Dictionary of DNA and Genome Technology

may be fused with a partner gene (= carrier gene) encoding a readily detectable product. The partner gene might encode, for example, glutathione S-transferase (GST) – in which case the fusion protein (with a GST affinity tail) can be isolated by using AFfiNITY CHROMATOGRAPHY with glutathione in the stationary phase. A target protein from a thermophilic organism might be isolated e.g. by fusion with a temperature-stable lectin-based affinity tail [BioTechniques (2006) 41(3):327– 332].

(See also flAG and PESC VECTORS.)

• To determine the intracellular location of a given protein using a reporter system such as GFP (GREEN flUORESCENT PROTEIN) as a fusion partner. Fusion with GFP permits the (intracellular) target protein to be detected by fluorescence microscopy. In one early study, the gene for FtsZ protein (see Z RING) was fused to that of GFP, enabling a remarkable photographic image of the Z ring of Escherichia coli to be obtained [Proc Natl Acad Sci USA (1996) 93:12998–13003].

The use of GFP (or other fluorescent protein partners such

as MONOMERIC RED flUORESCENT PROTEIN) places limitat-

ions on this technique in terms of excitation/emission wavelengths. In an alternative approach, a fusion protein consists of the protein of interest fused with O6-alkylguanine-DNA alkyltransferase (AGT). The advantage here is that AGT can be labeled in vivo, i.e. within living cells, by any of a range of fluorophores (derivatives of O6-benzylguanine) – of which many are cell-permeable; thus, an AGT fusion protein which is expressed intracellularly can be labeled by incubating the cells in a medium that contains one of the cell-permeable fluorophores. (O6-benzylguanine derivatives that are not cellpermeable may be internalized by microinjection.) Labeling of AGT fusion proteins with an O6-benzylguanine derivative involves a covalent bond. The AGT approach permits flexibility because the O6-benzylguanine derivatives are available with a wide range of emission spectra (from 472 nm to 673 nm) [BioTechniques (2006) 41(2):167–175].

AGT is a normal (eukaryotic) DNA repair enzyme which transfers alkyl groups from a guanine residue to a cysteine residue on the enzyme (thereby inactivating the enzyme). The (unwanted) labeling of endogenous molecules of AGT within the cells under test has been regarded as a disadvantage of the AGT technique, although such labeling apparently does not occur to any significant degree in at least some mammalian cell lines. Moreover, use can be made of a mutant form of AGT (MAGT); this mutant form of AGT can be labeled in the presence of N9-cyclopentyl-O6-(4-bromo-thenyl)guanine – a compound which blocks labeling of wild-type AGT. Another advantage of MAGT is that its affinity for alkylated DNA is lower than that of the wild-type protein; thus, it is less likely (than the wild-type AGT) to be inactivated by functioning as a repair enzyme.

•To increase the solubility of a target protein: see, for exam-

ple, CHAMPION PET SUMO VECTOR.

•To obtain a gene product which is normally not highly expressed. The relevant (target) gene may be fused downstream

of a highly expressed gene in an expression vector; given an appropriate choice, the partner gene may contribute increased solubility/stability to the fusion product.

•To modify the mobility of the target protein. For example, the fusion partner VP22 confers on a target protein the ability to translocate from a given mammalian cell to an adjacent cell in a cell culture (see VOYAGER VECTORS).

•To engineer the secretion of a given protein that normally remains intracellular. Fusion with a gene whose product is normally secreted may enable secretion of the target protein as part of the fusion product.

•To study the regulation of gene expression. For example, if there are problems in monitoring the expression of a given gene (perhaps due to difficulty in assaying the gene product), it may be possible to monitor activity of the gene’s promoter (rather than the whole gene). For this purpose, a promoterless reporter gene (encoding a readily detectable product) can be fused downstream of the target gene’s promoter; the promoter’s activity can then be monitored via the product of the reporter gene. Examples of reporter genes include those

encoding GFP, galactokinase and chloramphenicol acetyltransferase. Expression of the lacZ gene (encoding β-galacto- sidase) in a lacZ fusion product can be monitored on media containing X-GAL.

The use of reporter genes in this way generally works well. However, an early report [J Bacteriol (1994) 176:2128–2132] indicated that certain reporter genes are able to influence the activity of certain promoters; this serves as a useful reminder that unpredictable interactions between components may take place in recombinant DNA systems.

•To detect/identify specific organisms. In a novel approach, GFP was fused to the coat protein of a strain-specific phage and used for the rapid detection of the pathogenic bacterium Escherichia coli strain O157:H7 [Appl Environ Microbiol (2004) 70:527–534].

•See also ENZYME FRAGMENT COMPLEMENTATION.

Cleavage of fusion proteins

It may be possible to obtain a separate product from each of the two fusion genes by cleavage of the fusion protein. Given an appropriate and accessible sequence of amino acids at the junction region, cleavage may be achieved by a site-specific protease (see e.g. GST in GENE FUSION VECTOR).

(See also CHAMPION PET SUMO VECTOR and TEV protease in the entry TEV.)

Cleavage may also be carried out with chemical agents, e.g. hydroxylamine (which cleaves between adjacent residues of glycine and asparagine) or cyanogen bromide (which cleaves at methionine residues); the problems with chemical cleavage include unwanted modification (by the agent) and cleavage at sites within the target protein.

gene fusion vector An EXPRESSION VECTOR that includes a sequence encoding a partner gene – such as glutathione S- transferase (GST); the sequence from a target gene is inserted in the correct reading frame, adjacent to the partner gene, and both genes are jointly transcribed and translated as a single

product (a so-called fusion protein). The fusion protein may be cleavable to separate products; for example, a GST partner has a recognition site for a site-specific protease close to the fusion boundary.

gene gun A device used e.g. for the intraepidermal inoculation of a DNA VACCINE. The DNA is coated onto m-scale gold particles which are internalized via a pressure-based system. [Example of use (in mice): Infect Immun (2005) 73(5):2974– 2985.]

gene knock-out Syn. KNOCK-OUT MUTATION.

gene product (gp) The (final) product formed by the expression of a given gene: either a protein (formed via the mRNA intermediate) or an RNA molecule which is not translated – e.g. a molecule of rRNA or tRNA.

Specific gene products, i.e. products formed by the expression of specific genes, may be designated e.g. as gp32, gp120 etc., the numbers reflecting particular genes in a given organism.

gene shuffling See DNA SHUFflING.

gene silencing A term applied to any (natural or in vitro) process that tends to block gene expression (see, for example,

EXPONENTIAL SILENCING, MICRORNA, MORPHOLINO ANTI- SENSE OLIGOMER, PTGS, RIBOSWITCH, RNA INTERFERENCE).

(See also transcriptional gene silencing in the entry SIRNA.) In many eukaryotes (including plants), methylation of DNA sequences may promote the recruitment of specific binding proteins which – either by themselves or as part of a complex – recruit other agents (such as histone deacetylases), res-

ulting in a localized region of repressed chromatin.

DNA methylation is involved in gene silencing in various contexts, including e.g. imprinting, differentiation and tumor development

(See also DEMETHYLATION.)

gene targeting A procedure used to produce precise in vivo changes in genomic DNA at predetermined sites. Typically, this has been achieved by transfecting cells with a vector, e.g. a plasmid, containing an insert (and a selectable marker) that is flanked by sequences homologous to the targeted genomic DNA; replacement of a given sequence of genomic DNA by the insert relies on the (relatively rare) occurrence of homologous recombination.

Gene targeting can be used e.g. to inactivate a specific gene (a so-called knock-out mutation) or to modify the expression of a given gene e.g. by replacing part(s) of the gene’s coding sequence. In the context of therapy, gene targeting may be used to attempt to correct a mis-functioning gene.

The efficiency of gene targeting has been improved by the development of certain phage-encoded recombinase systems

(see e.g. RECOMBINEERING).

(cf. GENE TRAPPING; see also TARGETED TRAPPING.) (See also flP-IN CELL LINES.)

gene therapy Any form of therapy that involves genetic modification of (particular) cells with the object of correcting a genetic disorder or of treating certain types of infectious disease. Public awareness of gene therapy focuses primarily on

the treatment of inherited disorders (such as CYSTIC fiBROSIS

and ADENOSINE DEAMINASE DEfiCIENCY) but much work

has also been carried out on infectious diseases, such as those caused by hepatitis, influenza and human immunodeficiency viruses and by Mycobacterium tuberculosis.

Two basic approaches in gene therapy are: (i) the ex vivo mode, in which cells are removed from the body, genetically modified in vitro, and then returned to the body; (ii) the in vivo mode, in which genetic modification is carried out in situ, the modifying agent being internalized.

Inherited disease involving a single malfunctioning gene may be remediable by installing the normal (i.e. functional, wild-type) gene in order to counteract the specific deficiency. For example, successful therapy for ADENOSINE DEAMINASE DEfiCIENCY has been achieved by inserting the normal gene into STEM CELLS; in this case, adenosine deaminase is formed in the T cells derived from those stem cells. Stem cells suitable for gene therapy have been isolated e.g. by exploiting the CD34+ marker in IMMUNOMAGNETIC ENRICH- MENT techniques. Following in vitro modification, the target stem cells are isolated by appropriate selective procedures; selection that is not based on drug-resistance can be achieved e.g. with reporter genes encoding different fluorophores (i.e. fluorophores with different emission spectra) instead of drugresistance genes, the target cells being detected and separated by FACS (fluorescence-activated cell sorter) [Proc Natl Acad Sci USA (2005) 102(45):16357–16361].

Vectors in gene therapy

Vectors (i.e. vehicles used for inserting genetic material into cells) include certain types of virus which have been made replication-deficient and from which pathogenic potential has been eliminated. A gene which is to be inserted into cells (a transgene) is incorporated into the viral genome and enters a cell during viral infection of that cell.

Viral vectors include certain lentiviruses (a class of RETRO- VIRUSES), adenoviruses and adeno-associated viruses (the socalled AAVs); these viruses can infect both dividing and nondividing cells.

AAVs are able to carry small sequences (inserts), while the adenoviruses can carry larger sequences.

(See also ADEASY XL ADENOVIRAL VECTOR SYSTEM.)

AAVs have been used e.g. for introducing genes into mice, by intracranial inoculation, for studying GM2 gangliosidoses [Proc Natl Acad Sci USA (2006) 103 (27):10373–10378].

Some viral vectors infect many types of cell. This can be a disadvantage because, to deliver a transgene to a specific type of cell, it may be necessary to administer the vector in high doses in order to allow for wastage (i.e. the take-up by other types of cell); the use of high doses of the given vector may increase the risk of adverse reactions. For other viral vectors the range of host cells may be too narrow. In either case, the problem may be solved by PSEUDOTYPING (q.v.).

Virus-mediated transfer of genes into cells can be enhanced by certain peptides and also by certain types of polycationic compound. From in vitro studies on the effect of peptides and

polycationic agents on the transfer of genes from lentiviral and adenoviral vectors into human tumor cells, it was concluded that protamine sulfate may be the most suitable agent for clinical use [BioTechniques (2006) 40(5):573–576].

Strains of the Gram-positive bacterium Lactococcus lactis were reported to be able to deliver DNA into mammalian epithelial cells [Appl Environ Microbiol (2006) 72(11):7091– 7097], suggesting a possible use in this context. (So far, the use of bacteria as gene delivery vectors has been considered less frequently than viral vectors.)

Certain modified phages (see BACTERIOPHAGE) have been reported to act as vectors for gene delivery to mammalian cells, and a similar role has been reported for PHAGEMID particles. To fulfill this role, the coat proteins of phages or phagemid particles are modified to display certain targeting ligands which can facilitate uptake by eukaryotic cells. In the case of phagemid particles, coat proteins can be encoded by the helper phage so that more of the phagemid’s sequence can be used for the gene of interest; phagemid particles with a specific ligand have been prepared by using a coat-modified strain of phage M13 as helper phage [BioTechniques (2005) 39(4):493–497]. For phage or phagemid vectors, low levels of transgene expression in the recipient cells may reflect e.g. factors such as the need to convert ssDNA to dsDNA and the translocation of nucleic acid into the nucleus.

In cystic fibrosis, gene therapy is aimed at epithelial cells of the respiratory tract. So far, benefit has been achieved for only a limited period of time. Because extended benefit needs repeated dosing, this tends to exclude the use of viral vectors because repeat dosing with viral vectors can compromise the treatment as a result of a developing immune response to the vector. In this context, a novel, non-primate (caprine) adenoassociated virus (see AAVS) may be useful because it shows high resistance to neutralization by human antibodies and has a marked tropism for (murine) lung tissue [J Virol (2005) 79: 15238–15245].

For cystic fibrosis, attention has been focused on increasing the effectiveness of liposome-mediated delivery of the transgene, e.g. by facilitating transgene internalization, and escape from the endosome, as well as import into the nucleus [see e.g. Bioch J (2005) 387(1):1–15].

Gene therapy against infectious diseases

With infectious diseases, the possibilities for intervention by gene therapy are greater owing to the nature of infection and the diversity of infectious agents. Some approaches/ideas:

•Antisense oligonucleotides which bind to the pathogen’s mRNAs, blocking transcription (see e.g. VITRAVENE).

•Suicide genes whose expression – leading to cell death – occurs only in infected cells.

•RIBOZYMES targeting specific sequence(s) in an infected cell. A single vector may encode many different ribozymes. The ribozyme’s double requirement for correct binding and cleavage sites offers good specificity. The catalytic activity allows ongoing destruction of targets. To be useful, though, a ribozyme must be stabilized against intracellular degradation

by RNase. Useful results were reported in a simian immunodeficiency virus (SIV) model using an anti-SIV pol ribozyme [J Gen Virol (2004) 85:1489–1496].

[Catalytic nucleic acid enzymes for study and development of therapies in the central nervous system: Gene Ther Mol Biol (2005) 9A:89–106.]

• RNA INTERFERENCE (q.v.) in vitro can inhibit replication of e.g. HIV-1 and the viruses that cause hepatitis, influenza and poliomyelitis. Moreover, siRNA-mediated downregulation of a cell’s co-receptor for HIV can block infection of cells by HIV.

Synthetic siRNAs can be introduced into mammalian cells directly by the use of appropriate systems for internalization; alternatively, expression cassettes encoding siRNAs for intracellular expression can be transfected by an appropriate gene transfer vector.

(See also SHORT HAIRPIN RNA.)

A novel form of delivery of siRNA to mammalian cells has been carried out with an siRNA conjugated to an aptamer via a streptavidin bridge (the aptamer having been previously demonstrated to bind to prostate tumor cells); the conjugate (i.e. siRNA + aptamer) was internalized within 30 minutes of addition to the cells, and the siRNA was found to produce an efficient inhibition of gene expression [Nucleic Acids Res (2006) 34(10):e73].

Problems with RNAi in gene therapy

(a)Mutation in the target sequences can permit escape from RNAi, a consequence of the high-level specificity of RNAi cutting [e.g. escape of HIV-1 from RNAi: J Virol (2004) 78: 2601–2605]. One study indicated that RNAi can be effective against highly conserved sequences but was inhibited when mutations occur in the central, target-cleavage region [Blood (2005) 106(3):818–826].

[Computational design of RNAi strategies that resist escape of HIV: J Virol (2005) 79(3):1645–1654.]

(b)Non-specific (‘off-target’) modification of gene expression can occur, especially under high-dose conditions.

(c)There is a need for a highly efficient delivery system to avoid a residue of unprotected cells – in which target viruses

can replicate and mutate to RNAi-insensitivity.

• Intrabodies (= single-chain antibodies): molecules, encod-

ed by engineered antibody genes, which remain intracellular and which have antigen-binding properties.

[The de novo production of intracellular antibody libraries: Nucleic Acids Res (2003) 31(5):e23.]

An intrabody without disulfide bonds was found to perform better during in vitro studies on (non-infectious) Huntington’s disease [Proc Natl Acad Sci USA (2005) 101 (51):17616– 17621].

An intrabody has been used against vIL-6, a form of IL-6 (interleukin-6) encoded by human herpesvirus-8 (HHV-8) and associated with pathogenesis in certain types of disease, e.g. Kaposi’s sarcoma [J Virol (2006) 80(17):8510–8520].

• Transdominant negative proteins (TNPs): modified, inhibitory forms of a pathogen’s normal (regulatory or structural)

proteins; TNPs may block activity of wild-type proteins e.g. by forming inactive complexes or by competing for specific binding sites.

Negative immune reactions against cells containing TNPs must be avoided.

A transdominant negative version of the human immunodeficiency virus-1 (HIV-1) regulatory REV PROTEIN (which e.g. mediates the export of certain viral transcripts from the nucleus) was found to inhibit replication of HIV-1 in vitro.

• DNA VACCINES have shown promise in animal models. In some early studies a DNA vaccine encoding the 65-kDa heatshock protein of Mycobacterium tuberculosis (Hsp65) was able to act therapeutically as well as prophylactically in mice; a plasmid encoding interleukin-12 (IL-12) gave even better results.

DNA vaccines are being developed against e.g. papillomavirus type 16 (a virus associated with human cervical cancer) [J Virol (2004) 78(16):8468–8476]; the protozoa Leishmania donovani [Infect Immun (2005) 73(2):812–819] and Plasmodium falciparum [Infect Immun (2004) 72(1):253–259]; and Mycobacterium tuberculosis [Infect Immun (2005) 73 (9):5666–5674].

Genetic immunization (in animals) by intravenous delivery of antibody-specifying plasmid DNA was described as an efficient method for inducing antigen-specific antibodies in laboratory animals [BioTechniques (2006) 40(2):199–208].

gene transfer agent See CAPSDUCTION. gene-trap vector See GENE TRAPPING.

gene trapping A procedure which is used e.g. for introducing random insertional mutations into cultured mouse embryonic stem cells (ES cells); the inserts used for mutagenesis include a selectable marker or reporter which also acts as a ‘tag’ (i.e. a known sequence of nucleotides) so that a disrupted gene can be readily identified and investigated.

ES cells which have been genetically modified in this way can be injected into an (isolated) blastocyst which is then reimplanted in a mouse and allowed to develop. This procedure is therefore a valuable method for studying gene function, including studies on genetic modification of the germ line.

The insertional mutations are introduced by transfecting ES cells with a gene-trap vector that integrates randomly in the genome. Two common types of vector are described.

One type of gene-trap vector (used in ‘promoter trapping’) contains a promoter-less selectable sequence which is flanked on one side by an upstream splice acceptor sequence and on the other side by a polyadenylation signal; expression of the vector depends on chance integration into a host gene, the host gene supplying a promoter function. Transcription of the integrated vector produces an mRNA in which the poly(A) tail is transcribed from the vector’s sequence. Note that genes which are transcriptionally silent in the target cell will fail to express the vector.

The second type of gene-trap vector contains a promotercompetent selectable sequence which lacks a polyadenylation signal but which includes a downstream splice donor site; the

downstream splice donor site allows exploitation of the host’s polyadenylation signal.

mRNA formed by transcription of either type of integrated vector can be used to determine the site of integration of the vector.

(See also TARGETED TRAPPING and GENE TARGETING.) GeneChip® See MICROARRAY.

GeneMorph™ PCR mutagenesis kit A product (Stratagene, La Jolla CA) designed to generate random mutations during PCR-based amplification; the product includes an error-prone DNA polymerase (Mutazyme™) that is reported to generate from one to seven mutant bases per kilobase in a given PCR reaction.

Examples of use of GeneMorph: generation of temperaturesensitive mutants [Mol Biol Cell (2006) 17(4):1768–1778]; studies on the transcriptional regulator Mga of Streptococcus pyogenes [J Bacteriol (2006) 188(3):863–873].

(See also MUTAGENESIS.)

generalized transduction See TRANSDUCTION. GeneTailor™ site-directed mutagenesis system A system

(Invitrogen, Carlsbad CA) used for introducing specific point mutations, deletions, or insertions of up to 21 nucleotides in length. DNA of up to 8 kb, from any bacterial source, can be mutagenized.

(See also SITE-DIRECTED MUTAGENESIS.)

Initially, methylation of the (circular) molecule of DNA is carried out with a DNA methylase and S-adenosylmethionine as methyl donor.

A point mutation can be introduced by synthesizing copies of the DNA with a pair of primers which bind to overlapping sites, one of the primers incorporating the required mutation. Progeny strands anneal to form (nicked) circular molecules which are inserted into an appropriate strain of Escherichia coli, by transformation, for replication; the mcrBC+ strain of E. coli digests the (methylated) parental DNA (see the entry

MCRBC).

The GeneTailor™ system has been used e.g. in studies on the fatty-acid-binding protein aP2 [Proc Natl Acad Sci USA (2006) 103(18):6970–6975] and in studies on HIV-1 [J Virol (2006) 80(7):3617–3623].

genetic code The form of the information which enables a sequence of nucleotides in DNA (or, in some viruses, RNA) to direct the synthesis of a polypeptide – such direction usually involving the relay of information to an intermediate effector molecule: messenger RNA (mRNA); consecutive triplets of nucleotides in the mRNA (see CODON) encode consecutive AMINO ACID residues in the polypeptide, while certain of the codons (see the table) specify the termination of synthesis.

(In certain viruses with a positive-sense ssRNA genome the genomic RNA itself is translated into polyproteins – without the need for relaying information to an intermediate effector molecule.)

(Compare synthesis of oligopeptides by NON-RIBOSOMAL

PEPTIDE SYNTHETASE.)

In that a given amino acid may be specified by more than

GENETIC CODEa–e

aA = adenine; C = cytosine; G = guanine; U = uracil.

bAmino acid symbols are explained in the entry AMINO ACID (table).

cCodons are conventionally written in the 5′-to-3′ direction. dThe ochre, opal and amber codons are normally stop signals. eSee text for some variations in the code.

one codon (see e.g. Leu in the table), the genetic code is said to be degenerate.

A given amino acid may be specified preferentially by a certain codon in one species but by another codon in different species. (See also CODON BIAS.)

Although widely applicable, the code shown in the table – often called the ‘universal’ genetic code – is not invariable. In the mitochondria of at least some plants, animals and fungi the codon UGA specifies tryptophan (rather than acting as a stop codon), while codons UAA and UAG specify glutamine in ciliate macronuclei.

Among the arthropods, AGG is reported to specify serine in certain species and either lysine or arginine in others – with some species not using the codon at all [PLoS Biol (2006) 4(5):e127; PLoS Biol (2006) 4(5):e175].

genetic counseling Advice given to e.g. parents regarding the possibility/probability of genetically based problems in their existing or future offspring, or, in general, to others regarding their susceptibility to particular genetically based problem(s).

genetic disease A term generally used to refer specifically to a heritable disease or disorder, i.e. one which is determined by an individual’s genotype.

Some examples of genetic diseases are listed in the table.

(See also ONLINE MENDELIAN INHERITANCE IN MAN.)

genetic disorder Syn. GENETIC DISEASE.

genetic engineering The manipulation of genetic material – DNA and/or RNA – with the object of bringing about any desired change or innovation, either in vitro or in vivo, as carried out during the study, or modification for any purpose, of genes or genetic systems.

Genetic engineering therefore includes, for example, those in vitro techniques involved in the study of genes and their regulation; various techniques used in GENE THERAPY; and the creation of novel strains of existing microorganisms for medical or industrial use.

genetic footprinting A technique that can be used to examine gene function – e.g. to determine whether the expression of specific genes is required for growth under given conditions; the method is used in eukaryotic and prokaryotic microorganisms, including Saccharomyces cerevisiae and various Gramnegative bacteria.

Note. This technique is completely unrelated to the method, used for studying DNA–protein interactions, that is referred

to as FOOTPRINTING.

Genetic footprinting involves three basic stages. One of the formats for genetic footprinting in bacteria is as follows. The first step involves random, transposon-mediated mutagenesis of a population of bacteria. This may be carried out e.g. by (i) transposon mutagenesis of DNA fragments in vitro followed by insertion of the mutagenized fragments into cells by transformation, or (ii) use of the transposome approach (see TN5- TYPE TRANSPOSITION) in which mutagenesis occurs in vivo. (The latter approach has certain advantages which include the possibility of limiting insertions to one per cell, by using an appropriate transposome–cell ratio, and the lack of a requirement for preparing a special transposon-delivery vector.) An aliquot of the (mutagenized) population is set aside for subsequent analysis; it is labeled T0 (for time zero).

The remainder of the mutagenized population is then grown for many generations under conditions in which further transposition is inhibited.

Finally, genomic DNA from (i) T0 cells, and (ii) cells from the cultured, mutagenized population is isolated, and nucleotide sequences from specific genes are determined, e.g. by a PCR-based approach.

If, under the given conditions of growth, a specific gene is essential for cell viability, then transposon insertions in that gene should be easily detectable in cells of the T0 population but not in cells of the cultured population.

[Example of genetic footprinting in the bacterial pathogen Pseudomonas aeruginosa strain PA14: Proc Natl Acad Sci USA (2006) 103(8):2833–2838.]

genetic immunization See GENE THERAPY (DNA vaccine). genetic imprinting (genomic imprinting) The phenomenon in

which particular genes are methylated, at specific loci, as a marker of their origin – i.e. from the male or female parent; only one allele of an imprinted gene (from the male or female parent) is expressed under given circumstances.

Errors in imprinting may cause serious disorders – e.g. the Prader–Willi syndrome (which is characterized by obesity, mental retardation and hypogonadism).

Imprinting is an epigenetic phenomenon, that is, it directs a heritable phenotype that is not dependent on the sequence of nucleotides in the given genes. It occurs during the formation of germ cells and persists in somatic cells.

genetic screening Any procedure used for detecting/selecting individual cells or organisms with specific genetic characteristic(s) from among a population of those cells or organisms.

In a clinical context: test(s) carried out on patients in order to detect the risk of possible or probable genotype-based disorders in the patients themselves or in their offspring.

genetically modified food (GM food) Any food (such as GM soya) which has been produced after specific and intentional alteration of the genome of a given type of plant or animal.

The construction of a GM organism is not necessarily the accelerated production of an organism that could be obtained by a naturally occurring process. This is because it is possible to modify genomes with sequence(s) of nucleotides designed on a computer – sequences that do not necessarily reflect any that occur, have ever occured, or may ever occur, in nature.

Geneticin® A commercial brand of G418 SULFATE (Invitrogen, Carlsbad CA).

genistein 4′,5,7-Trihydroxyisoflavone: a strong inhibitor of the tyrosine kinases; it acts as a competitive inhibitor of ATP at the binding site.

(cf. TYRPHOSTIN.)

(See also TYROSINE KINASE.)

genome (1) The genetic content of a cell or virus – in terms of either (a) the information (coding) content or (b) the physical content of nucleic acid (i.e. a cell’s DNA or the DNA or RNA of a virus).

In this sense, DNA in mitochondria (including kinetoplast DNA) and chloroplasts forms part of the genome.

In bacteria, which often contain plasmids, genome is sometimes used specifically to refer to chromosomal DNA; this use of the term may be justified in that plasmids are often dispensable to the cell, and in that cells of a given species may contain different assortments of (dissimilar) plasmids (which would indicate a ‘variable genome’ for the species). However, ‘genome’ is also used to include bacterial plasmids, particularly when cells of a given species commonly contain a stable complement of plasmid(s) – or the plasmid(s) have a particular relevance to the organism’s biological activity or pathogenicity; the term has been used in this way e.g. for Bacillus anthracis [J Bacteriol (2000) 182(10):2928–2936], for Pseudomonas syringae pv. tomato [Proc Natl Acad Sci USA (2003) 100(18):10181–10186] for Rhizobium leguminosarum [Genome Biol (2006) 7(4):R34] and for Rhodococcus RHA1 [Proc Natl Acad Sci USA (2006) 103:15582–15587].

(2)The haploid set of genes or chromosomes in a cell.

(3)The complete set of different genes in an organism.

(4)The term ‘genome’ has even been used in the context of viroids (i.e. non-coding, single-stranded molecules of RNA that infect plants) [BMC Microbiol (2006) 6:24].

Genome Deletion Project (for Saccharomyces cerevisiae) A project that systematically replaced each open reading frame (ORF) in the genome of the yeast Saccharomyces cerevisiae with a kanamycin-resistance cassette – creating a collection of strains in which a different ORF has been replaced in each strain. This collection of (GDP) strains provides a comprehensive source of knock-outs for S. cerevisiae, facilitating studies on gene function.

In addition to their intended application, these strains can also be used e.g. to copy any desired S. cerevisiae promoter; a copy of this promoter can then be inserted into a strain of interest and used for controlling the expression of a given gene. For this purpose, one first identifies the particular GDP strain in which the desired promoter is flanked by a kanamycin knock-out immediately on its 5′ side. This promoter, together with its contiguous kanamycin cassette, is amplified by PCR, using primers having 5′ extensions homologous to sites of insertion in the strain of interest. The PCR products can then be inserted into the genome of the given strain by homologous recombination, replacing the existing promoter [Method: BioTechniques (2006) 40:728–734.]

genome walking Syn. CHROMOSOME WALKING.

genomic cloning See CLONING.

genomic imprinting Syn. GENETIC IMPRINTING.

genomic island In a bacterial genome: a discrete region of the chromosome, acquired from an exogenous source, that differs in GC% (and codon usage) from the rest of the chromosome and is often located near tRNA genes; genomic islands are typically flanked by direct repeat sequences and may include any of various genes as well as mobile genetic elements.

Recombination may result in fragmentation of a genomic island with re-location of part(s) of the island to different regions of the chromosome.

Genomic islands are reported to be generated, for example, at the sites of insertion of transposon Tn7 [J Bacteriol (2007) 189(5):2170–2173].

A 54-kb genomic island in the (Gram-negative) bacterium Leptospira interrogans serovar Lai (containing 103 predicted coding sequences) is reported to excise from the chromosome and to exist as a (replicative) plasmid [Infect Immun (2007) 75(2):677–683].

genomic library See LIBRARY.

genomic masking See PHENOTYPIC MIXING.

genospecies Any group of (microbial) strains between which genetic exchange can occur.

genotoxin Any agent that damages nucleic acid. [Detection of genotoxic compounds: Appl Environ Microbiol (2005) 71(5): 2338–2346.]

GenoType MTBDR See LINE PROBE ASSAY.

gentamicin An AMINOGLYCOSIDE ANTIBIOTIC used e.g. as a

selective agent for bacteria containing a vector which carries a gentamicin-resistance gene.

germ line cells Cells whose development leads to the formation of gametes (reproductive cells).

GFP See GREEN flUORESCENT PROTEIN.

GFP reactivation technique An approach designed to facilitate (drug-mediated) analysis of DNA methylation (see entry DEMETHYLATION); the protocol published in BioTechniques [(2006) 41(4):461–466] uses the methylation-inhibitory drug 5-aza-2′-deoxycytidine, but this approach may also be useful for studying the effects of some other chromatin-modifying drugs (such as TRICHOSTATIN A).

The method improves our ability to identify genes that have been activated by drug treatment – such activation resulting from the removal of methylation-induced repression. In this method, cells in which drug-induced activation has occurred are selected by using a reporter gene whose activation, by the drug, indicates ‘global’ drug-induced de-repression of genes in these cells.

This method uses transgenic female mice with an X-linked reporter gene for GREEN flUORESCENT PROTEIN (GFP). The relevant transgene in this method is the one present on the inactivated X chromosome.

The GFP reactivation technique involves several stages of screening of a population of cells isolated from the transgenic mouse. The final stage of screening isolates those cells in the population which exhibit drug-induced activation of the GFP reporter gene present on the inactive X chromosome. All the screening is carried out with a flUORESCENCE-ACTIVATED

CELL SORTER (FACS).

Cells from the animal were first sorted by flow cytometry to select for those containing an inactive, non-expressed gene for GFP (see X-INACTIVATION). After treatment with 5-aza- 2′-deoxycytidine this population of cells was again sorted by flow cytometry to separate those cells containing active GFP from those containing inactive GFP – i.e. separating cells in which the reporter gene had been activated by the drug from those in which no activation had occurred.

Subsequent MICROARRAY-based gene expression profiling on the three populations of cells – i.e. (i) the GFP-inactive untreated cells, (ii) GFP-inactive drug-treated cells and (iii) GFP-active drug-treated cells – indicated the potential of this method for identifying genes that were activated by global demethylation as a result of drug treatment.

giant chromosome See POLYTENIZATION.

Gigapack® Any of a range of products (Stratagene, La Jolla CA) used for packaging certain types of lambda-based vector molecules (‘packaging’ referring to the process in which each (DNA) vector molecule is enclosed in phage components to form an infective phage particle). Gigapack® products are used e.g. with the LAMBDA fiX II VECTOR.

GISA Strains of MRSA with decreased susceptibility to glycopeptide antibiotics (e.g. vancomycin), the abbreviation referring to ‘glycopeptide intermediately susceptible S. aureus’.

Glanzmann’s thrombasthenia An autosomal recessive disease

that is characterized by a deficiency in platelet agglutination and linked to abnormalities of the αIIbβ3 INTEGRIN.

[Review of Glanzmann’s thrombasthenia: Orphanet J Rare Dis (2006) 1:10.]

glargine See INSULIN GLARGINE. Glucuron™ See CHEMILUMINESCENCE. glucose effect See CATABOLITE REPRESSION.

glutathione S-transferase See GENE FUSION (uses). glycogen storage disorder Any of various human diseases that

involve dysfunctional glycogen metabolism.

The type I disease is characterized by deficiency of glucose 6-phosphatase in the liver. In infants it involves severe hypoglycemia and an enlarged liver containing abnormally high levels of glycogen.

The type VIII form of glycogen storage disorder, which is generally less severe, is characterized by inadequate levels of the enzyme phosphorylase kinase.

(See also GENETIC DISEASE (table).)

glycosylation (of recombinant proteins) Post-translational modification of recombinant proteins, carried out in cells capable of adding glycosyl residue(s) to the appropriate amino acid residue(s). In many cases such glycosylation is an essential requirement for normal activity of the recombinant protein.

Some types of cell (e.g. bacteria) do not normally carry out the patterns of glycosylation that are found in mammalian proteins; however, bacteria have been used for the production of certain mammalian proteins (e.g. interleukin-2) in which a lack of post-translational glycosylation apparently does not significantly affect biological activity.

(See also Neupogen® in the entry BIOPHARMACEUTICAL (table).)

GM food GENETICALLY MODIfiED FOOD.

GM2 gangliosidoses Certain GENETIC DISEASES in which nondegraded gangliosides accumulate in lysosomes.

One example of such diseases is TAY–SACHS DISEASE. goi Gene of interest.

gold standard Any well-established and trusted procedure (e.g. a particular form of test) regarded by workers in the field as being superior (in e.g. sensitivity/specificity/reliability) when compared to other (alternative or proposed) methods. In the diagnosis of infectious diseases a combined or expanded gold standard is generally taken to mean both culture and nucleic- acid-based methods.

Goldberg–Hogness box See CORE PROMOTER.

gp (1) GENE PRODUCT.

(2) A glycoprotein gene product encoded by retroviruses. gp41 (in HIV-1) See HIV-1.

gp64 gene (in baculoviruses) See BACULOVIRIDAE.

gp120 A major envelope glycoprotein of the HIV-1 virion. (See also DC-SIGN.)

Grace’s medium A liquid medium used for the culture of (i.e. growth of) insect cells. Grace’s medium includes a number of inorganic salts, sugars (fructose, D-glucose and sucrose) and a variety of amino acids.

(See also BACULOVIRUS EXPRESSION SYSTEMS.)