Dictionary of DNA and Genome Technology

and replication is mediated by the highly processive DNA polymerase from bacteriophage ϕ29 (omitting an initial stage of heat denaturation) [Proc Natl Acad Sci USA (2002) 99: 5261–5266]; average product length: >10 kb. Amplification bias is less than that reported in PCR-based methods, and the products are said to be useful for genomic studies and for SNP analysis.

Under-representation of terminal sequences in linear DNA may be overcome by ligation (forming a circular template) [BioTechniques (2005) 39(2):174–180].

Multiple displacement amplification (MDA) has been used for amplifying DNA in low-level sources: samples of plasm a stored at −40°C for ~10 years [BioTechniques (2005) 39(4): 511–515].

multiple loci VNTR analysis See MLVA.

multiplex amplifiable probe hybridization (MAPH) A technique used for assessing the copy number of target sequences and detecting deletion mutations. Essentially, genomic DNA is denatured and exposed to a range of probes; the probes are complementary to various target sequences in the genome, but all the probes have the same terminal 5′ and 3′ primerbinding sites. The probes are allowed to hybridize with the genomic DNA (which is immobilized on a filter), following which any unbound probes are removed by washing. Bound probes are then recovered (separated from genomic DNA) and amplified by PCR using a primer pair which is common to all the probes. The quantity of PCR product obtained from a given type of probe is assumed to correlate with the number of such probes present in the reaction mixture. A probe that is complementary to a deleted sequence in the genome is likely to yield no product by PCR.

multiplex ligation-dependent probe amplification (MLPA) A technique used for relative quantitation of specific sequences of nucleotides; the applications of MLPA include detection of, for example, Down’s syndrome (trisomy 21), gene duplications, exon deletions and SNPs.

Probes are added to a sample, each probe consisting of two oligonucleotides that bind adjacently on the target sequence. If both oligonucleotides hybridize correctly on the target sequence they can be ligated and the (now complete) probe can be amplified by PCR (each probe includes two primer-binding sites). Quantitation of the PCR products can be facilitated by using labeled primers in the PCR reaction. Products obtained from the target sequence in a given sample can be compared, quantitatively, with products obtained from the corresponding target sequence in other samples.

[Examples of multiplex ligation-dependent probe amplification: Nucleic Acids Res (2005) 33(14):e128 and PLoS Med (2006) 3(10):e431.]

(cf. COMPARATIVE GENOMIC HYBRIDIZATION.)

multiplex PCR A form of PCR for simultaneously amplifying two or more target sequences in the same assay; the reaction mixture contains primers for each target. PCR with several targets can be monitored by (target-specific) probes labeled with different types of fluorescent dye (i.e. dyes with differ-

ent emission spectra).

As multiple primers are used, extra care is required in order to prevent the formation of PRIMER–DIMERS.

In some cases, one primer can be shared by two targets. For example, a sequence in the 16S rRNA gene in Bacteroides forsythus and Prevotella intermedia has been amplified with

one FORWARD PRIMER (a BROAD-RANGE PRIMER common to

both species) and two species-specific reverse primers. Multiplex PCR has been used e.g. for detecting mecA and

coa genes in Staphylococcus aureus [J Med Microbiol (1997) 46:773–778]; for diagnostic virology [Clin Microbiol Rev (2000) 13:559–570]; for detecting toxin genes in Clostridium difficile [J Clin Microbiol (2004) 42:5710–5714] and for subspeciation of Campylobacter jejuni isolates [BMC Microbiol (2007) 7:11].

Multiplex PCR is also used e.g. for amplification of STRs in human DNA profiling (e.g. CODIS).

An alternative approach is

PCR.

multiplex QEXT See QEXT.

multiplex real-time PCR REAL-TIME PCR (q.v.) in which more than one target sequence is amplified in a given assay.

RECOMBINATION SYSTEM in which a range of vector molecules, each having att sites with distinct, modified sequences and specificities, offers the choice of diverse recombination partners and directional insertion of the sequence of interest.

Example of use

MultiSite technology was used e.g. for inserting a sequence (simultaneously) into two different locations in one plasmid. Initially, a target site was introduced into the plasmid at each of the two locations. Each target site contained a copy of the ccdB gene (see entry CCD MECHANISM) flanked by a pair of asymmetric attP sequences; the pair of asymmetric sequences flanking one target site was different to the asymmetric pair flanking the other target site.

Copies of the sequence to be inserted were prepared by PCR. One PCR reaction used primers that introduced terminal attB sequences corresponding to one pair of asymmetric attP

sites.

A separate PCR used primers that introduced terminal attB sequences corresponding to the other pair of asymmetric attP sites.

Thus, as a result of the two PCR reactions, there were two sets of inserts, each prepared for introduction into one of the two target sites.

The (recipient) plasmid was then incubated with the two sets of inserts (for 1 hour at 25°C) together with the Int and IHF proteins. During incubation, each insert was exchanged, by site-specific recombination, with its corresponding target sequence in the plasmid. (Note that elimination of each target sequence from the plasmid resulted in the removal of a ccdB

gene from the plasmid.)

All the above steps were carried out in vitro.

The recombinant plasmids were then used to transform a CcdB-sensitive strain of bacteria. As the plasmid included an ampicillin-resistance gene, positive selection of the (dual) recombinants was obtained by using an ampicillin-containing medium. Any cell containing a plasmid in which both (CcdBencoding) target sites had not been exchanged for inserts was killed by the CcdB toxin.

In this experiment, all copies of the insert were identical, except for their att sites, but it seems likely that this protocol can be easily adapted for the insertion of non-identical donor sequences at required sites.

[Approach used in the above experiment: BioTechniques (2005) 39(4):553–557.]

[Conversion of Gateway® vectors to MultiSite Gateway® vectors by means of a single recombination event: BMC Mol Biol (2006) 7:46.]

mung bean nuclease A SINGLE-STRAND-SPECIfiC NUCLEASE.

Mung bean nuclease differs from e.g. endonuclease S1 in that it cannot cleave the DNA strand opposite a nick in a DNA duplex.

(See also RIBONUCLEASE PROTECTION ASSAY.)

mutagen Any physical or chemical agent which can promote

MUTAGENESIS. Some mutagens are CARCINOGENS.

Mutagens typically interact with, and alter/damage, nucleic acid. In some cases a mutagen may alter a base such that, in a subsequent round of replication, incorrect base-pairing leads to the insertion of a different nucleotide.

Some mutagens are more effective with ssDNA than with dsDNA.

Physical mutagens include ionizing radiations and ULTRA- VIOLET RADIATION. Heat apparently increases the level of spontaneous mutation.

Chemical mutagens include compounds of a wide range of types – alkylating agents, base analogs, bisulfite, hydroxylamine, nitrous acid etc. Others include e.g. the AflATOXINS (associated with some instances of hepatocellular carcinoma) and β-naphthylamine (implicated in bladder cancer).

mutagenesis (in vitro) The creation of mutation(s) in one or more target sequences of nucleic acid. Mutations are created for various reasons: e.g. (i) to inactivate a gene, (ii) to modify the coding sequence of a gene, (iii) to modify the regulation of a gene’s expression. A gene may be inactivated e.g. to investigate its involvement (or otherwise) in a given function – cells with an inactivated gene being compared to those with a normal, wild-type gene. Modification of a gene’s coding sequence, or its regulation, has widespread applications in medicine and industry as well as in biological research.

There are two basic approaches to mutagenesis: (i) random mutagenesis (i.e. creating mutation(s) at random sites within a given target sequence) and (ii) site-directed (= site-specific) mutagenesis – in which mutations are created at known, predetermined sites.

Random mutagenesis

Random mutagenesis may be carried out in order to produce specific types of mutant (e.g. temperature-sensitive mutants) for use in various types of study; such mutants are recovered by the use of selective procedures.

Random, transposon-mediated mutagenesis has been used for the detection of virulence-associated genes in pathogenic

bacteria: see e.g. SIGNATURE-TAGGED MUTAGENESIS.

Transposition with Tn5 can be carried out in wholly in vitro systems, insertions occurring randomly in e.g. a population of plasmid vectors (see TN5-TYPE TRANSPOSITION).

Random mutations in bacteria can be created by exposing the cells to a mutagen, i.e. a physical or chemical agent such as ultraviolet radiation, X-rays, alkylating agents, bisulfite or hydroxylamine. Mutations develop in different genes (and/or non-coding sequences) in different cells. Those cells which have developed the required type of mutation may be isolated by using appropriate selective conditions – such as particular types of selective medium (see e.g. AUXOTROPHIC MUTANT for some approaches to methodology).

Random mutations can be introduced into an insert/gene in a vector molecule by replicating the vector within a so-called MUTATOR STRAIN; one advantage of this is that mutagenesis can be achieved without the use of mutagens.

Random mutations can be introduced into a target sequence in vitro by a PCR-based method involving the use of an errorprone DNA polymerase (see e.g. GENEMORPH PCR MUTA-

GENESIS KIT).

Site-directed mutagenesis (site-specific mutagenesis)

Mutation at specific site(s) is used e.g. for studies on gene expression, and also for modifying the products encoded by genes – for example, increasing the efficiency of an enzyme used in an industrial process by changing specific nucleotides in the coding region of the gene.

(See also SITE-DIRECTED MUTAGENESIS.)

Site-directed mutations can be single-nucleotide mutations (point mutations) or may involve insertion/deletion of short or long sequences of nuleotides.

For examples of methods used in site-directed mutagenesis

see entries EXSITE PCR-BASED SITE-DIRECTED MUTAGENESIS KIT, GENETAILOR SITE-DIRECTED MUTAGENESIS SYSTEM and QUIKCHANGE SITE-DIRECTED MUTAGENESIS KIT.

(See also CHIMERAPLAST.)

mutagenic repair See SOS SYSTEM.

mutasynthesis The synthesis of new type(s) of end-product by a mutant organism in which a normal biosynthetic pathway is blocked and the pathway is completed by an abnormal substrate. Mutasynthesis has been exploited e.g. for synthesizing novel products.

Mutatest Syn. AMES TEST.

mutator strain Any strain of bacteria that is deficient in DNA repair function(s) and which is used e.g. for creating random mutations (see MUTAGENESIS) in target sequences replicated within such strains.

One advantage of using a mutator strain is that mutagenesis can be achieved without the use of mutagens.

An example of a mutator strain is the XL1-Red strain of Escherichia coli (Stratagene, La Jolla CA); this strain lacks several of the normal repair functions and is reported to generate mutations at a rate which is several thousand times higher than is possible with a corresponding wild-type strain. The genotype of XL1-Red is endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, mutD5, mutS, mutT, Tn10 (Tetr ).

Examples of use of XL1-Red: generation of mutations in the lolC gene of Escherichia coli [J Bacteriol (2006) 188(8): 2856–2864] and investigation of the effect of mutation in the

RPB1 gene of Saccharomyces cerevisiae [Genetics (2006) 172(4):2201–2209].

A derivative of XL1-Red, containing the lacIq element, was used in studies on the E. coli ribonuclease E gene to control expression of rne [Genetics (2006) 172(1):7–15].

Mutazyme® See GENEMORPH and COMPETENT CELLS. MutH See MISMATCH REPAIR.

MutL See MISMATCH REPAIR.

MutM See 8-OXOG.

MutS See MISMATCH REPAIR.

MutT See 8-OXOG.

MutY See 8-OXOG.

Mx162 See RETRON.

myb (MYB) An ONCOGENE first identified in avian myeloblastosis virus (AMV). AMV causes rapidly fatal leukemia in chickens. The product of v-myb occurs in the nucleus. The c- myb product is a transcription factor that has roles e.g. in cell proliferation and apoptosis; it is strongly expressed e.g. in immature hemopoietic cells – but expression falls markedly during differentiation.

In chickens, insertional activation of c-myb is seen e.g. in lymphomas and myeloid leukemias.

myc (MYC) An ONCOGENE first detected in avian myelocytomatosis virus. The v-myc product (found in the nucleus) is a gag–myc fusion protein which has no protein kinase activity. Human c-myc normally occurs on chromosome 8; it encodes a transcription factor which may regulate activities such as e.g. proliferation, differentiation and apoptosis. Translocation of c-myc may be causally connected with Burkitt’s lymphoma.

The oncogenic activity of c-myc appears to arise mainly by overexpression.

In mice, experimental expression of c-myc results in the formation of tumors.

Mycobacterium tuberculosis A species of Gram-positive, acidfast bacteria; a causal agent of tuberculosis. [Genome: Nature (1998) 393:537–544.] Detection of M. tuberculosis in smear-

positive sputa may be achieved e.g. by the AMTDT; typing: e.g. by SPOLIGOTYPING. Some typing schemes have been based on the insertion sequence IS6110, which appears to be specific to members of the ‘M. tuberculosis complex’ (which includes M. tuberculosis, M. bovis and M. africanum). The number of copies of IS6110, per genome, varies according to strain, some strains apparently containing none; H37rv, the reference strain of M. tuberculosis, contains 16 copies. In at least some strains of M. tuberculosis the value of long-term IS6110-based typing may be compromised by a high rate of transposition of the element within the genome.

The detection of antibiotic resistance in clinical isolates of M. tuberculosis can be achieved e.g. by several commercial PROBE-based assay systems: see e.g. LINE PROBE ASSAY.

(See also W-BEIJING STRAIN.)

mycophenolate mofetil (MMF) A prodrug which is converted, intracellularly, to the active agent MYCOPHENOLIC ACID. mycophenolic acid An agent with antitumor and antimicrobial

activity synthesized e.g. by the fungus Penicillium brevicompactum; it inhibits the synthesis of guanosine monophosphate by inhibiting the formation of xanthosine monophosphate from inosine monophosphate – thus depleting the pool of guanine nucleotides.

The prodrug, mycophenolate mofetil (MMF), is converted, intracellularly, to mycophenolic acid.

Examples of use: a pilot study in which MMF was used for treating HIV-positive patients [AIDS Res Ther (2006) 3:16], and studies on the accumulation of RNAs of hepatitis delta virus (HDV) [J Virol (2006) 80(7):3205–3214].

mycovirus Any (commonly dsRNA) virus which can infect one or more species of fungus; in at least some cases mycoviruses encode an RNA-dependent RNA polymerase.

Transmissibility of mycoviruses seems to depend primarily on processes such as hyphal fusion and mating (as opposed to the passage of virions from one individual to another via an extracellular route).

Some mycoviruses do not form conventional virions, their nucleic acid being encapsulated in host-encoded vesicles.

myeloid lineage Cells that include e.g. monocytes and macrophages, granulocytes (e.g. neutrophils, basophils and eosinophils) and erythrocytes (red blood cells).

(See also LYMPHOID LINEAGE.)

myristylation membrane localization signal A function used

e.g. in the CYTOTRAP TWO-HYBRID SYSTEM.

myxophage Any bacteriophage that infects one or more species of gliding bacteria (bacteria of the order Myxobacterales).

N (1) An indicator of ambiguity in the recognition sequence of a RESTRICTION ENDONUCLEASE (or in any other sequence of nucleic acid); ‘N’ indicates A or C or G or T (in RNA: A or C or G or U), i.e. any nucleotide.

(2) L-Asparagine (alternative to Asn).

N-acetyl-L-cysteine See MUCOLYTIC AGENT. N-acetylmuramidase See LYSOZYME. N-acyl-homocysteine thiolactone See QUORUM SENSING. N-acyl-L-homoserine lactone See QUORUM SENSING. N-degron See N-END RULE.

N-end rule The observed rule that the half-life of a given protein is influenced by the identity of its N-terminal amino acid. The N-end rule applies in prokaryotes and eukaryotes.

The features which determine early degradation are signals (degrons) that are incorporated in the protein itself. One of these is the N-degron: a destabilizing N-terminal amino acid recognized by the cell’s specific degradation machinery. For example, in Escherichia coli, proteins with arginine or lysine at the N-terminal have characteristically short half-lives (see

AAT GENE).

The eukaryotic signals for protein degradation include not only an N-terminal amino acid residue but also internal lysine residue(s). A protein with the appropriate degradation signals binds a polyubiquitin chain (through multi-enzyme activity) as a prerequisite to degradation in a 26S PROTEASOME.

N15 phage See PHAGE N15.

NAAT Nucleic-acid-amplification test: any of various types of test (involving e.g. PCR) used in a clinical diagnostic setting.

NAD (former names: coenzyme I; cozymase; diphosphopyridine nucleotide, DPN) Nicotine adenine dinucleotide: a carrier of energy (and hydrogen) in many reactions; it is also involved in ADP-RIBOSYLATION. The oxidized form is written NAD+, the reduced form is written NADH.

from certain proteins or by a synthetic process. Movement of a DNA molecule through a nanopore, under appropriate control, may offer the possibility of using a nanopore device for sequencing the molecule. [Kinetics of translocation of DNA through synthetic nanopores: Biophys J (2004) 87(3):2086– 2097.]

NASBA Nucleic acid sequence-based amplification: a method used primarily for the isothermal amplification of specific sequences in RNA (at a temperature of e.g. 41°C/42°C).

Amplification of an RNA target sequence by NASBA is shown diagrammatically in the figure.

(For isothermal amplification of DNA see e.g. MOLECULAR

ZIPPER and SDA.)

NASBA and TMA both reflect an earlier technique used for the in vitro amplification of nucleic acids: see entry SELF-

SUSTAINED SEQUENCE REPLICATION.

The use of an RNA target sequence (as compared to DNA targets) is an advantage in some circumstances. For example, in a diagnostic assay of cellular pathogens, the abundance of rRNA molecules in every cell – as compared e.g. to (singlecopy) genes – increases the chances of detecting small numbers of pathogens in a clinical sample. Additionally, certain short-lived bacterial mRNAs are useful targets for detecting viable cells.

In the context of virology, NASBA has been useful e.g. for assaying genomic RNA of the retrovirus HIV-1 in a range of clinical specimens – including plasma, CSF (cerebrospinal fluid) and brain tissue.

NASBA is also useful for monitoring the appearance of transcriptional activity in latent DNA viruses. For example, transcripts of the late genes of cytomegalovirus (CMV) have been used as targets for monitoring patients at risk from CMV-related conditions. Recipients of transplants have been monitored by assaying transcripts of the CMV gene UL65, which encodes protein pp67.

The products of a NASBA assay can be detected in various ways. In one approach, products are examined by agarose gel electrophoresis, and blotting, followed by hybridization of a biotinylated probe and detection by an alkaline phosphatase– streptavidin conjugate and a chemiluminescent substrate.

A system involving electrochemiluminescence is available commercially (NucliSens™, bioMérieux, Boxtel, Holland).

Real-time detection of NASBA products can be achieved

e.g. with MOLECULAR BEACON PROBES.

Quantitation of NASBA products has been achieved e.g. by using several internal ‘calibrators’, i.e. RNA molecules which are co-amplified and co-extracted with target RNA; because the initial concentration of each of the calibrators is known accurately, the initial concentration of the target sequence can be calculated.

Examples of the use of NASBA include measurement of plasma levels of RNA from human immunodeficiency virus (HIV) [Clin Vaccine Immunol (2006) 13(4):511–519], and detection of 16S rRNA from Chlamydophila pneumoniae in respiratory specimens [J Clin Microbiol (2006) 44(4):1241– 1244]. NASBA was also reported to be useful for assaying noroviruses (common causal agents of viral gastroenteritis) in environmental waters – factors causing inhibition of reverse transcriptase PCR had little or no inhibitory effect on a realtime NASBA assay [Appl Environ Microbiol (2006) 72(8): 5349–5358].

National Bioforensics Analysis Center See NBFAC.

NBFAC National Bioforensics Analysis Center: the microbial forensics laboratory which forms part of the Department of Homeland Security in the USA; it works closely with the FBI (Federal Bureau of Investigation) and is a center specializing in the examination and analysis of material evidence in cases of actual or suspected criminal/terrorist activity.

(See also FORENSIC APPLICATIONS.)

negative control (in operons) See OPERON.

negative selection In general, selection based on the absence of a given feature, function or reaction.

(cf. POSITIVE SELECTION.)

neighbor exclusion principle See INTERCALATING AGENT.

nelfinavir See PROTEASE INHIBITORS. neomycin See AMINOGLYCOSIDE ANTIBIOTICS.

neoplasia Progressive, uncontrolled division of cells, resulting e.g. in a solid tumor or in leukemia.

(See also ONCOGENESIS.)

neoplasm A tumor. neoschizomer See ISOSCHIZOMER.

nested deletions Any process in which segments are progressively removed from the end of a DNA molecule. This type of procedure can be used e.g. when sequencing a long (>500nucleotide) molecule of DNA. Thus, the initial ~500-nucleo- tide length of the sample DNA may be sequenced (by the DI- DEOXY METHOD) from the first primer-binding site. Further sequencing of the molecule can be achieved by (i) removing that part of the (double-stranded) sample DNA which has already been sequenced, and (ii) sequencing the next section of single-stranded template from a new primer-binding site. This procedure is then repeated, so that sequencing of the entire molecule is achieved in a series of consecutive steps along the length of the sample DNA.

Unidirectional nested deletions in a cloned fragment within a circular vector can be prepared by first cutting the vector,

with appropriate RESTRICTION ENDONUCLEASES, at two sites

close to one end of the insert: (i) one enzyme to produce a 5′ overhang adjacent to the insert, and (ii) another enzyme to produce a 3′ overhang at the other end of the cleaved vector. The digested vector is then incubated with EXONUCLEASE III, and aliquots of the mixture are removed at intervals of time. Exonuclease III digests the 3′ end recessed under the 5′ overhang (but not the 3′ overhang at the other end of the cleaved vector). Increasing lengths of incubation with exonuclease III result in progressively greater digestion of the recessed 3′ end i.e. increasing the extent of deletion of the insert – so that aliquots removed later from the incubation will contain molecules in which more of the insert sequence has been deleted.

The 5′ single-stranded sequences (resulting from digestion by exonuclease III) – and the 3′ overhangs at the other end of the molecule – are removed by incubation with e.g. mung bean nuclease or ENDONUCLEASE S1. The resulting blunt-ended products can be ligated e.g. with T4 DNA ligase to re-form vector molecules containing inserts with a range of deletions.

neu In the rat: an ONCOGENE whose product is related to mem-

bers of the EPIDERMAL GROWTH FACTOR RECEPTOR FAMILY;

this product has TYROSINE KINASE activity associated with the intracellular domain.

Neu In the rat: the product of the neu gene (a homolog of the human HER2 protein).

Neulasta® See BIOPHARMACEUTICAL (table).

Neupogen® See BIOPHARMACEUTICAL (table).

nevirapine A NON-NUCLEOSIDE REVERSE TRANSCRIPTASE IN- HIBITOR.

Newcombe experiment An early (1949) classic experiment in which Newcombe demonstrated that pre-existing mutants are selected when a population of bacteria appears to adapt to a change in the environment.

A number of plates, containing nutrient agar, are each inoculated from a culture of phage-sensitive bacteria and are then incubated for a few hours. During the incubation period each viable cell gives rise to a number of progeny cells (a clone), forming a colony.

At this stage, the plates are divided into two groups: group A and group B.

In group A plates the bacterial growth is re-distributed over the surface of each plate by means of a sterile glass spreader. As a result, the cells of each clone (colony) are dispersed over the surface of the agar.

In group B plates the growth is left undisturbed.

Every plate (in both groups, A and B) is then sprayed with a suspension of virulent phage and re-incubated.

During the period of re-incubation all phage-sensitive cells are lysed. Any colonies that form on the plates necessarily arise from phage-resistant cells.

If resistance to phage developed adaptively (as a response to the presence of phage) it would be observed only after the cells had been exposed to phage. Were this the case, then redistribution of growth on group A plates should be irrelevant

because it was done before the cells were exposed to phage. Hence, if resistance to phage developed adaptively, similar numbers of phage-resistant colonies should develop on all the plates (in groups A and B).

If, however, phage-resistant cells had been present before exposure to phage (on both the A and B plates), then each phage-resistant cell (mutant) would form a clone of cells (i.e. a single colony) before exposure to phage. In this case, the re-distribution of growth on group A plates would spread the cells of each (phage-resistant) colony – so that each of these cells would itself give rise to a separate colony. By contrast, any colony of (phage-resistant) cells on group B plates would remain as a single colony because growth on these plates had not been re-distributed.

In practice, many more (phage-resistant) colonies are found on the group A plates, indicating the presence of pre-existing phage-resistant mutant cells in the original culture.

(cf. flUCTUATION TEST.)

NF-κB Nuclear factor κB: a major transcription factor found in mammalian cells. An inactive form of NF-κB, complexed with the inhibitory factor IκB, is found in the cytoplasm. On activation, NF-κB (no longer complexed with IκB) can enter the nucleus and regulate expression of various genes.

[An assay of NF-κB–DNA binding: BioTechniques (2006) 41(1):79–90.]

(See also PATHDETECT SYSTEMS.)

NHGRI National Human Genome Research Institute.

nick In dsDNA: a break in one strand of the duplex at a particular phosphodiester bond.

(cf. GAP.)

nick-closing enzyme See TOPOISOMERASE (type I).

nick translation A technique for labeling DNA (for example, cloned fragments) by replacing the existing nucleotides with nucleotides carrying a radioactive or other label. Essentially, the (ds) DNA is exposed to an endonuclease which makes a number of (random) nicks. Using each of the nicks as a starting point, DNA polymerase I extends the 3′ terminus – the newly synthesized strand incorporating labeled dNTPs (that are abundant in the reaction mixture); during synthesis, the polymerase exerts 5′-to-3′ exonucleolytic activity, i.e. it progressively removes nucleotides from the nick’s 5′ terminus.

nickel-charged affinity resin A resin (charged with nickel ions, Ni2+) which can bind proteins that have an (accessible) sequence of six consecutive histidine residues; it is used e.g. for the purification of 6XHis-tagged recombinant proteins – including those produced by expression vectors such as the

CHAMPION PET SUMO VECTOR and the VOYAGER VECTORS.

Elution of proteins bound to the resin can be achieved e.g. by using a low-pH buffer or by histidine competition.

(See also PROBOND NICKEL-CHELATING RESIN.) nicking enzyme See RESTRICTION ENDONUCLEASE.

nicotine adenine dinucleotide See NAD. Niemann–Pick disease See

nisin See LANTIBIOTIC.

nitrocefin An agent used, in vitro, to test for β-LACTAMASES;

nitrocefin is a CEPHALOSPORIN which changes from red to yellow when cleaved by a β-lactamase.

(See also PADAC.)

nitrous acid (as a mutagen) Nitrous acid (HNO2) brings about oxidative deamination of guanine (to xanthine), cytosine (to uracil) and adenine (to hypoxanthine). Replication of nitrous acid-treated DNA can lead to GC→AT mutation (deaminated cytosine) and AT→GC mutation (deaminated adenine).

(See also HYDROXYLAMINE.)

NMR Nuclear magnetic resonance, a phenomenon which has been exploited e.g. for monitoring gene expression in vitro and in vivo: see MRI.

NNRTIs NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIB-

ITORS (q.v.).

non-coding strand (of a gene) See CODING STRAND. non-isotopic labeling (of probes) See PROBE LABELING. non-Mendelian inheritance CYTOPLASMIC INHERITANCE. non-nucleoside reverse transcriptase inhibitors (NNRTIs) A

structurally diverse group of ANTIRETROVIRAL AGENTS that inhibit viral reverse transcriptase; they include delavirdine, efavirenz, GW678248 and nevirapine (see also SURAMIN).

(See also HAART.)

non-ribosomal peptide synthetase (NRPS) Any of a range of multisubunit enzymes which mediate ribosome-independent synthesis of peptides [e.g. NRPS from the bacterium Pseudomonas aeruginosa: J Bacteriol (2003) 185(9):2848–2855].

NRPSs have been genetically modified in order to generate new types of product; these and other advances have allowed the synthesis of e.g. various peptide antibiotics and integrin receptors.

[Chemoenzymatic and template-directed synthesis of bioactive macrocyclic peptides: Microbiol Mol Biol Rev (2006) 70(1):121–146.]

nonsense-associated disease Any of various diseases (such as Duchenne muscular dystrophy) which may develop through a NONSENSE MUTATION that affects a specific protein.

nonsense codon (chain-terminating codon, stop codon, termination codon) Any of the three codons – UAA (ochre codon), UAG (amber codon) and UGA (opal codon or umber codon) – which usually specify termination of polypeptide synthesis during translation.

nonsense mutation Any mutation that produces a NONSENSE CODON from a codon previously specifying a product.

nonsense suppressor See SUPPRESSOR MUTATION.

nopaline See CROWN GALL.

norfloxacin See QUINOLONE ANTIBIOTICS.

noroviruses See NASBA (uses).

Northern blotting A form of BLOTTING in which RNA is transferred from gel to matrix. (cf. SOUTHERN BLOTTING.) A Northern blot may be probed e.g. by labeled DNA.

(See also WESTERN BLOTTING.)

NotI A RARE-CUTTING RESTRICTION ENDONUCLEASE.

(See also GC% (figure).)

Novex® gels A range of gels (Invitrogen, Carlsbad CA) used in gel electrophoresis of nucleic acids and proteins.

Novex® TBE (Tris, boric acid, EDTA) gels are used for the separation of fragments of DNA of a wide range of sizes.

Novex® TBE-urea gels are optimized for fragments of DNA in the range ~20–800 bp.

novobiocin A coumarin derivative that binds to the B subunit of bacterial gyrase, blocking the enzyme’s ATPase activity and, hence, blocking DNA synthesis. (See also QUINOLONE ANTIBIOTICS.) Novobiocin is less effective against topoisomerase IV – apparently because of a one-residue difference in the target sequence [Antimicrob Agents Chemother (2004) 48:1856–1864].

A mutation in the B subunit of gyrase, causing hypersensitivity to novobiocin, was found to be partly suppressed by a further mutation in the A subunit [J Bacteriol (2005) 187 (19):6841–6844].

NovoRapid® See BIOPHARMACEUTICAL (table). NovoSeven® See BIOPHARMACEUTICAL (table). NPVs NUCLEAR POLYHEDROSIS VIRUSES.

nrdA gene (dnaF gene) In Escherichia coli: a gene encoding the enzyme ribonucleotide reductase (which converts ribonucleotides to deoxyribonucleotides).

NRPS NON-RIBOSOMAL PEPTIDE SYNTHETASE.

NRTIs NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS.

viruses which are distinguished from GRANULOSIS VIRUSES e.g. on the basis of characteristics of their occlusion bodies: see BACULOVIRIDAE. Those NPVs in which a single nucleocapsid is enveloped have been designated SNPV, while those in which multiple nucleocapsids are enveloped have been designated MNPV.

Unlike granulosis viruses, NPVs occur in various orders of arthropods, including Coleoptera, Diptera and Hymenoptera as well as Lepidoptera.

The type species of NPVs is Autographa californica NPV (AcMNPV) (genome: ~135 kb) which infects insects of the Lepidoptera. Other viruses in this group include the silkworm pathogen Bombyx mori NPV (BmSNPV) and the Douglas Fir tussock moth virus Orygia pseudotsugata NPV (OpMNPV).

5′-nuclease PCR Any of various forms of PCR in which the 5′- exonuclease activity of the polymerase is used to degrade a labeled probe which is bound to an amplicon; the signal (for example, fluorescence) generated by degradation of the probe is monitored.

nuclease protection assay See e.g. RIBONUCLEASE PROTECT-

ION ASSAY.

nuclease S1 Syn. ENDONUCLEASE S1.

nucleic acid A singleor double-stranded (hetero)polymer of NUCLEOTIDES in which adjacent nucleotides in a given strand are linked by a phosphodiester bond between the 5′ and 3′ positions of their pentose residues.

In a double-stranded nucleic acid the two strands are held together by base-pairing between complementary bases (i.e. between A in one strand and T (U in RNA) in the other, and between G in one strand and C in the other).

A nucleic acid molecule may be linear (with free 5′ and 3′ ends) or covalently closed circular (ccc) with no free 5′ or 3′ ends. (A ‘nicked’ molecule is a double-stranded molecule that contains one or more nicks – see NICK.)

Information is encoded in the sequence of the nucleotides in the polymer. In all organisms, the genome is composed of one or more molecules of nucleic acid.

DNA is a nucleic acid consisting of deoxyribonucleotides (in which the pentose residues carry −H, rather than −OH, at the 2′-position).

RNA is a nucleic acid consisting of ribonucleotides. nucleic acid amplification (in vitro) Any of various methods

used for the amplification (i.e. repeated copying) of specific sequences of nucleotides in RNA or DNA.

Some examples are listed in the table.

nucleic acid enzyme Any nucleic acid molecule (either RNA or DNA) which has enzymic capability – e.g. as a nuclease or

a ligase. See e.g. RIBOZYME and DEOXYRIBOZYME.

nucleic acid isolation (nucleic acid extraction) Any procedure which is used for removing and concentrating DNA or RNA from cells or tissues: see e.g. DNA ISOLATION, RNAQUEOUS

TECHNOLOGY and PAXGENE BLOOD RNA KIT.

Reagents commonly used in the isolation of nucleic acids are listed in the table. Note that some of these reagents may inhibit amplification in processes such as PCR – indicating the need for a pre-amplification clean-up. For example, lysozyme and proteinase K have both been reported as inhibitors of PCR.

nucleic acid sequence-based amplification See NASBA.

nucleic acid staining See DNA STAINING and RIBOGREEN. (See also ULTRAVIOLET ABSORBANCE.)

nucleocapsid A composite structure, consisting of nucleic acid and protein, that forms part, or all, of certain virions.

nucleoid In a prokaryotic cell: the body consisting mainly of genomic DNA (one or more chromosomes) and NUCLEOID- ASSOCIATED PROTEINS. The DNA in a nucleoid is highly compacted.

(See also A-TRACT and CHROMOSOME.)

The toroidal form of the nucleoid in Deinococcus radiodurans may contribute to this organism’s unusual resistance to ionizing radiation [Science (2003) 299:254–256].

Segregation of genetic material to daughter cells during cell division may require a force-generating system involving e.g. the actin-like cytoskeletal protein MreB and RNA polymerase in Escherichia coli [Genes Dev (2006) 20(1):113–124]. An alternative view is that segregation occurs in an entropydriven (i.e. spontaneous) manner due to the tendency of two nucleoids to exhibit mutual repulsion in order to maximize the conformational entropy [Proc Natl Acad Sci USA (2006) 103(33):12388–12393]. Whatever the mechanism, studies on segregation of the (paired) chromosomes in Vibrio cholerae have suggested the possibility of coordination between these chromosomes in order to ensure their correct transmission to daughter cells [J Bacteriol (2006) 188(3):1060–1070].

Note. The term nucleoid is also used to refer to the DNA–