Dictionary of DNA and Genome Technology

protein complex in a mitochondrion and to the electron-dense core of a peroxisome.

nucleoid-associated proteins Certain small proteins, such as H- NS PROTEIN and HU PROTEIN, which are generally found to be abundant in the (prokaryotic) nucleoid.

nucleolar phosphoprotein B23 Syn. NUCLEOPHOSMIN. nucleophosmin (nucleolar phosphoprotein B23; numatrin) A

multifunctional nuclear phosphoprotein with roles e.g. in ribosome synthesis and cell proliferation (see also histone ACETYLATION). It apparently regulates/stabilizes the tumor suppressors P53 and p19Arf [Mol Cell Biol (2005) 25(20): 8874–8886], and is formed in response to chemotherapy of lung cancer [Exp Cell Res (2007) 313(1):65–76].

nucleoprotein filament See HOMOLOGOUS RECOMBINATION.

nucleoside A compound consisting of the residue of a purine or pyrimidine base linked covalently to a pentose (i.e. 5-carbon sugar) – commonly ribose (in ribonucleosides) or 2′-deoxy- ribose in deoxyribonucleosides. A pyrimidine is linked via its 1-position to the sugar, a purine is linked via its 9-position.

A ribonucleoside which incorporates a residue of adenine, guanine, cytosine, uracil, thymine or hypoxanthine is called (respectively) adenosine, guanosine, cytidine, uridine, thymidine (or ribothymidine) or inosine. Corresponding deoxy-

ribonucleosides are deoxyadenosine, deoxyguanosine etc. In general, ‘thymidine’ is used to refer to deoxythymidine.

(cf. NUCLEOTIDE.)

nucleoside reverse transcriptase inhibitors (NRTIs) A class of synthetic nucleoside analogs used in antiretroviral therapy, e.g. anti-AIDS therapy; in cells, an NRTI is phosphorylated (to the triphosphate) and, when incorporated into DNA by the (viral) reverse transcriptase (RT), it blocks chain elongation by blocking formation of the next phosphodiester bond. All NRTIs have a similar mode of action. The NRTIs include e.g. ABACAVIR, adefovir, didanosine, LAMIVUDINE, stavudine, tenofovir, zalcitabine and ZIDOVUDINE.

The anti-HIV activity of an NRTI in vivo may be raised by raising the ratio of the NRTI to the cell’s own dNTPs; this can be done e.g. by using hydroxyurea to inhibit the host’s enzyme ribonucleotide reductase (thereby causing a decrease in endogenous dNTPs). This approach gave clinical benefit e.g. when used in combination with didanosine.

Resistance to NRTIs can be due to a mutant RT gene. In one in vitro study of the activity of various NRTIs against a mutant (K65R) strain of HIV-1, all NRTIs except those with a 3′-azido moiety were less active against the mutant virus, indicating the value of including AZT in drug combinations

[Antimicrob Agents Chemother (2005) 49(3):1139–1144]. Some NRTIs are also useful against DNA viruses: see e.g.

LAMIVUDINE.

nucleosome See CHROMATIN.

nucleotide A NUCLEOSIDE that carries one or more phosphate groups at the 5′ position of the sugar residue.

Nucleotides are subunits of the NUCLEIC ACIDS DNA and RNA.

Certain nucleotides, e.g. adenosine triphosphate (ATP) and guanosine triphosphate (GTP), have an important function in energy-transfer reactions.

Biosynthesis of nucleotides in microbial and/or tumor cells may be inhibited by various agents such as AZASERINE, DON,

HADACIDIN, MYCOPHENOLIC ACID and PSICOFURANINE. (See also UNIVERSAL NUCLEOTIDE.)

nucleotide excision repair Syn. UVRABC-MEDIATED REPAIR. NucliSens™ See NASBA.

numatrin Syn. NUCLEOPHOSMIN.

numts Nuclear mitochondria-like sequences: those mtDNAlike sequences (‘mitochondrial pseudogenes’) which occur in nuclear (chromosomal) DNA. Numts are a possible source of contamination when mtDNA specifically is being targeted for amplification by PCR (e.g. in certain forensic investigations).

A total of 46 fragments of nuclear DNA, representing the entire (human) mitochondrial genome, have been sequenced [BMC Genomics (2006) 7:185].

nutrient agar A general-purpose bacteriological MEDIUM that contains peptone, sodium chloride, beef extract and 1.5–2% agar. It supports the growth of various types of nutritionally undemanding heterotrophic bacteria such as Escherichia coli; many species of bacteria will not grow on such (basal) media unless they are supplemented with serum or other forms of enrichment.

oc mutant See LAC OPERON.

O6-alkylguanine-DNA alkyltransferase (AGT) See ‘Uses of gene fusion’ in the entry GENE FUSION.

OB Occlusion body: see BACULOVIRIDAE. occlusion body (OB) See BACULOVIRIDAE.

occlusion-derived virions (ODVs) See BACULOVIRIDAE.

ochre codon See NONSENSE MUTATION. ochre suppressor See SUPPRESSOR MUTATION.

OCT plasmid A large (~500 kb) plasmid, found in strains of Pseudomonas, which confers the ability to metabolize octane and decane.

octopine See CROWN GALL.

ODVs Occlusion-derived virions: see BACULOVIRIDAE. OFAGE Orthogonal-field-alternation gel electrophoresis: one

of a number of variant forms of PFGE which is used for the separation of large pieces of nucleic acid.

ofloxacin See QUINOLONE ANTIBIOTICS.

Okayama–Berg method An early method used for obtaining a full-length cDNA – in a known orientation within a vector – from an mRNA molecule with a poly(A) tail. Essentially, the vector was built around the mRNA in sequential steps. The initial step involved base-pairing between the poly(A) of the mRNA and a poly(T) overhang present on an added fragment of dsDNA. The fiRST STRAND was subjected to 3′ homopolymer tailing to enable base-pairing with (another) segment of dsDNA having a complementary 3′ overhang; the other end of this (second) segment was cut with a restriction endonuclease, enabling its ligation to the free end of the first fragment.

Okazaki fragment One of a large number of short, discontinuous sequences of deoxyribonucleotides synthesized during the formation of the lagging strand in DNA replication; each fragment is about 100–200 nt in eukaryotes (but reported to be ~1000–2000 nt in Escherichia coli). These fragments are joined together to form the lagging strand.

In a current model, the formation of an Okazaki fragment (in eukaryotes) begins with a hybrid RNA/DNA primer that is synthesized by the complex primase–DNA polymerase α. This primer consists of ~10 nt RNA and ~20 nt DNA (and has been referred to as iRNA/DNA); it is extended with deoxyribonucleotides, by DNA polymerase δ (pol δ), until it reaches the 5′ terminal of the last Okazaki fragment. Pol δ continues DNA synthesis, its strand-displacement activity displacing the iRNA/DNA as a 5′ ‘flap’; the flap is excised before the newly synthesized Okazaki fragment is joined to the previous one. In the yeast Saccharomyces cerevisiae, excision of the flap may involve mainly flap endonuclease 1 (FEN1) [J Biol Chem (2004) 279(15):15014–15024]; in a supplementary mechanism for flap removal in this yeast, the flap is coated with replication protein A (RPA), promoting flap cleavage by the Dna2p helicase/nuclease protein.

(See also IDLING.)

oligo Abbreviation for ‘oligonucleotide’ (used only when the

meaning is clear).

OliGreen® A fluorescent dye (Molecular Probes Inc., Eugene OR/Invitrogen, Carlsbad CA) used e.g. for the quantitation of ssDNA in solution. The sensitivity of the method is reported to be ~10000-fold greater than that obtainable with measurements made by the ULTRAVIOLET ABSORBANCE method.

(See also DNA STAINING and PICOGREEN ASSAY.)

omega (ω) protein See TOPOISOMERASE (type I).

OMIM See ONLINE MENDELIAN INHERITANCE IN MAN.

ompT gene In Escherichia coli: a gene encoding a protease, OmpT, which is part of the (wild-type) cell’s outer membrane – i.e. the outermost structure of the cell wall.

Some of the proteins secreted by E. coli consist of two parts, one of which forms a channel in the outer membrane through which the other part is translocated to the cell’s surface. This type of protein secretion mechanism is referred to as an autotransporter system (also called type IV secretion by some authors and type V secretion by different authors). The surface-exposed part of a secreted protein may be cleaved by OmpT and released into the environment [Autotransporters in E. coli: Appl Environ Microbiol (2007) 73(5):1553–1562; EMBO J (2007) 26:1942–1952.]

on-chip PCR See SOLID-PHASE PCR.

on-chip synthesis See MICROARRAY.

oncogene A gene which, if aberrantly expressed, may cause neoplastic transformation (‘cancer’). Oncogenes were initially found in acutely oncogenic retroviruses; subsequently, highly conserved homologs were found in a wide range of normal eukaryotic cells, including human, fruitfly (Drosophila) and yeast (Saccharomyces cerevisiae) cells. Retroviral oncogenes appear to have derived from cellular genes.

An oncogene is usually given a three-letter designation that is based on the name of the retrovirus in which it was first identified. Two examples of viral oncogenes are: v-fes from the feline sarcoma virus and v-myc from myelocytomatosis virus; the corresponding cellular homologs of these genes are designated, respectively, c-fes (or proto-fes) and c-myc (or proto-myc).

At least some oncogenes have role(s) in the regulation and/ or development of normal (i.e. non-neoplastic) cells (see e.g. MYB). Oncogenesis may arise as a result of dysregulation of these genes; it may occur e.g. through mutation, insertional activation, or chromosome rearrangements (e.g. ABL).

Viral oncogenes can cause cancer in various ways. In some cases the product of a viral gene may interfere with the cell’s growth-related reactions which normally involve factors such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). The v-sis product is almost identical to the B chain of PDGF. Other products may be active in the cell’s nucleus.

(See also entries for individual oncogenes: ABL, FES, FPS,

KIT, MYB, MYC, RAS, SIS, SRC.)

Cancers resulting from dysregulated tyrosine kinase activ-

ity have been treated with specific drugs, but resistance to these agents can arise through mutation in the oncogene. The problem of drug-resistance (similar to antibiotic-resistance in bacteria) requires the ongoing development of new drugs; a fruitful approach has been to screen existing clinically useful drugs for agents that are active against newly mutant targets [Proc Natl Acad Sci USA (2005) 102 (31):11011–11016].

Oncogenes also occur in certain DNA viruses (e.g. mastadenoviruses, polyomaviruses), although these genes appear to have no cellular homologs.

oncogenesis The development of a neoplastic condition such as a tumor or leukemia.

(See also NEOPLASIA.)

oncornaviruses The former name of viruses of the subfamily

ONCOVIRINAE.

Oncovirinae A subfamily of viruses of the family Retroviridae (see RETROVIRUSES) that includes all the oncogenic viruses in this family; some – apparently non-oncogenic but related – viruses have also been included in the subfamily. Members of the Oncovirinae were divided into types B, C and D on the basis of e.g. morphology and mode of development.

Online Mendelian Inheritance in Man (OMIM) A database of human genes and genetic disorders [see Nucleic Acids Res (2005) 33(Database Issue):D514–D517] accessible at: http://www.ncbi.nlm.nih.gov/omim/

ONPG The compound o-nitrophenyl-β-D-galactopyranoside; it is hydrolyzed by the enzyme β-galactosidase to galactose and the (yellow) compound o-nitrophenol.

ONPG is used e.g. as an indicator for the presence of β- galactosidase in the SOS CHROMOTEST and in other tests. In some species (e.g. Escherichia coli) ONPG can pass through the cell envelope (i.e. it can enter cells) without the need for a specific permease.

opa genes (of Neisseria) See OPA PROTEINS. (See also PHASE VARIATION.)

Opa proteins Cell-surface antigens encoded by the opa genes in pathogenic strains of Neisseria; they affect the opacity and color of the bacterial colonies and bind to epithelial cells and polymorphonuclear leukocytes (e.g. via eukaryotic CD66a, c, d or e adhesins).

opal codon See NONSENSE MUTATION.

open reading frame (ORF) A sequence of nucleotides, starting with an initiator codon and ending with a stop codon, which encodes an actual or potential polypeptide/protein or an RNA product. A reading frame is ‘blocked’ if it contains a stop codon close to the initiator codon.

Adjacent ORFs (which may be separated by a gap) may jointly encode a polypeptide; in such cases, translation of the polypeptide requires a mechanism for coupling the two ORFs [see e.g. EMBO J (2000) 19:2671–2680].

operon Two or more contiguous genes coordinately expressed from a common promoter and transcribed as a polycistronic mRNA (see e.g. LAC OPERON).

The operon is sometimes regarded as a regulatory system specific to prokaryotic gene organization but a similar type of

organization is also found in trypanosomes and (in some instances) in genes of the nematode Caenorhabditis elegans

(see also TRANS SPLICING).

The following refers to operons in bacteria.

The control mechanism varies according to operon. Some operons are controlled at the level of transcription, and some are controlled at the level of translation.

Some operons are regulated by CATABOLITE REPRESSION. Promoter control. This involves the synthesis of a regulator protein, which may be produced constitutively. One example of promoter control is the LAC OPERON of Escherichia coli; this operon is under negative control – meaning that the regulator protein inhibits or blocks expression of the operon. In the lac operon, the repressor protein binds to the operator region immediately downstream of the binding site of RNA polymerase, thus inhibiting transcription of the operon.

An example of positive control is found in the araBAD operon of E. coli which encodes enzymes involved in the metabolism of L-arabinose to D-xylulose 5′-phosphate. In the presence of arabinose the regulator protein, AraC (encoded by gene araC), acts as an activator of the araBAD operon – i.e. positive control. In the absence of arabinose (metabolic enzymes not needed) the regulator protein acts as a repressor (negative control).

Attenuator control. Operons under attenuator control are typically involved in the synthesis of amino acids. The upstream sequence of nucleotides in the mRNA transcript (the leader sequence) encodes a short leader peptide which is rich in the given amino acid whose synthesis is regulated by the operon. The leader sequence also includes a so-called attenuator: a rho-dependent terminator situated between the first gene of the operon and the sequence encoding the leader peptide.

Given adequate levels of the relevant amino acid (synthesis not required), transcription stops at the attenuator. If the level of amino acid is inadequate (synthesis needed), the ribosome stalls when it reaches that part of the transcript containing codons of the given amino acid within the leader peptide sequence; this permits downstream regions of the transcript to base-pair, preventing formation of the attenuator and, hence, permitting transcription of the genes.

Attenuator control is found e.g. in the his operon of E. coli – which regulates synthesis of histidine. This operon contains nine structural genes. The mRNA sequence that encodes the leader peptide includes a run of seven successive histidine codons.

The trp operon of E. coli (synthesis of tryptophan) is under both attenuator and (negative) promoter control. This operon may be more stable than other operons which have a less complex regulatory system [Microbiol Mol Biol Rev (2003) 67:303–342].

Translational control. In E. coli the genes infC–rpmI–rplT form an operon which encodes, respectively, the translation initiation factor IF3 and the two ribosomal proteins L35 and L20. High levels of L20 repress translation of both L35 and L20. L20 appears to bind to the mRNA upstream of the rpmI

region, and it has been suggested that it stabilizes an RNA pseudoknot that includes the initiator codon, thus inhibiting the translation of both rpmI and rplT.

operon fusion The engineered fusion of two operons such that genes of both operons are controlled by regulatory regions (promoter, operator etc.) of one of the operons.

opine See CROWN GALL.

OpMNPV See NUCLEAR POLYHEDROSIS VIRUSES.

Orc1–6 proteins See DNAA GENE.

ORF OPEN READING FRAME.

ORFeome A set of open reading frames (see ORF), representing many, most or all ORFs from a given species, that are stored as inserts in a collection of plasmid vectors. An ORFeome can be used as a source of material for functional analysis; thus, for example, a given ORF may be inserted into a protein expression vector or used to generate fusion products.

To facilitate use, ORFs may be made more accessible for transfer to other plasmids by using a protocol involving e.g.

the GATEWAY SITE-SPECIfiC RECOMBINATION SYSTEM.

ORFmer sets Commercially available (Sigma-Genosys) sets of primer pairs designed to enable the PCR-based amplification of sequences from most or all ORFs (open reading frames) in the genomic DNA of a particular organism. The primers contain ORF-specific sequences.

The 5′ terminus of each primer consists of an adaptamer: a sequence incorporating the recognition site of a particular restriction endonuclease; this facilitates insertion of a given PCR-amplified product (amplicon) into a cloning vector for re-amplification, if required.

orientation (DNA technol.) The direction in which a given sequence is found, or inserted, in relation to other sequence(s) or in relation to the other parts of a sequence.

For example, when using flP recombinase (q.v.), a target fragment which is bracketed by two recognition sequences (FRT sites) that are in the same orientation will be excised; however, the target will be inverted if the two FRT sites are in opposite orientation.

oriT The origin of transfer (in a conjugative plasmid): see CON-

JUGATION.

orthologous genes Functionally analogous genes in different species.

orthomere See MACRONUCLEUS.

Orygia pseudotsugata NPV A baculovirus within the category

NUCLEAR POLYHEDROSIS VIRUSES.

overexpression (of proteins) (syn. overproduction) Synthesis of high concentrations of a given protein, usually a heterologous (‘foreign’) protein, in a recombinant organism – either a prokaryotic or a eukaryotic organism; this procedure is used e.g. for manufacturing various products, especially for therapeutic or diagnostic use but also for research purposes (see e.g. BIO-

PHARMACEUTICAL).

Proteins are synthesized in cells from bacteria (particularly the Gram-negative species ESCHERICHIA COLI), yeasts (such as Pichia pastoris, Saccharomyces cerevisiae), insects (such as Bombyx mori, Spodoptera frugiperda) and mammals (such

as hamster: CHO (Chinese hamster ovary) and BHK (baby hamster kidney) cells).

Choice of cells

The choice of cells is influenced by factors such as the need for specific type(s) of post-translational modification in the recombinant product, and/or the preferred secretion of the recombinant protein by the producing cells (rather than intracellular accumulation) in order to facilitate the recovery and purification of the product.

For proteins produced on an industrial scale, the important factors include e.g. the need to maximize the yield and the ease of downstream processing of the product. Downstream processing may be simplified by choosing cells which secrete the recombinant protein (see below). Other considerations include e.g. the safety of the product; thus, for example, if recombinant proteins are synthesized in E. coli, or in other Gram-negative bacteria, there is a need for rigorous exclusion of endotoxin (see PYROGEN). Additionally, agents used for induction of transcription in the producing cells should not be associated with a risk of toxicity if the recombinant products are intended for therapeutic use.

Optimization of transcription

One obvious requirement is a strong promoter to control the expression of the target gene. Moreover, transcription should be tightly regulated – meaning that the target gene should not be expressed prior to the time of its intended induction or derepression. One reason for this is that the cells are commonly grown to high density before expression of the target gene in order to maximize the yield of product. Were the gene to be expressed before the appropriate time then there may be a depression in the growth rate with a possible decrease in the final yield of recombinant protein.

Another reason for tight regulation is found in the case in which the recombinant protein is toxic to the producing cells. In this case, early expression of the recombinant protein can be expected to decrease final cell density and, hence, yield.

Optimization of translation

In designing a recombinant gene there is a need to ensure that the characteristics of the transcript are optimal for the given translation system. Thus for example, the efficient translation of mRNA in Escherichia coli demands attention to factors such as the precise composition of the (ribosome-binding) Shine–Dalgarno (SD) sequence, and the number of nucleotides between the SD sequence and the start codon. Moreover, optimal translation may depend on the provision of a translational enhancer (‘downstream box’) located close to the start codon. Failure to optimize these features may result in a marked reduction in the yield of recombinant protein.

Codon bias

The synthesis of a heterologous protein (for example, when a mammalian gene is expressed in E. coli) may be inefficient owing to CODON BIAS (q.v.).

(See also CODON OPTIMIZATION.)

Inclusion bodies

Inclusion bodies are insoluble aggregates of the unfolded, or

incorrectly folded, heterologous protein which may develop within the cytoplasm of overexpressing cells. In E. coli, the formation of inclusion bodies can sometimes be inhibited e.g. by using a lower growth temperature (say, 30°C instead of 37°C) or by using the ‘thiofusion’ procedure (see below).

Another approach to the problem of inclusion bodies is to co-express chaperone proteins in overexpressing cells. These are normal constituents of cells that promote correct folding of nascent proteins.

If the inclusion bodies can be correctly folded in vitro (i.e. after isolation from the cells) then the formation of inclusion bodies can be an advantage in that they may facilitate purification of the product, i.e. by simplifying separation of product from cell debris etc.

The problem of proteolysis

Proteolysis of recombinant proteins in the cytoplasm of E. coli may be minimized in several ways. Thus, for example, the given protein may be targeted to the cell’s periplasm (i.e. the region between the cytoplasmic membrane and the cell wall) – in which there are fewer proteolytic enzymes. This is possible e.g. by fusing the gene of interest with the gene of a periplasmic protein (such as DsbA).

Alternatively, the target protein may be made secretable by fusing its gene to the gene of a secreted protein.

A further possibility is re-coding the gene to eliminate particular proteolytic sites in the protein.

Additionally, use may be made of rpoH mutant cells of E. coli. RpoH can promote synthesis of the Lon protease (which degrades abnormal proteins), and these mutants have been found to give increased yields of recombinant proteins in E. coli.

Post-translational modification

Many proteins (particularly those used therapeutically) are non-functional unless they have appropriate post-translational modification, such as the glycosylation of specific amino acid residues; hence, only cells that are capable of such activity are used for the synthesis of these proteins. For this reason, a number of the commercial therapeutic agents are produced in e.g. Chinese hamster ovary cells or baby hamster kidney cells – i.e. mammalian cells which can carry out the correct forms of post-translational modification.

In some cases the absence of glycosylation has little or no effect on normal biological activity; for example, unglycosylated recombinant interleukin-2 (IL-2) has essentially normal biological activity compared with the natural protein, and this agent can be produced satisfactorily in E. coli.

Secreted/intracellular proteins

The secretion of a recombinant protein can be advantageous in a production process because it tends to simplify isolation and purification of the product. Thus, if a protein remains intracellular it is necessary to lyse the cells in order to harvest the product; this entails (i) extra work to ensure efficient lysis of cells, and (ii) the need to separate the (one) target protein from the cell’s own natural proteins (as well as from the other types of cell debris).

The production of recombinant proteins intracellularly can be a problem particularly in E. coli and insect cells.

One approach to the problem of intracellular recombinant proteins is to design the expression vector in such a way that the recombinant gene is fused to another gene, or sequence, which promotes secretion/release of the fusion protein. When isolated in vitro, the product can be cleaved (with a suitable protease) to release the protein of interest.

In E. coli, the so-called thiofusion approach can yield extracellular recombinant products. In this process, the expression vector contains the gene of interest fused with the gene of THIOREDOXIN. This protein is found normally in the cell envelope, and the fusion protein can be released from the cells simply by subjecting the cells to osmotic shock (which disrupts the outer membrane).

Detection/isolation/purification of recombinant proteins

In vitro detection, isolation and purification of recombinant proteins can be facilitated by the use of expression vectors that encode certain types of peptide/protein tag which form a fusion product with the protein of interest. For example, the LUMIO tag provides fluorescent detection of the recombinant protein, while the calmodulin-binding peptide (CBP) of the Affinity® system (from Stratagene, La Jolla CA) helps in the isolation of a recombinant protein by binding the protein to a CALMODULIN-based adsorption resin (see also SIX-HISTIDINE TAG). The KEMPTIDE SEQUENCE permits robust labeling of the protein product.

Separation of the protein of interest from its tag may be achieved e.g. by using an endopeptidase which has a known cutting site (see e.g. ENTEROKINASE; see also TEV protease in the entry TEV).

(See also RAINBOW TAG.)

Quantitation of recombinant proteins

See e.g. Q TAG.

overhang See STICKY ENDS.

overlapping genes Two or more genes that share sequence(s) of nucleotides, part (or all) of a given gene being coextensive with part of another. This arrangement serves e.g. to maximize the amount of information carried by a genome. Such genes may differ e.g. in being translated in different reading frames, or they may be translated in the same reading frame but with different start/stop signals and/or a different pattern of splicing.

[Overlapping genes in mammals: Genome Res (2004) 14 (2):280–286; in microbial genomes: Genome Res (2004) 14 (11):2268–2272.]

overproduction (of proteins) Syn. OVEREXPRESSION.

8-oxoG 7,8-dihydro-8-oxoguanine: a mutagenic base produced in DNA e.g. by reactive oxygen species; if unrepaired (by the BASE EXCISION REPAIR system), it may cause a GC-to-TA transversion mutation as 8-oxoG (in the template strand) may pair with adenine during DNA replication.

During DNA replication, adenine misincorporated opposite a template-strand 8-oxoG can be excised by the Escherichia coli MutY DNA glycosylase (or by the human homolog of

p (1) A prefix often used to indicate a recombinant PLASMID (e.g. pBR322 or any of a large number of commercial vectors such as pBluescript®).

(2)An indicator of the short arm of a CHROMOSOME; it is used when giving a particular location on a specific chromosome – e.g. 4p16, which refers to a location (16) on the short arm of chromosome 4.

(3)A prefix used to denote a particular polypeptide product of a retroviral gene.

P Proline (alternative to Pro).

P element Any of a range of transposable elements (up to ~3 kb) found in certain strains of Drosophila. Excision occurs at the flanking 31-bp inverted repeats, and an 8-bp duplication is created at the insertion site. Transposition occurs in germ line cells. P elements are used e.g. for mutagenesis.

P site (of a ribosome) The ‘peptidyl’ site at which the initiator tRNA binds during translation.

(cf. A SITE.)

P1 plasmid The circular (extrachromosomal) form of the prophage of PHAGE P1.

pIII protein See PHAGE DISPLAY.

p21 protein See RAS.

p53 A gene (in human chromosome 17p) whose product arrests the cell cycle and induces apoptosis following damage to DNA. Loss of p53 activity leads to genetic instability and the survival of DNA-damaged cells. p53 is a tumor-suppressor gene; it can e.g. inhibit oncogene-mediated transformation in cultured cells. (See also NUCLEOPHOSMIN.)

p53 is frequently mutated in (human) cancers; details of mutations in p53 are saved in several databases (for example: www-p53.iarc.fr).

[Microarray-based assay for mutations in p53: BioTechniques (2005) 39(4):577–582 (578–580).]

p53 protein has a short half-life – and is often undetectable immunohistochemically. Mutation may extend the half-life, so that detection of p53 may indicate a mutant allele.

A scintillation proximity assay was used to assay binding between p53 and DNA [BioTechniques (2006) 41(3):303– 308].

P210 protein See ABL.

PACE PCR-assisted contig extension. Following SHOTGUN SEQUENCING and the construction of clone contigs, PACE is used for closing gaps (i.e. regions in the genome for which no cloned fragments of DNA are available). Essentially, PCR is used (with genomic DNA) to amplify a sequence that extends from near the end of a contig into the (unknown) gap region. The outward-facing primers bind specifically to the known region near the end of the contig. The inward-facing primers have arbitrary sequences. In some cases an arbitrary primer will bind (with mis-matches) to a sequence within the unknown region at an amplifiable distance from the specific primer; the products formed can be sequenced, thus extending the known region outwards from the end of the contig.

PACE 2C A PROBE-based test (Gen-Probe, San Diego CA) for detecting the bacterial pathogens Chlamydia trachomatis and Neisseria gonorrhoeae in clinical samples in a single assay; the probe includes sequences specific to each of these organisms, the target sequences being in rRNA. Hybridization of the probe to target(s) is indicated by a hybridization protection assay (see entry ACCUPROBE for details).

A positive PACE 2C screening test can be followed by separate tests for C. trachomatis and N. gonorrhoeae. This is a particularly useful approach because co-infection with both pathogens is quite common.

This is an example of a combination probe test. packaging (of phage DNA) Insertion of genomic DNA into the

phage capsid, and the formation of an infective phage virion: a process that differs in different phages and which, in some cases, is not fully understood.

In phage lambda (λ), genome-length pieces of DNA are cut from a concatemer formed by rolling circle replication. Cuts occur at the regularly repeated cos sites in the concatemer; a terminal cos site locates in the phage capsid, and DNA is fed into the capsid until the next cos site – when cleavage occurs. Thus, a capsid contains the DNA between two consecutive cos sites, and the packaged genome has terminal sticky ends.

Commercial products are available for packing the phage λ genome: see e.g. GIGAPACK.

In phage ϕ29, packaging is dependent on energy from ATP hydrolysis. Moreover, the mechanism of insertion involves a ‘motor’ which contains various phage-encoded products: (i) a connector (consisting of 12 subunits) with a central channel,

(ii)six molecules of RNA (which are designated pRNA), and

(iii)a protein (gp16) whose copy number is not known. The motor may wind DNA into the capsid rather like a bolt drawn

through a rotating nut. Two models for the packaging system in phage ϕ29 have been suggested. In one model the pRNA molecules are attached to the capsid, forming part of the stator; in another model a pRNA–connector complex forms the rotor. Studies using photoaffinity cross-linking indicate that pRNA molecules bind to the N-terminal amino acids of proteins in the connector [Nucleic Acids Res (2005) 33(8): 2640–2649].

packaging plasmid A plasmid encoding certain component(s) of a virion (e.g. capsid proteins) which is co-transfected into cells of a ‘producer cell line’ together with a plasmid that encodes (i) a replication-deficient part of a viral genome and (ii) the gene/fragment of interest. The (replication-deficient) viral particles produced in these cells are used to infect target cells, within which the gene of interest can be expressed.

Packaging plasmids are used e.g. in certain vector systems: see e.g. the lentiviral vector in entry GATEWAY SITE-SPECIfiC

RECOMBINATION SYSTEM (table).

(See also AAV HELPER-FREE SYSTEM.)

pactamycin An antibiotic which inhibits the initiation stage of translation in protein synthesis.

PADAC A violet-colored reagent (a cephalosporin derivative) which forms a yellow product when hydrolyzed e.g. by a β- lactamase.

pAd/CMV/V5-DEST™ vector A destination vector in the

GATEWAY SITE-SPECIfiC RECOMBINATION SYSTEM.

padlock probe A linear ssDNA PROBE in which the two end sections are complementary to adjacent regions of the target sequence; when bound to the target, the two end sections of the probe are therefore juxtaposed and can be ligated – thus circularizing the probe. Ligation is possible only if there is appropriate probe–target complementarity. Non-circularized probes can be eliminated by exonucleases. Padlock probes can be used e.g. to detect small variations in target sequences.

The central region of each padlock probe may include a unique identification sequence (the ZipCode or zipcode); all probes with the same target specificity have the same zipcode (and different zipcodes are found on probes with different target specificities). Universal primer-binding sites (that are the same on all the probes, regardless of zipcode) permit the (circularized) probes to be amplified; target-specific products can then be detected by a MICROARRAY of probes consisting of sequences complementary to the zipcode sequences.

This method has been used e.g. for simultaneous detection of multiple plant pathogens [Nucleic Acids Res (2005) 33(8): e70].

Padlock probes have also been used for the rapid and sensitive detection of the SARS virus in clinical samples [J Clin Microbiol (2005) 43(5):2339–2344].

(See also ZIPCODE ARRAY.)

painting (chromosome) See CHROMOSOME PAINTING.

palifermin See Kepivance® in BIOPHARMACEUTICAL (table). palindromic sequence In a double-stranded nucleic acid mole-

cule, a pair of INVERTED REPEAT sequences such as:

This region has twofold rotational symmetry; if a sequence of nucleotides separates the two inverted repeats, the region is said to have hyphenated dyad symmetry.

A palindromic sequence can exist as a linear molecule (as shown) or as a cruciform (cross-like) structure in which each strand forms a hairpin by intrastrand base-pairing.

Palindromic sequences are commonly present in various sites associated with protein–DNA binding – including e.g. operators and recognition sequences of certain RESTRICTION

Pandoraea apista See ERIC-PCR in the entry REP-PCR. panning (biopanning) See PHAGE DISPLAY.

PAP PYROPHOSPHOROLYSIS-ACTIVATED POLYMERIZATION.

papillomaviruses A category of small (~55 nm), icosahedral, non-enveloped viruses (genome: ccc dsDNA) which infect

epithelial cells. More than 100 types of human papillomavirus (HPV) have been identified; of these, some (e.g. HPV6) are considered low-risk (they give rise e.g. to warts) while others (such as HPV16 and HPV18) are considered high-risk because they can cause e.g. cervical cancer. These viruses (unlike the POLYOMAVIRUSES) are refractory to propagation in cultured cells. Vaccines against HPVs have been made e.g. by using virus-like particles (see VLP) composed of the major (immunogenic) capsid protein.

paramere See MACRONUCLEUS.

paranemic Refers to an unstable juxtaposition of two strands of DNA in which the strands are not wound around each other.

(cf. PLECTONEMIC.) parC See R1 PLASMID.

parD system In the R1 PLASMID: a system promoting stability of COPY NUMBER. The Kid protein is a nuclease which was believed to act solely by cleaving host-encoded mRNAs in plasmid-free cells – the (plasmid-encoded) Kis protein acting as an antidote to Kid. More recently, Kid has been reported to cleave both host mRNAs and a plasmid mRNA (encoding a repressor of plasmid replication) in plasmid-containing cells when the copy number falls [EMBO J (2005) 24(19):3459– 3469].

parenteral administration Administered by a non-oral route.

ParR See R1 PLASMID.

partial fill-in See e.g. LAMBDA fiX II VECTOR.

particle gun Syn. GENE GUN.

partition (of plasmids) (syn. segregation) See PLASMID. partitioning complex See R1 PLASMID.

partner gene See GENE FUSION and GENE FUSION VECTOR.

PathDetect® systems Reporter systems (Stratagene, La Jolla CA) used for investigating the regulatory function of a given, intracellularly expressed, gene product, or of an extracellular stimulus (e.g. a cytokine), on SIGNAL TRANSDUCTION PATH- WAY(S) within mammalian cells.

There are two forms of PathDetect® system. (i) The cis- reporting systems can be used e.g. to determine the ability of a given (intracellularly expressed) gene product to activate a signal transduction pathway, causing a factor (such as NFκB or p53) to bind to a sequence upstream of the promoter of a reporter gene – thus promoting transcription of that gene. (ii) The trans-reporting systems are used e.g. to determine the ability of a given (intracellularly expressed) gene product to activate – directly or indirectly – one of certain specific transcription factors (such as CREB or ELK-1).

In the cis-reporting system, a plasmid containing the gene of interest is co-transfected into mammalian cells with a plasmid containing the cis-reporter gene and a regulatory region. The cis-reporter plasmid contains (i) a reporter gene – either a gene encoding LUCIFERASE or a gene encoding the (lowtoxicity) variant of green fluorescent protein: the humanized Renilla GFP (see HRGFP); (ii) a promoter sequence for the reporter gene; and (iii) upstream of the promoter region, tandem repeats of a particular binding sequence (referred to

as the binding element or response element). Various cis– reporter plasmids are available, each plasmid with a different response element; thus, for example, one type of cis-reporter plasmid contains a response element consisting of 5 tandem repeats of the binding site of NFκB, while another contains a response element consisting of 15 tandem repeats of the binding site of p53. (A response element of choice can be inserted into a plasmid supplied with a multiple cloning site.) If the expressed protein of interest activates a signal transduction pathway which then releases (for example) the active form of NFκB, the NFκB binds to the response element and promotes expression of the reporter gene.

The trans-reporting system includes a reporter plasmid and a trans-activator plasmid – both of which are co-transfected into cells with the expression vector encoding the protein of interest. (There are also positive and negative control plasmids.)

The reporter plasmid of the trans-reporting system includes

(i)a reporter gene encoding e.g. luciferase or β-galactosidase;

(ii)a promoter region; and (iii) upstream of the promoter, a fivefold tandem repeat of a sequence which binds the GAL4 element.

The trans-activator plasmid of the trans-reporting system includes a sequence encoding a fusion protein: GAL4 fused to a transcription factor (e.g. CREB or Elk-1); transcription of this plasmid is mediated by a CMV promoter.

If the expressed protein of interest causes phosphorylation, directly or indirectly, of the activation domain of the fusion protein (i.e. the fusion partner of GAL4), then the activated GAL4 fusion protein binds to the tandem repeat binding site for GAL4 on the reporter plasmid, thus promoting expression of the reporter gene.

[Example of use of PathDetect®: J Neurochem (2006) 96 (1):65–77.]

pathogenicity island (PAI) In a microbial (usually bacterial) genome: a cluster of several to many genes encoding specific determinants of pathogenicity/virulence.

Commonly, the GC% of a PAI is different from that of the organism’s chromosome; this has been taken to indicate that, in general, PAIs were acquired by horizontal gene transfer, i.e. by a process such as conjugation.

Many PAIs are flanked by DIRECT REPEATS; the LEE PAI (see below) is one example of an exception. In Moraxella bovis (hemolytic strains) the mbx operon was found to be flanked by ~700-bp imperfect repeats – which, together with e.g. the GC% of mbx genes, suggested that this region of the genome is a PAI [J Med Microbiol (2006) 55:443– 449].

[PAIs in evolution: BMC Evol Biol (2007) 7(suppl l) S8; in Francisella: BMC Microbiol (2007) 7:1.]

The PAI designated LEE (locus of enterocyte effacement) is a 35-kb element found e.g. in all strains of enteropathogenic Escherichia coli (EPEC). Genes in the LEE PAI encode a socalled type III secretory system which apparently enables strains of EPEC to translocate effector proteins directly into (eukaryotic) target cells. [Type III systems and PAIs: J Med

Microbiol (2001) 50:116–126.] Proteins encoded by the LEE PAI are responsible for the typical ‘attaching and effacing’ lesions produced by this pathogen on intestinal epithelium. Transcription of at least some of the LEE genes is controlled from a regulatory region in the EAF plasmid (EAF = EPEC adherence factor) which occurs in all strains of EPEC.

The chromosomal site occupied by LEE is occupied by a different PAI in strains of UPEC (uropathogenic E. coli); the products of this 70-kb PAI include a hemolysin.

A number of distinct PAIs occur in Salmonella spp. One of them, SPI-1, contains the inv–spa genes which encode a type III secretory system involved in the invasion of intestinal epithelial cells. The PAI SPI-3 encodes the means of survival in macrophages.

In the gastric pathogen Helicobacter pylori, expression of the cagA gene (in the cag PAI), and the unlinked gene vacA, is linked to severe gastrointestinal disease. Apparently, CagA is injected into gastroepithelial cells by H. pylori and causes e.g. proliferation of these cells. VacA is a secreted cytotoxin (95 kDa) which, in vitro, blocks proliferation/activity of T cells.

The VPI pathogenicity island in Vibrio cholerae encodes e.g. cell-surface appendages (the so-called ‘toxin co-regulated pili’ – TCP) which function as receptors for phage CTXΦ – the phage whose genome encodes cholera toxin.

Certain strains of the Gram-positive pathogen Staphylococcus aureus contain a PAI which encodes the toxic shock syndrome toxin.

paucibacillary specimen Any specimen that contains a small number of target bacteria per unit volume. For example, a specimen may contain fewer than ~104 colony-forming units (cfu) per mL; such specimens usually permit detection of the target organism by culture, but nucleic-acid-based tests – which use a much smaller volume of the sample – may fail to detect the organism. Improved detection by a nucleic-acid- based test may be achieved if it is preceded by some form of concentration (such as immunomagnetic separation).

PAXgene™ blood RNA kit A product (from PreAnalytiX Hombrechtikon, Switzerland) used for the isolation of RNA from whole blood. The system was designed to avoid the problem of instability of the RNA profile in vitro (i.e. following collection of blood samples): during collection, storage and transport of blood – at ambient temperatures – the copy number of a given RNA species may change significantly owing to (i) degradation of the RNA, and/or (ii) the ongoing expression of genes following collection of the sample from the patient.

This approach involves collecting the blood in specialized tubes containing a mixture of reagents that stabilize the RNA profile by (i) protecting RNA from degradation by RNases and (ii) inhibiting gene expression.

Briefly, the method is as follows. The specialized collecting tube, containing sample, is centrifuged to pellet nucleic acids. The washed pellet is resuspended in buffers with the enzyme proteinase K and incubated. Following centrifugation, super-