Medicamentsen-ligne vous propose les traitements dont vous avez besoin afin de prendre soin de votre santé sexuelle. Avec plus de 5 ans d'expérience et plus de 80.000 clients francophones, nous étions la première clinique fournissant du acheter levitra original en France à vente en ligne et le premier vendeur en ligne de Levitra dans le monde. Pourquoi prendre des risques si vous pouvez être sûr avec Medicamentsen-ligne - Le service auquel vous pouvez faire confiance.

Ash 2000

Glossary of Molecular Biology Terminology
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 molecular biology is also included. The glossary is divided into sev- eral general sections. A cross-reference guide is included to direct readers to the terms they are interested in. The hope is that this addition to the Education Program will further the understanding of those who are less familiar with the discipline of molecular biology.
Competitive oligonucleotide hybridization * University of Washington School of Medicine, Division of He- matology, Box 357710, Seattle WA 98195-7710 FISH (fluorescence in situ hybridization) RDA (representational difference analysis) 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 translated 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 molecule con- tains an anti-codon, a three-nucleotide sequence by which the tRNA molecule recognizes the codon contained in the mRNA template, and an adapter onto which the amino 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 codonredundancy, and three codons signal termination of II. NUCLEIC ACIDS
DNA (deoxyribonucleic acid) The polymer constructed
ORF (open reading frame) The term given to any stretch
of successive nucleotides linked by phosphodiester bonds.
of a chromosome that could encode a polypeptide se- Some 3 x 109 nucleotides are contained in the human quence, i.e., the region between a methionine codon haploid genome. During interphase, DNA exists in a (ATG) that could serve to initiate proteins translation, nucleoprotein complex containing roughly equal amounts and the inframe stop codon downstream of it. Several of histones and DNA, which interacts with nuclear ma- features of the ORF can be used to judge whether it actu- trix proteins. This complex is folded into a basic struc- ally encodes an expressed protein, including its length, ture termed a nucleosome containing approximately 150 the presence of a “Kozak” sequence upstream of the ATG base pairs. From this highly ordered structure, DNA rep- (implying a ribosome might actually bind there and ini- lication requires a complex process of nicking, unfold- tiate protein translation), whether the ORF exists within ing, replication, and splicing. In contrast, gene transcrip- the coding region of another gene, the presence of exon/ tion requires nucleosomal re-organization such that sites intron boundary sequences and their splicing signals, and critical for the binding of transcriptional machinery re- the presence of upstream sequences that could regulate Branched chain DNA (b-DNA) A method that exploits
Plasmids Autonomously replicating circular DNA that
the formation of branched DNA to provide a sensitive are passed epigenetically between bacteria or yeast. In and specific assay for viral RNA or DNA. The assay is order to propagate, plasmids must contain an origin of performed in a microtiter format, in which partially ho- replication. Naturally occurring plasmids transfer genetic mologous oligodeoxynucleotides bind to target to create information between hosts; of these, the genes encoding a branched DNA. Enzyme-labeled probes are then bound resistance to a number of antibiotics are the most impor- to the branched DNA, and light output from a chemilu- tant clinically. The essential components of plasmids are minescence substrate is directly proportional to the used by investigators to introduce genes into bacteria and amount of starting target RNA. Standards provide quan- yeast and to generate large amounts of DNA for manipu- titation. The assay displays a 4 log dynamic range of de- tection, with greater sensitivity to changes in viral loadthan RT-PCR-based assays. It has been employed to quan- 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- Isoschizomer Restriction endonucleases that contain an
tion) efficiency into the bacterial host is usually two or- identical recognition site but are derived from different ders of magnitude greater for phage over that of plas- species of bacteria (and hence have different names).
Restriction endonuclease These enzymes are among the
Cosmid By combining the elements of phage and plas-
most useful in recombinant DNA technology, capable of mids, vectors can be constructed that carry up to 45 kb of introducing a single cleavage site into a nucleic acid. The site of cleavage is dependent on sequence; recognitionsites contain from 4 to 10 specific nucleotides. The re- cDNA A complementary copy of a stretch of DNA pro-
sultant digested ends of the nucleic acid chain may either duced by recombinant DNA technology. Usually, cDNA be blunt or contain a 5' or 3' overhang ranging from 1 to represents the mRNA of a given gene of interest.
Telomere A repeating structure found at the end of chro-
Ribonuclease These enzymes degrade RNA and exist as
mosomes, serving to prevent recombination with free- either exonucleases or endonucleases. The three most ended DNA. Telomeres of sufficient length are required commonly used ribonucleases are termed RNase A, to maintain genetic integrity, and they are maintained by RNase T1, and RNAse H (which degrades duplex RNA or the RNA portion of DNA•RNA hybrids).
CpG This under-represented (i.e. < 1/16 frequency) di-
Ribozyme A complex RNA molecule that possesses
nucleotide pair is a “hotspot” for point mutation. CpG endoribonuclease activity. A highly specific RNA se- dinucleotides are often methylated on cytosine. Should quence can generate secondary structure by virtue of Me-C undergo spontaneous deamination, uracil arises, intrachain base pairing. “Hairpin loops” and “hammer which is then repaired by cellular surveillance mecha- head” structures serve as examples of such phenomena.
nisms and altered to thymidine. The net result is a C to T When the proper secondary structure forms, such RNA molecules can bind a second RNA molecule (e.g. anmRNA) at a specific location (dependent on an approxi- III. ENZYMES OF RECOMBINANT DNA TECHNOLOGY
mately 20-nucleotide recognition sequence) and cleaveat a specific GUX triplet (where X = C, A, or U). These A. Nucleases
molecules will likely find widespread use as tools forspecific gene regulation or as antiviral agents but are evo- A number of common tools of recombinant DNA tech- lutionarily related to RNA splicing, which in its simplest 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- B. Polymerases
tect against the invasion of foreign DNA. Usually, theyspecifically methylate their own DNA and then express DNA polymerase The enzyme that synthesizes DNA
restriction endonucleases to degrade any DNA not ap- from a DNA template. The intact enzyme purified from propriately modified. From such systems come very use- bacteria (termed the holoenzyme) has both synthetic and ful tools. Today, most restriction endonucleases (and most editing functions. The editing function results from nu- other enzymes of commercial use) are highly purified from either natural or recombinant sources and are highlyreliable. Using these tools, the manipulation of DNA and Klenow fragment A modified version of bacterial DNA
RNA has become routine practice in multiple disciplines polymerase that has been modified so that only the poly- merase function remains; the 5'➝3' exonuclease activityhas been eliminated.
Exonuclease An enzyme that digests nucleic acids start-
ing from the 5' or 3' terminus and extending inward.
Thermostabile polymerases The prototype polymerase,
Taq, and newer versions such as Vent and Tth polymerase
Endonuclease An enzyme that digests nucleic acids from
are derived from microorganisms that normally reside at within the sequence. Usually, specific sequences are rec- high temperature. Consequently, their DNA polymerase ognized at the site where digestion begins.
enzymes are quite stable to heat denaturation, makingthem ideal enzymes for use in the polymerase chain re-action.
RNA polymerase II This enzyme is used by mamma-
Reverse transcriptase This enzyme, first purified from
lian cells to transcribe structural genes that result in retrovirus-infected cells, produces a cDNA copy from an mRNA. The enzyme interacts with a number of other mRNA molecule if first provided with an antisense primer proteins to correctly initiate transcription, including a (oligo dT or a random primer). This enzyme is critical number of general factors, and tissue-specific and induc- for converting mRNA into cDNA for purposes of clon- ing, PCR amplification, or the production of specificprobes.
RNA polymerase III This enzyme is used by the cell to
transcribe ribosomal RNA genes.
Topoisomerase A homodimeric chromosomal unwind-
ing enzyme that introduces a double-stranded nick in
Kinases These enzymes transfer the ␥-phosphate group
DNA, which allows the unwinding necessary to permit from ATP to the 5' hydroxyl group of a nucleic acid chain.
DNA replication, followed by religation. Inhibition oftopoisomerases leads to blockade of cell division, the tar- Viral-derived kinases These enzymes are utilized in re-
get of several chemotherapeutic agents (e.g., etoposide).
combinant DNA technology to transfer phosphate groups(either unlabeled or 32P-labeled) to oligonucleotides or Telomerase A specialized DNA polymerase that protects
DNA fragments. The most commonly used kinase is T4 the length of the terminal segment of a chromosome.
Should the telomere become sufficiently shortened (byrepeated rounds of cell division), the cell undergoes Mammalian protein kinases These enzymes transfer
apoptosis. The holoenzyme contains both a polymerase phosphate groups from ATP to either tyrosine, threonine, and an RNA template; only the latter has been character- or serine residues of proteins. These enzymes are among ized, although the gene for the enzymatic activity has the most important signaling molecules present in mam- IV. MOLECULAR METHODS
Farnesyl protein transferase (FTPase) FTPase adds 15
carbon farnesyl groups to CAAX motifs, such as one
A number of molecular techniques have found widespread present in ras, allowing their insertion into cellular mem- application in the biomedical sciences. This section of the glossary provides general concepts and is not intendedto convey adequate details. The interested reader is re- Terminal deoxynucleotidyl This lymphocyte-specific
ferred to the excellent handbook of J. Sambrook and co- enzyme normally transfers available (random) nucleotides workers (Molecular Cloning, A Laboratory Manual, 2nd to the 3' end of a growing nucleic acid chain. In recombi- nant DNA technology, these enzymes can be used to adda homogeneous tail to a piece of DNA, thereby allowing Maxam-Gilbert sequencing A method to determine the
its specific recognition in PCR reactions or in cloning sequence of a stretch of DNA based on its differential cleavage pattern in the presence of different chemicalexposures. A nucleic acid chain can be cleaved follow- Ligases These enzymes utilize the g-phosphate group of
ing G, A, C, or C and T by exposure of 32P-labeled DNA ATP for energy to form a phosphodiester linkage between to neutral dimethylsulfate, dimethylsulfate-acid, hydra- two pieces of DNA. The nucleotide contributing the 5' zine-NaCl-piperidine or hydrazine-piperidine alone, re- hydroxyl group to the linkage must contain a phosphate, which is then linked to the 3' hydroxyl group of the grow-ing chain.
Dideoxynucleotide (ddN) chain termination sequenc-
Also termed “Sanger sequencing,” this method relies
DNA methylases These enzymes are normally part of a
on the random incorporation of dideoxynucleotides into bacterial host defense against invasion by foreign DNA.
a growing enzyme-catalyzed DNA chain. As no 3' hy- The enzyme normally methylates endogenous (host) droxyl group is present on the ddN, chain synthesis halts DNA and thereby renders it resistant to a series of en- following its incorporation into the chain. If 32P or 35S dogenous restriction endonucleases. In recombinant DNA nucleotides are also incorporated into the reaction, a fam- work, methylation finds use in cDNA cloning to prevent ily of DNA fragments will be generated that can be visu- subsequent digestion by the analogous restriction endonu- alized on a polyacrylamide gel. This method is presently the most commonly used chemistry to determine the se-quence of DNA.
DNAse footprinting This technique depends on the abil-
S nuclease analysis This technique is used to identify
ity of protein specifically bound to DNA to block the the start of RNA transcription. The DNAse enzyme S1 activity of the endonuclease DNAse I. 32P-labeled DNA cleaves only at sites of single-stranded DNA. Therefore, is mixed with nuclear proteins, which potentially con- if 32P-labeled DNA is hybridized with mRNA, the result- tain specific DNA-binding proteins, and the reaction is ing heteroduplex can be digested with S , and the result- then subjected to limited DNAse digestion. If a given ing DNA fragment will be of length equivalent to the site site of DNA is free of protein, it will be cleaved by the at which the piece of DNA begins through the mature 5' DNAse. In contrast, regions of DNAse specifically bound by proteins (transcription factors or enhancers) will beprotected from digestion. The resultant mixture of DNA RNAse protection assay This assay is in many ways
fragments from control and protein-containing reactions similar to the S nuclease analysis. In this case, a 35S- or are then separated on a polyacrylamide gel. As the site of 32P-labeled antisense RNA probe is synthesized and hy- 32P labeling of the original DNA fragment is known, sites bridized with mRNA of interest. The duplex RNA is then that were protected from DNAse digestion will be repre- subjected to digestion with RNAse A and T , both of sented on the gel as a region devoid of that length frag- which will cleave only single-stranded RNA. Following ment. Therefore, in comparison to naked DNA, regions digestion, the remaining labeled RNA is size-fraction- that bind specific proteins will be represented as a “foot- ated, and the size of the protected RNA probe then gives an indication of the size of the mRNA present in the origi-nal sample. This assay can also be used to quantitate the DNAse hypersensitivity site mapping This technique
amount of specific RNA in the original sample.
is designed to uncover regions of DNA that are in an“active” transcriptional state. It depends on the hyper- PCR (polymerase chain reaction) This technique finds
sensitivity of such sites (because of the lack of the highly use in several arenas of recombinant DNA technology. It compact nucleosome structure) to limited digestion with is based on the ability of sense and antisense DNA prim- DNAse. Intact nuclei are subjected to limited DNAse di- ers to hybridize to a cDNA of interest. Following exten- gestion. The resultant large DNA fragments are then ex- sion from the primers on the cDNA template by DNA tracted, electrophoretically separated, and hybridized with polymerase, the reaction is heat-denatured and allowed a 32P-labeled probe from a known site within the gene of to anneal with the primers once again. Another round of interest. If, for example, the probe were located at the extension leads to a multiplicative increase in DNA prod- site of transcription initiation, and should DNA fragments ucts. Therefore, a minute amount of cDNA can be effi- of 2 kb and 5 kb be detected with this probe, hypersensi- ciently amplified in an exponential fashion to result in tive sites would thereby be mapped to 2kb and 5 kb up- easily manipulable amounts of cDNA. By including criti- stream of the start of transcription initiation. By extrapo- cal controls, the technique can be made quantitative. Im- lation, these sites would then be assumed important in portant clinical examples of the use of PCR or reverse the transcriptional regulation of the gene of interest, es- transcription PCR (see below) include (1) detection of pecially if such a footprint were only detected using cells 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 lymphoma], or (2) de- Mobility shift (or band shift) assays Like DNAse
tection of minimal residual disease following treatment.
footprinting, this technique is also utilized to determine The level of sensitivity is one in 104 to 105 cells.
whether a fragment of DNA binds specific proteins. 32P-labeled DNA (either duplex oligonucleotides or small RT-PCR (reverse transcription PCR) This technique
restriction fragments) are incubated with nuclear protein allows the rapid amplification of cDNA starting with extracts and subjected to native acrylamide gel electro- RNA. The first step of the reaction is to reverse-transcribe phoresis. Should specific DNA-binding proteins that rec- the RNA into a first strand cDNA copy using the enzyme ognize the oligonucleotide or restriction fragment probe reverse transcriptase. The primer for the reverse transcrip- be present in the nuclear extracts, a DNA-protein com- tion can either be oligo dT, to hybridize to the plex will be formed and its migration through the native polyadenylation tail, or the antisense primer that will be gel will be retarded compared to the unbound DNA.
used in the subsequent PCR reaction. Following this first Hence, the labeled band will be shifted to a more slowly step, standard PCR is then performed to rapidly amplify migrating position. The specificity of their reaction can large amounts of cDNA from the reverse transcribed be demonstrated by also incubating, in separate reactions, competitor DNA that contains the presumed binding siteor irrelevant DNA sequence.
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 theRNA sample for Northern blot), the techniques are es- Figure 1. Nested PCR.
sentially identical. The probe for Northern blotting mustbe antisense.
Nested PCR By using an independent set of PCR prim-
Western blotting This technique is designed to detect
ers located within the sequence amplified by the primary specific protein present in a heterogenous sample. Pro- set, the specificity of a PCR reaction can be greatly en- teins are denatured and size-fractionated by polyacryla- hanced. In Figure 1, should the first PCR reaction yield
mide gel electrophoresis, transferred to nitrocellulose or a product of 600 nucleotides, a second PCR reaction us- other synthetic membranes, and then probed with an an- ing the first product as template and a different set of tibody to the protein of interest. The immune complexes primers will produce a smaller, “nested” PCR product, present on the blot are then detected using a labeled sec- the presence of which acts to confirm the identity of the ond antibody (for example, a 125I-labeled or biotinylated goat anti-rabbit IgG). As the original gel electrophoresiswas done under denaturing and reducing conditions, the Real-time automated PCR During PCR, a fluorogenic
precise size of the target protein can be determined.
probe, consisting of an oligodeoxynucleotide with bothreporter and quencher dyes attached, anneals between the Southwestern blotting This technique is designed to
two standard PCR primers. When the probe is cleaved detect specific DNA-binding proteins. Like the Western during the next PCR cycle, the reporter is separated from blot, proteins are size-fractionated and transferred to ni- the quencher so that the fluorescence at the end of PCR trocellulose. The probe in this case, however, is a double- is a direct measure of the amplicons generated through- stranded labeled DNA that contains a putative protein- out the reaction. Such a system is amenable to automa- binding site. Should the DNA probe hybridize to a spe- tion and gives precise quantitative information.
cific protein on the blot, that protein can be subsequentlyidentified by autoradiography. This technique often suf- Allele-specific PCR By using generic PCR primers flank-
fers from nonspecificity, so that a number of critical con- ing the immunoglobulin or T cell receptor genes, the pre- trols must be included in the experiment for the results to cise rearranged gene characteristic of a B or T cell neo- plasm can be amplified and sequenced. Once so obtained,new PCR primers can then be designed that are unique In situ hybridization This technique is designed to de-
to the patient’s tumor. Such allele-specific PCR can then tect specific RNA present in histological samples. Tissue be used to detect blood cell contamination by tumor and is prepared with particular care not to degrade RNA. The to detect minimal residual disease following therapy.
cells are fixed on a microscope slide, allowed to hybrid-ize to probe, and then washed and overlaid with photo- Southern blotting This technique is used to detect spe-
graphic emulsion. Following exposure for one to four cific sequences within mixtures of DNA. DNA is size- weeks, the emulsion is developed and silver grains over- fractionated by gel electrophoresis and then transferred lying cells that contain specific RNA are detected. The by capillary action to nitrocellulose or another suitable most useful probes for this purpose are metabolically 35S- synthetic membrane. Following blocking of nonspecific labeled riboprobes generated by in vitro transcription of binding sites, the nitrocellulose replica of the original gel a cDNA using viral RNA polymerase. These probes give electrophoresis experiment is then allowed to hybridize the lowest background and are preferable to using termi- with a cDNA or oligonucleotide probe representing the nal deoxynucleotidyl transferase or alternative methods specific DNA sequence of interest. Should specific DNA be present on the blot, it will combine with the labeledprobe and be detectable by autoradiography. By co-elec- FISH (fluorescence in situ hybridization) A general
trophoresing DNA fragments of known molecular weight, method to assign chromosomal location, gene copy num- the size(s) of the hybridizing band(s) can then be deter- ber (both increased and decreased), or chromosomal re- mined. For gene rearrangement studies, Southern blot- arrangements. Biotin-containing nucleotides are incor- ting is capable of detecting clonal populations that repre- porated into specific cDNA probes by nick-translation.
sent approximately 1% of the total cellular sample.
Alternatively, digoxigenin or fluorescent dyes can be in- corporated by enzymatic or chemical methods. The probes Mutagenesis, site-specific Several methods are now
are then hybridized with solubilized, fixed metaphase available to intentionally introduce specific mutations into cells, and the copy number of specific chromosomes or a cDNA sequence of interest. Most are based on design- genes are determined by counter-staining with fluores- ing an oligonucleotide that contains the desired mutation cein isothiocyanate (FITC)-labeled avidin or other de- in the context of normal sequence. This oligonucleotide tector reagents. The number and location of detected fluo- is then incorporated into the cDNA using DNA poly- rescent spots correlates with gene copy number and chro- merase, either using a single-stranded DNA template (ph- mosomal location. The method also allows chromosomal age M13) or in a PCR format to produce a heteroduplex analysis in interphase cells, allowing extension to condi- DNA containing both wild type and mutant sequences.
Using M13, recombinant phage are then produced andmutant cDNA are screened for on the basis of the differ- CGH (comparative genome hybridization) In CGH,
ence in wild type and mutant sequences; using the PCR DNA is extracted from tumor and from normal tissues format, the exponential amplification of the mutant se- and differentially labeled with fluorescent dyes. Once the quence results in its overwhelming numerical advantage DNA samples are mixed and hybridized to normal over wild type sequence, resulting in nearly all clones metaphase chromosome spreads, chromosomal regions containing mutant sequence. Both of these methods re- that are under-represented or over-represented in the tu- quire that the entire cDNA insert synthesized in vitro be mor sample can be identified. This method can be ap- sequenced in its entirety to guarantee the fidelity of mu- plied to extremely small tumor samples (by using PCR tagenesis and synthesis of the remaining wild type se- 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 Chromatography, gel filtration This technique is de-
malignancies. The sensitivity of the technique approaches signed to separate proteins based on their molecular weight. It is dependent on the exclusion of proteins froma matrix of specific size. Proteins that are too large to fit Nick-translation This technique is used to label cDNA
into the matrix of the gel bed run to the bottom of the to high specific activity for the purpose of probing South- column more quickly than smaller proteins, which are ern and Northern blots and screening cDNA libraries.
included in the volume of the matrix. Therefore, using The cDNA fragment is first nicked with a limiting con- appropriate size markers, the approximate molecular centration of DNAse, then DNA polymerase is used to weight of a given protein can be determined and it can be both digest and fill in the resulting gaps with labeled separated from proteins of dissimilar size. Typical sepa- ration media for gel filtration chromatography includeSephadex and Ultragel.
Random priming This technique is also used to pro-
duce labeled cDNA probes and is dependent on using
Chromatography, ion exchange This separation meth-
random 6- to 10-base oligonucleotides to sit down on a odology depends on the preferential binding of positively single-stranded cDNA and then using DNA polymerase charged proteins to a matrix containing negatively charged to synthesize the complementary strand using labeled groups or a negatively charged protein binding to a ma- nucleotides. This technique usually produces more fa- trix containing positively charged groups. Increases in vorable results than nick-translation.
the buffer concentration of sodium chloride are then usedto break the ionic interaction between protein and matrix Riboprobes These labeled RNA molecules are produced
and elute off-bound proteins. Examples of such separa- by first cloning the cDNA of interest into a plasmid vec- tion media include DEAE and CM cellulose.
tor that contains promoters for viral RNA polymerases.
Following cloning, the viral RNA polymerase is added, Chromatography, hydrophobic This methodology sepa-
and labeled nucleotides are incorporated into the result- rates proteins based on their hydrophobicity. Proteins ing RNA transcript. This molecule is then purified and preferentially bind to the matrix based on the strength of used in probing reactions. Many such cloning vectors (for this interaction; proteins are then eluted off using sol- example, pGEM) have different RNA polymerase pro- vents of increasing hydrophobicity. Separation media moters on either side of the cloning site, allowing the include phenyl-sepharose and octyl-sepharose.
generation of both sense and antisense probes from thesame construct.
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 either orientation and can operate up to 50 kb or more bind glycoproteins or using covalently coupled mono- from the gene of interest. Enhancers are cis-acting in that clonal antibodies to bind specific protein ligands.
they must lie on the same chromatin strand as the struc-tural gene undergoing transcription. These cis-acting se- Chromatography, high performance liquid (HPLC)
quences function by binding specific proteins, which then A general methodology to improve the separation of com- interact with the RNA polymerase complex.
plex protein mixtures. The types of HPLC columns avail-able are the same as for conventional chromatography, Silencer These elements are very similar to enhancers
such as those based on size exclusion, hydrophobicity, except that they have the function of binding proteins and ionic interaction, but the improved flow rates result- ing from the high pressure system provide enhanced sepa-ration capacity and improved speed.
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 silencers.
The regulation of gene expression is central to physiol- The proteins are brought together by the looping of DNA ogy. Complex organisms have evolved multiple mecha- strands so that protein binding sites, which may range up nisms to accomplish this task. The first step in protein to tens of kb apart, can be brought into close juxtaposi- expression is the transcription of a specified gene. The tion. Specific protein•protein interactions then allow as- rate of initiation and elongation of this process is the most commonly used mechanism for regulating gene expres-sion. Once formed, the primary transcript must be spliced, Polyadenylation Following transcription of a gene, a
polyadenylated, and transported to the cytoplasm. These specific signal near the 3' end of the primary transcript mechanisms are also possible points of regulation. In the (AATAAA) signals that a polyadenine tail be added to cytoplasm, mRNA can be rapidly degraded or retained, the newly formed transcript. The tail may be up to sev- another potential site of control. Protein translation next eral hundred nucleotides long. The precise function of occurs on the ribosome, which can be free or membrane- the poly A tail is uncertain but it seems to play a role in associated. Secreted proteins take the latter course, and stability of the mRNA and perhaps in its metabolism the trafficking of the protein through these membranes through the nuclear membrane to the ribosome.
and ultimately to storage or release makes up anotherimportant point of potential regulation. Individual gene Splicing The primary RNA transcript contains a number
expression is often controlled at multiple levels, making of sequences that are not part of the mature mRNA. These investigation and intervention a complex task.
regions are called introns and are removed from the pri-mary RNA transcript by a process termed splicing. A Transcription Transcription is the act of generating a
complex tertiary structure termed a lariat is formed and primary RNA molecule from the double-stranded DNA the intron sequence is eliminated bringing the coding se- gene. Regulation of gene expression is predominantly at quences (exons) together. Specific sequences within the the level of regulating the initiation and elongation of primary transcript dictate the sites of intron removal.
transcription. The enzyme RNA polymerase is the keyfeature of the system, which acts to generate the RNA Exons These are the regions of the primary RNA tran-
copy of the gene in combination with a number of im- script that, following splicing, form the mature mRNA portant proteins. There is usually a fixed start to tran- species, which encodes polypeptide sequence.
Introns These are the regions of the primary RNA tran-
TATA Many genes have a sequence that includes this
script that are eliminated during splicing. Their precise tetranucleotide close to the beginning of gene transcrip- function is uncertain. However, several transcriptional tion. RNA polymerase binds to the sequence and begins regulatory regions have been mapped to introns, and they transcription at the cap site, usually located approximately are postulated to play an important role in the generation of genetic diversity (exon shuffling mechanism).
Enhancer An enhancer is a segment of DNA that lies
Nucleosomes When linear, the length of a specific chro-
either upstream, within, or downstream of a structural mosome is many orders of magnitude greater than the gene that serves to increase transcription initiation from diameter of the nucleus. Therefore, a mechanism must that gene. A classical enhancer element can operate in exist for folding DNA into a compact form in the inter- phase nucleus. Nucleosomes are complex DNA protein Missense mutation Mutation of the mRNA sequence to
polymers in which the protein acts as a scaffold around generate an altered codon, which results in an amino acid which DNA is folded. The mature chromosomal struc- change, is termed a missense mutation.
ture then appears as beads on a string; within each bead(nucleosome) are folded DNA and protein. Nucleosome Nonsense mutation This type of mutation results in the
structure is quite fluid, and internucleosomal stretches of generation of a premature termination codon and hence DNA are thought to be sites that are important for active Transcriptional regulation Gene regulation is deter-
Trans-acting factors Proteins that are involved in the
mined by the rate of transcriptional initiation. This usu- transcriptional regulation of a gene of interest.
ally results from alteration in the level of activity of trans-acting proteins, which, in turn, are regulated either by Cis-acting factors These are regions at a gene either
the amount of the transcriptionally active protein or by upstream, within, or downstream of the coding sequence that contains sites to which transcriptionally importantproteins may bind. Sequences that contain 5 to 25 nucle- Leucine zipper proteins A family of DNA-binding pro-
otides are present in a typical cis-acting element.
teins that require a dimeric state for activity and thatdimerize by virtue of an alpha helical region that con- Transcription factors Specific proteins that bind to con-
tains leucine at every seventh position. Because 3.4 amino trol elements of genes. Several families of transcription acids reside in each turn of an alpha helix, the occur- factors have been identified and include helix-loop-helix rence of leucine at every seventh position results in a strip proteins, helix-turn-helix proteins, and leucine zipper of highly hydrophobic residues on one surface of the al- proteins. Each protein includes several distinct domains pha helix. Such a domain on one polypeptide can such as activation and DNA-binding regions.
intercollate with a similar domain on a second polypep-tide, resulting in the formation of a stable homodimer or LCR (locus control region) Cis-acting sites are occa-
heterodimer. Examples of the leucine zipper family in- sionally organized into a region removed from the struc- clude the proto-oncogenes c-jun and c-fos.
tural gene(s) they control. Such locus control regions(LCRs) are best described for the b globin and a globin Basic helix-loop-helix proteins These transcriptional
loci. First recognized by virtue of clustering of multiple proteins are characterized by two alpha helical regions DNAse hypersensitive sites, the ␤ globin LCR is required separated by a loop structure; this domain is involved in for high level expression from all of the genes and ap- protein dimerization. Examples of this family of tran- pears to be critical for their stage-specific developmental scription factors include E12/E47 of the immunoglobu- lin promoter or Myo D of muscle cell regulation.
Protein translation This term is applied to the assembly
Helix-turn-helix This family of transcriptionally active
of a polypeptide sequence from mRNA.
proteins depends on the helix-turn-helix motif for dimer-ization. Examples include the homeodomain genes such 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- Master switch genes These polypeptide products are
tion is located three nucleotides upstream from the ini- thought to regulate a whole family of genes and result in tiation ATG and is almost always an adenine nucleotide.
a cell undergoing a new program of differentiation. An When multiple potential initiation codons are present in example of such a system is Myo D, in which activation an open reading frame, the ATG codon, which contains a is thought to lead to differentiation along the muscle cell strong consensus KOZAK sequence, is likely the true Zinc finger domain proteins The presence of conserved
Initiation codon The ATG triplet is used to begin
histidine and cysteine residues allows chelation of a zinc polypeptide synthesis. This is usually the first ATG codon, atom and results in the formation of a loop structure called located approximately 30 nucleotides downstream of the the zinc finger domain. This feature is present in a large site of transcription initiation (cap site). However, the family of transcriptionally active proteins such as the ste- context in which the ATG resides is also important (see Post-transcriptional regulation Mechanisms of gene
machinery. Several classes of cyclins (A through E) exist regulation that do not involve transcriptional enhance- that regulate different aspects of the cell cycle (G , G , S, ment or silencing and include altering the rate of mRNA G , M). Altered expression of some cyclins is associated degradation, the efficiency of translation or post-transla- with hematologic malignancy, e.g., t(11;14) in mantle cell tional modification, or transportation of the polypeptide lymphoma leads to over-expression of cyclin D , a G Actinomycin D pulse experiments The application of
Cdk (cyclin-dependent kinase) A related group of cel-
actinomycin D to actively metabolizing cells results in lular kinases, present in virtually all cells, that are regu- the cessation of new RNA transcription. Consequently, lated both positively and negatively by specific phospho- serial determinations of specific RNA levels will allow rylation events and negatively by association with other one to calculate the mRNA half life. Should this vary proteins, and are dependent on cyclins, present only dur- between control and stimulated conditions, evidence is ing certain phases of the cell cycle (cdk1-activated dur- garnered that a gene of interest is regulated at the level of ing G /M phase, cdk2-G /S phases, cdk4-G /S phases, cdk6-G phase, cdk7-throughout the cell cycle).
Reporter genes In order to determine how a gene pro-
CdkI (cdk inhibitors) Proteins that inhibit the cdks by
moter or enhancer works in vitro, that genetic element is stoicheometric combination, arresting cells in G phase, often linked to a gene for which a simple assay is readily and include p27, p21 and the p16 Ink 4A family of pro- available and whose regulation is not affected by post- teins. The latter are implicated as tumor suppressor genes, transcriptional processes. Such reporter genes include as their deficiency in mice leads to rapid cellular prolif- chloramphenicol acetyl transferase, ␤ galactosidase, and eration and a high rate of spontaneous tumor develop- firefly luciferase. The first is the most commonly used re- ment. Moreover, deficiency of p16 family members has porter; however, more recent studies have emphasized the been associated with numerous types of human tumors, use of the latter two reporters, as these are more sensitive to including a fraction of cases of B cell ALL and T cell minimal changes in promoter or enhancer activity.
CAT (chloramphenicol acetyl transferase) The bacte-
rial gene for chloramphenicol resistance, chlorampheni-col acetyl transferase (CAT) is commonly used as a re- In order to exploit the techniques of recombinant DNA porter gene for investigating physiologic gene regulation.
research, one must possess a system to manufacture the The assay depends on the ability of transfected cellular protein of interest. After identifying the gene encoding cytoplasm to convert 14C chloramphenicol to its acety- the protein and obtaining a cDNA representation of it lated form in the presence of acetyl CoA. The acetylated (“cloning”), the cDNA must be placed in a vector ca- forms are separated from the 14C substrate using thin pable of driving high levels of RNA transcription in a host system capable of translating and appropriatelymodifying the polypeptide to produce fully functional ␤ galactosidase The presence of ␤ galactosidase activ-
protein. And just like obtaining a protein of interest from ity in the cytoplasm of transfected cells can be readily natural sources, one must purify protein from the final detected by its ability to convert a colorless substrate to a expression system. Because of the nature of the highly blue-colored product. This is usually assayed using a fluo- engineered systems and high levels of expression, this latter task is usually considerably easier using recombi-nant methods than from natural sources. The methods Luciferase This gene, which is the most recent reporter
used to generate expression vectors are described in Sec- gene to be used, has gained increasing acceptance be- tion IV, but the methods to purify proteins are discussed cause of its ease of assay and extreme sensitivity. The in only a rudimentary way and are beyond the scope of assay is based on the ability of the protein to undergo chemiluminescence and transmit light, detected with aluminometer.
Expression vector A plasmid that contains all of the el-
ements necessary to express an inserted cDNA in the host
Cyclins A group of proteins that vary in expression
of interest. For a mammalian cell host, such a vector typi- throughout the cell cycle. Once a threshold level is at- cally contains a powerful promoter coupled to an en- tained, interaction with specific cellular kinases results hancer, a cloning site, and a polyadenylation signal. In in phosphorylation of critical components of the mitotic addition, several expression vectors also contain a select- able marker gene such as DHFR or NeoR, which aids in impairment of translation efficiency. Most successful at- the generation of stable cell lines. The plasmid also re- tempts using antisense ODN have targeted sequences quires a bacterial origin of replication and an antibiotic surrounding and including the initiation codon. To re- resistance gene (AmpR) to allow propagation and expan- duce nuclease attack, the antisense ODN are often syn- thesized using an altered chemistry involving thiol ratherthan phosphodiester linkages.
Transfection Once the expression vector has been as-
sembled, it must be inserted into the host of interest. Sev-
Transgenic animals By introducing an intact or manipu-
eral methods are available for such transfections and in- lated gene into the germline of mice, the effects of pro- clude calcium/phosphate/DNA complexes, DEAE Dext- moter expression in specific cell lineages can be investi- ran, electroporation, liposome, and retrovirus-mediated 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 Calcium phosphate This method relies on the produc-
methods for production of transgenic mice have been tion of a calcium/phosphate/DNA microprecipitate, which extensively reviewed and are based on the microinjec- is then taken up by cells by pinocytosis. The method is tion of linear DNA into the pronucleus of a fertilized egg.
very effective for a number of commonly used mamma- Several types of experiments can be performed. First, the lian cell expression systems including COS, BHK, 293, effect of aberrant expression of a gene can be investi- gated, as was recently performed by expressing GM-CSFin a wide variety of tissues. Second, the necessary ele- DEAE dextran This method depends on the formation
ments for tissue- and developmental level-specific ex- of a complex between the insoluble positively charged pression of a gene can be studied, as has been performed dextran and the DNA to be transfected. Like calcium for the ␤-globin locus. Third, the tissue distribution of a phosphate, this method is highly successful with many specific gene can be determined by engineering a marker gene adjacent to a specific promoter. A specific exampleof this strategy employs a “suicide gene,” the herpes vi- Electroporation When cells are suspended in buffer be-
rus thymidine kinase (TK). When animals carrying such tween two electrodes, discharge of an electrical impulse genes are exposed to gancyclovir, cells expressing the momentarily creates pores in the cell membrane. During promoter of interest will express TK, be killed, and be this time, DNA in solution is free to diffuse into the cells.
This method is highly successful in transfecting a largenumber of cell types, including cells previously thought Gene knock-out experiments Specific genes in the mam-
to be difficult to transfect with other methods, such as malian genome can now be targeted for interruption or correction based on the technique of homologous recom-bination. By generating DNA constructs that contain an Liposomes By encapsulating the DNA to be transfected
interrupted gene of interest, or a corrected gene, in the in an artificial lipid carrier, foreign DNA can be intro- setting of adequate flanking sequences to allow for tar- duced into the cell. This method, like electroporation, geting to the genetic locus of interest, the endogenous has been successful in transfecting cells previously gene can be replaced or corrected. The methods involve thought difficult to manipulate. Its only drawback is its introduction of the gene into an embryonic stem (ES) cell line, selection for subclones of cells that have hadsuccessful homologous recombination events, and then Transduction The act of transferring a foreign gene into
introduction of the ES subclone into the blastocyst of a developing embryo. A chimeric animal results, and shouldthe newly introduced gene become part of the germline, VII. EXPERIMENTAL GENE MANIPULATION
it can be bred to the homozygous state. Using these tech-niques, investigators can now determine whether a single Antisense oligonucleotides By introducing short single-
genetic locus is responsible for a given disease, deter- stranded deoxyribonucleic acids (ODN) into a cell, spe- mine the significance of specific cytokines or growth fac- cific gene expression can be interrupted. Several mecha- tors, and generate model systems useful investigation of nisms have been postulated to account for these results including interruption of ribosome binding to mRNA,enhanced degradation of mRNA mediated by the double- Gene knock-in experiments A similar technology to
strand specific RNAseH, DNA triplex formation, and knock-out strategy, but rather than simply obliterating function of the targeted gene, the knock-in is designed to sired genes has become common. Insertion of the gene replace the locus with a specific mutation of interest.
into target cells and high (adequate) level expression ismore problematic. Several types of transfer vehicles have Homologous recombination When a manipulated gene
found use, including viral vectors and chemical agents.
is introduced into a cell, it can be incorporated into thegenome either randomly or at a specific locus. By incor- Viral transduction vectors Retroviral vectors are based
porating sequences that normally flank the desired lo- on murine retroviruses. They can carry 6 to 7 kb of for- cus, a manipulated gene can be specifically (albeit rarely) eign DNA (promoter + cDNA) but suffer from the draw- introduced into the genome. Selection for this unlikely backs of requiring the development of high titer packag- event can be enhanced by introduction of the herpes thy- ing lines, requiring that target cells be dividing, and are midine kinase (TK) gene into the original targeting con- subject to host cell down-modulation. Adenoviral vec- struct. Should the construct be randomly incorporated tors can be produced at high levels and do not require a into the genome, the TK gene will also be introduced, dividing target cell, but they do not normally integrate, rendering the cell sensitive to gancyclovir. If homolo- resulting in only transient expression. Adeno-associated gous recombination occurs, the TK gene will be elimi- viral vectors are defective parvoviruses that integrate into nated, as there are no homologous sequences at the spe- a non-dividing host cell at a specific location (19q). Dis- cific genetic locus of interest and the resultant cell will advantages are genetic instability, small range of insert size (2–4.5 kb), and thus far, only transient expression.
YAC (yeast artificial chromosome) A yeast artificial
Ecotropic vectors Many retroviruses are host cell spe-
chromosome (YAC) utilizes centromeric and telomeric cific, i.e. they will only infect a specific species of cells.
elements from yeast chromosomes to construct genetic An example is the widely used Maloney virus, and its elements that can be propagated in yeast and transferred basis lies in the species-specific expression of the viral into mammalian cells. Such vehicles allow the introduc- tion of up to 200 kb or more of genetic material into thehost cells. YACs are now being used to study the physi- Ecotropic viruses Murine retroviruses that contain coat
ologic regulation of large genetic loci such as the ␤-globin proteins that can only bind to murine cellular receptors.
Amphotropic viruses Retroviruses whose coat proteins
Contig The jargon term used to describe the assembly of
bind to a receptor found throughout multiple species, clones necessary to include all of the DNA in a specific usually including man, making these vectors suitable for stretch of chromosome. Such maps are usually assembled human use. Problems related to the level of receptor ex- from overlapping YAC (yeast artificial chromosome) or pression on cells of hematologic interest (e.g. stem cells) BAC (bacterial artificial chromosome) clones. Once the “genome project” is complete, it will consist of 24 (verylarge) contigs (22 autosomal, an X and a Y).
Pseudotyped virus These take advantage of the power-
ful expression levels obtainable by murine retroviral back-
Transposon Naturally occurring genetic elements that
bones, yet are packaged in an envelope that allows dock- are naturally easily removed and inserted into the genome, ing and uptake by human target cells. An example is the allowing for the recombination of genetic segments, giv- popular MFG vector that utilizes a murine leukemia ing rise to genetic diversity. These same elements can be 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
Gene therapy takes many forms. To treat malignancy, it may involve the insertion of an adjuvant substance (suchas GM-CSF) into tumor cells to generate a tumor vac- Positional variegation Refers to the observation that the
cine, transfer of a gene that renders tumor cells suscep- site of vector integration into the genome often results in 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., Chimeraplasty A technique of gene therapy dependent
MDR). For gene deficiencies, insertion of the wild type on construction of a DNA:RNA oligonucleotide hybrid allele is the therapeutic goal. Obtaining cDNA for de- that once introduced into a cell relies upon DNA repair mechanisms to introduce a (corrective) change in the tar- 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 Chitosan-DNA A chemical means of packaging foreign
strand synthesis using terminal deoxynucleotide trans- DNA to allow introduction into cells; the complexes ex- ferase, then second strand priming can occur using a syn- ist as nanospheres and have been tested in factor IX defi- thetic oligonucleotide complementary to the TdT tail.
Should the former technique be used, an extra step tonick the hairpin loop using the enzyme S1 nuclease would Long terminal repeat (LTR) This segment of a retroviral
be required prior to inserting the cDNA into its vector.
genome carries the genetic information for both transcrip-tion of downstream viral structural genes and the mecha- cDNA blunting First and second strand synthesis usu-
nisms of viral replication. It is often used in retroviral ally results in nonflush ends. To prepare the cDNA for applications to drive the exogenous therapeutic gene as insertion into a cloning vector, the ends must be made it carries a powerful (but non-tissue specific) promoter.
flush with one another. Such blunting reactions can beconducted with a DNA polymerase, such as the Klenow Interference The mechanisms by which infection of a
fragment of DNA polymerase I or T4 DNA polymerase.
cell by one virus excludes infection by others. Interfer-ence is often due to the cellular production of coat pro- Linkering To efficiently insert the cDNA library into a
teins, which bind to and block the cells’ remaining viral cloning vector, synthetic duplex oligonucleotides that contain a restriction endonuclease site are attached to theblunted ends of the cDNA. A restriction endonuclease is Nonviral transduction methods Nonviral methods in-
chosen that rarely cuts DNA (such as the 8 bp recogni- clude polylysine-ligand DNA complexes, where the tion sequence for Not I, or if a more common restriction ligand (e.g., transferrin) allows access to the cell through site is used such as Eco RI, the cDNA should first be normal receptor-mediated uptake, and phospholipid methylated in order to prevent subsequent cDNA diges- vesicles. Both methods suffer from not providing a tion with the enzyme) and is used to generate “sticky mechanism for genomic integration, precluding long-term cDNA library preparation Once the cDNA has been
prepared and sticky ends generated, the library is insertedinto a convenient cloning vector. Because of high clon- Obtaining cDNA representing a protein of interest is usu- ing efficiency, most cDNA libraries are constructed in a ally the first step in the process of applying the techniques ␭ phage vector. Typically, if screening is to be performed of recombinant DNA research to an important physiologic using a monoclonal antibody, ␭ gt 11 is used. If screen- question. A suitable cDNA library must first be con- ing is to be performed using oligonucleotide probes, ␭ gt structed starting with RNA abundant (or as abundant as 10 can be used. If larger DNA fragments are to be pre- possible) in the transcripts for the gene of interest. Fol- pared, such as from genomic fragments of DNA, ␭ vec- lowing library construction a probe must be developed tors that can accommodate up to 20 kb are available (e.g., that can specifically recognize the gene or cDNA of in- terest, or the expressed protein product of the specificcDNA.
Subtractive library The purpose of generating a sub-
tractive library is to enrich for cDNA that are expressed
First strand synthesis The retroviral enzyme reverse
under one condition but are not expressed under a sec- transcriptase is used along with an antisense primer to ond condition. This facilitates screening for the cDNA of produce a complementary DNA strand of mRNA ex- interest in that the complexity of the library is much re- tracted from a cellular source known to express the gene duced, requiring one to screen far fewer clones. At its of interest. Two types of primers are used, either oligo extreme, investigators have used subtractive libraries to dT, in which the poly A tail begins the cDNA synthesis, generate a very highly select group of clones (in the range or random primers, in which a whole range of start sites of 100) and then have sequenced all of the resulting cDNA. The principle behind a subtractive library is theelimination of cDNA common to induced and control Second strand synthesis The enzyme DNA polymerase
conditions. By eliminating such clones, only cDNA that is used to generate the sense strand of cDNA. Priming of are present under the induced conditions will remain in the second strand can occur spontaneously, as the anti- the library. Those techniques depend on the differential elimination of duplex mRNA/cDNA or cDNA/cDNA Reverse genetics Often, large families of homologous pro-
hybrids, which form between genes expressed under both teins exist and multiple previously unknown members of conditions, leaving the single-stranded mRNA or cDNA the family can be obtained by screening cDNA libraries under low stringency using cDNA or oligonucleotide probesfrom regions highly conserved amongst members of the RDA (representational difference analysis) A molecu-
family. In this case, genes are identified before their func- lar method to amplify genes that are expressed in an RNA tion is known, a situation referred to as reverse genetics.
sample of interest, that are not present, or present at very Examples in hematology include identifying members of reduced levels, in a comparison RNA sample (e.g. cyto- the tyrosine kinase family of receptor proteins using a probe kine induced and control cells). The method relies on derived from the conserved kinase domain of the cytoplas- RT-PCR amplification of the RNA that does not contain mic region of src or other tyrosine kinase proto-oncogenes, the gene(s) of interest to produce a “driver” cDNA, and or the identification of transcription factors important in RT-PCR to produce “tester” cDNA from the RNA popu- hematopoiesis using conserved motifs present in zinc fin- lation in which you hope to find new genes. After liga- tion of different oligonucleotides to the ends of each popu-lation, both are denatured and an excess of the driver is X. ONCOGENESIS AND ANTI-ONCOGENES
hybridized to the tester and PCR performed with primersthat will amplify only sequences present in the tester that Oncogenes have usually been identified in the context of are not in the driver, thereby “removing” cDNA com- a tumor-inducing virus. Such viral oncogenes (v-onc) are mon to both populations. The resultant cDNA are en- thought to be derived from host cells, but have been al- tered such that abnormal regulation of production or func-tion has ensued during the transfer process. Subsequent Directional cloning To improve efficiency when screen-
reintroduction of the altered gene into a host cell leads to ing functional expression libraries, many investigators transformation. Proto-oncogenes, the normal cellular construct cDNA libraries in which the proper coding ori- counterpart of viral oncogenes, can contribute to cellular entation of the cDNA is maintained in the library. In con- transformation by mechanisms that disturb normal gene ventional library preparation, the 5' and 3' ends of the function. Such mechanisms include mutation (resulting DNA are identical; thus, cDNA can be inserted into the in abnormal function), amplification (resulting in abnor- cloning vector in either orientation. If screening is de- mal levels of expression), rearrangement (resulting in a pendent on the production of a functional protein, one- new function), or promoter mutation (again resulting in half of the library will be useless, as those cDNA inserted abnormal levels of expression). Most or all proto- in an inverse orientation will not produce functional pro- oncogenes are involved in normal cellular processes such tein. Directional cloning is dependent on producing sticky as growth factor signal transduction, mitogenic signal- ends that differ on the 5' and 3' termini. The cloning vec- ing, or regulation of DNA transcription or cellular pro- tor has the appropriate pair of complementary cloning liferation. The nomenclature convention is to indicate the cellular version of the proto-oncogene as “c-onc” andthe viral version, which is transforming, as “v-onc.” Most Library screening Three major methods are available
altered proto-oncogenes act in a dominant genetic fash- to obtain cDNA of interest. The classic technique uti- ion. Anti-oncogenes, or tumor suppressor genes, usually lizes DNA probes (such as oligonucleotides or intact act in a recessive genetic fashion and function to slow cDNA from a homologous gene) to screen cDNA librar- processes involved in cellular proliferation. Most of the ies. An oligonucleotide probe is usually derived from a identified anti-oncogenes have been involved in gene tran- reverse translation of known protein sequence. By ex- scription, presumably acting to enhanced differentiation pressing cDNA as a fusion protein with ␤ galactosidase, programs over those of proliferation.
various antisera can be used to screen for fusion proteinsencoded by the cDNA of interest. Finally, cDNA librar- c-abl This gene, present on human chromosome 9, en-
ies may be constructed in cloning vectors that allow for codes a tyrosine kinase whose role in normal hemato- expression of the cDNA insert in E. coli or a mammalian poiesis is unclear; however, its fusion to the BCR gene cell host. If a highly sensitive assay for the desired on human chromosome 22, the functional counterpart of protein’s function can be developed, pools of cDNA the Ph1 chromosome strongly associated with the dis- clones can be expressed and then assayed together; a ease chronic myelogenous leukemia, eliminates the first positive assay from a pool would allow one to subdivide two or three exons of c-abl and results in unregulated into smaller pools and eventually at clonal density.
tyrosine kinase activity. The resultant fusion protein iseither 210 kDa or 195 kDa. The latter version is more acutely transforming in experimental settings; it is also IRF-1 (interferon regulatory factor-1) IRF-1 is a tran-
associated with acute lymphoblastic leukemia and with a scription factor that activates the expression of IFN ␣ worse prognosis in both disease settings. One of the ways and ␤ and maps to chromosome 5q31.1. As it is thought in which the unregulated kinase activity may be manifest to act as a tumor suppressor gene, its role in the patho- is through phosphorylation of SHC and/or GRB-2, logic consequences of the 5q- syndrome is under active adopter proteins necessary for coupling growth factor IRF-2 (interferon regulatory factor-2) Interferon regu-
c-jun This proto-oncogene encodes a ~45 kDa transcrip-
latory factor-2 is a gene which binds to a promoter ele- tion factor that is a member of AP1 family of transcrip- ment shared by IFN ␣ and ␤ and many IFN-inducible tional proteins. c-jun must form dimers to function and genes; unlike IRF-1, which stimulates such genes, IRF-2 does so through the leucine zipper motif. Although c- represses transcription at the site. It is felt that the ratio jun-c-jun homodimers do form, they do so with low af- of IRF-1 to IRF-2 might be a critical event in the regula- finity and are not thought to be critical in gene transcrip- tion. Rather, a second partner, usually c-fos, generatesthe transcriptionally active heterodimer.
Rb The prototypical tumor suppressor gene Rb behaves
in a genetically recessive fashion. Elimination or inacti-
c-fos This ~62 kDa leucine zipper protein cannot homo-
vation of both Rb gene copies is required for manifesta- dimerize but rather functions in heterodimeric complex tion of the tumorgenic phenotype, first recognized in chil- with c-jun and other members of the AP1 family of tran- dren with retinoblastoma. Such children inherit only a single functional copy; subsequent mutagenic inactiva-tion of the remaining allele results in tumor susceptibil- c-myc This proto-oncogene plays a critical role in he-
ity. Rb acts to sequester a group of transcription factors, matopoietic cell proliferation. Like the leucine zipper termed E2F, which regulate genes critical for DNA syn- protein, it too functions as a heterodimer. One of its part- thesis. Alterations of Rb alleles are found in approximately ners is termed Max. The myc-related protein, Mad, also dimerizes with Max; the myc/Max complex stimulatesproliferation, the Mad/Max complex inhibits myc-func- SCL This proto-oncogene, first identified in a stem cell
tion. The importance of dysregulated myc function can leukemia at the site of t(1;14), is a member of the helix- be seen in Burkitt lymphoma in which a t(8;14) brings loop-helix group of transcriptionally active proteins. The myc, on chromosome 8, into juxtaposition with the im- gene, also termed Tal 1, is expressed in erythroid and munoglobulin enhancer on chromosome 14. Such mast cell lineages but not in T cells. The association of upregulation of myc in a B lymphocyte setting results in t(1;14) with up to 25% of T cell ALL suggests that its a proliferative advantage and represents one important ectopic expression is associated with transformation.
step in the genesis of this lymphoma. Myc has both leu-cine zipper and helix-loop-helix domains.
Bcl-1 This gene, located on chromosome 11 q13, was
first identified at the site of translocation
c-myb This gene encodes a transcription factor not be-
p(11;14)(q13;q32), has a strong association with central longing to any other class previously described and is acinar/mantle cell lymphoma and functions in normal expressed primarily in immature hematopoietic cells and cells as the G cyclin termed CCND1 or cyclin D . Nor- declines as cells differentiate. Forced expression of c- mally, lymphocytes lack cyclin D1 expression; its aber- myb tends to block hematopoietic differentiation. Clini- rant expression resulting from chromosomal transloca- cally, high levels of myb are noted in acute leukemia, and tion of the Bcl-1 locus to an immunoglobulin locus is such patients are less likely to enter remission or tend to thought to be associated with aberrant proliferation.
Bcl-2 This gene product normally functions to suppress
c-rel This gene belongs to the NF-␬B family of transcrip-
programmed cell death (apoptosis). Its overexpression is tion factors and can act to enhance or repress transcrip- associated with the most common molecular abnormal- tion from selected genes. This family of proteins includes ity in non-Hodgkin’s lymphoma, t(14;18)(q32;q21), p50 and its precursor p105, p65, p49 and its precursor present in 80% of follicular small cleaved cell lymphoma.
p100, and Bcl-3, one of the I␬B family.
Presumably, suppression of apoptosis leads to extendedcell survival, a characteristic of low-grade lymphomas.
Bcl-3 This gene is a member of the I␬B family. Pres-
not contain DNA-binding domains, they are thought to ently, it is unclear how this protein acts in tumorigenesis, be involved in protein-protein interactions. Neither but it is likely that its involvement in transcriptional pro- Rhomb 2 or 1 are normally expressed in T cells; trans- formation involving these genes, like SCL or Hox 11 isthought to be due to ectopic expression of the protein in Bcl-6 A zinc finger transcription factor, expression of
which is altered in approximately one-third of diffuse Blarge cell lymphomas as a consequence of 3q27 translo- ALK (anaplastic lymphoma kinase) A large propor-
cations. Its target genes are unknown.
tion of Ki-1 positive lymphomas are characterized by at(2;5). The breakpoint involves nucleophosmin, a ubiq- RAR (retinoic acid receptor) The retinoic acid receptor
uitously expressed gene, and ALK. The chimeric mRNA is a member of the steroid hormone group of transcrip- and protein are thought to be responsible for transforma- tionally active proteins and contains a steroid hormone- tion. ALK is a member of the insulin receptor family of binding domain, a zinc finger DNA-binding domain, and transmembrane receptor kinases, which is not normally a transcriptional activation domain. RAR is located at expressed in hematopoietic tissues; the fusion gene is no the t(15;17) present in the majority of cases of acute longer membrane bound, which may underlie its patho- promyelocytic leukemia. Its fusion partner in the trans- location is termed pml. Normally, RAR formsheterodimers with members of the RXR family of tran- 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 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 ETO Located on chromosome 8, ETO is involved in
suppression of cell growth, consistent with our under- t(8:21) of AML type M2. Based on the presence of two standing of the mechanisms of anti-oncogenes. Mutations zinc-finger motifs. ETO possibly encodes a transcription of p53 may not only inactivate its growth-suppression factor, but its role in the pathogenesis of AML is unknown.
function, but can actually generate a genetically domi-nant, functional oncogene. Human tumors associated with AML-1 Located on chromosome 21, AML-1 is the fu-
p53 mutations include those of hematopoietic tissues (e.g., sion partner of ETO in t(8:21). The gene is homologous 20% of myelomas), bladder, liver, brain, breast, lung, and to the runt gene of Drosophila and encodes a transcrip- colon. It is likely the most frequently mutated gene in tion factor. Normal hemopoietic targets include the CD13, GM-CSF, MPO, IL-3, and the T cell antigen receptorpromoters. AML-1 binds as a heterodimer, partnered with ras This gene encodes a critical signalling intermediate
CBF␤. It is unclear if its mechanism of action is to en- involved in the response to multiple growth factors. There hance aberrant transcription or to blunt transcription by are several related proteins (Ha-ras, Ki-ras, N-ras). N- acting in a dominant negative fashion.
ras and K-ras are mutated in many cancers, including45% of myelomas and > 50% of CMML cases. Consti- CBF␤ Located on chromosome 16, CBF␤ is one of the
tutive activation of ras can mimic chronic stimulation by fusion partners in the inv(16) associated with AML type the corresponding lineage-specific growth factor.
M4Eo. As with AML-1, it is unclear whether the alteredtranscription factor enhances or blocks transcription.
Hox 11 A homeobox containing transcription factor dis-
rupted by translocation to the T cell receptor locus
MLL Located on chromosome 11, MLL (mixed lineage
[t(10;14)] in 10% of cases of T cell ALL/lymphoblastic leukemia) is frequently altered in ALL, 1oAML, and es- lymphoma. The Hox 11 gene is critical to the develop- pecially in AML secondary to the use of topoisomerase ment of the spleen but its role in hematopoiesis is un- 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.
Rhomb 2 Like Hox 11, Rhomb 2 is translocated in T
cell ALL/lymphoma associated with t(11;14). Rhomb 1
Tel A helix-loop-helix transcription factor fused to the
may play a similar role in additional cases of T cell ALL.
PDGF␤-receptor in CMML t(5:12) and to other genes in The Rhomb gene products are members of a family of AML or MDS. Like most other translocation oncogenes, transcription factors, but as Rhomb 2 and Rhomb 1 do the mechanism of leukemogenesis is unknown. More recently, a Tel/AML1 fusion gene representing a t(12;21) pared to that of a similar assessment of both parents. The has been found in a large number of cases of childhood presence of multiple family members allows a complete ALL. As the translocation is not detected by routine cy- genetic pedigree to be constructed. For example, globin togenetics, molecular analysis (FISH, etc.) is required to gene mutations such as sickle hemoglobin can be ana- identify this favorable chromosomal rearrangement.
lyzed. The ␤6 mutation in hemoglobin, which results inHgb S, destroys an Mst II site. Therefore, a larger than DEK Located on chromosome 6, DEK is involved in
normal DNA fragment is generated by digestion of ge- t(6:9) of AML. This translocation is usually seen in young nomic DNA with Mst II, which can be easily detected by patients and carries a poor prognosis. Its normal func- Southern blot hybridization. In this specific case, the Mst tion is unknown, but DEK localizes to the nucleus.
II polymorphism is absolutely specific for the mutant geneand family studies are not necessary. If the RFLP had not CAN Located on chromosome 9, CAN is part of t(6:9).
been specific for the mutation, but only existed close to CAN forms part of the nuclear pore. As it has two differ- the specific disease-producing mutation, then family stud- ent fusion partners but a consistent phenotype, CAN is ies would have been required to determine which pattern likely the critical component of t(6:9).
(presence or absence of restriction site) tracks with themutant (disease) allele.
Fas (CD95 or Apo-1) A transmembrane glycoprotein
expressed on a wide variety of primitive and mature he-
Allele-specific hybridization If the nucleotide basis for
matopoietic cells, which, upon binding to its natural ligand 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 NF-1 The gene responsible for neurofibromatosis. The
genomic DNA. The pattern of hybridization thus gives spe- normal protein functions to negatively regulate ras cific information regarding which alleles are present. In a proteins, key intermediates in cytokine-induced polymorphic disease such as ␤ thalassemia (in which mul- tiple mutations can give rise to the same disease pheno-type), multiple probes might be required to detect all pos- XI. GENETIC SCREENING
sible causes. In addition, new mutations causing the samedisease would be missed. However, should a specific probe X-linked methylation patterns Several loci present on the prove useful for one population group or be positive in one X chromosome become highly methylated when inac- family member, that probe becomes very useful for the in- tive but remain unmethylated on the active X chromo- some (Lyon hypothesis). Should a polymorphic site for amethylation-sensitive restriction endonuclease exist at Reverse allele-specific hybridization This automated
such an X-linked locus, one can distinguish between the variant of allele-specific hybridization couples unlabeled active and inactive X chromosome by the pattern of re- synthetic oligonucleotides specific for a wild type or striction endonuclease digestion of that gene. However, mutant sequence to a solid support that is then allowed in order to be widely useful for determining clonality of to bind genomic sequences of the locus of interest, which hematopoietic cells, the allelic frequency must be close have been amplified by PCR. The use of highly stringent to equality. Several X-linked genes meet these criteria conditions of hybridization allows differential binding of and include phosphoglycerate kinase (PGK), hypoxan- the amplified DNA to the wild type or mutant specific thine phosphoribosyltransferase (HPRT), the human an- oligonucleotide and thereby allows genotypic determi- drogen receptor gene (HUMARA), and the hyper-vari- able DXS255 locus. Both Southern blotting and PCRmethods can be applied to this type of analysis.
Competitive oligonucleotide hybridization Mutant or
wild type-specific oligonucleotide primers are used in a
RFLP (restriction fragment length polymorphism) If
PCR reaction with genomic DNA. The primers and strin- a mutation of one allele of a genetic locus either gener- gency of PCR are chosen so that single-based mismatches ates or destroys a restriction endonuclease site, the het- between genomic DNA and PCR primer fail to yield an erogeneity present within or very close to a gene of inter- amplified product. Thus, the PCR detection of a locus- est can be used to track which allele an individual has specific product allows the genotyping of the individual.
inherited from each parent. When genomic DNA is di-gested with a restriction enzyme that recognizes a poly- Color complementation assay This method is an ad-
morphic site and then hybridized with a probe specific vancement over competitive oligonucleotide hybridiza- for the gene of interest, the allelic pattern can be com- tion in that the wild type and mutation specific PCR prim- ers are labeled with different color fluorescent tags, andboth 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 whetherwild type, mutant, or both specific alleles were presentin the original DNA sample.



Tips from Toni Hasler, Natascha Badmann’s Red Bull is definitely more than just a party drink. The original energy drink also has a lot to offer for athletes. What’s in it, how does it work and how can it be used effectively for sports? Toni Hasler, trainer and nutritional counsellor, provides the answers. There is no doubt for Toni Hasler: suppliers. Cons

Y bont: 2004: january: pdf (english)

As we enter a New Year we hope you all had a good Christmas and have recovered from the New Year celebrations. Our last edition caused us some problems and only through the persistence of A5 Publishing did we succeed in producing edi-tion three. Because of these difficulties the photograph with the caption ‘Who do you recognise?’ might have resulted in the answer being ‘Nobody’, due to

Copyright © 2010-2014 Pharmacy Pills Pdf