分子生物学术语

Glossary of Molecular Biology Terminology

Kenneth Kaushansky, MD*

This glossary is designed to help the reader with the ter-minology of molecular biology. Each year, the glossary will be expanded to include new terms introduced in the Education Program. The basic terminology of molecu-lar biology is also included. The glossary is divided into several general sections. A cross-reference guide is in-cluded to direct readers to the terms they are interested in. The hope is that this addition to the Education Pro-gram will further the understanding of those who are less familiar with the discipline of molecular biology.

C ROSS-R EFERENCE G UIDE

Term Section Actinomycin D pulse experiments V Adeno-associated viral vectors VIII Adenoviral vectors VIII

ALK X Allele-specific hybridization XI Allele-specific PCR IV

AML-1X Amphotropic virus VIII Anaplastic lymphoma kinase X Antisense oligonucleotides VIII Basic helix-loop-helix proteins V

Bcl-1X

Bcl-2X

Bcl-3X

Bcl-6X

β galactosidase V Branched chain DNA signal

amplification assay II

c-abl X

c-fos X

c-jun X

c-myb X

c-myc X

c-ras X Term Section c-rel X Calcium phosphate VI CAN X CAT V cDNA II cDNA blunting IX cDNA library preparation IX cdk V cdkI V CFBβIX Chimeraplasty VIII Chitosan-DNA VIII Chloramphenicol transferase V Chromatography, gel filtration IV Chromatography, ion exchange IV Chromatography, hydrophobic IV Chromatography, affinity IV Chromatography, high performance

liquid (HPLC)IV Cis-acting factors V Codon II Color complementation assay XI Comparative gene hybridization IV Competitive oligonucleotide hybridization XI Concatamerization VI Cyclin-dependent kinase V Contig VII Cosmid II CpG nucleotide II Cyclins V DEAE dextran VI DEK X Dideoxynucleotide (ddN) chain

termination sequencing IV Directional cloning IX DNA (deoxyribonucleic acid)II DNA methylases III DNA microarrays IV DNA polymerase III DNAse footprinting IV DNAse hypersensitivity site mapping IV

* University of Washington School of Medicine, Division of Hematology, Box 357710, Seattle W A 98195-7710

Term Section Ecotropic vectors VIII Ecotropic virus VIII Electroporation VI Endonuclease III Enhancer V Episomal VIII ETO X Evi-1X Exons V Exonuclease III Farnesyl protein transferase III Fas X First strand synthesis IX FISH (fluorescence in situ hybridization)IV FTPase III Gene knock-in experiments VII Gene knock-out experiments VII Helix-turn-helix V Homologous recombination VII Hox II IX HPLC IV Immunoglobulin somatic hypermutation V

In situ hybridization IV Initiation codon V Initiation complex V Interferon regulatory factor X Introns V IRF-1X IRF-2X Isoschizomer III Kinases III Klenow fragment III KOZAK sequence V LCR V Leucine zipper proteins V Library screening IX Ligases III Linkering IX Liposomes VI Locus control region V Long terminal repeat VIII Luciferase V Mammalian protein kinases III Master switch genes V Max X Maxam-Gilbert sequencing IV Minimal residual disease IV Missense mutation V MLL X Mobility shift (or band shift) assays IV mRNA II Mutagenesis, site-specific IV Term Section Nested PCR IV NF-1X Nick-translation IV Nonsense mutation V Nonviral transduction methods VIII Northern blotting IV Nucleases III Nucleosomes V ORF (open reading frame)II

p53X PCR (polymerase chain reaction)IV Phage II Plasmids II Polyadenylation V Polylysine-ligand DNA IX Polymerases III Positional variegation VIII Post transcriptional regulation V Protein translation V Proteomics IV Proteosome V Pseudotype retroviral vectors IV Pseudotyped viruses VIII Random priming IV RAR X Rb X RDA (representational difference analysis)IX Real-time PCR IV Reporter genes V Restriction endonuclease III Restriction fragment length polymorphism XI Retinoic acid receptor X Retroviral vectors VIII Reverse allele-specific hybridization XI Reverse genetics IX Reverse PCR IV Reverse transcriptase III RFLP XI Ribonuclease III Riboprobes IV Ribozymes III RNA (Ribonucleic acid)II RNA polymerase II III RNA polymerase III III RNAse protection assay IV S

1

nuclease analysis IV SCL X Second strand synthesis IX Silencer V Southern blotting IV Southwestern blotting IV Splicing V

Term Section Subtractive library IX

Tal-1X TATA V

Tel X Telomere II Telomerase III Terminal deoxynucleotidyl III Thermostabile polymerases III Topoisomerase III Trans-acting factors V Transcription V Transcription factors V Transcriptional regulation V Transduction VI Transfection VI Transgenic animals VIII Transposon VII tRNA II Ubiquitin V

Viral-derived kinases III

Viral-derived transduction vectors VIII Western blotting IV

X-linked methylation patterns XI

YAC VII Yeast artificial chromosome VII Yeast 2-hybrid screens IV

Zinc finger domain proteins V

II. N UCLEIC A CIDS

DNA (deoxyribonucleic acid) The polymer constructed of successive nucleotides linked by phosphodiester bonds. Some 3 x 109 nucleotides are contained in the human haploid genome. During interphase, DNA exists in a nucleoprotein complex containing roughly equal amounts of histones and DNA, which interacts with nuclear matrix proteins. This complex is folded into a basic structure termed a nucleosome containing approxi-mately 150 base pairs. From this highly ordered struc-ture, DNA replication requires a complex process of nicking, unfolding, replication, and splicing. In contrast, gene transcription requires nucleosomal re-organization such that sites critical for the binding of transcriptional machinery reside at internucleosomal junctions. Branched chain DNA (b-DNA) A method that exploits the formation of branched DNA to provide a sensitive and specific assay for viral RNA or DNA. The assay is performed in a microtiter format, in which partially ho-mologous oligodeoxynucleotides bind to target to cre-ate a branched DNA. Enzyme-labeled probes are then bound to the branched DNA, and light output from a chemiluminescence substrate is directly proportional to the amount of starting target RNA. Standards provide quantitation. The assay displays a 4 log dynamic range of detection, with greater sensitivity to changes in viral load than RT-PCR-based assays. It has been employed to quantitate levels of HIV, HCV, and HBV.

RNA (ribonucleic acid) Three varieties of RNA are eas-ily identified in the mammalian cell. Most abundant is ribosomal RNA (rRNA), which occurs in two sizes, 28S (approximately 4600 nucleotides) and 18S (approxi-mately 1800 nucleotides); together they form the basic core of the eukaryotic ribosome. Messenger RNA (mRNA) is the term used to describe the mature form of the primary RNA transcript of the individual gene once it has been processed to eliminate introns and to contain a polyadenylated tail. mRNA links the coding sequence present in the gene to the ribosome, where it is trans-lated into a polypeptide sequence. Transfer RNA (tRNA) is the form of RNA used to shuttle successive amino acids to the growing polypeptide chain. A tRNA mol-ecule contains an anti-codon, a three-nucleotide se-quence by which the tRNA molecule recognizes the codon contained in the mRNA template, and an adapter onto which the amino acid is attached.

Codon Three successive nucleotides on an mRNA that encode a specific amino acid in the polypeptide. Sixty-one codons encode the 20 amino acids, leading to codon redundancy, and three codons signal termination of polypeptide synthesis.

ORF (open reading frame) The term given to any stretch of a chromosome that could encode a polypep-tide sequence, i.e., the region between a methionine codon (ATG) that could serve to initiate protein transla-tion, and the inframe stop codon downstream of it. Sev-eral features of the ORF can be used to judge whether it actually encodes an expressed protein, including its length, the presence of a “Kozak” sequence upstream of the ATG (implying a ribosome might actually bind there and initiate protein translation), whether the ORF exists within the coding region of another gene, the presence of exon/intron boundary sequences and their splicing signals, and the presence of upstream sequences that could regulate expression of the putative gene. Plasmids Autonomously replicating circular DNA that are passed epigenetically between bacteria or yeast. In order to propagate, plasmids must contain an origin of replication. Naturally occurring plasmids transfer genetic information between hosts; of these, the genes encoding

resistance to a number of antibiotics are the most impor-tant clinically. The essential components of plasmids are used by investigators to introduce genes into bacteria and yeast and to generate large amounts of DNA for manipulation.

Phage A virus of bacteria, phage such as lambda have been used to introduce foreign DNA into bacteria. Be-cause of its infectious nature, the transfection (introduc-tion) efficiency into the bacterial host is usually two or-ders of magnitude greater for phage over that of plas-mids.

Cosmid By combining the elements of phage and plas-mids, vectors can be constructed that carry up to 45 kb of foreign DNA.

cDNA A complementary copy of a stretch of DNA pro-duced by recombinant DNA technology. Usually, cDNA represents the mRNA of a given gene of interest.

Telomere A repeating structure found at the end of chro-mosomes, serving to prevent recombination with free-ended DNA. Telomeres of sufficient length are required to maintain genetic integrity, and they are maintained by telomerase.

CpG This under-represented (i.e. < 1/16 frequency) di-nucleotide pair is a “hotspot” for point mutation. CpG dinucleotides are often methylated on cytosine. Should Me-C undergo spontaneous deamination, uracil arises, which is then repaired by cellular surveillance mecha-nisms and altered to thymidine. The net result is a C to T mutation.

III. E NZYMES OF R ECOMBINANT DNA T ECHNOLOGY

A.Nucleases

A number of common tools of recombinant DNA tech-nology have been developed from the study of the basic enzymology of bacteria and bacteriophage. For example, most unicellular organisms have defense systems to pro-tect against the invasion of foreign DNA. Usually, they specifically methylate their own DNA and then express restriction endonucleases to degrade any DNA not ap-propriately modified. From such systems come very use-ful tools. Today, most restriction endonucleases (and most other enzymes of commercial use) are highly puri-fied from either natural or recombinant sources and are highly reliable. Using these tools, the manipulation of DNA and RNA has become routine practice in multiple disciplines of science.Exonuclease An enzyme that digests nucleic acids start-ing from the 5' or 3' terminus and extending inward.

Endonuclease An enzyme that digests nucleic acids from within the sequence. Usually, specific sequences are rec-ognized at the site where digestion begins.

Isoschizomer Restriction endonucleases that contain an identical recognition site but are derived from different species of bacteria (and hence have different names). Restriction endonuclease These enzymes are among the most useful in recombinant DNA technology, capable of introducing a single cleavage site into a nucleic acid. The site of cleavage is dependent on sequence; recogni-tion sites contain from 4 to 10 specific nucleotides. The resultant digested ends of the nucleic acid chain may either be blunt or contain a 5' or 3' overhang ranging from 1 to 8 nucleotides.

Ribonuclease These enzymes degrade RNA and exist as either exonucleases or endonucleases. The three most commonly used ribonucleases are termed RNase A, RNase T1, and RNAse H (which degrades duplex RNA or the RNA portion of DNA?RNA hybrids). Ribozymes are based on a catalytic RNA characterized by a hammerhead-like secondary structure, and by in-troducing specific sequences into its RNA recognition domain, destruction of specific mRNA species can be accomplished. Ribozymes thus represent a tool to elimi-nate expression of specific genes, and are being tested in several hematological disease states, including neo-plasia. A highly specific RNA sequence can generate secondary structure by virtue of intrachain base pairing.“Hairpin loops” and “hammer head” structures serve as examples of such phenomena. When the proper second-ary structure forms, such RNA molecules can bind a second RNA molecule (e.g. an mRNA) at a specific lo-cation (dependent on an approximately 20-nucleotide recognition sequence) and cleave at a specific GUX trip-let (where X = C, A, or U). These molecules will likely find widespread use as tools for specific gene regulation or as antiviral agents but are evolutionarily related to RNA splicing, which in its simplest form is autocatalytic.

B.Polymerases

DNA polymerase The enzyme that synthesizes DNA from a DNA template. The intact enzyme purified from bacteria (termed the holoenzyme) has both synthetic and editing functions. The editing function results from nu-clease activity.

Klenow fragment A modified version of bacterial DNA polymerase that has been modified so that only the poly-merase function remains; the 5'?3' exonuclease activ-ity has been eliminated.

Thermostabile polymerases The prototype polymerase, Taq, and newer versions such as Vent and Tth polymerase are derived from microorganisms that normally reside at high temperature. Consequently, their DNA poly-merase enzymes are quite stable to heat denaturation, making them ideal enzymes for use in the polymerase chain reaction.

RNA polymerase II This enzyme is used by mamma-lian cells to transcribe structural genes that result in mRNA. The enzyme interacts with a number of other proteins to correctly initiate transcription, including a number of general factors, and tissue-specific and in-duction-specific enhancing proteins.

RNA polymerase III This enzyme is used by the cell to transcribe ribosomal RNA genes.

Kinases These enzymes transfer the γ-phosphate group from ATP to the 5' hydroxyl group of a nucleic acid chain.

Viral-derived kinases These enzymes are utilized in re-combinant DNA technology to transfer phosphate groups (either unlabeled or 32P-labeled) to oligonucleotides or DNA fragments. The most commonly used kinase is T4 polynucleotide kinase.

Mammalian protein kinases These enzymes transfer phosphate groups from ATP to either tyrosine, threo-nine, or serine residues of proteins. These enzymes are among the most important signaling molecules present in mammalian cell biology.

Farnesyl protein transferase (FTPase) F TPase adds 15 carbon farnesyl groups to CAAX motifs, such as one present in ras, allowing their insertion into cellular mem-branes.

Terminal deoxynucleotidyl This lymphocyte-specific enzyme normally transfers available (random) nucle-otides to the 3' end of a growing nucleic acid chain. In recombinant DNA technology, these enzymes can be used to add a homogeneous tail to a piece of DNA, thereby allowing its specific recognition in PCR reac-tions or in cloning efforts.

Ligases These enzymes utilize the γ-phosphate group of ATP for energy to form a phosphodiester linkage be-tween two pieces of DNA. The nucleotide contributing the 5' hydroxyl group to the linkage must contain a phos-phate, which is then linked to the 3' hydroxyl group of the growing chain.

DNA methylases These enzymes are normally part of a bacterial host defense against invasion by foreign DNA. The enzyme normally methylates endogenous (host) DNA and thereby renders it resistant to a series of en-dogenous restriction endonucleases. In recombinant DNA work, methylation finds use in cDNA cloning to prevent subsequent digestion by the analogous restriction endonuclease.

Reverse transcriptase This enzyme, first purified from retrovirus-infected cells, produces a cDNA copy from an mRNA molecule if first provided with an antisense primer (oligo dT or a random primer). This enzyme is critical for converting mRNA into cDNA for purposes of cloning, PCR amplification, or the production of spe-cific probes.

Topoisomerase A homodimeric chromosomal unwind-ing enzyme that introduces a double-stranded nick in DNA, which allows the unwinding necessary to permit DNA replication, followed by religation. Inhibition of topo-isomerases leads to blockade of cell division, the target of several chemotherapeutic agents (e.g., etoposide). Telomerase A specialized DNA polymerase that pro-tects the length of the terminal segment of a chromo-some. Should the telomere become sufficiently short-ened (by repeated rounds of cell division), the cell un-dergoes apoptosis. The holoenzyme contains both a poly-merase and an RNA template; only the latter has been characterized, although the gene for the enzymatic ac-tivity has recently been cloned.

IV. M OLECULAR M ETHODS

A number of molecular techniques have found wide-spread application in the biomedical sciences. This sec-tion of the glossary provides general concepts and is not intended to convey adequate details. The interested reader is referred to the excellent handbook of J. Sambrook and coworkers (Molecular Cloning, A Labo-ratory Manual, 2nd Ed., CSH Laboratory Press, 1989). Maxam-Gilbert sequencing A method to determine the sequence of a stretch of DNA based on its differential cleavage pattern in the presence of different chemical exposures. A nucleic acid chain can be cleaved follow-ing G, A, C, or C and T by exposure of 32P-labeled DNA

to neutral dimethylsulfate, dimethylsulfate-acid, hydra-zine-NaCl-piperidine or hydrazine-piperidine alone, re-spectively.

Dideoxynucleotide (ddN) chain termination sequenc-ing Also termed “Sanger sequencing,” this method re-lies on the random incorporation of dideoxynucleotides into a growing enzyme-catalyzed DNA chain. As no 3' hydroxyl group is present on the ddN, chain synthesis halts following its incorporation into the chain. If 32P or 35S nucleotides are also incorporated into the reaction, a family of DNA fragments will be generated that can be visualized on a polyacrylamide gel. This method is pres-ently the most commonly used chemistry to determine the sequence of DNA.

DNAse footprinting This technique depends on the abil-ity of protein specifically bound to DNA to block the activity of the endonuclease DNAse I. 32P-labeled DNA is mixed with nuclear proteins, which potentially con-tain specific DNA-binding proteins, and the reaction is then subjected to limited DNAse digestion. If a given site of DNA is free of protein, it will be cleaved by the DNAse. In contrast, regions of DNAse specifically bound by proteins (transcription factors or enhancers) will be protected from digestion. The resultant mixture of DNA fragments from control and protein-containing reactions are then separated on a polyacrylamide gel. As the site of 32P labeling of the original DNA fragment is known, sites that were protected from DNAse digestion will be represented on the gel as a region devoid of that length fragment. Therefore, in comparison to naked DNA, re-gions that bind specific proteins will be represented as a “footprint.”

DNAse hypersensitivity site mapping This technique is designed to uncover regions of DNA that are in an “active” transcriptional state. It depends on the hyper-sensitivity of such sites (because of the lack of the highly compact nucleosome structure) to limited digestion with DNAse. Intact nuclei are subjected to limited DNAse digestion. The resultant large DNA fragments are then extracted, electrophoretically separated, and hybridized with a 32P-labeled probe from a known site within the gene of interest. If, for example, the probe were located at the site of transcription initiation, and should DNA fragments of 2 kb and 5 kb be detected with this probe, hypersensitive sites would thereby be mapped to 2 kb and 5 kb upstream of the start of transcription initiation. By extrapolation, these sites would then be assumed im-portant in the transcriptional regulation of the gene of interest, especially if such a footprint were only detected using cells that express that gene.Mobility shift (or band shift) assays Like DNAse footprinting, this technique is also utilized to determine

whether a fragment of DNA binds specific proteins. 32P-

labeled DNA (either duplex oligonucleotides or small

restriction fragments) are incubated with nuclear pro-

tein extracts and subjected to native acrylamide gel elec-

trophoresis. Should specific DNA-binding proteins that

recognize the oligonucleotide or restriction fragment

probe be present in the nuclear extracts, a DNA-protein

complex will be formed and its migration through the

native gel will be retarded compared to the unbound

DNA. Hence, the labeled band will be shifted to a more

slowly migrating position. The specificity of their reac-

tion can be demonstrated by also incubating, in separate

reactions, competitor DNA that contains the presumed

binding site or irrelevant DNA sequence.

S

1

nuclease analysis This technique is used to identify the start of RNA transcription. The DNAse enzyme S

1 cleaves only at sites of single-stranded DNA. Therefore,

if 32P-labeled DNA is hybridized with mRNA, the re-

sulting heteroduplex can be digested with S

1

, and the

resulting DNA fragment will be of length equivalent to

the site at which the piece of DNA begins through the

mature 5' end of the RNA.

RNAse protection assay This assay is in many ways similar to the S

1

nuclease analysis. In this case, a 35S- or 32P-labeled antisense RNA probe is synthesized and hy-bridized with mRNA of interest. The duplex RNA is then subjected to digestion with RNAse A and T

1

, both of

which will cleave only single-stranded RNA. Following

digestion, the remaining labeled RNA is size-fraction-

ated, and the size of the protected RNA probe then gives

an indication of the size of the mRNA present in the

original sample. This assay can also be used to quanti-

tate the amount of specific RNA in the original sample. PCR (polymerase chain reaction) This technique finds use in several arenas of recombinant DNA technology. It is based on the ability of sense and antisense DNA primers to hybridize to a cDNA of interest. Following extension from the primers on the cDNA template by DNA polymerase, the reaction is heat-denatured and al-lowed to anneal with the primers once again. Another round of extension leads to a multiplicative increase in DNA products. Therefore, a minute amount of cDNA can be efficiently amplified in an exponential fashion to result in easily manipulable amounts of cDNA. By in-cluding critical controls, the technique can be made quan-titative. Important clinical examples of the use of PCR or reverse transcription PCR (see below) include (1) detection of diagnostic chromosomal rearrangements

[e.g., bcr/abl in CML, t(15;17) in AML-M3, t(8;21) in AML-M2, or bcl-2 in follicular small cleaved cell lym-phoma], or (2) detection of minimal residual disease following treatment. The level of sensitivity is one in 104 to 105 cells.

RT-PCR (reverse transcription PCR) This technique allows the rapid amplification of cDNA starting with RNA. The first step of the reaction is to reverse-tran-scribe the RNA into a first strand cDNA copy using the enzyme reverse transcriptase. The primer for the reverse transcription can either be oligo dT, to hybridize to the polyadenylation tail, or the antisense primer that will be used in the subsequent PCR reaction. Following this first step, standard PCR is then performed to rapidly amplify large amounts of cDNA from the reverse transcribed RNA.

Nested PCR By using an independent set of PCR prim-ers located within the sequence amplified by the primary set, the specificity of a PCR reaction can be greatly en-hanced. In Figure 1, should the first PCR reaction yield a product of 600 nucleotides, a second PCR reaction us-ing the first product as template and a different set of primers will produce a smaller, “nested” PCR product, the presence of which acts to confirm the identity of the primary product.

Real-time automated PCR During PCR, a fluorogenic probe, consisting of an oligodeoxynucleotide with both reporter and quencher dyes attached, anneals between the two standard PCR primers. When the probe is cleaved during the next PCR cycle, the reporter is separated from the quencher so that the fluorescence at the end of PCR is a direct measure of the amplicons generated through-out the reaction. Such a system is amenable to automa-tion and gives precise quantitative information.

Allele-specific PCR By using generic PCR primers flanking the immunoglobulin or T cell receptor genes, the precise rearranged gene characteristic of a B or T cell neoplasm can be amplified and sequenced. Once so obtained, new PCR primers can then be designed that are unique to the patient’s tumor. Such allele-specific PCR can then be used to detect blood cell contamina-tion by tumor and to detect minimal residual disease fol-lowing therapy.

Southern blotting This technique is used to detect spe-cific sequences within mixtures of DNA. DNA is size-fractionated by gel electrophoresis and then transferred by capillary action to nitrocellulose or another suitable synthetic membrane. Following blocking of nonspecific binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hy-bridize with a cDNA or oligonucleotide probe represent-ing the specific DNA sequence of interest. Should spe-cific DNA be present on the blot, it will combine with the labeled probe and be detectable by autoradiography. By co-electrophoresing DNA fragments of known mo-lecular weight, the size(s) of the hybridizing band(s) can then be determined. F or gene rearrangement studies, Southern blotting is capable of detecting clonal popula-tions that represent approximately 1% of the total cellu-lar sample.

Northern blotting This modification of a Southern blot is used to detect specific RNA. The sample to be size-fractionated in this case is RNA and, with the exception of denaturation conditions (alkali treatment of the South-ern blot versus formamide/formaldehyde treatment of the RNA sample for Northern blot), the techniques are essentially identical. The probe for Northern blotting must be antisense.

Western blotting This technique is designed to detect specific protein present in a heterogenous sample. Pro-teins are denatured and size-fractionated by polyacryla-mide gel electrophoresis, transferred to nitrocellulose or other synthetic membranes, and then probed with an an-tibody to the protein of interest. The immune complexes present on the blot are then detected using a labeled sec-ond antibody (for example, a 125I-labeled or biotinylated goat anti-rabbit IgG). As the original gel electrophore-sis was done under denaturing and reducing conditions, the precise size of the target protein can be determined. Southwestern blotting This technique is designed to detect specific DNA-binding proteins. Like the Western blot, proteins are size-fractionated and transferred to ni-trocellulose. The probe in this case, however, is a double-stranded labeled DNA that contains a putative protein-binding site. Should the DNA probe hybridize to a spe-cific protein on the blot, that protein can be subsequently identified by autoradiography. This technique often suf-fers from nonspecificity, so that a number of critical con-trols must be included in the experiment for the results to be considered rigorous.

Figure 1. Nested PCR.

In situ hybridization This technique is designed to de-tect specific RNA present in histological samples. Tis-sue is prepared with particular care not to degrade RNA. The cells are fixed on a microscope slide, allowed to hybridize to probe, and then washed and overlaid with photographic emulsion. Following exposure for one to four weeks, the emulsion is developed and silver grains overlying cells that contain specific RNA are detected. The most useful probes for this purpose are metaboli-cally 35S-labeled riboprobes generated by in vitro tran-scription of a cDNA using viral RNA polymerase. These probes give the lowest background and are preferable to using terminal deoxynucleotidyl transferase or alterna-tive methods using 32P as an isotope.

FISH (fluorescence in situ hybridization) A general method to assign chromosomal location, gene copy num-ber (both increased and decreased), or chromosomal re-arrangements. Biotin-containing nucleotides are incor-porated into specific cDNA probes by nick-translation. Alternatively, digoxigenin or fluorescent dyes can be in-corporated by enzymatic or chemical methods. The probes are then hybridized with solubilized, fixed metaphase cells, and the copy number of specific chro-mosomes or genes are determined by counter-staining with fluorescein isothiocyanate (F ITC)-labeled avidin or other detector reagents. The number and location of detected fluorescent spots correlates with gene copy number and chromosomal location. The method also allows chromosomal analysis in interphase cells, allow-ing extension to conditions of low cell proliferation. CGH (comparative genome hybridization) In CGH, DNA is extracted from tumor and from normal tissues and differentially labeled with fluorescent dyes. Once the DNA samples are mixed and hybridized to normal metaphase chromosome spreads, chromosomal regions that are under-represented or over-represented in the tu-mor sample can be identified. This method can be ap-plied to extremely small tumor samples (by using PCR methods) of formalin-fixed or frozen tissue. It has been applied to detect loss of chromosome 18q or 17p in co-lon cancer and is likely to be applied to hematologic malignancies. The sensitivity of the technique ap-proaches 1 cell in 100.

Nick-translation This technique is used to label cDNA to high specific activity for the purpose of probing South-ern and Northern blots and screening cDNA libraries. The cDNA fragment is first nicked with a limiting con-centration of DNAse, then DNA polymerase is used to both digest and fill in the resulting gaps with labeled nucleotides.Random priming This technique is also used to pro-duce labeled cDNA probes and is dependent on using random 6- to 10-base oligonucleotides to sit down on a single-stranded cDNA and then using DNA polymerase to synthesize the complementary strand using labeled nucleotides. This technique usually produces more fa-vorable results than nick-translation.

Riboprobes These labeled RNA molecules are produced by first cloning the cDNA of interest into a plasmid vec-tor that contains promoters for viral RNA polymerases. Following cloning, the viral RNA polymerase is added, and labeled nucleotides are incorporated into the result-ing RNA transcript. This molecule is then purified and used in probing reactions. Many such cloning vectors (for example, pGEM) have different RNA polymerase promoters on either side of the cloning site, allowing the generation of both sense and antisense probes from the same construct.

Mutagenesis, site-specific Several methods are now available to intentionally introduce specific mutations into a cDNA sequence of interest. Most are based on designing an oligonucleotide that contains the desired mutation in the context of normal sequence. This oligo-nucleotide is then incorporated into the cDNA using DNA polymerase, either using a single-stranded DNA template (phage M13) or in a PCR format to produce a heteroduplex DNA containing both wild type and mu-tant sequences. Using M13, recombinant phage are then produced and mutant cDNA are screened for on the ba-sis of the difference in wild type and mutant sequences; using the PCR format, the exponential amplification of the mutant sequence results in its overwhelming numeri-cal advantage over wild type sequence, resulting in nearly all clones containing mutant sequence. Both of these methods require that the entire cDNA insert synthesized in vitro be sequenced in its entirety to guarantee the fi-delity of mutagenesis and synthesis of the remaining wild type sequences.

Chromatography, gel filtration This technique is de-signed to separate proteins based on their molecular weight. It is dependent on the exclusion of proteins from a matrix of specific size. Proteins that are too large to fit into the matrix of the gel bed run to the bottom of the column more quickly than smaller proteins, which are included in the volume of the matrix. Therefore, using appropriate size markers, the approximate molecular weight of a given protein can be determined and it can be separated from proteins of dissimilar size. Typical separation media for gel filtration chromatography in-clude Sephadex and Ultragel.

Chromatography, ion exchange This separation meth-odology depends on the preferential binding of positively charged proteins to a matrix containing negatively charged groups or a negatively charged protein binding to a matrix containing positively charged groups. In-creases in the buffer concentration of sodium chloride are then used to break the ionic interaction between protein and matrix and elute off-bound proteins. Examples of such separation media include DEAE and CM cellulose. Chromatography, hydrophobic This methodology separates proteins based on their hydrophobicity. Pro-teins preferentially bind to the matrix based on the strength of this interaction; proteins are then eluted off using solvents of increasing hydrophobicity. Separation media include phenyl-sepharose and octyl-sepharose. Chromatography, affinity This separation method de-pends on using any molecule that can preferentially bind to a protein of interest. Typical methodologies include using lectins (such as wheat germ or concanavalin A) to bind glycoproteins or using covalently coupled mono-clonal antibodies to bind specific protein ligands. Chromatography, high performance liquid (HPLC).

A general methodology to improve the separation of complex protein mixtures. The types of HPLC columns available are the same as for conventional chromatogra-phy, such as those based on size exclusion, hydropho-bicity, and ionic interaction, but the improved flow rates resulting from the high pressure system provide enhanced separation capacity and improved speed. Proteomics. The general term used in the study of the display of all proteins present in cells under defined con-ditions. By deciphering which proteins are differentially displayed in tumor cells compared to their normal coun-terparts, or in cells stimulated to grow, vs. their quies-cent state, one can determine the proteins that are re-sponsible for the cellular phenotype. In essence, proteomics is to proteins what genomics is to genes. DNA microarrays(gene expression arrays or gene chips) Multiple (presently up to tens of thousands) gene fragments or oligonucleotides representing distinct genes spotted onto a solid support. Theoretically, microarrays could be used to determine the totality of the genome expressed in a given cell under specific growth condi-tions, if the entire genome were present on the microarray. At present, gene chips are available that rep-resent about 1/3 of the human genome. The microarray is hybridized with a labeled probe (either radioactive or fluoresceinated) representing all the mRNA species in a given cell grown under a certain condition. By compar-ing the hybridization patterns produced by probes pro-duced from cells under two different growth conditions, one can determine which genes are increased and which are decreased in response to the growth stimulus. In a similar way, comparison of the expression profiles of a malignant cell type and its normal counterpart, poten-tially allows one to determine the genes responsible for transformation.

Yeast 2-hybrid screens A strategy designed to deter-mine the binding partners for a protein of interest. The gene (or a fragment of the gene) representing a protein of interest (the “bait”) is fused in frame to DNA binding domain (DBD) of yeast transcription factor and then in-troduced into a yeast strain. A cDNA library is then con-structed from the cells in which the bait is normally ex-pressed, and fused in frame to the activation domain (AD) of the same yeast transcription factor. When the library is introduced into the yeast expressing the bait/DBD fu-sion, any yeast cell expressing a cDNA encoding a bind-ing partner of the bait protein will have that cDNA/AD fusion protein bind to the bait/DBD fusion, bringing the AD and DBD together, thereby creating a fully func-tional transcription factor that now drives a reporter gene, allowing the yeast carrying such interacting proteins to be identified and the cDNA recovered.

V. P HYSIOLOGIC G ENE R EGULATION

The regulation of gene expression is central to physiol-ogy. Complex organisms have evolved multiple mecha-nisms to accomplish this task. The first step in protein expression is the transcription of a specified gene. The rate of initiation and elongation of this process is the most commonly used mechanism for regulating gene ex-pression. Once formed, the primary transcript must be spliced, polyadenylated, and transported to the cyto-plasm. These mechanisms are also possible points of regulation. In the cytoplasm, mRNA can be rapidly de-graded or retained, another potential site of control. Pro-tein translation next occurs on the ribosome, which can be free or membrane-associated. Secreted proteins take the latter course, and the trafficking of the protein through these membranes and ultimately to storage or release makes up another important point of potential regulation. Indi-vidual gene expression is often controlled at multiple lev-els, making investigation and intervention a complex task. Transcription Transcription is the act of generating a primary RNA molecule from the double-stranded DNA gene. Regulation of gene expression is predominantly at the level of regulating the initiation and elongation of

transcription. The enzyme RNA polymerase is the key feature of the system, which acts to generate the RNA copy of the gene in combination with a number of im-portant proteins. There is usually a fixed start to tran-scription and a fixed ending.

TATA Many genes have a sequence that includes this tetranucleotide close to the beginning of gene transcrip-tion. RNA polymerase binds to the sequence and begins transcription at the cap site, usually located approxi-mately 30 nucleotides downstream.

Enhancer An enhancer is a segment of DNA that lies either upstream, within, or downstream of a structural gene that serves to increase transcription initiation from that gene. A classical enhancer element can operate in either orientation and can operate up to 50 kb or more from the gene of interest. Enhancers are cis-acting in that they must lie on the same chromatin strand as the structural gene undergoing transcription. These cis-act-ing sequences function by binding specific proteins, which then interact with the RNA polymerase complex. Silencer These elements are very similar to enhancers except that they have the function of binding proteins and inhibiting transcription.

Initiation complex This multi-protein complex forms at the site of transcription initiation and is composed of RNA polymerase, a series of ubiquitous transcription factors (TF II family), and specific enhancers and/or si-lencers. The proteins are brought together by the loop-ing of DNA strands so that protein binding sites, which may range up to tens of kb apart, can be brought into close juxtaposition. Specific protein?protein interactions then allow assembly of the complex. Polyadenylation F ollowing transcription of a gene, a specific signal near the 3' end of the primary transcript (AATAAA) signals that a polyadenine tail be added to the newly formed transcript. The tail may be up to sev-eral hundred nucleotides long. The precise function of the poly A tail is uncertain but it seems to play a role in stability of the mRNA and perhaps in its metabolism through the nuclear membrane to the ribosome. Splicing The primary RNA transcript contains a num-ber of sequences that are not part of the mature mRNA. These regions are called introns and are removed from the primary RNA transcript by a process termed splic-ing. A complex tertiary structure termed a lariat is formed and the intron sequence is eliminated bringing the cod-ing sequences (exons) together. Specific sequences within the primary transcript dictate the sites of intron removal.

Exons These are the regions of the primary RNA tran-script that, following splicing, form the mature mRNA species, which encodes polypeptide sequence.

Introns These are the regions of the primary RNA tran-script that are eliminated during splicing. Their precise function is uncertain. However, several transcriptional regulatory regions have been mapped to introns, and they are postulated to play an important role in the genera-tion of genetic diversity (exon shuffling mechanism).

Nucleosomes When linear, the length of a specific chro-mosome is many orders of magnitude greater than the diameter of the nucleus. Therefore, a mechanism must exist for folding DNA into a compact form in the inter-phase nucleus. Nucleosomes are complex DNA protein polymers in which the protein acts as a scaffold around which DNA is folded. The mature chromosomal struc-ture then appears as beads on a string; within each bead (nucleosome) are folded DNA and protein. Nucleosome structure is quite fluid, and internucleosomal stretches of DNA are thought to be sites that are important for active gene transcription.

Trans-acting factors Proteins that are involved in the transcriptional regulation of a gene of interest.

Cis-acting factors These are regions at a gene either upstream, within, or downstream of the coding sequence that contains sites to which transcriptionally important proteins may bind. Sequences that contain 5 to 25 nucle-otides are present in a typical cis-acting element. Transcription factors Specific proteins that bind to con-trol elements of genes. Several families of transcription factors have been identified and include helix-loop-he-lix proteins, helix-turn-helix proteins, and leucine zip-per proteins. Each protein includes several distinct do-mains such as activation and DNA-binding regions. LCR (locus control region) Cis-acting sites are occa-sionally organized into a region removed from the struc-tural gene(s) they control. Such locus control regions (LCRs) are best described for the β globin and α globin loci. First recognized by virtue of clustering of multiple DNAse hypersensitive sites, the β globin LCR is required for high level expression from all of the genes and ap-pears to be critical for their stage-specific developmen-tal pattern of expression.

Protein translation This term is applied to the assem-bly of a polypeptide sequence from mRNA.

KOZAK sequence This five-nucleotide sequence resides just prior to the initiation codon and is thought to repre-sent a ribosomal-binding site. The most consistent posi-tion is located three nucleotides upstream from the ini-tiation ATG and is almost always an adenine nucleotide. When multiple potential initiation codons are present in an open reading frame, the ATG codon, which contains a strong consensus KOZAK sequence, is likely the true initiation codon.

Initiation codon The ATG triplet is used to begin polypeptide synthesis. This is usually the first ATG codon, located approximately 30 nucleotides down-stream of the site of transcription initiation (cap site). However, the context in which the ATG resides is also important (see KOZAK sequence).

Missense mutation Mutation of the mRNA sequence to generate an altered codon, which results in an amino acid change, is termed a missense mutation.

Nonsense mutation This type of mutation results in the generation of a premature termination codon and hence creates a truncated polypeptide.

Transcriptional regulation Gene regulation is deter-mined by the rate of transcriptional initiation. This usu-ally results from alteration in the level of activity of trans-acting proteins, which, in turn, are regulated either by the amount of the transcriptionally active protein or by their state of activation.

Leucine zipper proteins A family of DNA-binding pro-teins that require a dimeric state for activity and that dimerize by virtue of an alpha helical region that con-tains leucine at every seventh position. Because 3.4 amino acids reside in each turn of an alpha helix, the occurrence of leucine at every seventh position results in a strip of highly hydrophobic residues on one surface of the alpha helix. Such a domain on one polypeptide can intercollate with a similar domain on a second polypeptide, resulting in the formation of a stable homodimer or heterodimer. Examples of the leucine zip-per family include the proto-oncogenes c-jun and c-fos. Basic helix-loop-helix proteins These transcriptional proteins are characterized by two alpha helical regions separated by a loop structure; this domain is involved in protein dimerization. Examples of this family of tran-scription factors include E12/E47 of the immunoglobu-lin promoter or Myo D of muscle cell regulation.

Helix-turn-helix This family of transcriptionally active proteins depends on the helix-turn-helix motif for dimer-ization. Examples include the homeodomain genes such as the Hox family.

Master switch genes These polypeptide products are thought to regulate a whole family of genes and result in a cell undergoing a new program of differentiation. An example of such a system is Myo D, in which activation is thought to lead to differentiation along the muscle cell lineage.

Zinc finger domain proteins The presence of conserved histidine and cysteine residues allows chelation of a zinc atom and results in the formation of a loop structure called the zinc finger domain. This feature is present in a large family of transcriptionally active proteins such as the steroid hormone receptors.

Post-transcriptional regulation Mechanisms of gene regulation that do not involve transcriptional enhance-ment or silencing and include altering the rate of mRNA degradation, the efficiency of translation or post-trans-lational modification, or transportation of the polypep-tide out of the cell.

Actinomycin D pulse experiments The application of actinomycin D to actively metabolizing cells results in the cessation of new RNA transcription. Consequently, serial determinations of specific RNA levels will allow one to calculate the mRNA half life. Should this vary between control and stimulated conditions, evidence is garnered that a gene of interest is regulated at the level of mRNA stability.

Reporter genes In order to determine how a gene pro-moter or enhancer works in vitro, that genetic element is often linked to a gene for which a simple assay is readily available and whose regulation is not affected by post-transcriptional processes. Such reporter genes include chloramphenicol acetyl transferase, β galactosi-dase, and firefly luciferase. The first is the most com-monly used reporter; however, more recent studies have emphasized the use of the latter two reporters, as these are more sensitive to minimal changes in promoter or enhancer activity.

CAT (chloramphenicol acetyl transferase) The bac-terial gene for chloramphenicol resistance, chloram-phenicol acetyl transferase (CAT) is commonly used as a reporter gene for investigating physiologic gene regu-

lation. The assay depends on the ability of transfected cellular cytoplasm to convert 14C chloramphenicol to its acetylated form in the presence of acetyl CoA. The acety-lated forms are separated from the 14C substrate using thin layer chromatography.

β galactosidase The presence of β galactosidase activ-ity in the cytoplasm of transfected cells can be readily detected by its ability to convert a colorless substrate to a blue-colored product. This is usually assayed using a fluorimeter.

Luciferase This gene, which is the most recent reporter gene to be used, has gained increasing acceptance be-cause of its ease of assay and extreme sensitivity. The assay is based on the ability of the protein to undergo chemiluminescence and transmit light, detected with a luminometer.

Cyclins A group of proteins that vary in expression throughout the cell cycle. Once a threshold level is at-tained, interaction with specific cellular kinases results in phosphorylation of critical components of the mitotic machinery. Several classes of cyclins (A through E) ex-ist that regulate different aspects of the cell cycle (G 0,G 1, S, G 2, M). Altered expression of some cyclins is as-sociated with hematologic malignancy, e.g., t(11;14) in mantle cell lymphoma leads to over-expression of cyclin D 1, a G 1 phase cyclin.

Cdk (cyclin-dependent kinase) A related group of cel-lular kinases, present in virtually all cells, that are regu-lated both positively and negatively by specific phos-phorylation events and negatively by association with other proteins, and are dependent on cyclins, present only during certain phases of the cell cycle (cdk1-activated during G 2/M phase, cdk2-G 1/S phases, cdk4-G 1/S phases,cdk6-G 1 phase, cdk7-throughout the cell cycle).CdkI (cdk inhibitors) Proteins that inhibit the cdks by stoicheometric combination, arresting cells in G 1 phase,and include p27, p21 and the p16 Ink 4A family of pro-teins. The latter are implicated as tumor suppressor genes,as their deficiency in mice leads to rapid cellular prolif-eration and a high rate of spontaneous tumor develop-ment. Moreover, deficiency of p16 family members has been associated with numerous types of human tumors,including a fraction of cases of B cell ALL and T cell leukemia.

Proteosome A large multiprotein complex designed to digest proteins that have been targeted for destruction,usually based on the presence of multiple sites of ubiquination. The proteosome is critical for many cellu-lar processes, including cell growth, where it eliminates a series of brakes on cell cycle progression, or elimi-nates growth factor receptors following their internal-ization following ligand binding.

Ubiquitin A small molecular weight protein that can be coupled to lysine residues of proteins targeted for de-struction. There are ubiquitin ligases and deubiquinases,which add or remove ubiquitin from proteins, usually in a fairly specific manner. Once polyubiquinated, proteins are subject to destruction by the proteosome.

Immunoglobulin somatic hypermutation Immunoglo-bulin variable region gene sequences are further diver-sified in mature B cells during clonal expansion that occurs following antigen stimulation. Mutations clustered within V regions typically involve nucleotide substitu-tion, and less frequently small deletions or insertions.This event usually follows immunoglobulin class switch-ing, which by itself does little to alter Ig specificity; only the effector functions of the molecule are altered by the change from IgM to IgG, etc.

VI. E XPRESSION OF R ECOMBINANT P ROTEINS

In order to exploit the techniques of recombinant DNA research, one must possess a system to manufacture the protein of interest. After identifying the gene encoding the protein and obtaining a cDNA representation of it (“cloning”), the cDNA must be placed in a vector ca-pable of driving high levels of RNA transcription in a host system capable of translating and appropriately modifying the polypeptide to produce fully functional protein. And just like obtaining a protein of interest from natural sources, one must purify protein from the final expression system. Because of the nature of the highly engineered systems and high levels of expression, this latter task is usually considerably easier using recombi-nant methods than from natural sources. The methods used to generate expression vectors are described in Sec-tion IV , but the methods to purify proteins are discussed in only a rudimentary way and are beyond the scope of this glossary.

Expression vector A plasmid that contains all of the elements necessary to express an inserted cDNA in the host of interest. For a mammalian cell host, such a vec-tor typically contains a powerful promoter coupled to an enhancer, a cloning site, and a polyadenylation sig-nal. In addition, several expression vectors also contain a selectable marker gene such as DHFR or NeoR, which aids in the generation of stable cell lines. The plasmid

also requires a bacterial origin of replication and an an-tibiotic resistance gene (AmpR) to allow propagation and expansion in a bacterial host.

Transfection Once the expression vector has been as-sembled, it must be inserted into the host of interest. Sev-eral methods are available for such transfections and in-clude calcium/phosphate/DNA complexes, DEAE Dex-tran, electroporation, liposome, and retrovirus-mediated gene transfer.

Calcium phosphate This method relies on the produc-tion of a calcium/phosphate/DNA microprecipitate, which is then taken up by cells by pinocytosis. The method is very effective for a number of commonly used mammalian cell expression systems including COS, BHK, 293, and CHO cells.

DEAE dextran This method depends on the formation of a complex between the insoluble positively charged dextran and the DNA to be transfected. Like calcium phosphate, this method is highly successful with many cell types.

Electroporation When cells are suspended in buffer be-tween two electrodes, discharge of an electrical impulse momentarily creates pores in the cell membrane. Dur-ing this time, DNA in solution is free to diffuse into the cells. This method is highly successful in transfecting a large number of cell types, including cells previously thought to be difficult to transfect with other methods, such as endothelial cells and fibroblasts.

Liposomes By encapsulating the DNA to be transfected in an artificial lipid carrier, foreign DNA can be intro-duced into the cell. This method, like electroporation, has been successful in transfecting cells previously thought difficult to manipulate. Its only drawback is its expense.

Transduction The act of transferring a foreign gene into a host genome.

VII. E XPERIMENTAL G ENE M ANIPULATION Antisense oligonucleotides By introducing short single-stranded deoxyribonucleic acids (ODN) into a cell, spe-cific gene expression can be interrupted. Several mecha-nisms have been postulated to account for these results including interruption of ribosome binding to mRNA, enhanced degradation of mRNA mediated by the double-strand specific RNAseH, DNA triplex formation, and impairment of translation efficiency. Most successful at-tempts using antisense ODN have targeted sequences surrounding and including the initiation codon. To re-duce nuclease attack, the antisense ODN are often syn-thesized using an altered chemistry involving thiol rather than phosphodiester linkages.

Transgenic animals By introducing an intact or manipu-lated gene into the germline of mice, the effects of pro-moter expression in specific cell lineages can be investi-gated. In contrast to highly artificial in vitro studies us-ing reporter gene analysis, such transgenic animals pro-vide an important in vivo model of gene function. The methods for production of transgenic mice have been extensively reviewed and are based on the microinjec-tion of linear DNA into the pronucleus of a fertilized egg. Several types of experiments can be performed. First, the effect of aberrant expression of a gene can be investigated, as was recently performed by expressing GM-CSF in a wide variety of tissues. Second, the nec-essary elements for tissue- and developmental level-spe-cific expression of a gene can be studied, as has been performed for the β-globin locus. Third, the tissue dis-tribution of a specific gene can be determined by engi-neering a marker gene adjacent to a specific promoter.

A specific example of this strategy employs a “suicide gene,” the herpes virus thymidine kinase (TK). When animals carrying such genes are exposed to gancyclovir, cells expressing the promoter of interest will express TK, be killed, and be readily detected.

Gene knock-out experiments Specific genes in the mammalian genome can now be targeted for interrup-tion or correction based on the technique of homolo-gous recombination. By generating DNA constructs that contain an interrupted gene of interest, or a corrected gene, in the setting of adequate flanking sequences to allow for targeting to the genetic locus of interest, the endogenous gene can be replaced or corrected. The methods involve introduction of the gene into an em-bryonic stem (ES) cell line, selection for subclones of cells that have had successful homologous recombina-tion events, and then introduction of the ES subclone into the blastocyst of a developing embryo. A chimeric animal results, and should the newly introduced gene become part of the germline, it can be bred to the ho-mozygous state. Using these techniques, investigators can now determine whether a single genetic locus is re-sponsible for a given disease, determine the significance of specific cytokines or growth factors, and generate model systems useful investigation of human disease. Gene knock-in experiments A similar technology to knock-out strategy, but rather than simply obliterating

function of the targeted gene, the knock-in is designed to replace the locus with a specific mutation of interest. Homologous recombination When a manipulated gene is introduced into a cell, it can be incorporated into the genome either randomly or at a specific locus. By incor-porating sequences that normally flank the desired lo-cus, a manipulated gene can be specifically (albeit rarely) introduced into the genome. Selection for this unlikely event can be enhanced by introduction of the herpes thy-midine kinase (TK) gene into the original targeting con-struct. Should the construct be randomly incorporated into the genome, the TK gene will also be introduced, rendering the cell sensitive to gancyclovir. If homolo-gous recombination occurs, the TK gene will be elimi-nated, as there are no homologous sequences at the spe-cific genetic locus of interest and the resultant cell will be resistant to the antibiotic.

YAC (yeast artificial chromosome) A yeast artificial chromosome (Y AC) utilizes centromeric and telomeric elements from yeast chromosomes to construct genetic elements that can be propagated in yeast and transferred into mammalian cells. Such vehicles allow the introduc-tion of up to 200 kb or more of genetic material into the host cells. YACs are now being used to study the physi-ologic regulation of large genetic loci such as the β-globin region of chromosome 11.

Contig The jargon term used to describe the assembly of clones necessary to include all of the DNA in a specific stretch of chromosome. Such maps are usually assembled from overlapping Y AC (yeast artificial chromosome) or BAC (bacterial artificial chromosome) clones. Once the “genome project” is complete, it will consist of 24 (very large) contigs (22 autosomal, an X and a Y). Transposon Naturally occurring genetic elements that are naturally easily removed and inserted into the ge-nome, allowing for the recombination of genetic seg-ments, giving rise to genetic diversity. These same ele-ments can be utilized for gene therapy.

VIII. G ENE T HERAPY

Gene therapy takes many forms. To treat malignancy, it may involve the insertion of an adjuvant substance (such as GM-CSF) into tumor cells to generate a tumor vac-cine, transfer of a gene that renders tumor cells suscep-tible to eradication with an antitumor agent (e.g., herpes thymidine kinase), or insertion of a gene that makes by-stander cells resistant to the effects of chemotherapy (e.g., MDR). For gene deficiencies, insertion of the wild type allele is the therapeutic goal. Obtaining cDNA for de-sired genes has become common. Insertion of the gene into target cells and high (adequate) level expression is more problematic. Several types of transfer vehicles have found use, including viral vectors and chemical agents.

Viral transduction vectors Retroviral vectors are based on murine retroviruses. They can carry 6 to 7 kb of for-eign DNA (promoter + cDNA) but suffer from the draw-backs of requiring the development of high titer pack-aging lines, requiring that target cells be dividing, and are subject to host cell down-modulation. Adenoviral vectors can be produced at high levels and do not re-quire a dividing target cell, but they do not normally in-tegrate, resulting in only transient expression. Adeno-associated viral vectors are defective parvoviruses that integrate into a non-dividing host cell at a specific loca-tion (19q). Disadvantages are genetic instability, small range of insert size (2–4.5 kb), and thus far, only tran-sient expression.

Ecotropic vectors Many retroviruses are host cell spe-cific, i.e. they will only infect a specific species of cells. An example is the widely used Maloney virus, and its basis lies in the species-specific expression of the viral cell surface receptor.

Ecotropic viruses Murine retroviruses that contain coat proteins that can only bind to murine cellular receptors.

Amphotropic viruses Retroviruses whose coat proteins bind to a receptor found throughout multiple species, usually including man, making these vectors suitable for human use. Problems related to the level of receptor ex-pression on cells of hematologic interest (e.g. stem cells) remain for amphotropic viruses.

Pseudotyped virus These take advantage of the power-ful expression levels obtainable by murine retroviral backbones, yet are packaged in an envelope that allows docking and uptake by human target cells. An example is the popular MFG vector that utilizes a murine leuke-mia retroviral backbone and an amphotropic packaging cell line to produce infectious particles.

Episomal Episomal refers to gene therapy vectors that remain free in the target cell without being taken into the host genome.

Positional variegation Refers to the observation that the site of vector integration into the genome often results in variable levels of gene expression.

Chimeraplasty A technique of gene therapy dependent on construction of a DNA:RNA oligonucleotide hybrid that once introduced into a cell relies upon DNA repair mechanisms to introduce a (corrective) change in the targeted gene.

Chitosan-DNA A chemical means of packaging foreign DNA to allow introduction into cells; the complexes ex-ist as nanospheres and have been tested in factor IX de-ficiency in animals.

Long terminal repeat (LTR) This segment of a retro-viral genome carries the genetic information for both tran-scription of downstream viral structural genes and the mechanisms of viral replication. It is often used in retroviral applications to drive the exogenous therapeutic gene as it carries a powerful (but non-tissue specific) promoter. Interference The mechanisms by which infection of a cell by one virus excludes infection by others. Interfer-ence is often due to the cellular production of coat pro-teins, which bind to and block the cells’ remaining viral receptors.

Nonviral transduction methods Nonviral methods in-clude polylysine-ligand DNA complexes, where the ligand (e.g., transferrin) allows access to the cell through normal receptor-mediated uptake, and phospholipid vesicles. Both methods suffer from not providing a mechanism for genomic integration, precluding long-term expression.

IX. C LONING AND L IBRARY S CREENING Obtaining cDNA representing a protein of interest is usu-ally the first step in the process of applying the tech-niques of recombinant DNA research to an important physiologic question. A suitable cDNA library must first be constructed starting with RNA abundant (or as abun-dant as possible) in the transcripts for the gene of inter-est. Following library construction a probe must be de-veloped that can specifically recognize the gene or cDNA of interest, or the expressed protein product of the spe-cific cDNA.

First strand synthesis The retroviral enzyme reverse transcriptase is used along with an antisense primer to produce a complementary DNA strand of mRNA ex-tracted from a cellular source known to express the gene of interest. Two types of primers are used, either oligo dT, in which the poly A tail begins the cDNA synthesis, or random primers, in which a whole range of start sites will be used.Second strand synthesis The enzyme DNA polymerase is used to generate the sense strand of cDNA. Priming of the second strand can occur spontaneously, as the anti-sense first cDNA strand can form a hairpin loop at its 3' end bending back to prime second strand synthesis. Al-ternatively, a polynucleotide tail can be added to the first strand synthesis using terminal deoxynucleotide trans-ferase, then second strand priming can occur using a synthetic oligonucleotide complementary to the TdT tail. Should the former technique be used, an extra step to nick the hairpin loop using the enzyme S1 nuclease would be required prior to inserting the cDNA into its vector. cDNA blunting First and second strand synthesis usu-ally results in nonflush ends. To prepare the cDNA for insertion into a cloning vector, the ends must be made flush with one another. Such blunting reactions can be conducted with a DNA polymerase, such as the Klenow fragment of DNA polymerase I or T4 DNA polymerase. Linkering To efficiently insert the cDNA library into a cloning vector, synthetic duplex oligonucleotides that contain a restriction endonuclease site are attached to the blunted ends of the cDNA. A restriction endonu-clease is chosen that rarely cuts DNA (such as the 8 bp recognition sequence for Not I, or if a more common restriction site is used such as Eco RI, the cDNA should first be methylated in order to prevent subsequent cDNA digestion with the enzyme) and is used to generate “sticky ends” on the cDNA.

cDNA library preparation Once the cDNA has been prepared and sticky ends generated, the library is in-serted into a convenient cloning vector. Because of high cloning efficiency, most cDNA libraries are constructed in a λ phage vector. Typically, if screening is to be per-formed using a monoclonal antibody, λ gt 11 is used. If screening is to be performed using oligonucleotide probes, λ gt 10 can be used. If larger DNA fragments are to be prepared, such as from genomic fragments of DNA, λ vectors that can accommodate up to 20 kb are available (e.g., λ Charon 4A).

Subtractive library The purpose of generating a sub-tractive library is to enrich for cDNA that are expressed under one condition but are not expressed under a sec-ond condition. This facilitates screening for the cDNA of interest in that the complexity of the library is much reduced, requiring one to screen far fewer clones. At its extreme, investigators have used subtractive libraries to generate a very highly select group of clones (in the range of 100) and then have sequenced all of the resulting cDNA. The principle behind a subtractive library is the

elimination of cDNA common to induced and control conditions. By eliminating such clones, only cDNA that are present under the induced conditions will remain in the library. Those techniques depend on the differential elimination of duplex mRNA/cDNA or cDNA/cDNA hybrids, which form between genes expressed under both conditions, leaving the single-stranded mRNA or cDNA of interest.

RDA (representational difference analysis) A molecu-lar method to amplify genes that are expressed in an RNA sample of interest, that are not present, or present at very reduced levels, in a comparison RNA sample (e.g. cyto-kine induced and control cells). The method relies on RT-PCR amplification of the RNA that does not contain the gene(s) of interest to produce a “driver” cDNA, and RT-PCR to produce “tester” cDNA from the RNA popu-lation in which you hope to find new genes. After liga-tion of different oligonucleotides to the ends of each population, both are denatured and an excess of the driver is hybridized to the tester and PCR performed with prim-ers that will amplify only sequences present in the tester that are not in the driver, thereby “removing” cDNA com-mon to both populations. The resultant cDNA are en-riched in uniquely expressed genes.

Directional cloning To improve efficiency when screen-ing functional expression libraries, many investigators construct cDNA libraries in which the proper coding ori-entation of the cDNA is maintained in the library. In conventional library preparation, the 5' and 3' ends of the DNA are identical; thus, cDNA can be inserted into the cloning vector in either orientation. If screening is dependent on the production of a functional protein, one-half of the library will be useless, as those cDNA in-serted in an inverse orientation will not produce func-tional protein. Directional cloning is dependent on pro-ducing sticky ends that differ on the 5' and 3' termini. The cloning vector has the appropriate pair of comple-mentary cloning sites.

Library screening Three major methods are available to obtain cDNA of interest. The classic technique uti-lizes DNA probes (such as oligonucleotides or intact cDNA from a homologous gene) to screen cDNA librar-ies. An oligonucleotide probe is usually derived from a reverse translation of known protein sequence. By ex-pressing cDNA as a fusion protein with β galactosidase, various antisera can be used to screen for fusion pro-teins encoded by the cDNA of interest. Finally, cDNA libraries may be constructed in cloning vectors that al-low for expression of the cDNA insert in E. coli or a mammalian cell host. If a highly sensitive assay for the desired protein’s function can be developed, pools of cDNA clones can be expressed and then assayed to-gether; a positive assay from a pool would allow one to subdivide into smaller pools and eventually at clonal density.

Reverse genetics Often, large families of homologous pro-teins exist and multiple previously unknown members of the family can be obtained by screening cDNA libraries under low stringency using cDNA or oligonucleotide probes from regions highly conserved amongst members of the family. In this case, genes are identified before their function is known, a situation referred to as reverse genet-ics. Examples in hematology include identifying members of the tyrosine kinase family of receptor proteins using a probe derived from the conserved kinase domain of the cytoplasmic region of src or other tyrosine kinase proto-oncogenes, or the identification of transcription factors important in hematopoiesis using conserved motifs present in zinc finger or homeodomain proteins.

X. O NCOGENESIS AND A NTI-O NCOGENES Oncogenes have usually been identified in the context of a tumor-inducing virus. Such viral oncogenes (v-onc) are thought to be derived from host cells, but have been altered such that abnormal regulation of production or function has ensued during the transfer process. Subse-quent reintroduction of the altered gene into a host cell leads to transformation. Proto-oncogenes, the normal cellular counterpart of viral oncogenes, can contribute to cellular transformation by mechanisms that disturb normal gene function. Such mechanisms include muta-tion (resulting in abnormal function), amplification (re-sulting in abnormal levels of expression), rearrangement (resulting in a new function), or promoter mutation (again resulting in abnormal levels of expression). Most or all proto-oncogenes are involved in normal cellular pro-cesses such as growth factor signal transduction, mito-genic signaling, or regulation of DNA transcription or cellular proliferation. The nomenclature convention is to indicate the cellular version of the proto-oncogene as “c-onc” and the viral version, which is transforming, as “v-onc.” Most altered proto-oncogenes act in a domi-nant genetic fashion. Anti-oncogenes, or tumor suppres-sor genes, usually act in a recessive genetic fashion and function to slow processes involved in cellular prolifera-tion. Most of the identified anti-oncogenes have been in-volved in gene transcription, presumably acting to enhanced differentiation programs over those of proliferation.

c-abl This gene, present on human chromosome 9, en-codes a tyrosine kinase whose role in normal hemato-

poiesis is unclear; however, its fusion to the BCR gene on human chromosome 22, the functional counterpart of the Ph1 chromosome strongly associated with the dis-ease chronic myelogenous leukemia, eliminates the first two or three exons of c-abl and results in unregulated tyrosine kinase activity. The resultant fusion protein is either 210 kDa or 195 kDa. The latter version is more acutely transforming in experimental settings; it is also associated with acute lymphoblastic leukemia and with a worse prognosis in both disease settings. One of the ways in which the unregulated kinase activity may be manifest is through phosphorylation of SHC and/or GRB-2, adopter proteins necessary for coupling growth factor signals to ras.

c-jun This proto-oncogene encodes a ~45 kDa transcrip-tion factor that is a member of the AP1 family of tran-scriptional proteins. c-jun must form dimers to function and does so through the leucine zipper motif. Although c-jun-c-jun homodimers do form, they do so with low affinity and are not thought to be critical in gene tran-scription. Rather, a second partner, usually c-fos, gener-ates the transcriptionally active heterodimer.

c-fos This ~62 kDa leucine zipper protein cannot homo-dimerize but rather functions in heterodimeric complex with c-jun and other members of the AP1 family of tran-scription factors.

c-myc This proto-oncogene plays a critical role in he-matopoietic cell proliferation. Like the leucine zipper protein, it too functions as a heterodimer. One of its part-ners is termed Max. The myc-related protein, Mad, also dimerizes with Max; the myc/Max complex stimulates proliferation, the Mad/Max complex inhibits myc-func-tion. The importance of dysregulated myc function can be seen in Burkitt lymphoma in which a t(8;14) brings myc, on chromosome 8, into juxtaposition with the im-munoglobulin enhancer on chromosome 14. Such upregulation of myc in a B lymphocyte setting results in a proliferative advantage and represents one important step in the genesis of this lymphoma. Myc has both leu-cine zipper and helix-loop-helix domains.

c-myb This gene encodes a transcription factor not be-longing to any other class previously described and is expressed primarily in immature hematopoietic cells and declines as cells differentiate. Forced expression of c-myb tends to block hematopoietic differentiation. Clini-cally, high levels of myb are noted in acute leukemia, and such patients are less likely to enter remission or tend to have a short remission duration.c-rel This gene belongs to the NF-κB family of tran-scription factors and can act to enhance or repress tran-scription from selected genes. This family of proteins includes p50 and its precursor p105, p65, p49 and its precursor p100, and Bcl-3, one of the IκB family.

IRF-1 (interferon regulatory factor-1) IRF-1 is a tran-scription factor that activates the expression of IFN αand β and maps to chromosome 5q31.1. As it is thought to act as a tumor suppressor gene, its role in the patho-logic consequences of the 5q- syndrome is under active investigation.

IRF-2 (interferon regulatory factor-2) Interferon regu-latory factor-2 is a gene which binds to a promoter ele-ment shared by IFN α and β and many IFN-inducible genes; unlike IRF-1, which stimulates such genes, IRF-2 represses transcription at the site. It is felt that the ra-tio of IRF-1 to IRF-2 might be a critical event in the regulation of cellular proliferation.

Rb The prototypical tumor suppressor gene Rb behaves in a genetically recessive fashion. Elimination or inacti-vation of both Rb gene copies is required for manifesta-tion of the tumorgenic phenotype, first recognized in chil-dren with retinoblastoma. Such children inherit only a single functional copy; subsequent mutagenic inactiva-tion of the remaining allele results in tumor susceptibil-ity. Rb acts to sequester a group of transcription factors, termed E2F, which regulate genes critical for DNA syn-thesis. Alterations of Rb alleles are found in approxi-mately 30% of human acute leukemias.

SCL This proto-oncogene, first identified in a stem cell leukemia at the site of t(1;14), is a member of the helix-loop-helix group of transcriptionally active proteins. The gene, also termed Tal 1, is expressed in erythroid and mast cell lineages but not in T cells. The association of t(1;14) with up to 25% of T cell ALL suggests that its ectopic expression is associated with transformation. Bcl-1 This gene, located on chromosome 11 q13, was first identified at the site of translocation p(11;14)(q13;q32), has a strong association with central acinar/mantle cell lymphoma and functions in normal cells as the G

1

cyclin termed CCND1 or cyclin D

1

. Nor-mally, lymphocytes lack cyclin D

1

expression; its aber-rant expression resulting from chromosomal transloca-tion of the Bcl-1 locus to an immunoglobulin locus is thought to be associated with aberrant proliferation. Bcl-2 This gene product normally functions to suppress programmed cell death (apoptosis). Its overexpression

is associated with the most common molecular abnor-mality in non-Hodgkin’s lymphoma, t(14;18)(q32;q21), present in 80% of follicular small cleaved cell lymphoma. Presumably, suppression of apoptosis leads to extended cell survival, a characteristic of low-grade lymphomas.

Bcl-3 This gene is a member of the IκB family. Pres-ently, it is unclear how this protein acts in tumorigen-esis, but it is likely that its involvement in transcriptional processes is critical.

Bcl-6 A zinc finger transcription factor, expression of which is altered in approximately one-third of diffuse B large cell lymphomas as a consequence of 3q27 translo-cations. Its target genes are unknown.

RAR (retinoic acid receptor) The retinoic acid recep-tor is a member of the steroid hormone group of tran-scriptionally active proteins and contains a steroid hor-mone-binding domain, a zinc finger DNA-binding do-main, and a transcriptional activation domain. RAR is located at the t(15;17) present in the majority of cases of acute promyelocytic leukemia. Its fusion partner in the translocation is termed pml. Normally, RAR forms heterodimers with members of the RXR family of tran-scription factors.

p53 Wild-type p53 is a sequence-specific DNA-binding nuclear protein that acts to induce gene expression. Over-all, the program of p53-activated genes is associated with suppression of cell growth, consistent with our under-standing of the mechanisms of anti-oncogenes. Muta-tions of p53 may not only inactivate its growth-suppres-sion function, but can actually generate a genetically dominant, functional oncogene. Human tumors associ-ated with p53 mutations include those of hematopoietic tissues (e.g., 20% of myelomas), bladder, liver, brain, breast, lung, and colon. It is likely the most frequently mutated gene in human cancer.

ras This gene encodes a critical signalling intermediate involved in the response to multiple growth factors. There are several related proteins (Ha-ras, Ki-ras, N-ras). N-ras and K-ras are mutated in many cancers, including 45% of myelomas and > 50% of CMML cases. Consti-tutive activation of ras can mimic chronic stimulation by the corresponding lineage-specific growth factor. Hox 11 A homeobox containing transcription factor dis-rupted by translocation to the T cell receptor locus [t(10;14)] in 10% of cases of T cell ALL/lymphoblastic lymphoma. The Hox 11 gene is critical to the development of the spleen but its role in hematopoiesis is unclear.Rhomb 2 Like Hox 11, Rhomb 2 is translocated in T cell ALL/lymphoma associated with t(11;14). Rhomb 1 may play a similar role in additional cases of T cell ALL. The Rhomb gene products are members of a family of transcription factors, but as Rhomb 2 and Rhomb 1 do not contain DNA-binding domains, they are thought to be involved in protein-protein interactions. Neither Rhomb 2 or 1 are normally expressed in T cells; transformation involving these genes, like SCL or Hox 11 is thought to be due to ectopic expression of the protein in T cells.

ALK (anaplastic lymphoma kinase) A large propor-tion of Ki-1 positive lymphomas are characterized by a t(2;5). The breakpoint involves nucleophosmin, a ubiq-uitously expressed gene, and ALK. The chimeric mRNA and protein are thought to be responsible for transfor-mation. ALK is a member of the insulin receptor family of transmembrane receptor kinases, which is not nor-mally expressed in hematopoietic tissues; the fusion gene is no longer membrane bound, which may underlie its pathogenesis.

Evi-1 A transcription factor whose rearrangement in t(3;21) is implicated as contributing to MDS. Over-expression of evi-1 blocks differentiation in response to hematopoietic growth factors.

ETO Located on chromosome 8, ETO is involved in t(8:21) of AML type M2. Based on the presence of two zinc-fin-ger motifs. ETO possibly encodes a transcription factor, but its role in the pathogenesis of AML is unknown.

AML-1 Located on chromosome 21, AML-1 is the fu-sion partner of ETO in t(8:21). The gene is homologous to the runt gene of Drosophila and encodes a transcrip-tion factor. Normal hemopoietic targets include the CD13, GM-CSF, MPO, IL-3, and the T cell antigen re-ceptor promoters. AML-1 binds as a heterodimer, partnered with CBFβ. It is unclear if its mechanism of action is to enhance aberrant transcription or to blunt transcription by acting in a dominant negative fashion.

CBFβLocated on chromosome 16, CBFβ is one of the fusion partners in the inv(16) associated with AML type M4Eo. As with AML-1, it is unclear whether the altered transcription factor enhances or blocks transcription.

MLL Located on chromosome 11, MLL (mixed lineage leukemia) is frequently altered in ALL, 1o AML, and es-pecially in AML secondary to the use of topoisomerase II inhibitors. MLL is homologous to the trithorax gene of Drosophila and displays many features of a transcrip-tion factor and of a DNA methyl transferase.

Tel A helix-loop-helix transcription factor fused to the PDGFβ-receptor in CMML t(5:12) and to other genes in AML or MDS. Like most other translocation oncogenes, the mechanism of leukemogenesis is un-known. More recently, a Tel/AML1 fusion gene repre-senting a t(12;21) has been found in a large number of cases of childhood ALL. As the translocation is not de-tected by routine cytogenetics, molecular analysis (FISH, etc.) is required to identify this favorable chromosomal rearrangement.

DEK Located on chromosome 6, DEK is involved in t(6:9) of AML. This translocation is usually seen in young patients and carries a poor prognosis. Its normal function is unknown, but DEK localizes to the nucleus.

CAN Located on chromosome 9, CAN is part of t(6:9). CAN forms part of the nuclear pore. As it has two dif-ferent fusion partners but a consistent phenotype, CAN is likely the critical component of t(6:9).

Fas (CD95 or Apo-1) A transmembrane glycoprotein expressed on a wide variety of primitive and mature he-matopoietic cells, which, upon binding to its natural ligand triggers programmed cell death.

NF-1 The gene responsible for neurofibromatosis. The normal protein functions to negatively regulate ras proteins, key intermediates in cytokine-induced cellular proliferation.

XI. G ENETIC S CREENING

X-linked methylation patterns Several loci present on the X chromosome become highly methylated when in-active but remain unmethylated on the active X chro-mosome (Lyon hypothesis). Should a polymorphic site for a methylation-sensitive restriction endonuclease ex-ist at such an X-linked locus, one can distinguish be-tween the active and inactive X chromosome by the pat-tern of restriction endonuclease digestion of that gene. However, in order to be widely useful for determining clonality of hematopoietic cells, the allelic frequency must be close to equality. Several X-linked genes meet these criteria and include phosphoglycerate kinase (PGK), hypoxanthine phosphoribosyltransferase (HPRT), the human androgen receptor gene (HUMARA), and the hyper-variable DXS255 locus. Both Southern blotting and PCR methods can be applied to this type of analysis.

RFLP (restriction fragment length polymorphism) If a mutation of one allele of a genetic locus either gener-ates or destroys a restriction endonuclease site, the het-erogeneity present within or very close to a gene of in-terest can be used to track which allele an individual has inherited from each parent. When genomic DNA is di-gested with a restriction enzyme that recognizes a poly-morphic site and then hybridized with a probe specific for the gene of interest, the allelic pattern can be com-pared to that of a similar assessment of both parents. The presence of multiple family members allows a com-plete genetic pedigree to be constructed. For example, globin gene mutations such as sickle hemoglobin can be analyzed. The β6 mutation in hemoglobin, which results in Hgb S, destroys an Mst II site. Therefore, a larger than normal DNA fragment is generated by digestion of genomic DNA with Mst II, which can be easily detected by Southern blot hybridization. In this specific case, the Mst II polymorphism is absolutely specific for the mu-tant gene and family studies are not necessary. If the RFLP had not been specific for the mutation, but only existed close to the specific disease-producing mutation, then family studies would have been required to deter-mine which pattern (presence or absence of restriction site) tracks with the mutant (disease) allele.

Allele-specific hybridization If the nucleotide basis for a specific genetic abnormality is known, oligonucleotides specific for wild type and for mutant sequence can be de-signed and used to probe Southern blots of an individual’s genomic DNA. The pattern of hybridization thus gives spe-cific information regarding which alleles are present. In a polymorphic disease such as β thalassemia (in which mul-tiple mutations can give rise to the same disease pheno-type), multiple probes might be required to detect all pos-sible causes. In addition, new mutations causing the same disease would be missed. However, should a specific probe prove useful for one population group or be positive in one family member, that probe becomes very useful for the individual under study.

Reverse allele-specific hybridization This automated variant of allele-specific hybridization couples unlabeled synthetic oligonucleotides specific for a wild type or mutant sequence to a solid support that is then allowed to bind genomic sequences of the locus of interest, which have been amplified by PCR. The use of highly strin-gent conditions of hybridization allows differential bind-ing of the amplified DNA to the wild type or mutant specific oligonucleotide and thereby allows genotypic determination of the individual.

Competitive oligonucleotide hybridization Mutant or wild type-specific oligonucleotide primers are used in a PCR reaction with genomic DNA. The primers and strin-

gency of PCR are chosen so that single-based mismatches between genomic DNA and PCR primer fail to yield an amplified product. Thus, the PCR detection of a locus-specific product allows the genotyping of the individual. Color complementation assay This method is an ad-vancement over competitive oligonucleotide hybridiza-tion in that the wild type and mutation specific PCR prim-ers are labeled with different color fluorescent tags, and both are used in a PCR reaction with genomic DNA. When highly stringent conditions are met, the fluores-cent colors of the resultant PCR product indicate whether wild type, mutant, or both specific alleles were present in the original DNA sample.

最新分子生物学名词解释

分子生物学名词解释

名词解释 1. 基因（gene）： 2. 结构基因（structural gene）： 3. 断裂基因（split gene）： 4. 外显子（exon）: 5. 内含子（intron）: 6. 多顺反子RNA（polycistronic/multicistronic RNA）: 7. 单顺反子RNA（monocistronic RNA）: 8. 核不均一RNA（heterogeneous nuclear RNA, hnRNA）: 9. 开放阅读框（open reading frame, ORF）: 10. 密码子（codon）: 11. 反密码子（anticodon）: 12. 顺式作用元件（cis-acting element）: 13. 启动子（promoter）: 14. 增强子（enhancer）: 15. 核酶（ribozyme） 16. 核内小分子RNA（small nuclear RNA, snRNA） 17. 信号识别颗粒（signal recognition particle, SRP） 18. 上游启动子元件（upstream promoter element） 19. 同义突变（same sense mutation） 20. 错义突变（missense mutation） 21. 无义突变（nonsense mutation）

22. 移码突变（frame-shifting mutation） 23. 转换（transition） 24. 颠换（transversion） （三）简答题 1. 顺式作用元件如何发挥转录调控作用？ 2. 比较原核细胞和真核细胞mRNA的异同。 3. 说明tRNA分子的结构特点及其与功能的关系。 4. 如何认识和利用核酶？ 5. 若某一基因的外显子发生一处颠换，对该基因表达产物的结构和功能有什么影响？ 6. 举例说明基因突变如何导致疾病。 （四）论述题 1. 真核生物基因中的非编码序列有何意义？ 2. 比较一般的真核生物基因与其转录初级产物、转录成熟产物的异同之处。 3. 真核生物的基因发生突变可能产生哪些效应？ （二）名词解释 1.基因组（genome） 2. 质粒（plasmid） 3.内含子（intron） 4.外显子（exon） 5.断裂基因（split gene） 6.假基因（pseudogene）

分子生物学词汇(S)

分子生物学词汇(S) 分子生物学词汇(S)分子生物学词汇(S)saccharomyces cerevisiae 酿酒酵母 saccharopine 酵母氨酸 saccluse [高尔基体]扁平膜囊 safranine 番红 safrole 黄樟素 sagamicin 相模霉素 saikoside 柴胡皂苷 sakaguchi reaction 坂口反应 salicin 水杨苷 salicylate 水杨酸盐 salinomycin 盐霉素 salmin 鲑精蛋白 salmonella 沙门氏菌属 salmonella typhi 伤寒（沙门氏）杆菌 saltatory conduction 跳跃传导 salvelin 鲑精蛋白 sapogenin 皂（角）苷配基，皂（角）苷元 saran wrap [商]saran包装膜，莎伦包装膜，偏氯伦包装膜，

[dow chemical公司商标，是用强韧而易弯曲的热敏塑料制成的包装用薄膜] sarcina 八叠球菌 sarcodina 肉足总纲 sarcomere 肌（原纤维）节 sarcomycin 肉瘤霉素 sarcoplasmic 肌质 sarcosinate 肌氨酸盐 sarcosine 肌氨酸 sarcosyl 十二烷基肌氨酸钠 sarcotubule 肌小管 sarin 萨林 satellite dna 卫星dna[真核细胞染色体dna经氯化铯密度梯度离心，其高度重复序列因组成不同而在主带旁自成一条或数条条带] satellitism 卫星现象[可特指一种微生物在另一种微生物菌落附近才能旺盛地生长繁殖的现象] satiety center 饱中枢 saturation hybridization 饱和杂交[一种组分大大过量而使另一组分的互补序列全部掺入双链中] saturation mutagenesis 饱和诱变[对基因的某一小区域内碱基进行多种形式的置换]

现代分子生物学_复习笔记完整版.doc

现代分子生物学 复习提纲 第一章绪论 第一节分子生物学的基本含义及主要研究内容 1 分子生物学Molecular Biology的基本含义 ?广义的分子生物学：以核酸和蛋白质等生物大分子的结构及其在遗传信息和细胞信息传递中的作用为研究 对象，从分子水平阐明生命现象和生物学规律。 ?狭义的分子生物学：偏重于核酸（基因）的分子生物学，主要研究基因或DNA的复制、转录、表达和调控 等过程，也涉及与这些过程相关的蛋白质和酶的结构与功能的研究。 1.1 分子生物学的三大原则 1) 构成生物大分子的单体是相同的 2) 生物遗传信息表达的中心法则相同 3) 生物大分子单体的排列（核苷酸、氨基酸）的不同 1.3 分子生物学的研究内容 ●DNA重组技术（基因工程） ●基因的表达调控 ●生物大分子的结构和功能研究(结构分子生物学） ●基因组、功能基因组与生物信息学研究 第二节分子生物学发展简史 1 准备和酝酿阶段 ?时间：19世纪后期到20世纪50年代初。 ?确定了生物遗传的物质基础是DNA。 DNA是遗传物质的证明实验一：肺炎双球菌转化实验 DNA是遗传物质的证明实验二：噬菌体感染大肠杆菌实验 RNA也是重要的遗传物质-----烟草花叶病毒的感染和繁殖过程 2 建立和发展阶段 ?1953年Watson和Crick的DNA双螺旋结构模型作为现代分子生物学诞生的里程碑。 ?主要进展包括： ?遗传信息传递中心法则的建立 3 发展阶段 ?基因工程技术作为新的里程碑，标志着人类深入认识生命本质并能动改造生命的新时期开始。 ? 第三节分子生物学与其他学科的关系 思考 ?证明DNA是遗传物质的实验有哪些？ ?分子生物学的主要研究内容。 ?列举5～10位获诺贝尔奖的科学家，简要说明其贡献。

分子生物学名词解释全

1. 半保留复制（semiconservative replication）：DNA复制时，以亲代DNA的每一股做模板，以碱基互补配对原则，合成完全相同的两个双链子代DNA，每个子代DNA中都含有一股亲代DNA链，这种现象称为半保留复制。 2.复制子replicon：由一个复制起始点构成的DNA复制单位。 57. 复制起始点(Ori C)DNA在复制时，需在特定的位点起始，这是一些具有特定核苷酸序列顺序的片段，即复制起始点。 24.（35）复制叉（replication fork）是DNA复制时在DNA链上通过解旋、解链和SSB蛋白的结合等过程形成的Y字型结构称为复制叉。 3. Klenow 片段klenow fragment：DNApol I（DNA聚合酶I）被酶蛋白切开得到的大片段。 4. 外显子exon、extron：真核细胞基因DNA中的编码序列，这部分可转录为RNA，并翻译成蛋白质，也称表达序列。 5.（56）核心启动子core promoter：指保证RNA聚合酶Ⅱ转录正常起始所必需的、最少的DNA序列，包括转录起始位点及转录起始位点上游TATA区。（Hogness区） 6. 转录（transcription）：是在DNA的指导下的RNA聚合酶的催化下，按照硷基配对的原则，以四种核苷酸为原料合成一条与模板DNA互补的RNA 的过程。 7. 核酶（ribozyme）：是具有催化功能的RNA分子，是生物催化剂，可降解特异的mRNA序列。 8.（59）信号肽signal peptide：常指新合成多肽链中用于指导蛋白质的跨膜转移（定位）的N-末端的氨基酸序列（有时不一定在N端）。 9.顺式作用元件（cis-acting element）：真核生物DNA中与转录调控有关的核苷酸序列，包括增强子、沉默子等。 10.错配修复（mismatch repair，MMR）：在含有错配碱基的DNA分子中，使正常核苷酸序列恢复的修复方式；主要用来纠正DNA双螺旋上错配的碱基对，还能修复一些因复制打滑而产生的小于4nt的核苷酸插入或缺失。修复的过程是：识别出正确的链，切除掉不正确的部分，然后通过DNA聚合酶III和DNA连接酶的作用，合成正确配对的双链DNA。 直接修复direct repair：是将被损伤碱基恢复到正常状态的修复。有三种修复方式：1光复活修复2、O6-甲基鸟嘌呤-DNA甲基转移酶修复3单链断裂修复。

分子生物学名词解释

重要名词：（下划线的尤其重要） 1.常染色质：细胞间期核内染色质折叠压缩程度较低，碱性染料着色浅而均匀的区域， 是染色质的主体部分。DNA主要是单拷贝和中度重复序列，是基因活跃表达部分。2.异染色质：细胞间期核内染色质压缩程度较高，碱性染料着色较深的区域。着丝粒、端 粒、次缢痕，DNA主要是高度重复序列，没有基因活性。 3.核小体：核小体是染色体的基本组成单位，它是由DNA和组蛋白构成的，组蛋白H3、 H4、H2B、H2A各两份，组成了蛋白质八聚体的核心结构，大约200bp的DNA盘绕在蛋白质八聚体的外面，相邻两个核小体之间结合了1分子的H1组蛋白。 4.组蛋白：是染色体的结构蛋白，其与DNA组成核小体。根据其凝胶电泳性质可将其分 为H1、H2A、H2B、H3及H4。 5.转座子：是在基因组中可以移动和自主复制的一段DNA序列。 6.基因：原核、真核生物以及病毒的DNA和RNA分子中具有遗传效应的核苷酸序列，是 遗传的基本单位。它包括结构蛋白和调控蛋白。 7.基因组：每个物种单倍体染色体的数目及其所携带的全部基因称为该物种的基因组。 8.顺反子：由顺/反测验定义的遗传单位，与基因等同，都是代表一个蛋白质的DNA 单 位组成。一个顺反子所包括的一段DNA与一个多肽链的合成相对应。 9.单顺反子和多顺反子： 真核基因转录的产物是单顺反子mRNA，即一个基因一条多肽链，每个基因转录都有各自的调控原件。 多顺反子是指原核生物一个mRNA分别编码多条多肽链，而这些多肽链对应的DNA片段位于一个转录单位内，享用同一对起点和终点。 10.转录单位：即转录时，DNA上从启动子到终止子的一段序列。原核生物的转录单位往 往可以包括一个以上的基因，基因之间为间隔区，转录之后形成多顺反子mRNA，可以编码不同的多肽链。真核生物的转录单位一般只有一个基因，转录产物为单顺反子RNA，只编码一条多肽链。 11.重叠基因：是指两个或两个以上的基因共有一段DNA序列重叠基因有多种重叠方式， 比如说大基因内包含小基因，几个基因重叠等等。 12.断裂基因：在真核生物基因组中，基因是不连续的，在基因的编码区域内部含有大量的 不编码序列，从而隔断了对应于蛋白质的氨基酸序列。这种不连续的基因又称断裂基因或割裂基因 13.限制性内切酶：限制性内切酶是一类能够识别双链DNA分子中的某种特定核苷酸序列， 并在相关位置切割DNA双链结构的核酸内切酶。 14.超螺旋：如果固定DNA分子的两端，或者本身是共价闭合环状DNA或与蛋白质结合 的DNA分子，DNA分子两条链不能自由转动，额外的张力不能释放，DNA分子就会发生扭曲，用以抵消张力。这种扭曲称为超螺旋(supercoil)，是双螺旋的螺旋。 15.拓扑异构酶：通过切断DNA的一条或两条链中的磷酸二酯键，然后重新缠绕和封口来 改变DNA连环数的酶。拓扑异构酶I主要消除负超螺旋，作用一次超螺旋交叉数变化+1；拓扑异构酶II主要引入负超螺旋，作用一次L变化-2。TOPO I催化DNA的单链

分子生物学词汇(E1)

分子生物学词汇(E1) 分子生物学词汇(E1)分子生物学词汇(E1)e rosette e（玫瑰）花结[e表示红细胞erythrocyte] e rosette test e（玫瑰）花结试验 ea rossette ea（玫瑰）花结[e表示红细胞erythrocyte,a表示抗体antibody] eac rossette eac（玫瑰）花结[e表示红细胞erythrocyte,a表示抗体antibody，c表示补体complement] eadie plot eadie图 eadie plotting eadie作图法[用于酶促反应动力学] early gene 早期基因[可特指病毒] early hypersensitivity 早发型超敏反应 early phage 早期[有时特指病毒复制的早期] early promoter 早期启动子[有时特指病毒] early protein 早期蛋白[有时特指病毒] early transcription 早期转录[有时特指病毒] eburicoic acid 齿孔酸 ecdysis 蜕皮 ecdyson 蜕皮激素[见于昆虫等节肢动物] ecdyson response element 蜕皮激素效应元件

ecdysteroid hormone 蜕皮类固醇激素[见于血吸虫] echinomycin 棘霉素 echinonectin 海胆粘连蛋白 echiststin 锯鳞（蝮蝰）血抑（环）肽[可抑制血小板凝集] echo virus echo病毒，艾柯病毒 eclipse period [细胞生长的]隐蔽期 eclipsed conformation 重叠构象 eclosion hormone 蜕壳激素[见于昆虫] ecological isolation 生态隔离 ecology 生态学 ecosphere 生态圈 ecotropic retrovirus 亲嗜性逆转录病毒[只能在原始宿主细胞引起产毒性感染] ecotype 生态型 ecteinascidin 海鞘素 ectendomycorrhiza 内外生菌根 ectoderm 外胚层 ectodesma 胞外连丝 ectodesmata （复）胞外连丝 ectodomain 胞外（结构）域 ectoenzyme 胞外酶 ectomycorrhiza 外生菌根

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ilamycin 岛霉素 ilarvirus 等轴不稳定环斑病毒组 illegitimate recombination 非常规重组 imago 成虫 imbibant 吸涨体 imbibition 吸涨（作用） imipramine 丙咪唑 immediate early gene 立即早期基因[有时特指病毒] immobiline [商]固定化电解质[pharmacia公司商标，为丙烯酰胺衍生物，带有官能团，可在凝胶上自动形成固定的ph梯度] immortalization 无限增殖化，永生化[使细胞长期不断维持增殖状态] immunoadhesin 免疫粘附素 immunoadjuvant 免疫佐剂 immunoadsorbent 免疫吸附剂 immunoadsorption 免疫吸附 immunoassay 免疫测定 immunobiology 免疫生物学 immunoblot 免疫印迹 immunoblotting 免疫印迹（法） immunocapture 免疫捕捉，免疫捕获 immunochemiluminescence 免疫化学发光

(完整版)分子生物学总结完整版

分子生物学 第一章绪论 分子生物学研究内容有哪些方面？ 1、结构分子生物学； 2、基因表达的调节与控制； 3、DNA重组技术及其应用； 4、结构基因组学、功能基因组学、生物信息学、系统生物学 第二章DNA and Chromosome 1、DNA的变性：在某些理化因素作用下，DNA双链解开成两条单链的过程。 2、DNA复性：变性DNA在适当条件下，分开的两条单链分子按照碱基互补原则重新恢复天然的双螺旋构象的现象。 3、Tm(熔链温度)：DNA加热变性时，紫外吸收达到最大值的一半时的温度，即DNA分子内50%的双链结构被解开成单链分子时的温度） 4、退火：热变性的DNA经缓慢冷却后即可复性，称为退火 5、假基因：基因组中存在的一段与正常基因非常相似但不能表达的DNA序列。以Ψ来表示。 6、C值矛盾或C值悖论:C值的大小与生物的复杂度和进化的地位并不一致，称为C值矛盾或C值悖论(C-Value Paradox)。 7、转座：可移动因子介导的遗传物质的重排现象。 8、转座子：染色体、质粒或噬菌体上可以转移位置的遗传成分 9、DNA二级结构的特点：1）DNA分子是由两条相互平行的脱氧核苷酸长链盘绕而成；2）DNA分子中的脱氧核苷酸和磷酸交替连接，排在外侧，构成基本骨架，碱基排列在外侧；3）DNA分子表面有大沟和小沟；4）两条链间存在碱基互补，通过氢键连系，且A=T、G ≡ C（碱基互补原则）；5）螺旋的螺距为3.4nm，直径为2nm，相邻两个碱基对之间的垂直距离为0.34nm，每圈螺旋包含10个碱基对；6）碱基平面与螺旋纵轴接近垂直，糖环平面接近平行 10、真核生物基因组结构：编码蛋白质或RNA的编码序列和非编码序列，包括编码区两侧的调控序列和编码序列间的间隔序列。 特点：1）真核基因组结构庞大哺乳类生物大于2X109bp；2）单顺反子（单顺反子：一个基因单独转录，一个基因一条mRNA，翻译成一条多肽链；）3）基因不连续性断裂基因（interrupted gene）、内含子(intron)、外显子(exon)；4）非编码区较多，多于编码序列(9:1) 5）含有大量重复序列 11、Histon(组蛋白)特点：极端保守性、无组织特异性、氨基酸分布的不对称性、可修饰作用、富含Lys的H5 12、核小体组成：由组蛋白和200bp DNA组成 13、转座的机制：转座时发生的插入作用有一个普遍的特征，那就是受体分子中有一段很短的被称为靶序列的DNA会被复制，使插入的转座子位于两个重复的靶序列之间。 复制型转座：整个转座子被复制，所移动和转位的仅为原转座子的拷贝。 非复制型转座：原始转座子作为一个可移动的实体直接被移位。 第三章DNA Replication and repair 1、半保留复制：DNA生物合成时，母链DNA解开为两股单链，各自作为模板(template)按碱

分子生物学名词解释

1, 错义突变:DNA分子中碱基对的取代,使得mRNA的某一密码子发生变化,由它所编码 的氨基酸就变成另一种的氨基酸,使得多肽链中的氨基酸顺序也相应的发生改变的突变. 2 无义突变:由于碱基对的取代,使原来可以翻译某种氨基酸的密码子变成了终止密码子的突变. 3 同义突变:碱基对的取代并不都是引起错义突变和翻译终止,有时虽然有碱基被取代,但 在蛋白质水平上没有引起变化,氨基酸没有被取代,这是因为突变后的密码子和原来的密码子代表同一个氨基酸的突变. 4移码突变:在编码序列中,单个碱基,数个碱基的缺失或插入以及片段的缺失或插入等均 可以使突变位点之后的三联体密码阅读框发生改变,不能编码原来的蛋白质的突变. 1转化:指质粒DNA或以它为载体构建的重组DNA导入细菌的过程. 2 感染:以噬菌体,粘性质粒和真核细胞病毒为载体的重组DNA分子,在体外经过包装成 具有感染能力的病毒或噬菌体颗粒,才能感染适当的细胞,并在细胞内扩增. 3转导:指以噬菌体为载体,在细菌之间转移DNA的过程,有时也指在真核细胞之间通过 逆转录病毒转移和获得细胞DNA的过程. 4转染:指病毒或以它为载体构建的重组子导入真核细胞的过程. 5.癌基因:是细胞内控制细胞生长的基因,具有潜在的诱导细胞恶性转化的特性.当癌基因 结构或表达发生异常时,其产物可使细胞无限制增殖,导致肿瘤的发生.包括病毒癌基因和细胞癌基因. 6., 细胞癌基因:存在于正常的细胞基因组中,与病毒癌基因有同源序列,具有促进正常细胞 生长,增殖,分化和发育等生理功能.在正常细胞内未激活的细胞癌基因叫原癌基因,当其受到某些条件激活时,结构和表达发生异常,能使细胞发生恶性转化. 7. 病毒癌基因:存在于病毒(大多是逆转录病毒)基因组中能使靶细胞发生恶性转化的基因. 它不编码病毒结构成分,对病毒无复制作用,但是当受到外界的条件激活时可产生诱导肿瘤发生的作用. 8. 基因诊断:以DNA或RNA为诊断材料,通过检查基因的存在,结构缺陷或表达异常, 对人体的状态和疾病作出诊断的方法和过程. 9 RFLP:即限制性片段长度多态性,个体之间DNA的核苷酸序列存在差异,称为DNA多 态性.若因此而改变了限制性内切酶的酶切位点则可导致相应的限制性片段的长度和数量发生变化,称为RFLP. 10 基因治疗:一般是指将限定的遗传物质转入患者特定的靶细胞,以最终达到预防或改变特殊疾病状态为目的治疗方法. 11, 反义RNA:碱基序列正好与有意义的mRNA互补的RNA称为反义RNA.可以作为一种 调控特定基因表达的手段. 12, 核酶:是一种可以催化RNA切割和RNA剪接反应的由RNA组成的酶,可以作为基因表 达和病毒复制的抑制剂. SSCP:单链构象多态性检测是一种基于DNA构象差别来检测点突变的方法.相同长度的 单链DNA,如果碱基序列不同,形成的构象就不同,这样就形成了单链构象多态性. 13, 管家基因:在生物体生命的全过程都是必须的,且在一个生物个体的几乎所有细胞中持续表达的基因. 14, 细胞全能性:指同一种生物的所有细胞都含有相同的DNA,即基因的数目和种类是一样的,但在不同阶段,同一个体的不同组织和器官中基因表达的种类和数目是不同的. 15, SD序列:转录出的mRNA要进入核糖体上进行翻译,需要一段富含嘌呤的核苷酸序列与

分子生物学名词解释1

分子生物学名词解释 第二章(主要的:核小体、半保留复制、复制子、单链结合蛋白、岗崎片段、错配修复、DNA的转座、C值矛盾、前导链与后随链。) 1. C值反常现象(C值矛盾C-value paradox): C值是一种生物的单倍体基因组DNA的总量。 真核细胞基因组的最大特点是它含有大量的重复 序列，而且功能DNA序列大多被不编码蛋白质的非 功能DNA所隔开，这就是著名的“C值反常现象”。 C值一般随着生物进化而增加，高等生物的C值一般大于低等生物。某些两栖动物的C值甚至比哺乳动物还大，而在两栖动物里面，C值变化也很大。 2.DNA的半保留复制: 由亲代DNA生成子代DNA时，每个新形成的子代DNA中，一条链来自亲代DNA，而另一条链则是新合成的，这种复制方式称半保留复制。 3.DNA聚合酶: ●以DNA为模板的DNA合成酶 ●以四种脱氧核苷酸三磷酸为底物 ●反应需要有模板的指导 ●反应需要有3 -OH存在 ●DNA链的合成方向为5 3 4.DNA连接酶（1967年发现）：若双链DNA中一条链有切口，一端是3’-OH，另一端是5‘-磷酸基，连接酶可催化这两端形成磷酸二酯键，

而使切口连接。但是它不能将两条游离的DNA单链连接起来 DNA连接酶在DNA复制、损伤修复、重组等过程中起重要作用5.DNA 拓扑异构酶(DNA Topisomerase)： 拓扑异构酶?：使DNA一条链发生断裂和再连接，作用是松解负超螺旋。主要集中在活性转录区，同转录有关。例：大肠杆菌中的ε蛋白 拓扑异构酶Π：该酶能暂时性地切断和重新连接双链DNA，作用是将负超螺旋引入DNA分子。同复制有关。 例：大肠杆菌中的DNA旋转酶 6. DNA 解螺旋酶/解链酶（DNA helicase) 通过水解ATP获得能量来解开双链DNA。 E.coli中的rep蛋白就是解螺旋酶，还有解螺旋酶I、II、III。rep蛋白沿3 ’ 5’移动，而解螺旋酶I、II、III沿5 ’ 3’移动。 7. 单链结合蛋白(SSBP-single-strand binding protein)：稳定已被解开的DNA单链，阻止复性和保护单链不被核酸酶降解。 8. 从复制原点到终点，组成一个复制单位，叫复制子.每个DNA复制的独立单元被称为复制子（replicon），主要包括复制起始位点（Origine of replication）和终止位点 9.复制时，解链酶等先将DNA的一段双链解开，形成复制点，这个复制点的形状象一个叉子，故称为复制叉 10.DNA的半不连续复制:DNA复制时其中一条子链的合成是连续的，而另一条子链的合成是不连续的，故称半不连续复制。

分子生物学与基因工程复习资料

分子生物学与基因工程 绪论 1、分子生物学与基因工程的含义 从狭义上讲，分子生物学主要是研究生物体主要遗传物质-基因或DNA的结构及其复制、转录、表达和调节控制等过程的科学。 基因工程是一项将生物的某个基因通过载体运送到另一种生物的活体细胞中，并使之无性繁殖和行使正常功能，从而创造生物新品种或新物种的遗传学技术。 2、分子生物学与基因工程的发展简史，特别是里程碑事件，要求掌握其必要的理由 上个世纪50年代，Watson和Crick提出了的DNA双螺旋模型； 60年代，法国科学家Jacob和Monod提出了的乳糖操纵子模型； 70年代，Berg首先发现了DNA连接酶，并构建了世界上第一个重组DNA分子； 80年代，Mullis发明了聚合酶链式反应（Polymerase Chain Reaction，PCR）技术； 90年代，开展了“人类基因组计划”和模式生物的基因组测序，分子生物学进入“基因组时代” 3、分子生物学与基因工程的专业地位与作用。 核酸概述 1、核酸的化学组成 2、核酸的种类与特点：DNA和RNA的区别 （1）DNA含的糖分子是脱氧核糖，RNA含的是核糖；

（2）DNA含有的碱基是腺嘌呤（A）、胞嘧啶（C）、鸟嘌呤（G）和胸腺嘧啶（T），RNA含有的碱基前3个与DNA完全相同，只有最后一个胸腺嘧啶被尿嘧啶（U）所代替； （3）DNA通常是双链，而RNA主要为单链； （4）DNA的分子链一般较长，而RNA分子链较短。 3、DNA作为遗传物质的直接和间接证据； 间接： （1）一种生物不同组织的细胞，不论年龄大小，功能如何，它的DNA含量是恒定的，而生殖细胞精子的DNA含量则刚好是体细胞的一半。多倍体生物细胞的DNA含量是按其染色体倍数性的增加而递增的，但细胞核里的蛋白质并没有相似的分布规律。 （2）DNA在代谢上较稳定。 （3）DNA是所有生物的染色体所共有的，而某些生物的染色体上则没有蛋白质。（4）DNA通常只存在于细胞核染色体上，但某些能自体复制的细胞器，如线粒体、叶绿体有其自己的DNA。 （5）在各类生物中能引起DNA结构改变的化学物质都可引起基因突变。 直接：肺炎链球菌试验、噬菌体侵染实验 4、DNA的变性与复性：两者的含义与特点及应用 变性：它是指当双螺旋DNA加热至生理温度以上（接近100oC）时，它就失去生理活性。这时DNA双股链间的氢键断裂，最后双股链完全分开并成为无规则线团的过程。简而言之，就是DNA从双链变成单链的过程。

分子生物学名词解释

Central dogma （中心法则）：DNA 的遗传信息经RNA 一旦进入蛋白质就不能再输出了。Reductionism （还原论）：把问题分解为各个部分，然后再按逻辑顺序进行安排的研究方法。Genome （基因组）：单倍体细胞的全部基因。 transcriptome（转录组）：一个细胞、组织或有机体在特定条件下的一组完整基因。roteome （蛋白质组）：在大规模水平上研究蛋白质特征，获得蛋白质水平上的关于疾病的发生、细胞代谢等过程的整体而全面的认识。 Metabolome （代谢组）：对生物体内所有代谢物进行定量分析并寻找代谢物与生病理变化的相关关系的研究方法。 Gene （基因）：具有遗传效应的DNA 片段。 Epigenetics （表观遗传学现象）：DNA 结构上完全相同的基因，由于处于不同染色体状态下具有不同的表达方式，进而表现出不同的表型。 Cistron （顺反子）：即结构基因，决定一条多肽链合成的功能单位。 Muton（突变子）：顺反子中又若干个突变单位，最小的突变单位被称为突变子。 recon（交换子）：意同突变子。 Z DNA(Z型DNA) ：DNA 的一种二级结构，由两条核苷酸链反相平行左手螺旋形成。Denaturation （变性）：物质的自然或非自然改变。 Renaturation （复性）：变形的生物大分子恢复成具有生物活性的天然构想的现象。egative superhelix （负超螺旋）：B-DNA 分子被施加左旋外力，使双螺旋体局部趋向松弛，ＤＮＡ分子会出现向右旋转的力的超螺旋结构。 C value paradox （Ｃ值矛盾）：生物 overlapping gene（重叠基因）：不同的基因公用一段相同的ＤＮＡ序列。体的大Ｃ值与小ｃ值不相等且相差非常大。 interrupted gene （断裂基因）：由若干编码区和非编码区连续镶嵌而成的基因。 splitting gene（间隔基因）：意思与断裂基因相同。 jumping gene（跳跃基因）：一段可以从原位上单独复制并断裂下来，环化后插入另一位点并对其后的基因起调控作用。 Transposon （转座子）：与跳跃基因意思相同。 eudo gene（假基因）：与功能基因相似却失去基因活性的基因。 Retro-transposon（反转录转座子）：转座子从ＤＮＡ到ＲＮＡ再到ＤＮＡ的转移过程。Replicon （复制子）：从复制起点到复制终点的ＤＮＡ区段。 emiconservative replication（半保留复制）：ＤＮＡ复制过程中亲代ＤＮＡ双链分开作为模板合成两条新生子链，每条新生链均含有一条母链和一条新合成的链。 emi-discontinuous replication（半不连续复制）：前导链以连续复制的方式完成子代ＤＮＡ的合成，而后随链以不连续复制的方式完成冈崎片段的合成。 leading strand（前导链）：随着复制叉的分开，以显露的单链ＤＮＡ为模板聚合ｄＮＴＰ而延伸的链。 lagging strand （后随链）：复制叉的延伸与新生链的延伸背道而驰的链。 dUMP fragment （ｄＵＭＰ片段）：约１２００个核苷酸中有一个错配而引起的DNA 链被切断而形成的大小形似冈崎片段的DNA 分子片段。 replisome （复制体）：连接酶等内在的酶分子集中于复制叉处组成一个复合体协同互作，完成DNA 复制的复合体。 Telomerase （端粒酶）：端粒酶是参与真核生物染色体末端的端粒DNA 复制的一种核糖核蛋白酶。由RNA 和蛋白质组成，其本质是一种逆转录酶。它以自身的RNA 作为端粒DNA 复制的模版，合成出富含脱氧单磷酸鸟苷Deoxyguanosine Monophosphate(dGMP)

分子生物学名词解释最全

第一章名词解释 1.基因（gene）是贮存遗传信息的核酸（DNA或RNA）片段，包括编码RNA和蛋白质的结构基因以及转录调控序列两部分。 2. 结构基因（structural gene）指基因中编码RNA和蛋白质的核苷酸序列。它们在原核生物中连续排列，在真核生物中则间断排列。 3.断裂基因（split gene真核生物的结构基因中，编码区与非编码区间隔排列。 4. 外显子（exon）指在真核生物的断裂基因及其成熟RNA中都存在的核酸序列。 5.内含子（intron）指在真核生物的断裂基因及其初级转录产物中出现，但在成熟RNA中被剪接除去的核酸序列。 6.多顺反子RNA（polycistronic/multicistronic RNA）一个RNA分子上包含几个结构基因的转录产物。原核生物的绝大多数基因和真核生物的个别基因可转录生成多顺反子RNA。 7.单顺反子RNA（monocistronic RNA）一个RNA分子上只包含一个结构基因的转录产物。真核生物的绝大多数基因和原核生物的个别基因可转录生成单顺反子RNA。 8. 核不均一RNA（heterogeneous nuclear RNA, hnRNA）是真核生物细胞核内的转录初始产物，含有外显子和内含子转录的序列，分子量大小不均一，经一系列转录后加工变为成熟mRNA。 9. 开放阅读框（open reading frame, ORF）mRNA分子上从起始密码子到终止密码子之间的核苷酸（碱基）序列，编码一个特定的多肽链。 10.密码子（codon） mRNA分子的开放读框内从5' 到3' 方向每3个相邻的核苷酸（碱基）为一组，编码多肽链中的20种氨基酸残基，或者代表翻译起始以及翻译终止信息。

分子生物学词汇(N)

分子生物学词汇(N) 分子生物学词汇(N)分子生物学词汇(N)n nucleotide n核苷酸[见于免疫球蛋白和t细胞受体的重链基因，系重排过程中随机插入] n orientation 正向（插入），同向（插入）[插入片段与载体同向] n region n区[见于免疫球蛋白和t细胞受体，系重排过程中所插入] nalidixic acid 萘啶酮酸 naloxone 纳洛酮[阿片样肽拮抗剂] naphthol 萘酚 narcotic 麻醉药 narrow groove [dna双螺旋的]窄沟 narrow heribatility 狭义遗传率[加性遗传方差在总表型方差中所占的比例] nasopharyngeal carcinoma 鼻咽癌 nastic movement 感性运动[见于植物] nasty 感性 natamycin 游霉素 native gel 非变性凝胶 natriuretic hormone 利尿钠激素[血浆量增多时，体内生成的

一类能抑制钠泵功能和促进尿钠排出的物质] natriuretic peptide 钠尿肽，利尿钠肽 nebramycin 暗霉素 nebularin 水粉蕈素 nebulin 伴肌动蛋白[骨骼肌的一种肌节基质蛋白，与肌动蛋白等长，可作用于细肌丝] nebulization 雾化 necroparasite 致坏死寄生物[藉分泌物杀死寄生组织后在死组织上生存] necrosis virus 坏死病毒 necrovirus 坏死病毒组[一组植物病毒，模式成员是烟草坏死病毒] nectar 花蜜 nectary 蜜腺 negative interference 负干涉[染色体上一处发生重组，使同一染色体另一处重组率升高] neighborhood correlation 相邻相关（效应），邻位相关（效应）[残基在一级结构上的距离与其三维结构中的距离相关] neisseria 奈瑟菌属 neisseria gonorrhoeae 淋球菌 neisseria meningitidis 脑膜炎球菌 nematoda 线虫纲

分子生物学复习题(有详细答案)

绪论 思考题：（P9） 1.从广义和狭义上写出分子生物学的定义？ 广义上讲的分子生物学包括对蛋白质和核酸等生物大分子结构与功能的研究，以及从分子水平上阐明生命的现象和生物学规律。 狭义的概念，即将分子生物学的范畴偏重于核酸（基因）的分子生物学，主要研究基因或DNA结构与功能、复制、转录、表达和调节控制等过程。其中也涉及与这些过程相关的蛋白质和酶的结构与功能的研究。 2、现代分子生物学研究的主要内容有哪几个方面？什么是反向生物学？什么是 后基因组时代？ 研究内容： DNA的复制、转录和翻译；基因表达调控的研究；DNA重组技术和结构分子生物学。 反向生物学：是指利用重组DNA技术和离体定向诱变的方法研究已知结构的基因相应的功能，在体外使基因突变，再导入体内，检测突变的遗传效应，即以表型来探索基因结构。 后基因组时代：研究细胞全部基因的表达图式和全部蛋白质图式，人类基因组研究由结构向功能转移。 3、写出三个分子生物写学展的主要大事件（年代、发明者、简要内容） 1953年Watson和Click发表了?脱氧核糖核苷酸的结构?的著名论文，提出了DNA的双螺旋结构模型。 1972~1973年，重组DNA时代的到来。H.Boyer和P.Berg等发展了重组DNA 技术，并完成了第一个细菌基因的克隆，开创了基因工程新纪元。 1990~2003年美、日、英、法、俄、中六国完成人类基因组计划。解读人类遗传密码。 4、21世纪分子生物学的发展趋势是怎样的？ 随着基因组计划的完成，人类已经掌握了模式生物的所有遗传密码。又迎来了后基因组时代，人类基因组的研究重点由结构向功能转移。相关学说理论相应诞生，如功能基因组学、蛋白质组学和生物信息学。生命科学又进入了一个全新的时代。 第四章 思考题：（P130） 1、基因的概念如何？基因的研究分为几个发展阶段？ 概念：基因是原核、真核生物以及病毒的DNA和RNA分子中具有遗传效应的核苷酸序列，是遗传的基本单位和突变单位以及控制形状的功能单位。 发展阶段：○120世纪50年代以前，主要从细胞的染色体水平上进行研究，属于基因的染色体遗传学阶段。 ○220世纪50年代以后，主要从DNA大分子水平上进行研究，属于分