Dictionary of DNA and Genome Technology

exogenote See MEROZYGOTE.

exon See SPLIT GENE and SPLICING.

exon amplification PCR-based amplification using, as template, cDNA copies of the spliced RNA produced during EXON TRAPPING. This approach may be used e.g. to recover exon(s) derived from the sample DNA.

exon-prediction program Computer software that is designed to detect exons within sequences of nucleotides from cloned DNA, the object being to detect the presence of gene(s).

exon skipping See ALTERNATIVE SPLICING.

exon trapping A method used e.g. for demonstrating/detecting the presence of exon(s) – with functional splice sites – within a given fragment of sample DNA. Essentially, the fragment is inserted into a construct (a minigene) which already contains known exon and intron sequences; when the minigene (which is part of a plasmid) is transcribed in vivo, exons in the minigene may be spliced to any exon(s) that may be present in the fragment. If cDNA copies of the spliced transcript are then amplified by PCR, the length and composition of the amplicons can indicate whether the transcript contains only minigene exons or includes exon(s) from the sample fragment. If required, specific amplified exon(s) can then be recovered by the use of appropriate PCR primers.

In one scheme for exon trapping, the minigene forms part of a 6-kb plasmid shuttle vector which includes an origin of replication and an ampicillin-resistance gene for replication (and selection) in Escherichia coli. For replication and RNA splicing in suitable mammalian cells, one terminal region of the minigene consists of an origin of replication and promoter region from simian virus 40 (SV40), while the other terminal region consists of the SV40 polyadenylation signal (encoding the 3′ end of the transcript). The sequence between these two regions consists of two exons that are separated by an intron; the intron includes a multiple cloning site (i.e. POLYLINKER) into which the sample DNA fragment is inserted. Following transcription of the minigene in COS cells (recombinant cells prepared from a monkey cell line), several types of splicing pattern may be detected by amplifying cDNA copies of the transcripts. In the absence of (functional) exon(s) in the DNA sample fragment, the two exons in the minigene are spliced together (forming a sequence of known length). However, a functional exon present in the fragment may be spliced to the minigene’s exons; in this case, primers that are complementary to sequences in the minigene’s exons can be used to produce amplicons that indicate the composition of these transcripts.

This general approach – using minigenes in an expression system – has also been valuable for investigating the effect on splicing of mutations or SNPs and for studying the overall regulation of pre-mRNA splicing, including ALTERNATIVE SPLICING (which includes various tissue-specific forms of splicing). A recently proposed minigene plasmid vector may be useful e.g. for (i) assessing the effect of disease-associated mutations on the splicing of relevant gene(s); (ii) identifying cis-acting elements that are involved in normal regulation of

splicing; and (iii) examining the effects of specific regulatory factors and regions on alternative splicing [BioTechniques

exonuclease Any enzyme which cleaves terminal nucleotides from a nucleic acid molecule.

The lambda exonuclease acts preferentially 5′-to-3′ on a 5′- phosphorylated strand of blunt-ended dsDNA. This enzyme is therefore useful e.g. for preparing single-stranded products (e.g. for SSCP ANALYSIS) from PCR amplicons. To prepare ssDNA products, PCR is carried out with one of the primers having a 5′-terminal phosphate – so that one strand of the amplicon will be susceptible to digestion by the lambda exonuclease.

(See also subsequent entries.)

exonuclease III An enzyme (product of the Escherichia coli gene xthA) which degrades blunt-ended dsDNA (or dsDNA with 5′ overhangs) from the 3′ end of each strand, leaving the 5′ end intact and producing 5′-phosphomononucleotides; the enzyme, which does not degrade 3′ single-stranded overhangs, has been used e.g. for preparing NESTED DELETIONS in cloned DNA.

The enzyme also has activity similar to that of RNASE H and it can function as an AP endonuclease in the BASE EXCISION REPAIR process.

(See also EMEA.)

exonuclease VII An enzyme (product of the Escherichia coli gene xseA) which degrades the ssDNA of 3′ and 5′ overhangs but does not degrade dsDNA.

exonuclease-mediated ELISA-like assay See EMEA. 5′-exonuclease PCR Syn. 5′-NUCLEASE PCR. exoribonuclease See RIBONUCLEASE.

exosome (1) In eukaryotic cells: a multi-protein complex containing 3′-to-5′ exoribonucleases involved e.g. in degrading aberrant pre-mRNA, processing snRNAs and mediating the maturation of 5.8S rRNA; the RNA-binding protein MPP6 in HEp-2 cells is apparently needed for the latter role [Nucleic Acids Res (2005) 33(21):6795–6804].

(2) The term has also been used to refer to a DNA fragment that may replicate, and be expressed, when taken up by a cell but which may not be integrated in the host’s DNA.

expanded gold standard See GOLD STANDARD.

exponential silencing Downregulation of promoters of specific bacterial gene(s) associated with rapid growth in a rich medium [EMBO J (2001) 20:1–11].

exportins See KARYOPHERINS.

expressed sequence tag (EST) A recorded sequence of nucleotides (often ~200–400 nt), derived e.g. from a cDNA library, which is kept in a database as a record of the expression of an unknown gene from a particular type of cell under given conditions.

A given EST, with a sequence of interest, may be located in a database by computer sequence-matching programs.

A pair of primers, specific for a given EST, can be design-

ed and then used in a PCR assay of a large, cloned random fragment of genomic DNA; if this assay yields products that correspond to the given EST (i.e. if the fragment contains a homologous sequence), then the relevant sequence within the random fragment of DNA is referred to as a sequence-tagged site (STS).

Given an assortment of large, cloned random fragments of genomic DNA (collectively, covering a large region of the genome), each of these fragments can be assayed for STSs corresponding to a large number of different ESTs. If, as a result of all these assays, various STSs are located in each of the random fragments, this permits alignment of fragments with overlapping sequences; thus, for example, if fragment A has STSs X and Y, and fragment B has STSs Y and Z, then fragments A and B probably overlap at Y (‘probably’ because the Y STS may be a sequence that occurs more than once in the genome).

Analysis of 10000 ESTs, derived from a cDNA library, has been used to increase understanding of the immune system in the cynomolgus monkey (Macaca fascicularis) and – given the similarities between the genome of M. fascicularis and the human genome – may also prove to be useful for revising annotations of relevant genes in the human genome [BMC Genomics (2006) 7:82].

[Assembly of approximately 185000 ESTs from the cotton plant (Gossypium): Genome Res (2006) 16(3):441–450.]

expression bacmid See BAC-TO-BAC. expression platform See RIBOSWITCH. expression site (of trypanosomes) See VSG.

expression vector A VECTOR which encodes functions for the transcription/translation of a gene (or other sequence) carried by the vector (or subsequently inserted into the vector).

An expression vector may include: (i) an appropriate origin of replication; (ii) a marker gene (e.g. an antibiotic-resistance gene); (iii) one or more promoters (allowing e.g. transcription of the insert in either direction); (iv) a segment corresponding to the Shine–Dalgarno sequence in the transcript (for translation in bacteria); (v) a POLYLINKER (allowing flexibility when inserting target DNA); (vi) a transcription terminator (to avoid unwanted readthrough); (vii) an appropriate control system for initiating transcription of the insert.

Target DNA may be initially cloned by replication in the expression vector. Transcription can be initiated, as needed, e.g. via a lac operator (see LAC OPERON) located upstream of the coding sequence and controlled by the LacI repressor protein; in this case, repression can be lifted (i.e. transcription initiated) by adding IPTG.

Cloning and expression can be carried out as separate processes, in different organisms, in a SHUTTLE VECTOR.

Expression of a gene carried by an expression vector may be regulated by a chromosomally located control system (see e.g. CASCADE EXPRESSION SYSTEM) – or by a control system located on the vector itself.

The use of an EPITOPE TAGGING system may facilitate the detection/isolation of proteins.

(See also PESC VECTORS.)

The EXCHANGER SYSTEM of expression vectors is useful e.g. for epitope tagging of proteins and also for changing the selectable marker in a vector (by site-specific recombination) without the need to re-locate the insert/gene of interest.

Some expression vectors include a sequence that encodes a fusion partner for the gene of interest in order e.g. to increase the solubility of the target protein – the fusion product being more soluble than the target protein: see e.g. CHAMPION PET

SUMO VECTOR.

Engineering gene expression in trypanosomes – a class of eukaryotic microorganisms – is associated with an additional problem. Apparently, trypanosomes generally do not regulate expression of their protein-encoding genes by controlling the initiation of transcription. Consequently, for studies on gene expression with an inducible transcription system in these organisms there is a requirement to construct such a system from exogenous sources. For Trypanosoma cruzi (the causal agent of Chagas’ disease in Central and South America), a stable tetracycline-inducible expression vector (pTcINDEX), containing a multiple cloning site (MCS), was designed to integrate into the genome at a specific, transcriptionally silent location, thus avoiding some of the problems of integration within an endogenously transcribed site. [pTcINDEX: BMC Biotechnol (2006) 6:32.]

(See also the table of destination vectors in entry GATEWAY

SITE-SPECIfiC RECOMBINATION SYSTEM.)

Expressway™ Plus expression system A system (Invitrogen, Carlsbad CA) for in vitro protein synthesis (see CELL-FREE

PROTEIN SYNTHESIS).

ExSite™ site-directed mutagenesis kit A kit (Stratagene, La

Jolla CA) used for PCR-based SITE-DIRECTED MUTAGENESIS

in which deletions, insertions or point mutations can be introduced into almost any double-stranded plasmid, eliminating the need for e.g. phage M13-based vectors.

Essentially, the type of mutation produced depends on the design of the primers. A point mutation can be introduced by using primers with a single-base substitution. A deletion can be produced by primers which bind at sites separated by the sequence to be deleted. An insertion can be introduced by the use of primers with a 5′ extension.

Following PCR, the reaction mixture includes the following molecules: original template DNA (which had been methylated in vivo); linear, newly synthesized (unmethylated) DNA; and linear, hybrid DNA which consists of parental and newly synthesized strands. The reaction mixture is then subjected to restriction enzyme DpnI (see table in the entry RESTRICTION ENDONUCLEASE for details). Blunt-ended ligation of newly synthesized DNA is then carried out with T4 DNA ligase – this providing circular, mutagenized molecules suitable for transformation of Escherichia coli.

ExSite™ has been used e.g. to replace a GTC codon with a GAG codon [J Bacteriol (2006) 188(7):2604–2613], and to introduce mutations into the hmuR gene (encoding a hemin receptor) in a periodontitis-associated Gram-negative patho-

gen, Porphyromonas gingivalis [Infect Immun (2006) 74(2): 1222–1232].

extein See INTEIN.

extendase activity (template-independent polymerase activity) During PCR: the addition of an extra residue of an adenosine nucleotide to the 3′ end of the product strand (i.e. beyond the final, template-determined 3′ nucleotide) when synthesis is mediated e.g. by TAQ DNA POLYMERASE. Single-nucleotide (adenosine) 3′ overhangs can be exploited e.g. in facilitating insertion of the amplicons into certain types of vector which have terminal single-nucleotide thymidine overhangs – see

e.g. TOPOISOMERASE I CLONING and CHAMPION PET SUMO VECTOR. (See also TOPO TA CLONING KIT.)

The expression of extendase activity is strongest when the last (template-determined) residue at the 3′ end of the product strand is a pyrimidine; if required, extendase activity can be

promoted by including a purine (rather than a pyrimidine) in the relevant position in the template. If the last residue at the 3′ end of the product strand is a purine, extendase activity is less efficient.

If blunt-ended products are required from PCR, amplicons with the 3′ overhang can be subjected to exonuclease action:

see POLISHING.

(See also END-IT DNA END-REPAIR KIT.)

extended BiFC See BIMOLECULAR flUORESCENCE COMPLEM-

extrachromosomal inheritance Syn. CYTOPLASMIC INHERIT-

ANCE.

extranuclear genes Syn. CYTOPLASMIC GENES.

EYFP Enhanced YELLOW flUORESCENT PROTEIN (q.v.).

F L-Phenylalanine (alternative to Phe).

F− recipient See F PLASMID.

F+ donor See F PLASMID.

F factor See F PLASMID. F-like pili See PILUS.

F plasmid (formerly F factor) A specific, IncFI, conjugative, low-copy-number plasmid (~95 kb) found e.g. in Escherichia coli; the F plasmid can exist either as an extrachromosomal replicon or integrated in the host chromosome – and is able to mediate CONJUGATION in either location.

Cells containing the free, circular plasmid are conjugative donor cells (F+ cells); potential recipient cells (F− cells) lack the plasmid. The F+ cells express (plasmid-encoded) donor functions (e.g. a PILUS) and are derepressed for conjugation – meaning that all, or nearly all, cells in an F+ population can function as donors (see fiNOP SYSTEM); hence, in a mixed population of F+ and F− cells, most or all F− cells receive an F plasmid (through conjugation) and are converted to F+ cells.

Chromosomal integration of the F plasmid gives rise to an Hfr donor (Hfr means high frequency of recombination); Hfr donors transfer chromosomal (as well as plasmid) DNA to F− recipients, the amount of DNA transferred increasing with increased length of uninterrupted mating. (The F plasmid in an Hfr cell is said to mobilize the chromosome for transfer.)

(See also INTERRUPTED MATING.)

An F plasmid may excise from the chromosome aberrantly to form an F′ plasmid (see PRIME PLASMID).

A protein, SopA, involved in the partitioning of plasmids to daughter cells during cell division, was reported to polymerize into filaments, in vitro; these filaments elongated at a rate similar to the rate of plasmid separation in vivo [Proc Natl Acad Sci USA (2005) 102(49):17658–17663].

Transcription from the PY promoter of the tra (transfer) operon of the F plasmid was reported to be repressed by the host’s H-NS protein [J Bacteriol (2006) 188(2):507–514].

(See also CCD MECHANISM and PIF.)

F′ plasmid See F PLASMID and PRIME PLASMID.

f1 phage See PHAGE F1.

Fab fragment (of an antibody) See ZENON ANTIBODY-LABEL- ING REAGENTS.

Fabry disease An X-linked disease involving a deficiency in activity of the enzyme α-galactosidase A. Various manifestations are reported, including e.g. vascular-type skin lesions and cardiac symptoms; the cause of death is reported to be usually renal failure.

A recombinant enzyme, Fabrazyme®, is used for treatment (see entry BIOPHARMACEUTICAL (table)).

facile (of procedures) Able to be carried out easily – i.e. with no specific or unspecified difficulties.

FACS flUORESCENCE-ACTIVATED CELL SORTER.

factor VIIa A factor involved in the blood-clotting mechanism (see NovoSeven® in entry BIOPHARMACEUTICAL (table)). factor VIII A factor involved in the blood-clotting mech-

anism – see ‘hemophilia’ in the table in entry GENETIC DIS-

EASE, and see also KOGENATE.

factor IX A factor involved in the blood-clotting mechanism – see CHRISTMAS FACTOR, and see also ‘hemophilia’ in entry

GENETIC DISEASE (table).

factor essential for methicillin resistance ( fem) See MRSA.

FAM 6-Carboxyfluorescein: a fluorescent reporter dye with an emission maximum at 525 nm and an excitation maximum at 493 nm.

farnesol A sesquiterpene alcohol reported to act as a QUORUM SENSING molecule in Candida albicans [autoregulation and quorum sensing in fungi: Eukaryotic Cell (2006) 5(4):613– 619].

Farnesol and the antibiotic gentamicin were reported to act synergistically against the bacterium Staphylococcus aureus [Antimicrob Agents Chemother (2006) 50(4):1463–1469].

FBS Fetal bovine serum.

Fc portion (of an antibody) See ZENON ANTIBODY-LABELING REAGENTS.

fd phage See PHAGE FD.

feature The exact location (on a coated glass surface) where a given DNA probe is synthesized during the preparation of a GeneChip® (Affymetrix) MICROARRAY; a feature is ~5 µm in size.

feeder cells Cells which may be included in cultures of STEM CELLS in order to promote growth of the latter. For mouse ES cells in culture the engineered expression of Bcl-2 has been reported to override the requirement for feeder cells [Proc Natl Acad Sci USA (2005) 102(9):3312–3317].

feline immunodeficiency virus See LENTIVIRINAE. feline syncytial virus See SPUMAVIRINAE.

fem genes See MRSA.

female-specific phage Any of certain phages (e.g. T7, W31) whose replication is inhibited in cells containing particular plasmids; for example, production of intermediate and late phage proteins of T7 is inhibited in cells of Escherichia coli containing the F PLASMID.

Note that cells containing conjugative plasmids are said to be ‘male’ cells; cells lacking these plasmids are ‘female’ cells.

In Pseudomonas aeruginosa, the DNA of phages F116 and G101 is degraded by RESTRICTION ENDONUCLEASE activity in cells containing plasmid pMG7.

(See also PIF.)

femto- In SI (Système International) units: a prefix meaning 10−15.

For a list of prefixes see the ‘Ready reference’ section at the front of the dictionary.

FEN1 (FEN-1) See flAP ENDONUCLEASE.

Fenton reaction The reaction in which ferrous iron is oxidized to ferric iron by hydrogen peroxide with the production of hydroxyl radical (·OH):

Fe(II) + H2O2 → Fe(III) + ·OH + OH−

A Fenton reaction at the endoplasmic reticulum is reported to influence the expression of hypoxia-inducible genes [Proc Natl Acad Sci USA (2004) 101(12):4302– 4307].

The highly reactive hydroxyl radical, produced in a Fenton reaction, can be employed to obtain fine resolution in FOOT- PRINTING studies [Nucleic Acids Res (2006) 34(6):e48].

In vivo footprinting (in frozen cells) has been achieved with a brief exposure to a synchrotron X-ray beam that produces hydroxyl radical footprints similar to those obtained in vitro using the Fenton reaction [Nucleic Acids Res (2006) 34(8): e64].

fermentation (1) In an industrial/commercial context: any process involving the large-scale culture (i.e. growth) of microorganisms, regardless of whether metabolism is fermentative or respiratory (oxidative). This use of the term ‘fermentation’ is in sharp distinction to that given in meaning (2), below.

(2) (energy metab.) A specific energy-converting process in which a substrate is metabolized without the involvement of an external electron acceptor, oxidation and reduction within the process being balanced. ATP is characteristically produced by substrate-level phosphorylation.

(cf. RESPIRATION.)

fes (FES) An ONCOGENE in strains of feline sarcoma virus. The v-fes product has TYROSINE KINASE activity.

FHA The filamentous hemagglutinin produced by the (Gramnegative) bacterium Bordetella pertussis.

(See also INTEGRINS.)

FhuA protein See TONA GENE.

field inversion electrophoresis See PFGE. filament pyrolyser See PYROLYSIS.

filamentous hemagglutinin (FHA) In Bordetella pertussis: a cell-surface ligand which binds the αMβ2 INTEGRIN.

filgrastim See Neupogen® in BIOPHARMACEUTICAL (table). fimbria (bacteriol.) A term often regarded (illogically) as a

synonym of PILUS (q.v.). Fimbriae are thin, proteinaceous filaments which project from the surface of certain types of cell and which have roles e.g. in cell–cell adhesion, cell–substrate adhesion and (in some cases) in a type of motility referred to as ‘twitching motility’.

fingerprint (DNA) A pattern formed by a number of bands of stained (or labeled) fragments of DNA, of different sizes, that are present within a gel medium (generally following electrophoresis of a restriction-digested sample) or, subsequent to a blotting procedure, on a nitrocellulose (or other) membrane; the nature of any given fingerprint will depend on the particular sample of DNA which is being examined and on the method used to produce the fingerprint.

Fingerprints are generated by various TYPING procedures (which often produce strain-specific fingerprints).

FinOP system A regulatory system, involving plasmid genes finO and finP, which controls conjugative transfer in most F- like plasmids. The products of finO and finP jointly inhibit the expression of traJ, a gene in the ‘transfer operon’ which has a key role in the initiation of conjugative transfer.

The product of finP is a molecule of ANTISENSE RNA which

binds to the traJ transcript. The product of finO, a polypeptide referred to as an ‘RNA chaperone’, stabilizes the activity of the antisense RNA. With a fully active FinOP system in operation the transfer system remains repressed and conjugation remains inhibited.

The F PLASMID is (constitutively) derepressed for transfer, i.e. it is permanently conjugation-competent. Earlier, it had been thought that this was due to the absence of a finO locus in this plasmid. Later it was shown that finO is present but is inactivated by an insertion sequence, IS3, which disrupts the coding sequence of the gene. As a consequence, FinO is not available to act jointly with the finP product.

first strand (of cDNA) The strand of DNA synthesized on an mRNA molecule by reverse transcriptase. If only a few types of mRNA are being targeted then gene-specific primers may be used. If many, or all, mRNAs are being targeted (e.g. for a microarray experiment) then oligo(dT) primers may be used; these primers bind to the 3′ poly(A) tails that are commonly present on eukaryotic mRNAs. Another possibility is the use of random primers (e.g. random hexamers); in one study it was found that the use of random pentadecamer (i.e. 15-mer) primers resulted in an improved range and yield of cDNAs compared with the use of random hexamers [BioTechniques (2006) 40(5):649–657].

mRNA is removed from the RNA/DNA hybrid – e.g. by RNASE H or by alkaline hydrolysis – and the second strand of DNA is synthesized, making ds cDNA.

In the Gubler–Hoffmann procedure, the hybrid RNA/DNA structure is treated by controlled enzymic action – leaving short sequences of RNA; DNA polymerase I (an enzyme with 5′-to-3′ exonuclease activity) is then used to synthesize the second strand. Hence, the second strand is produced without the use of an exogenous primer.

[Example of the Gubler–Hoffmann method: BioTechniques (2005) 38(3):451–458.]

(See also entry CDNA.)

FISH Fluorescence in situ hybridization: a particular form of in situ hybridization (ISH) in which the label is a fluorophore. In one approach, BIOTINylated probes, bound to their target, are detected by a STREPTAVIDIN-conjugated fluorophore.

In an alternative approach, the probe itself incorporates a fluorophore (for example, ChromaTide™: see entry PROBE

LABELING).

Increased sensitivity may be achieved by using TYRAMIDE

SIGNAL AMPLIfiCATION.

FLAG® A peptide (sequence: DYKDDDDK) used for tagging proteins by creating protein–peptide fusions; a protein tagged with this sequence can be detected/recovered by monoclonal antibodies specific for the tagged region of the protein.

[Uses of FLAG® (examples): Mol Biol Cell (2006) 17(8): 3356–3368; Mol Cell Biol (2006) 26(7):2697–2715.]

nuclease involved e.g. in DNA replication (see OKAZAKI FRAGMENT). FEN1 was also reported to exhibit a gap endo-

nuclease function which may have a part to play e.g. in the resolution of stalled replication forks [mode of FEN1 activity with gap substrates: Nucleic Acids Res (2006) 34(6):1772– 1784].

FLASH® chemiluminescent gene mapping kit A kit (Stratagene, La Jolla CA) designed for high-resolution restriction mapping.

Essentially, the gene (or fragment) – flanked by T3 and T7 promoter sites – is initially cloned in a vector. The gene is then excised from the vector, still flanked by the T3 and T7 sites. (Excision occurs at two NotI recognition sites; the NotI sites are used because they are the recognition sites of a ‘rarecutting’ enzyme – an enzyme which is unlikely to interfere with the activity of the particular enzyme whose target sites are being mapped.)

The excised gene is exposed to partial digestion with the given RESTRICTION ENDONUCLEASE whose target sites are to be mapped.

The fragments produced by the restriction enzyme are subjected to electrophoresis in an agarose gel, and the bands of fragments are transferred to a hybridization membrane.

Bands of fragments on the membrane are probed with a T3 sequence that is conjugated to ALKALINE PHOSPHATASE. The hybridization membrane is then incubated with a chemiluminescent substrate (see CHEMILUMINESCENCE), and the light signal from the target–probe complex is detected within 30 minutes on radiographic film.

The position of each band in the resulting ladder reflects the distance between one end of the gene (i.e. the T3 site) and one of the cutting sites of the given restriction enzyme. The bands in the ladder therefore indicate the various cutting sites of the given enzyme within the gene.

By replicating the procedure with an alkaline phosphataseconjugated T7 probe, a complementary set of bands are seen – each band reflecting the distance between one end of the gene (i.e. the T7 end in this case) and one of the cutting sites of the restriction enzyme. Hence, the second determination (with the T7 probe) serves to confirm the results obtained in the first determination.

flood plate A PLATE whose surface has been flooded with a (concentrated) liquid suspension of a given organism (usually a suspension of bacteria) and excess liquid removed e.g. with a sterile Pasteur pipet.

A (‘dried’) flood plate is used e.g. in PHAGE TYPING. floral dip method A method first used for the transfection of

Arabidopsis thaliana with Agrobacterium tumefaciens, which involves dipping developing floral tissues (including floral buds) into a solution containing 5% sucrose, 500 µL/L of the surfactant Silwet L-77 and cells of A. tumefaciens. [See also Methods Mol Biol (2005) 286:91–102, (2005) 286:103–110.] [Use of method in transcriptional profiling of the Arabidopsis embryo: Plant Physiol (2007) 143(2):924–940.]

floxed DNA A segment of DNA flanked, on both sides, by a

loxP site (see CRE–LOXP SYSTEM).

Flp (FLP) A site-specific recombinase (encoded by the two-

micron plasmid) first isolated from the yeast Saccharomyces; Flp is a member of the integrase family of recombinases, and a tyrosine residue is found at the active site which mediates phosphodiester bond cleavage.

Within S. cerevisiae Flp catalyzes the inversion of a section of the plasmid flanked by a pair of (inverted) recognition sequences.

In recombinant DNA technology, Flp is used e.g. for the excision of specific sequences in vivo. The enzyme is active in both eukaryotes (e.g. Drosophila) and bacteria (including Escherichia coli). The enzyme can excise a sequence that is flanked, at both ends, with a so-called Flp recognition target (FRT) sequence – both FRTs being in the same orientation; the excised fragment is in the form of a circular molecule.

A plasmid containing one FRT site can be inserted, by the action of the Flp recombinase, into another replicon that also contains a copy of the FRT site.

[Example of use of the Flp recombinase: BioTechniques (2006) 40(1):67–72.]

[Modular and excisable molecular switch for the induction of gene expression by the Flp recombinase: BioTechniques (2006) 41(6):711–713.]

(See also the vector pEF5/FRT/V5-DEST™ in the table for

entry GATEWAY SITE-SPECIfiC RECOMBINATION SYSTEM.)

Certain peptides which inhibit Flp have been reported from studies on the HOLLIDAY JUNCTION (q.v.).

(See also CRE–LOXP SYSTEM.)

Flp-In™ cell lines Cell lines from Invitrogen (Carlsbad CA) in which the target site (FRT) of the Flp recombinase (see flP) has been inserted stably into a transcriptionally active region of the genome. These cells are designed to be co-transfected with an FRT-containing Flp-In™ expression vector, carrying the gene of interest, and a plasmid (pOG44) encoding the Flp recombinase; within these cells, targeted integration of the expression vector occurs at the same location in each cell – promoting uniform levels of expression of the given gene of interest.

Flp-In™ cells have been prepared from various types of parental cell, including BHK, CHO, Jurkat and 293. In some of these cells a CMV promoter works well, but in others (e.g. BHK) expression from a CMV promoter is reported to be downregulated; in the latter cells, Flp-In™ vectors with the EF-1α promoter are recommended.

(See also vector pEF5/FRT/V5-DEST™ in GATEWAY SITE-

SPECIfiC RECOMBINATION SYSTEM (table).)

fluctuation test A classic, early (1943) test, devised by Luria and Delbrück, for examining the way in which populations of bacteria respond to changes in the environment.

Two hypotheses co-existed at the time: (i) genetic change is induced by environmental influences (i.e. an adaptive mechanism), and (ii) genetic change occurs spontaneously (that is, independently of environmental influences) – cells in which such changes have occurred being selected (and proliferating) if conditions become suitable.

Essentially the (statistics-based) test measured the variance

between numbers of phage-resistant (mutant) cells in each of a set of individual, separate liquid cultures of phage-sensitive bacteria; this was compared with the variance in numbers of phage-resistant mutants in each of a number of samples taken from a single (bulk) liquid culture. (The numbers of phageresistant mutants were ascertained by plating aliquots from the cultures on separate plates and then exposing the plates to virulent phage.)

The high variance (wide fluctuation) in numbers of mutants from the separate cultures indicated that the (phage-resistant) mutant cells had arisen spontaneously – at different times in different cultures – i.e. that the appearance of mutants was unrelated to exposure of the bacteria to phage.

(See also NEWCOMBE EXPERIMENT.) fluorescence (in vivo) See IN VIVO flUORESCENCE.

fluorescence-activated cell sorter (FACS) An instrument used for flow cytometry in which individual, fluorophore-labeled cells are counted by recording the fluorescence they emit, on excitation, when passing through a narrow aperture.

fluorescence in situ hybridization See fiSH. fluorescence resonance energy transfer See FRET.

fluoroquinolones See QUINOLONE ANTIBIOTICS.

FluoroTrans® PVDF membranes See PVDF.

5-FOA See YEAST TWO-HYBRID SYSTEM.

foamy viruses Viruses of the subfamily SPUMAVIRINAE. footprinting A technique that is used to determine the location

(i.e. sequence) at which a given ligand (e.g. a DNA-binding protein) binds to DNA.

Note. An entirely unrelated technique is known as GENETIC

FOOTPRINTING.

This account refers specifically to in vitro footprinting – see also the entry for IN VIVO FOOTPRINTING.

Initially, two identical populations of end-labeled sample fragments of DNA are prepared, and the particular ligand is allowed to bind to fragments in one of the populations. Each of the populations is then subjected to either enzymatic or chemical cleavage under conditions in which each fragment is (ideally) cut at only one of a large number of possible sites. In each population of fragments, the result is a collection of subfragments, present in a range of different lengths, that are produced by cleavage at different sites.

In the population of ligand-bound fragments, cleavage will not have occurred at those cleavage sites which had been shielded from the enzyme (or chemical agent) by the bound ligand; this population will therefore lack a certain range of subfragments that are present in the other population. The absence of certain subfragments in the ligand-bound population will be seen as a gap when the two populations of subfragments are compared by gel electrophoresis; this gap is the ligand’s footprint, and its position in the gel (in relation to the bands of subfragments) indicates the ligand’s binding site on the DNA.

In one approach, cleavage of fragments is carried out by the enzyme DNase I. This tends to produce a large and clear footprint because DNase I is a large protein that will not cut

the DNA at sites close to a large DNA-bound ligand (such as a DNA-binding protein).

Resolution can be improved (i.e. the footprint can be better defined) by using a small chemical agent to cleave the DNA. One such agent is hydroxyl radical (·OH) which brings about sequence-independent cleavage of DNA: any phosphodiester bond which is unprotected by the bound ligand is susceptible to cleavage. Footprinting with the hydroxyl radical can be achieved by means of the FENTON REACTION [Nucleic Acids Res (2006) 34(6):e48]. Footprinting with hydroxyl radical in frozed cells (i.e. in vivo) can be achieved by using a brief pulse of X-radiation from a synchrotron – which can generate hydroxyl radical footprints similar to those generated in vitro by the Fenton reaction [Nucleic Acids Res (2006) 34(8):e64].

forced cloning (directional cloning) Any procedure designed to ensure that target DNA is inserted into a vector molecule in a definite, known orientation; insertion in this way is required e.g. when the target DNA is subsequently to be transcribed from a promoter sequence within the vector.

forensic applications (of DNA-based technology) DNA-based technology can offer a range of possibilities for assisting with investigations. Moreover, given that DNA may survive for long periods of time, particularly in the right kind of tissue under appropriate conditions, current methods can sometimes resolve crimes that have remained unsolved for many years.

DNA-based identification

DNA is naturally important in the context of identification – including the identification of those killed in natural disasters such as earthquakes, floods and tsunamis as well as victims (or perpetrators) of crime; moreover, this technology also has a role e.g. in cases involving paternity issues.

Each investigation starts with one or more samples that are sources of DNA. The quality and quantity of DNA available from a given sample can vary enormously according to (i) the type of sample, (ii) the conditions under which the sample existed prior to collection, and (iii) the time for which it was exposed to those conditions.

Positive, unambiguous analysis of DNA requires a sample of at least a certain minimum quantity – e.g. of the order of nanograms – which has not been degraded to an extent that precludes its use. Fortunately, modern methods permit truly minute quantities of DNA to be characterized, and a number of commercial methods (e.g. CHARGESWITCH TECHNOLOGY) can efficiently recover the DNA from various types of crude sample. Even poor quality/damaged DNA may still be of use if it is susceptible to PCR amplification by the Y FAMILY of DNA polymerases [Nucleic Acids Res (2006) 34 (4):1102– 1111].

Degraded DNA in small amounts is reported to be susceptible to isothermal whole-genome amplification with primers optimized, bioinformatically, for coverage of the human genome. Degraded DNA, pre-amplified by this procedure, was reported to be satisfactory for STR-based genotyping [DNA Res (2006) 13(2):77–88].

A system for quantifying the level of damage in a sample of

DNA has been devised in a zoological context and may be useful for forensic investigations involving domestic animals or wildlife [Frontiers Zool (2006) 3:11].

Both nuclear DNA and MITOCHONDRIAL DNA are of value for identification purposes. In general, analysis of nuclear (as opposed to mitochondrial) DNA is frequently the preferred option because nuclear DNA has a potentially greater power to discriminate between individuals. Samples of mtDNA may be unsatisfactory in certain cases, e.g. in that they can fail to distinguish between individuals of the same maternal line. Nevertheless, in the USA there is an extensive database of mtDNA – known as the mtDNA Popstats Population Database – containing over 5000 mtDNA profiles; this database is intended primarily for law-based and academic use. A given mtDNA profile in the database is described in relation to the (revised) standard ‘Cambridge Reference Sequence’. Ongoing scrutiny continues to reduce errors in this database, which is considered to be a reliable resource for forensic case analysis [Forensic Sci Comm (2005) 7(10)].

A potential problem with the use of mtDNA targets is the existence of so-called ‘mitochondrial pseudogenes’: mtDNAlike sequences which occur in nuclear (chromosomal) DNA. Thus, unless appropriate precautions are taken, these nuclear sequences could be co-amplified by PCR when attempting specifically to amplify particular mtDNA targets. The importance of such potential contamination may be underestimated if it is assumed that the high copy number of mtDNA genes, compared with nuclear pseudogenes, may mask the effect of these nuclear targets: some pseudogenes are represented at multiple sites in nuclear DNA. Some 46 fragments of nuclear DNA – covering all of the human mitochondrial DNA – have been sequenced [BMC Genomics (2006) 7:185].

Voluntary samples of DNA are obtainable e.g. by BUCCAL CELL SAMPLING or from blood etc. Other sources of DNA include hairs, teeth and cells from the skin.

(See also DNA ISOLATION.)

Teeth as a source of DNA

Given adverse conditions, the remains of dead individuals, including isolated body parts, may be poor sources of DNA. However, the more-durable hard tissues, such as teeth, can be a valuable source; moreover, compared with other types of sample, hard-tissue samples may be easier to free from contamination by extraneous DNA.

Hair as a source of DNA

Hair can be useful as a source of DNA, and is particularly valuable when the origin of a given sample is a major factor in an investigation. Prior to DNA analysis, it is important to carry out a thorough macroscopic and microscopic examination of the hair sample because (particularly with a very small sample) the portion used for DNA analysis will no longer be available for physical characterization.

A hair in the active phase of growth is likely to provide a good source of both nuclear and mitochondrial DNA from cells in the root and sheath regions. However, hairs at the end of the growth cycle – lacking follicular components, and shed

from the body – are likely to be poor/inadequate sources of nuclear DNA, although they may be adequate for the analysis of mitochondrial DNA.

[Guidelines for the forensic examination of human hair (including a glossary of descriptive terms): Forensic Sci Comm (2005) 7(2).]

Types of investigation used for identification purposes

Given adequate samples, human and non-human DNA can be readily distinguished (if necessary).

Identification can involve e.g. the analysis of genomic short tandem repeats (see STR) – an approach used in CODIS (q.v.). Male-specific STRs (i.e. those found on the Y chromosome) may be useful for investigations in sex-based cases.

The gender of a subject may be ascertained e.g. by a PCRbased assay of sequences from alleles of the amelogenin gene on the X and Y chromosomes: see DNA SEX DETERMINATION

ASSAY. (See also BARR BODY.)

Results that exclude suspect(s) can be as valuable as those which incriminate.

The area of DNA sampling and identification has important ethical aspects. [Balancing crime detection, human rights and privacy (UK National DNA Database): EMBO Rep (2006) 7 (Special Issue):S26–S30.]

Disasters with mass fatalities

In exceptional events, e.g. major earthquakes and tsunamis, the problem of identification may be exacerbated by an overwhelming number of victims and, in many cases, by the lack of adequate technical facilities. Moreover – in any situation – dental, fingerprint and DNA-based data can be useful only if comparative data (e.g. in a database) are also available.

In the South Asian tsunami of December, 2004, an international collaborative effort assisted with the identification of victims. At the TTVI (Thai Tsunami Victim Identification) center, some 2010 victims had been identified 7 months posttsunami. Of these victims, 1.3% had been identified on the basis of DNA analyses – as compared with 61% by dental records and 19% from fingerprints (some of the victims were identified by a combination of methods). In this scenario it has been considered that DNA-based identification should be reserved for those cases in which attempts at identification by other methods have been unsuccessful (instead of being used as a first-line approach).

[Management of mass fatality following the South Asian tsunami of 2004: PLoS Medicine (2006) 3(6):e195.]

Microbial forensics

Microbial forensics is a multi-disciplinary field of endeavor created in response to the ongoing threat of attack, on populations and/or individuals, by terrorists/criminals using pathogenic microorganisms or their toxins. A high-profile example of such attacks was the letter containing spores of Bacillus anthracis (causal agent of anthrax) sent to a member of the US government in 2001. A specialized laboratory facility, the National Bioforensics Analysis Center (NBFAC), was set up to examine and analyze relevant material.

[Sample collection, handling and preservation for an effect-

ive program of microbial forensics: Appl Environ Microbiol (2006) 72(10):6431–6438.]

Guidance is available for those physicians who believe that a given patient is a victim of bioterrorism (or other type(s) of biocrime). [Biocrimes, microbial forensics and the physician: PLoS Medicine (2005) 2(12):e337.]

The role of DNA-based technology in this field could be seen as central to tasks such as the identification of pathogens and determination of their source(s). Ideally, methods should be available to characterize a given isolate of a pathogen and to link it indisputably to a specific origin. However, this was not possible in the case of letter-borne spores of B. anthracis; in this case, TYPING based on the analysis of tandem repeats in DNA pointed to a laboratory source – but could not offer information that was more specific regarding the origin of the strain. A paper summarizing the requirements of the nascent field of microbial forensics [Appl Environ Microbiol (2005) 71(5):2209–2213] referred to a more informative and highly sophisticated (but not yet realized) typing system that could exploit our expanding knowledge of genome plasticity and bioinformatics. A future system for comparing genomic sequence data (suitable for detecting differences among isolates of a pathogen) was seen in terms of an approach that could accomodate genomic variation due to recombinational events and also possible genome-wide patterns of mutation.

Reliance on selected sequences of nucleotides for identification purposes can be problematic – as was shown e.g. by the finding of genes of B. anthracis virulence plasmid pXO2 in plasmids from other species of Bacillus [J Clin Microbiol (2006) 44(7):2367–2377]. Precision-based identification and comparison of isolates by whole-genome sequencing would require the availability of techniques that are faster and less expensive than those in current use; moreover, even with the development of such techniques, maximum use of the sequencing data is possible only if accessible databases on the pathogens, and related species, are in place.

Typing of B. anthracis on the basis of single-nucleotide repeats (see SNR) has been reported to permit differentiation of isolates with extremely low levels of genetic diversity – i.e. very closely related strains – even when such isolates could not be distinguished by other methods of typing [J Clin Microbiol (2006) 44(3):777–783].

Any typing procedure involving copying specific sequences of genomic DNA is likely to be facilitated by techniques that accelerate amplification – such as an ultra-fast form of PCR [Nucleic Acids Res (2006) 34:e77].

B. anthracis is, of course, only one of a number of possible microorganisms that can be considered in this context. Other, more common, types of pathogen, including those associated with natural outbreaks of disease, are also candidates. Less dramatic, covert events caused by this kind of pathogen may be correspondingly more difficult to detect against the background of routine, natural outbreaks.

It was suggested [Appl Environ Microbiol (2005) 71(5): 2209–2213] that nucleic-acid-based techniques are unlikely

to be able to pinpoint the unique source of a given isolate of a pathogen (a requirement e.g. for tracing perpetrators of bioterrorism). This pointed to the need for chemical and physical analyses to supplement nucleic-acid-based methods in order to supply additional information; in particular, it was thought that the (chemical) composition of microbial cells might be able to reveal more information about their origin if more information were available on the way in which the chemical nature of miroorganisms reflects the type of environment in which they were grown.

Plant pathogen forensics

Bioterrorism directed at agricultural targets has the potential for creating widespread nutritional and economic damage as well as political instability. The diffuse nature of agriculture, and the (relatively) lower level of sophistication needed to target this area, makes this a particularly difficult problem to tackle. Moreover, the majority of plant pathogens are species of fungi which are, in general, technically less amenable than bacteria to laboratory investigation. Additionally, plant pathogens are generally harmless to humans, so that any wouldbe bioterrorist in the agricultural sphere is exposed to minimal risk. The total prevention of an attack on agricultural targets has been assessed to be impossible by the Banbury Microbial Forensics Group, so that vigilence and an effective response to any incident remain the optimal approach.

Information, technology and resources currently available in the agricultural sphere have been assessed for potential use in plant pathogen forensics [Microbiol Mol Biol Rev (2006) 70(2):450– 471].

formamide (H.C=O.NH2) A denaturing agent which lowers Tm

in the THERMAL MELTING PROfiLE of dsDNA.

forward mutation Any mutation that gives rise to a mutant phenotype, commonly by inactivating or modifying a gene. forward primer A PRIMER that binds to a non-coding strand

(see CODING STRAND).

The fragment shown below identifies a forward primer as a sequence of nucleotides – in heavy type – within the coding strand. As an isolated oligonucleotide, this eight-mer primer, 5′-GCTATGCT-3′, would bind to the underlined sequence in the non-coding strand:

5′-GCTATGCTCTGCC/......./GGGACTCGGCACGA-3′ (coding strand )

3′-CGATACGAGACGG/......./CCCTGAGCCGTGCT-5′ (non-coding strand )

Consequently, when extended, this primer would give rise to a sequence identical to the coding strand.

A reverse primer binds to the coding strand. In the fragment shown, a reverse primer is identified (in heavy type) as a sequence of nucleotides within the non-coding strand. As an isolated oligonuleotide, 5′-TCGTGCCG-3′ would bind to the underlined sequence in the coding strand.

fosmid A COSMID based on the F PLASMID; intracellularly, it