K4me3were purchased from Mil-lipore.Anti-NFATc1and anti-NFATc2were purchased from Santa Cruz Biotechnology.Following the IP and subsequent washing steps,DNA was isolated and subjected to real-time PCR analysis and quantitated based on a standard curve to that primer set.The values for ChIP-immunoprecipi-tated DNA were normalized to that of input DNA and then plotted as fold over the normalized values of irrelevant Ab control (anti-TCR).Each ChIP analysis was performed a minimum of three times from independent chro-matin preparations and SE bars were calculated.EMSAsNuclear extracts were prepared from EL4cells and probes,and binding reactions were prepared and conducted as described (36–38).Ab super-shifts were conducted with an anti-NFATc1Ab (Santa Cruz Biotechnol-ogy).DNA protein complexes were resolved on 5%polyacrylamide gels (69:1::acrylamide:bisacrylamide)in a taurine-based gel buffer system.Dried gels were exposed and the results were visualized using autoradiography.StatisticsStudent t tests were used to assess the significance of differences in all quantitative results.ResultsPD-1is transiently induced on activated CD8T cells and is con-stitutively expressed in cells exhibiting the exhausted phenotype (8–11).To develop systems in which PD-1expression could be studied,a number of lymphocytic cell lines were analyzed for ex-pression of PD-1.The EL4T lymphoma cell line was identified as constitutively expressing high levels of PD-1mRNA,which could be further induced following stimulation with PMA and ionomycin (Fig.1A ).In contrast,the A20B lymphoma line was found to be negative for PD-1expression (Fig.1A ).Additionally,ex vivo iso-lated CD8T cells from C57BL/6mice could achieve significant levels of PD-1expression following stimulation with PMA and ionomycin (Fig.1B ).In the experiments that follow,the cell lines EL4and A20will be used to initially identify and assess PD-1regulatory elements,while ex vivo stimulated CD8T cells will be used to confirm the findings in primary cells.Two hypersensitive sites are located in the 5ЈCRs of PD-1Complex gene regulation involves the combinatorial action of cis -acting elements,including promoters,enhancers,locus control re-gions,and boundary elements.To identify potential conserved reg-ulatory elements encoded in the PD-1locus,the University of California Santa Cruz genome browser ()3Abbreviations used in this paper:CsA,cyclosporine A;ChIP,chromatin immuno-precipitation;CR,conserved region;DHS,DNase I hypersensitive site.4The online version of this article contains supplemental material.FIGURE 1.Analysis of PD-1transcription in murine cell lines and primary T cells.A ,Expression levels of pdcd1transcripts were determined in cell lines by quanti-tative RT-PCR.Where indicated,EL4cells were treated with PMA/ionomycin overnight (ϩP/I).Expression levels were normalized to quantitative RT-PCR values obtained for 18s rRNA.B ,CD8T cells were isolated as described under Materials and Methods and left untreated or treated with PMA/ionomycin overnight.Following incubation,the cells were harvested and the levels of PD-1mRNA were determined as above.The data are expressed as fold PD-1expression over untreated/control CD8T cells.Each experiment was performed a minimum of three times and SE bars were calculated.4833The Journal of Immunologywas used to screen for regions that encode high mammalian se-quence homology.Two highly CRs located 5Јto the transcrip-tional start site were identified through basic local alignment search tool homology sequences and were termed CR-B and CR-C with CR-A representing the first exon of PD-1(Fig.2A ).Sensitivity of intact chromatin to DNase I digestion is a hall-mark of accessible chromatin and potential regulatory function.To ascertain the significance of the conserved sequences,DNase I hypersensitivity assays were performed on A20(PD-1negative)and EL4(PD-1positive)lymphocytes.The results highlighted two DNase I hypersensitive sites (termed DHS-B and DHS-C),which were located within four kilobases of the transcriptional start site.These sites mapped to CR-B and CR-C (Fig.2B ).Importantly,the hypersensitive sites were present in the PD-1-expressing cell line EL4but not in the null cell line A20,suggesting that these CRs may regulate PD-1expression.To evaluate these sites in primary CD8T cells,a quantitative-PCR based assay was used (34)to analyze DNase I hypersensi-tivity in ex vivo isolated cells where total cell numbers can be limiting.In this assay,DNase I hypersensitivity is measured by the inability to amplify a region of DNA following purification from nuclei treated with nuclease.A reduction or loss of PCR product/signal signifies that a region was sensitive to DNase I.As com-pared with unstimulated ex vivo isolated CD8T cells,amplicons spanning CR-B and CR-C were diminished significantly following ex vivo stimulation (Fig.3,B and C ).The intensity of the control amplicon remained relatively unchanged following stimulation.CRs B and C encode transcriptionally active sequences To determine the functional relevance of the regions encoding the hypersensitive sites identified by DNase I hypersensitivity analy-sis,luciferase promoter reporter vectors containing the PD-1pro-moter region with either CR-B or CR-B and CR-C were con-structed and transiently transfected into A20and EL4cells (Fig.4).Cells transfected with CR-B induced a moderate level of lu-ciferase activity in both cell types,while a more robust level of promoter activity was seen only in EL4cells transfected with CR-B and CR-C.In contrast,the addition of CR-C to CR-B led to a notable decrease in transcriptional activity of the reporter vector in A20cells.In conjunction with the data in Figs.2and 3,these results strongly suggest that CRs B and C contain regulatory ele-ments necessary for PD-1promoter activity in EL4cells.Active chromatin modifications are present at the PD-1regulatory regions and promoter in PD-1expressing cellsInduction of reporter activity in the PD-1-negative A20cells sug-gested that an additional mechanism(s)might play a role in the regulation of PD-1.Transcriptional regulatory regions are associ-ated with a combination of specific posttranslational modifications on their nucleosomes (39).These modifications are indicative of the potential activation state of the local chromatin structure.To identify such marks for PD-1,ChIP assays for histone modifica-tions associated with gene expression were conducted in PD-1ex-pressing and nonexpressing cell lines (Fig.5A ).Compared with A20cells,the upstream sequences of the PD-1gene in EL4cells displayed high levels of histone acetylation at lysines 9and 18of histone 3(H3K9Ac and H3K18Ac ).Histone acetylation levels were highest at CR-B but still robust at CR-C and the sequences sepa-rating the two regions.H3K4me3marks were highest at CR-B,the site closest to the transcriptional start site.This latter mark is often associated with actively transcribing genes and typically peaks at transcription start sites (40).To examine the chromatin structure of the PD-1locus in pri-mary cells,CD8T cells were isolated from the spleens of C57BL/6mice and ChIP assays were performed with chromatin obtained from PMA/ionomycin treated and untreated cells.High levels of H3K9Ac and H3K4me3could be detected in the CD8T cells stim-ulated to produce PD-1when compared with untreated controls (Fig.5B ).These data indicate that the 5Јupstream regions of the pdcd1locus exhibit hallmarks of active genes in both cell lines and primary CD8T cells.NFAT inhibition results in a reduction of transcriptional activation and PD-1expressionSeveral pieces of information suggested that NFAT might be in-volved in PD-1expression.First,agents that block T cell activa-tion such as the NFAT inhibitor CsA have previously been found to cause a decrease in PD-1expression in mice undergoing trans-plantation responses (41).Second,transcripts of NFATc1were observed at higher levels in exhausted CD8T cells when comparedACBHProbeC o n s e r v a t i o nFIGURE 2.Two DHS are located in 5ЈCRs of the pdcd1locus.A ,The University of California Santa Cruz genome browser was used to identify regions of high DNA sequence conservation in the region 5Јof the PD-1transcriptional start site (arrow).Two regions were identified and termed B and C (as seen labeled above with capital letters).B ,Nuclei from EL4and A20cells were isolated and treated with increasing concentrations of DNase I.Genomic DNA was obtained and digested with Hin dIII (H)to generate the full-length genomic DNA fragment (line below schematic).Digested DNA was then subjected to Southern blot analysis with a probe specific for the 3Јend of the Hin dIII fragment (box labeled in schematic).The resulting DHS map to CRs B and C located 5Јof the pdcd1gene and are indicated by arrows.4834REGULATION OF PD-1with naive,effector,and memory phenotypes (32).Lastly,se-quence analysis of the 5Јupstream region of PD-1,including CR-B and CR-C,using Genomatix (www.genomatix.de)revealed the oc-currence of numerous potential NFAT binding sites.To begin to examine a role for NFAT in PD-1regulation,CsA was used.CsA inhibits the transcription of NFAT regulated genes by preventing the dephosphorylation of NFAT by calcineurin and its subsequenttranslocation to the nucleus (28–31).EL4cells,which constitu-tively produce PD-1,displayed a robust decrease in transcription after CsA treatment (Fig.6A ).Similarly,primary CD8T cells,which produce PD-1upon stimulation with PMA/ionomycin,showed a marked reduction of expression when treated with CsA (Fig.6A ).Because CsA inhibits calcineurin function,there was the possibility that the inhibitory effect was not directly attributable to NFAT.To circumvent this issue,a cell permeable form of the NFAT-specific inhibitor peptide VIVIT was used (42).The results showed that VIVIT specifically inhibited the induction of PD-1in both EL4and primary CD8T cells (Fig.6A ).To further define the role of T cell stimulation and NFAT in-hibitors on PD-1expression,EL4cells were transfected with lu-ciferase promoter reporter vectors containing either CR-B or CR-B and CR-C and the transcriptional response of these constructs to PMA/ionomycin,CsA,and VIVIT was assessed (Fig.6B ).While CR-B displayed little response to PMA/ionomycin,CsA,or VIVIT,a robust increase in transcriptional activation was observed with the reporter vector containing CR-B and CR-C upon cell treatment with PMA/ionomycin.Importantly,this increase was at-tenuated upon addition of CsA or VIVIT.Taken together,these results suggest that PD-1may be an NFAT-regulated gene.NFATc1binds to a PD-1regulatory element in vitro and in vivo The above data implicate NFAT as having a role in the regulation of PD-1expression.However,which NFAT family member was responsible and whether this role was through direct binding to the locus or indirect by influencing the regulation of another trans -acting factor was unclear.Software analysis of potential transcrip-tion factor binding sites (Genomatix)identified five NFAT con-sensus-binding sites within the 5ЈCR of the PD-1gene.Notably,FIGURE 3.DNase I hypersensi-tive sites are located in the 5ЈCR of the pdcd1locus in primary CD8T cells expressing PD-1.A ,A schematic of CRs of PD-1and primer set ampli-cons are shown.B ,Nuclei from A20,EL4,and (C )primary CD8T (ϮPMA/ionomycin)cells were isolated and treated with increasing concentra-tions of DNase I.Genomic DNA was isolated and subjected to real-time PCR analysis with primer sets (I,II,and III)spanning portions of the pdcd1gene.Relative hypersensitivity was assayed by normalization of DNase I-treated DNA to that of the genomic (untreated)DNA control,and the data are plotted as fold sensi-tivity.Increasing DNase I concentra-tion is indicated along the x -axis.The data shown are the average (ϮSE)of three independent experiments.pPD1B pPD1B/CControlA20EL4A20EL4A20EL4CBpPD1B/C pPD1BLUC LUCPD-1FIGURE 4.CRs B and C encode transcriptionally active sequences.Lu-ciferase reporter gene (pGL3)constructs containing CR-B,CR-B and CR-C,or vector alone were transiently cotransfected into EL4or A20cells with a constitutive renilla luciferase expression vector.Following 24h,cells were lysed and assayed for luciferase activity as described in Mate-rials and Methods .Samples were normalized to the renilla luciferase ac-tivity and the average values from three independent experiments were plotted as fold over vector alone.4835The Journal of Immunologyseveral of these sites fall within CR-B and CR-C.To provide a direct association between NFAT and PD-1expression,ChIP assays for NFAT family members NFATc1and NFATc2were conducted across the upstream portion of the pdcd1locus.Assays conducted using EL4cells treated with PMA/ionomycin to ensure full NFAT induction demonstrated that NFATc1(NFAT2)binds to CR-C in EL4cells (Fig.7A )and that NFATc2(NFAT1)does not bind to any of the sequences tested (Fig.7B and data not shown).The location of the ChIP amplicon that was bound by NFATc1was closest to DHS-C.To determine whether NFATc1bound to DHS-C in primary CD8T cells,ChIP assays were con-ducted on ex vivo stimulated primary CD8T cells.As with EL450100150200250AH3K9AcA20 EL4BCD8H3K9AcH3K18IIIA20 EL4C B PD-1I50100150200250300350020406080100120140160 A20 EL4H3K4020406080100120III Con P/I1020304050H3K4Con P/ICD8FIGURE 5.High levels of H3acetylation and H3K4trimethylation are found at the PD-1regulatory regions in PD-1expressing cells.Chromatin was isolated from (A )A20and EL4;or (B )primary CD8T cells (Con)and primary CD8T cells treated with PMA/ionomycin (P/I).ChIP assays were conducted with the indicated Abs,and quantitative real-time PCR was used to determine the levels of chromatin modifications at CR-B,the intervening region between CR-B and CR-C and CR-C.The locations of the PCR amplicons are shown in the schematic.Quantitative PCR results were normalized to input DNA amounts and plotted as fold over an irrelevant Ab control (anti-TCR).The results presented were the average (ϮSE)of three independent experiments.FIGURE 6.NFAT inhibition re-sults in a reduction of PD-1transcrip-tion.A ,NFAT inhibitors reduce PD-1expression.EL4cells and primary CD8T cells (Control,Con;and PMA/ionomycin treated,P/I)were treated with CsA and VIVIT for 2and 5days,respectively.Following cell treatment,total RNA was harvested and the level of pdcd1transcripts de-termined as in Fig.1.B ,NFAT activ-ity is required for activation of re-porter genes encoding CR-C.Luciferase reporter gene (pGL3)con-structs containing CR-B,CR-B and CR-C,or vector alone were tran-siently cotransfected into EL4cells with a constitutive renilla luciferase expression vector.Transfected cells were then left untreated,treated with PMA/ionomycin (P/I)alone,or in combination with CsA or VIVIT.Fol-lowing overnight incubation,cells were lysed and assayed for luciferase activity as in Fig.4.These data rep-resent an average of at least three in-dependent experiments.4836REGULATION OF PD-1cells,NFATc1binding was limited to PD-1expressing cells,oc-curred only within CR-C (Fig.7A ),and NFATc2binding was not detected (Fig.7B ).To determine whether NFATc1binding to the above identified NFAT consensus binding site could be detected in vitro,EMSAs using nuclear extracts from EL4cells stimulated with PMA/iono-mycin were performed with a probe containing the portion of DHS-C encompassing the NFAT binding site (Fig.7C ).A protein/DNA complex was observed using the NFAT binding site from CR-C (termed N1).This complex was supershifted using an NFATc1Ab but not by an irrelevant antiserum (Fig.7C ).Impor-tantly,visualization of the N1complex was abrogated by compe-tition with unlabeled N1DNA but not with a mutant N1DNA sequence.These results demonstrate that PD-1is regulated in part by the direct binding of NFATc1to DHS-C located within con-served regulatory region C.The N1NFAT cis -element is necessary for PD-1transcriptional regulationTo demonstrate that the N1element is necessary for PD-1pro-moter activity,we mutated the NFATc1consensus binding site located in DHS-C and performed reporter assays in primary CD8T cells.When compared with its wild-type counterpart,the mu-tated reporter showed a complete loss of promoter activity follow-ing cell stimulation (Fig.8).Importantly,a reporter construct car-rying a mutation of another consensus NFAT binding site within CR-C showed no loss of promoter activity.A construct carrying1234Con P/IAIVIIIVVIIII CBGCTTGGTGGGGAA GGAAA CATTACTTTGAANFATN1mutN1GCTTGGTGGGGAC TTAAA CATTACTTTGAANFATN 1m u t N 1Comp.α-N f a t c 1α-I g G0123456789BP/ICon 5PD-1//C12345ConP/ICD8EL4*{{FIGURE 7.NFATc1binds to the CR-C regulatory element in vivo and in vitro.ChIP was performed on EL4and CD8T cells with antisera specific to (A )NFATc1,(B )NFATc2,or an irrelevant antiserum (anti-TCR).Cells were either untreated (con)or treated with PMA/ionomycin (P/I)as indi-cated.Quantitative real-time PCR was used to determine the levels of NFATc1binding at locations throughout the 5ЈCR in each cell type.The locations of the PCR amplicons are shown on the schematic below the figures.PCR results were normalized to input DNA amounts and plotted as fold over an irrelevant Ab control (anti-TCR).The results shown representan averaging of three independent experiments.In B ,data are shown for PCR amplicon II.C ,EL4nuclear extracts from cells treated with PMA/ionomycin were incubated with the N1NFAT-encoding binding site probe and Abs where indicated.DNA binding competition assays were conducted with unlabeled N1DNA or the same sequence containing a mutated NFAT binding site (mutN1).An asterisk marks the location of the anti-NFATc1supershifted complex.FIGURE 8.NFATc1binding is necessary for PD-1transcriptional reg-ulation.PD-1promoter reporter constructs (pGL3)containing vector alone,CR-B,CR-B and CR-C,or CR-B and CR-C with the indicated NFAT site mutation were transiently transfected into primary CD8T cells using nucleofection.Cells were left untreated or treated with PMA/ionomycin for 6h.Following incubation,cells were lysed and assayed for luciferase ac-tivity as above.The results presented represent the average of three inde-pendent transfection assays.4837The Journal of Immunology。