DNA sequence analysis of the imp UV protection and mutation operon of the plasmid TP110: identification of a third gene

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1 1990 Oxfod Univesity Pess Nucleic Acids eseach, Vol. 18, No DNA sequence analysis of the imp UV potection and mutation opeon of the plasmid TP110: identification of a thid gene David Lodwick, Daeca Owen and Pete Stike* Depatment of Genetics and Micobiology, Univesity of Livepool, PO Box 147, Livepool L69 3BX, UK eceived June 19, 1990; evised and Accepted July 31, 1990 ABSTACT The sequence of the Imp opeon of the plasmid TP110 (which belongs to the Incl, incompatibility goup) has been detemined, and is shown to contain thee open eading fames. This opeon, involved in UV potection and mutation, is functionally analogous to the umudc opeon of E.coli and the muc AB opeon of the plasmid pkm101, which belongs to the quite unelated IncN incompatibility goup. The umu and muc opeons howeve contain only two open eading fames, coding fo poteins of appoximately 16kD and 46kD. The high degee of homology between the two 16kD poteins (UmuD and MucA) and between the two 46kD poteins (UmuC and MucB) clealy shows thei elatedness. This is shown also to extend to the Imp gene poducts, with ImpA shaing homology with UmuD and MucA, and ImpB shaing homology with UmuC and MucB. Howeve, the two Imp genes ae peceded in the opeon by a thid gene, impc, which encodes a small potein of 9.5kD and which has no equivalent in the umu and muc opeons. INTODUCTION In Escheichia coli, the mutagenic esponses to UV light and to a lage numbe of chemical mutagens ae lagely dependent upon the inducible pathway commonly efeed to as 'eo-pone epai' (see (1) fo a eview). The essential components of this pathway include both the eca potein and the poducts of the umu opeon, UmuC and UmuD. These poteins, in some as yet unchaacteised way, may modify the DNA eplication machiney to allow bypass synthesis acoss a damaged template. Seveal lines of evidence (2,3,4) suggest that the umu gene poducts may facilitate the pompt esumption of chain elongation at sites whee the eplication foks have been stalled by DNA damage. Phenotypically, umucd mutants show a geatly educed mutagenic esponse to UV and many chemical mutagens, and they ae also slightly UV sensitive. Although thei gene poducts play an impotant ole in mutation in E.coli, it has become appaent that many bacteial species do not cay equivalent genes EMBL accession no. X53528 (5). Even in the closely elated species Salmonella typhimuium, the umu gene equivalents appea to be ineffective, and mutation in this species depends to a consideable extent on the pesence of plasmids fom a vaiety of incompatibility (Inc) goups, caying functional analogues of the umu genes. Thus the sensitivity of the stains employed in the Ames mutagenicity test is geatly inceased by the pesence of the IncN plasmid pkmlol, which caies the muc analogue of the umu opeon (6). Sequence analysis of both the umu and muc opeons has evealed that although thee is a wide divegence between these systems at the level of DNA sequence, they ae closely elated in tems of thei gene poducts. Both opeons contain two genes encoding poteins of MW15,064 and 47,681 in the case of umu (umud and umuc espectively), and MW and 46,362 in the case of muc (muca and mucb espectively) (7,8). In addition to being vey simila in size, the poteins of these opeons show consideable amino acid homology. UmuD and MucA poteins ae 41 % homologous, UmuC and MucB poteins ae 55% homologous, these aeas of homology being lagely confined to blocks of amino acids which ae almost completely conseved. The implication of these obsevations is clealy that the IncN plasmid muc genes and the chomosomal umu genes ae evolutionaily elated, and that they have pobably evolved fom a common ancesto. The similaities in the gene poducts leave little doubt that these two opeons pefom simila if not identical functions. In this pape, we descibe the sequence analysis of a second plasmid-encoded «/n«-analogue, encoded in this case by the imp genes of the IncI, plasmid TP110 (9). Although both this plasmid and pkmlol wee oiginally deived fom clinical isolates of Salmonella typhimuium, they can both be tansfeed vey efficiently by conjugation to E.coli, whee thei UV potection and mutation popeties ae again manifested. We wished to detemine whethe the UV potection and mutation functions of this plasmid, which is completely unelated to the IncN plasmids such as pkm 101, wee also evolutionaily elated to the umu system, o whethe this was a functionally analogous system with no stuctual similiaities. The esults clealy indicate that all thee systems, umu, muc, and imp, shae a common Downloaded fom at Pennsylvania State Univesity on Septembe 18, 2016 * To whom coespondence should be addessed

2 5046 Nucleic Acids eseach, Vol. 18, No. 17 ancesty, but that thee ae some majo diffeences between imp and the othe two opeons. The most obvious of these is that the imp opeon contains a thid gene, encoding a small potein of MW MATEIALS AND METHODS Bacteia] Stains used included TGI -8(lac-po), supe, thi, hsdd5 [ ' tad36 poa + B+ lacb laczsmis] (10); and JL652-as AB1157, F~ thi-1, th-1, leu-6, poa2, age3, his4, lacyl, galk2, xyl-5, aa-14, psl, sup37, but containing also F' lacfi and pjl59 (J.Little, pes.comm) Plasmids: pkglo (5.9kb (11)) contains the 3.2kb Ecol -BglU fagment of TP110 caying the imp genes, clonedinto the vecto pt7-l. pdl2 (4.8kb; this wok) contains the 2.4kb Clal-Xbal fagment of pkglo caying the impb gene, cloned into Accl-Xbal digested pt7-2. pgpl-2, pt7-l, and pt7-2 make up the T7 NA polymease / pomote contolled expession system of Tabo and ichadson (12). pjl59 (Amp lexa + ) is a LexA ove-poducing plasmid kindly povided by D J. Little. DNA Sequencing Sequencing of the imp genes was achieved by diected subcloning of fagments fom pkglo into M13 mpl8 and mpl9 as appopiate (13), the clones used being shown in Figue 1. The subclones wee sequenced by the dideoxy chain temination method of Sange et al (14), initially using the standad Klenow (E. coli DNA polymease 1 lage fagment) method and lattely the modified T7 DNA polymease (Sequenase) method (United States Biochemical Copoation), (15). C band compessions wee esolved by the substitution of dttp fo dgtp as necessay. The poducts of the eactions wee labelled with [a- 35 S]dATP (Amesham) and wee subsequently analysed on buffe gadient gels (16). Single standed DNA templates wee pepaed fom PEG pecipitated phage paticles fom the supenatent of an infected cultue of stain TGI (10). LexA Binding LexA potein was puified fom the ovepoducing stain JL652 (the geneous gift of D John Little) using the method of Schna et al (17). Cells wee lysed by sonication, cleaed by centifugation, and nucleic acid in the supenatent pecipitated with Polymin P. Total potein was then pecipitated with ammonium sulphate, dissolved, and subjected to chomatogaphy on phosphocellulose. The peak factions contained >95% pue LexA potein, as judged by stained SDS-PAGE. These factions wee pooled and used fo subsequent analysis. LexA DNA binding eactions contained the end-labelled imp DNA fagment, pepaed as descibed below (20-50,000 cpm pe eaction), and puified LexA potein to give the equied final concentation of between 10~ 5 and 10~"M. Final eaction volumes of 120/xl contained DNA, potein, and eaction buffe (loomm Tis-HCl, ph7.4; 1.6mM NaEDTA; 0.04mg/ml bovine seum albumen; 20% glyceol; 25mM NaCl). The eaction was incubated at 37 C fo 5mins, and then on ice fo 15mins. 100/tl samples wee loaded onto non-denatuing 5% polyacylamide gels, made up in TBE buffe (0.09M Tis-boate, 0.002M EDTA, ph 8.0). The gel was un in 1 x TBE at a constant cuent of 50mA fo 2 3 hous, died and then exposed to X-ay film at 70 C ovenight o longe if equied. Labelling of DNA fagments DNA fagments fo use in gel etadation expeiments wee endlabelled using [a- 35 S]dATP and T4 DNA polymease in a eplacement synthesis eaction, as descibed by Maniatis et al (18). Unincopoated mateial was emoved by passage though Sephadex G50 (nucleic acid gade, Phamacia-LKB). Identification of Plasmid-Coded Gene Poducts Poteins encoded by the imp genes wee identified using the T7 NA polymease / pomote system as devised by Tabo and ichadson (12, and pesonal communication). Cells containing both the bacteiophage T7 NA polymease plasmid pgpl-2, and a pt7 ecombinant plasmid, wee gown in Luia both to mid-log phase. A 0.2ml sample was taken, washed twice in M9 buffe, and esuspended in M9 buffe supplemented with 20/tg/ml thiamine and 0.01% (w/v) 18 amino acids (excluding cysteine and methionine). The cells wee gown with shaking at 30 C fo 60 mins, then the tempeatue shifted to 43 C fo 15 mins. ifampicin was then added to a final concentation of 200/tg/ml, to inhibit host diected tansciption, and the cells wee incubated at 42 C fo a futhe 10 mins. The tempeatue was then shifted down to 30 C fo 20 mins, when 10/iCi 35 S-methionine (Amesham) was added. Incubation was continued fo 5 mins, the cells wee pelleted, and labelled poteins analysed by SDS- PAGE as descibed peviously (9). ESULTS Sequencing the imp Opeon We have peviously descibed the location of the imp genes of TP110 within a 3.2kb col -Bglil fagment which is capable of fully expessing UV potection and mutation functions when cloned in high copy numbe vectos (9). Futhe subcloning defined the imp coding egion as being completely contained on a ~ 2.6kb Ecol EcoV fagment (11), and this fagment was chosen as the stating mateial fo the detemination of the DNA sequence. The stategy adopted fo die sequence detemination is shown in Figue 1. The complete sequence was detemined on both stands using the dideoxy chain temination method of Sange et al (14), as descibed above. The esulting sequence PvuII Clal ilc I Hiel PatI C EcoV j Bgll t B Nael Hlndlll Sphl Kpnl 1 1 Ecofl U^ Figue 1. Gene oganisation, estiction map and sequencing stategy of the imp opeon of TP110. Sub-clones deived fom the Ecol-EcoV fagment (-2.6kb) wee used to detemine the sequence on both stands fo a distance of 2527 bp. fom the Ecol site. The extent of the sequenced fagments is shown as aows. The positions of a numbe of six base pai estiction sites ae shown, as ae the positions of the thee imp open eading fames, C, A, and B. Tansciption acoss the opeon poceeds in the left to ight diection. Downloaded fom at Pennsylvania State Univesity on Septembe 18, 2016

3 Nucleic Acids eseach, Vol. 18, No is shown in Figue 2. The estiction sites pedicted fom the DNA sequence wee in complete ageement with those found ealie by estiction analysis (9), with the exception of an additional Pstl site at position 1685 (numbeed fom the Ecol site). This site, togethe with the site at 1607, geneates a small Pstl fagment of 78 bp. which had not peviously been detected: a futhe pai of Pstl sites (at bases 629 and 790 in the sequence) also geneate a small Pstl fagment. As the sub-clone caying the imp opeon was oiginally isolated as a Pstl patial digestion poduct, we felt it impotant to ensue that these small fagments wee tue components of the imp opeon, and wee not atefacts of the initial cloning stategy. EcoUEcoV estiction digests of TP110 DNA wee theefoe hybidised with labelled M13 clones containing these two small fagments, cloned individually. In each case, hybidisation was obseved to the appopiate band, confiming that the sequenced fagment epesented an uninteupted egion of TP110. Analysis of the sequence shown in Figue 2 eveals thee majo l * S5S OJU«TCtCCT CTCACATGCG GCCTCCATCA GGCCGTGCCG CACCTCTTAC COGCCGACCG CATACGAATC CAGGTGCCGC CCCGAAOCCG CAOCCCOTW AOCAOGAACC ACGAAACCTG ACCCGGTGAG GAGCCCGGCG ACACCGACAC ACTTGCATCC GATGCAGCCC OGTTAACTGC CQQCACQQCC TGQCTAACCA OGTATTTTCT CCACACAACC GTGCACAAAA TGCTGTGGAT AAGCTGGACA CAGCACCCCA CACCACCCAT ACAOCCACAC ACTCAGAGAA TTGACCGTTT TTTACTCACA ACAATCACAT TTCACTOTGT GCGGATTCAC TTATTACAAT CATACaSCAA AACAAAMOC^ACATGAGAAC 390 ACTOTATATA ATC ATC CGC ATT GAA ATT CTT TTC GAC CGC CAG AGC ACA AAA AAC CTC AAA TCA GGC ACA CTT CAG GCG CTA CAG AAT M I I B I L F D Q S T K M L K S G T L Q A L Q N GAA ATC GAA CAA CGT CTG AAA CCA CAC TAC CCG GAA ATC TGG CTG CGT ATA GAC CAG GGC AGC GCC CCG TCT GTA TCT B I H Q L K P H T P E I H L I D Q G S A P 8 V S CTC ACA CCA GCC CGC AAC GAC AAG GAT AAA GAA CGT ATC CTG TCT CTG CTG GAA GAA ATC TGG CAG GAC GAC AGC TGG V T C A N D K D K B I L S L L E E I H Q D D S H CTG CCT CCA CCA TGA 639 L P A A X 636 ATG ACT ACC GTT TAT CAC CGT CCG GCT GAC CCT TCA GGC GAT GAC AGT TAC GTG CGA CCG TTG TTT M S T V T H P A D P 8 G D D 3 T V P L P GCC GAT CGT TGC CAG GCC GCT TTT CCT TCG CCT GCC ACT GAT TAT GCT GAG CAG GAA CTG GAT CTG AAC AGC TAT TGC A D C Q A G F P S P A T D T A E Q B L D L N S Y C ATC AGC AGA CCT GCA GCC ACC TTC TTT CTG CGC GCC AGC GGT GAA TCG ATG AAC CAG GCT GGC GTG CAG AAT GGT GAT I S P A A T F F L A 8 G E S M N Q A G V Q N G D CTG CTG GTA GTG GAC AGG GCC GAA AAA CCA CAA CAC GGG GAC ATC GTT ATC GCT GAG ATC GAC GGT GAG TTC ACC GTC L L V V D A E K P Q H G O I V I A E I D G E F T V AAA CGA CTG CTG TTG CGC CCA CGC CCG GCA CTG GAG CCG GTT TCA GAC AGC CCG GAG TTC CGC ACA CTG TAT CCG GAA K L L L P P A L E P V S D 8 P E F T L Y P E AAC ATC TGT ATT TTT GGT GTT GTC ACT CAC GTG ATA CAC AGC ACC CGG GAG TTA CGC TGA 1073 N I C I F G V V T H V I H,...T, E L Z " D 1073 ATG TTT GCA CTG GCT GAT M F A L A D ATC AAC AGT TTC TAC GCC TCA TGT GAA AAA GTT TTC CGC CCG GAC CTT CGC AAC GAA CCG GTC ATC GTA CTC AGC AAT I N S F Y A S C E K V P P D L N E P V I V L S N AAC GAT GGC TGT GTG ATC GCG CGC AGC CCG GAG GCA AAA GCC CTTGGC ATC AGA ATG GGG CAG CCC TGG TTT CAG GTG N D G C V I A S P E A K A L G I M G Q P H F Q V AGA CAA ATG CGC CTG GAG AAG AAA ATA CAT GTA TTT TCC AGC AAT TAT GCG CTG TAC CAC AGC ATG AGC CAA CGG GTT Q M L B K K I H V F S S N Y A L Y H S M S 0 V ATG GCT GTT CTG GAG TCG CTT TCT CCC GCA GTT GAG CCC TAC TCA ATT GAT GAA ATG TTC ATT GAT TTG CGG GGG ATA M A V L B S L S P A V E P Y S I D E M F I D L G I AAT CAT TGC ATC TCT CCG GAG TTT TTT GGT CAT CAG CTC AGG GAA CAG GTA AAG AGC TGC ACA GGA CTC ACC ATG GGG N H C I S P E F F G H Q L E Q V K S W T G L T H G GTG GGC ATT GCG CCT ACA AAA ACG CTG GCT AAA AGT GCA CAG TGG GCA ACA AAG CAA TGG CCA CAG TTT TCC GGA GTG V G I A P T K T L A K S A Q W A T K Q W P Q P S G V GTC GCG CTG ACG GCA GAA AAC CGT AAT CGG ATC TTG AAG CTA CTG GGG CTG CAG CCA GTT GGT GAG GTC TGG GGA GTA V A L T A B N N I L K L L G L Q P V G E V W G V GGA CAC AGA CTG ACG GAA AAG CTG AAT GCG CTG GGT ATT AAC ACA GCA CTG CAG CTG GCG CAG GCT AAC ACG GCA TTC G H L T E K L N A L G I N T A L 0 L A Q A N T A F ATC CGG AAA AAC TTC AGC GTC ATT CTT GAG CGT ACG GTA CGC GAA CTC AACGGC GAG TCC TGC I K H F S V I L E T V E L N G E S C ATA TCC CTG GAA GAA I S L E E GCA CCA CCG GCA AAA CAG CAG ATT GTC TGT AGT CGC AGT TTT GGT GAA CGA ATC ACA GAC AAA GAT GCC ATG CAC CAG A P P A K Q Q I V C S 8 F G E I T D K D A M H Q GCT GTT GTT CAG TAT GCA GAG CGG GCC GCA GAG AAA CTA CGT GGG GAG CGT CAG TAT TGC CGG CAG GTG ACG ACA TTT 1871 A V V Q Y A E A A E K L G B Q Y C Q V T T F 1949 GTA CGG ACA TCA CCC TTT GCA GTA AAA GAA CCC TGT TAC AGC AAT GCC GCT GTG GAA AAG CTT CCA TTG CCC ACA CAG V T S P F A V K E P C Y S N A A V E K L P L P T Q 2027 GAC AGC CGG GAC ATT ATT GCC GCC GCA TGC AGA GCC TTA AAC CAT GTCTGG CGT GAA GGG TACCGC TAT ATG AAG GCA D S D I I A A A C A L N H V W E G Y Y H K A 2105 GGT GTC ATG CTG GCT GAT TTC ACA CCA TCG GGT ATA GCG CAG CCG GGA TTA TTT GAT GAA ATC CAG CCC CGT AAA AAC G V M L A D S G L F D E I Q P K N 2183 AGT GAA AAG TTA ATG AAA ACA CTC GAT GAA CTG AAC CAG TCG GGA AAA GGG AAA GTG TGG TTT GCG GGG CGA GGA ACC S E K L M K T L D E L N Q S G K G K V W F A G G T 2261 AAA CGG GAA ATG TTG AGT CAG TGT TAT ACA ACT AAA TGG CGA GAT ATT CCC CTG GCC AGC K B M L S Q C Y T T K W D I P L A GCC CCT GAA TGG CAA ATG A P E W Q M CTG GGT TAG 2347 L G Z 2348 TTCAGTCATC TCCGAACATA TTTTCAGCGT TTCTTCTGGT CTGGTCTTCA CCGTCATTTC TCGACAGACT CTGCTCTGTA AGAGGAGTAC 2438 TGCAATCCAC AGACGTATGG AGACCAGATT GTTTTTCCAG TAATTCAAGC AATTTCTTTT CAACCGAAAA AGCACCTGGT ATTACGCTAA Downloaded fom at Pennsylvania State Univesity on Septembe 18, 2016 Figue 2. DNA sequence of the imp opeon and pedicted amino acid sequence of the gene poducts. The sequence stats at the Ecol site shown in Figue 1. Stuctual featues indicated in bold type include the likely pomote sequence (-35 and -10), the ibosome binding site (SD), and the two LexA binding sites (LexAl and LexA2). The tanslation poducts ae shown fo each of the thee imp open eading fames (impa, ; impb, ; impc, ).

4 5048 Nucleic Acids eseach, Vol. 18, No. 17 Open eading Fames Within The Sequence The fist open eading fame of the imp opeon, designated tmpc, potentially encodes a potein of MW 9,491. The pedicted amino acid sequence (Figue 2) shows no homology to the poducts of eithe the muc o umu opeons. The second open eading fame, designated impa, potentially encodes a potein of MW 16,201. This is simila in size to both UmuD (MW 15,064) and MucA (MW 16,371). Although thee is little nucleotide sequence homology between these thee genes, the degee of amino acid homology is stiking (Figue 4). ImpA is 43% homologous to MucA, and 49% homologous to UmuD. The homology between MucA and UmuD is slightly lowe at 40%. Thity one pe cent of amino acids ae common to all thee poteins, this figue ising to fifty one pe cent if consevative substitutions ae allowed. As might be expected, this high degee of homology is achieved by the etention of blocks of amino acids which ae essentially 100% conseved, these blocks being sepaated by moe vaiable egions. It seems easonable to assume that these blocks of conseved sequence epesent sites essential fo the mutagenic function of these poteins, while the vaiable sequences have othe oles. Pey et al (7) demonstated consideable amino acid homology between UmuD, MucA and the egion of LexA potein suounding its Ala Gly (esidues 84/85) cleavage site. This homology, as shown in Figue 4, extends to the amino acid sequence pedicted fo ImpA, and includes the esidues Seine and Lysine (esidues 119 and 156 in LexA; 61 and 98 in ImpA), as well as the Ala and Gly (esidues 27/28 in ImpA). In this latte espect, the ImpA potein esembles the MucA potein athe than UmuD in having an Ala-Gly as opposed to a Cys Gly cleavage site. The seine and lysine esidues in LexA have been shown to be impotant fo the autolytic cleavage of the potein (19), and this consevation of sequence indicates that ImpA may undego eca mediated self cleavage in a manne simila to that demonstated fo LexA and UmuD (20,21). The thid imp open eading fame, designated impb, potentially encodes a potein of MW 47,731, simila in size to UmuC (47,681) and MucB (46,362). Again, although thee is little nucleotide homology between the coding egions, thee is significant homology between the pedicted amino acid sequences of the gene poducts (Figue 5). ImpB is 51% homologous to MucB, and 54% homologous to UmuC. Homology between MucB and UmuC is slightly highe at 56%. In total, 43% of the amino acids ae common to all thee poteins, and if substitutions of simila amino acids is allowed, the figue ises to 57%. The impa and impb coding egions ovelap by one base pai (Figue 2), in the same way as those of umud and umuc (7), which may have implications fo tanslational coupling. Thee is an excellent match to the Shine-Dalgano consensus sequence in the appopiate position (within impa) to pomote efficient tanslation of impb. The impc and impa coding egions also ovelap, but thee is no obvious Shine-Dalgano sequence upsteam of the imp A initiation codon. This may account fo the appaent low level of tanslation of imp A which has peviously made it difficult to identify its poduct. OBUD I-pA MucA 1 f i : k : p a d 1 e i v t : : f s t v y h p a d p a g d d s y v k v d i : : : e s a q a s v h a i d 1 v g c G F P 8 P A a d 3 c q a G F P 8 P A t 3 q i B a_ G P P S P A q g 1 i q h c T a c v h G D G D G D K t T VK B F T VK E f T VK A T A T A T V id e 1 3 a 1 n a e e k v k a S C I a S 6 1 v SG 8 M 8 M S M V D B a i t a s HG V D a e k p q BG V D B 1 t a s HG L P t v q L P p a L P _g c G V V G V V G V V 04* Figue 3. Electophoetic mobility of the 413 bp EcoI Taq\ fagment of the imp opeon in the pesence of puified LexA potein. Lanes A and B contain the end-labelled DNA fagment, and no LexA potein. Lanes C I contain deceasing amounts of LexA, the concentation educing ten-fold in each tack fom 10~*M in tack C to 10~ "M in tack I. Aows adjacent to tack A indicate the positions of the molecula weight makes, and aows labelled B and NB indicate the positions of the DNA band when the potein is eithe bound o not bound espectively. Electophoesis conditions ae descibed in the main text. 1 f B a f y 1 n q 1 nay h e v. i d g a i a n q a g v q e d g i h I V I V I V a v d e i 3 c i h v B d mn k d f p v v k am v i h t «1 i e h : : : : : e 1 p v c X Figue 4. Sequence homology compaisons of the ImpA potein with UmuD and MucA. Amino acid esidues which ae conseved in all thee equivalent gene poducts ae shown in bold type and ae flanked by bas. Sequences common to eithe UmuD and ImpA, o MucA and ImpA, ae undelined. The Ala-Gly (Cys Gly in UmuD) Se Lys esidues involved in the autocleavage eaction ae staed (*). Othe esidues common to ImpA and LexA ae indicated by points ( ). Sequence data fo UmuD and MucA ae taken fom Pey et al (7) Downloaded fom at Pennsylvania State Univesity on Septembe 18, 2016 ovelapping eading fames, downsteam of a good consensus pomote sequence which is ovelapped by two potential LexA binding sites. One of these ovelaps the pomote sequence, while the othe ovelaps the 10 egion. Gel etadation studies using puified LexA potein and the 392 bp. col -Mbo\ imp fagment evealed only a single-step binding eaction (Figue 3), and it appeas likely that only one of these potential binding sites is stongly bound by LexA potein. This is most likely to be the site ovelapping the pomote sequence, since the cental (N,o) of the CTG (N10) CAG motif is in this instance vey AT ich (90%), whilst the site which ovelaps the box contains only 50% AT base pais. It is howeve possible that the Lex box does play a ole, paticulaly as a simila box with a 50% GC content also exists in font of the umudc opeon (7). If LexA binding to such sites is weak, it is likely not to be detected unde the conditions of the gel etadation assay.

5 Nucleic Acids eseach, Vol. 18, No Identification of Gene Poducts In ode to identify the poducts of the imp open eading fames, the coding egion was inseted into suitable pt7 vectos and Dnuc impb MucB a f T A S C E t v F P D L stt A S C B k v P P D L g m Y A S C E q a F P D L M P A L M F A L M F A L V L S N M D G c V i A n a e A K a 1 V L S N M D G c V I A ape A K a 1 V L S N N D G n i V A n y 1 A K k a g k n e p n a k q k d l f s g v v c F 8 q v q a l e k k i h v F S k v p i i e h n ^ a i F S v k M G i M G 1 k M G 1 t T c k I S I D! Y S I DB Y S I DB e h q v n c d 1 t d i n h c i s p e f i t a a m s 1 d a 1 q k s w h a t e q spa ash t v G V G I A q T t m G V G I A p T v c G V G I A T TLA TLA TLA K K K Ian h s a q w 1 c n h n 1 e a e n d g a q k L a n a l p v d d v w g i n i L k 1 1 g l q p v g e v w p v a e v w g v 1 k k L m 8 i q a t g G V V d : p q s G V V a : a t g G V V a A a K k A t K q A a K t s k k l d a m g i k t v l d l t e k l n a l g i n t a e k a l a t m g i k t v l d I K h I K n I K t L a d t d i L q 1 a q a n L a a d t L L L i v c h h t : : t a n V V L E T D E L GE P C 1 q a V i L E T U E L n GE 8 C i s g V V L B T D E L GB a c P l-h a p t K 0 e I i c S S F G e i t 4 VP E B t B B a P p a K Q q I V C S S F G E B n E p a K Q 3 I V V S S F G q V e tit M Q A i M h Q A V M q Q A V i q V V i 8 t F F 8 V F t t c v t i v i a q g k A a A A E K L s E h Q Y C y A e A A E K L g E Q Y C f A a A A E K L n E Q Y C 1 T S P f a 1 n e p y T S P f a v k e p c T s P y s v d t q K L 1 t E T Q D 8 K L pip T Q D S K L t v a T Q D S q a h e g y e d 1 a 1 n L F D d n a p g L F D e i q 1 d L F D sat a k e q s : : I I I I I I K A G V N L K A G V H L K A G V M L p g S E k n S E sag S E t 1 y FAG k g k v w FAG k n q 1 f FAG MLS P Y T T MLS q c Y T T k w MLS P d Y T T d w i q q q v_ t a p e wq i d n s a s s d l l v k d i p T a l g s i p i a t i k Figue 5. Sequence homology compaisons of the ImpB potein with UmuC and MucB. Amino acid esidues which ae conseved in all thee equivalent genes ae shown in bold type and ae flanked by bas. Sequences common to eithe UmuC and ImpB, o MucB and ImpB, ae undelined. Sequence data fo UmuC and MucB ae taken fom Pey et al (7). Figue 6. Polyacylamide gel electophoesis analysis of imp encoded poteins poduced fom T7 expession plasmids. Lanes 1, 2, and 3 show poteins poduced by the plasmid pdl2 (impc~a"b + ), lanes 5, 6, and 7 show poteins poduced by pkglo (z/npc+a + B + ). The constuction of the two plasmids is descibed in the main text. Lanes 3 and 7 show poteins pepaed fom cultues which have not been induced fo T7 NA polvmease. Lanes 2 and 6 contain samples pepaed fom induced cells, and lanes 1 and 5 contain samples fom induced cells to which ifampicin (200(jg/ml) has been added to inhibit host mediated tansciption. Lane 4 contains molecula weight makes, the sizes of which ae indicated in kilodattons. Aows indicate the thee imp gene poducts in lane 5, with appoximate sizes of lokd (ImpC), l6kd (ImpA) and 47kD (ImpB). Downloaded fom at Pennsylvania State Univesity on Septembe 18, 2016 v ma t 1 v a a v T f a a v v L Y a d M S L Y h s H S L Y a a M S SHY S N Y SHY expession was induced by poviding T7 NA polymease in tans (12). Figue 6 shows the esults of an analysis using two plasmid constucts, namely pkglo, in which the 3.2kb Ecol BglH fagment is cloned in pt7-l, and pdl2, in which the Clal EcoV fagment encoding the 3' end of imp A and the entie sequence of impb (Figue 1), is cloned in pt7-2. Compaison of the lanes eveals plasmid coded poteins of appoximately lokd, 16kD and 50kD in the pkglo tack, but supisingly no plasmid coded poducts in the pdl2 tack. We have peviously identified poteins of appoximately llkd and 51kD as imp poducts (9), which wee at that time suggested to be the poducts of the impa and imp genes espectively. It is clea fom the sequence data that this is unlikely to be the case, and that while the assignment of the ~ 50kD potein to imp is still valid, the _ lokd potein is almost cetainly the poduct of the impc gene. The use of the pt7 system allows the visualisation of a 16kD potein not peviously seen, which is consistent in size with the potential poduct of the impa gene. Additional expeiments using impc+ 8(impAB) deivatives (containing eithe the Ecol-Clal o Ecol-Pstl fagments (Figue 1) confimed unequivocally that the lokd potein is poduced not by imp A, but by impc (data not shown).

6 5050 Nucleic Acids eseach, Vol. 18, No. 17 The lack of visible imp-coded gene poducts in the pdl2 tack was an unexpected obsevation, since this plasmid contains an intact impb gene which is known fom genetic complementation to be completely functional (Lodwick and Stike, manuscipt in pepaation). It seems likely that the high level expession of this gene cannot be achieved in this constuct, pehaps due to the lack of tanslational coupling which nomally occus when the impc and imp A genes ae pesent. ACKNOWLEDGEMENTS This wok was suppoted by a poject gant fom the Medical eseach Council to P.S. The excellent technical assistance of Jenni Jones is gatefully acknowledged. We ae paticulaly gateful to D John Little fo the supply of LexA ovepoducing stains, fo the supply o puified LexA potein, and fo help and advice on puification pocedues. Thanks ae also due to D Stan Tabo fo kindly supplying the pt7 expession plasmids. DISCUSSION The DNA sequence of the imp opeon eveals a close elationship to the two peviously chaacteised UV potection / mutation opeons muc and umu. Howeve, despite the fact that both imp and muc ae found on plasmids oiginally isolated fom Salmonella typhimuium, while umu occupies a chomosomal location in E.coli, imp and muc show no geate homology to each othe than to umu. Each of these thee opeons codes fo poteins of ~ 16kD and ~47kD, which ae essential fo the UV potection / mutation effect. The high degee of consevation of blocks of amino acids amongst the thee ~ 16kD poteins and amongst the thee ~47kD poteins clealy indicates those egions of the potein essential fo function. Howeve, it is also clea that while these systems ae closely elated, they ae quite distinct fom each othe. The low level of homology between the opeons at the DNA level pevents coss-hybidisation, and gives a clea indication of the evolutionay divegence which must have taken place fom thei (pesumed) common ancesto. This divegence is also indicated by the failue of the component genes of the thee opeons to effectively coss complement. Thee is no coss complementation between die muc and umu component genes (1), and only a vey limited complementation between imp and umu (Lodwick and Stike, manuscipt in pepaation). The majo diffeence between the imp opeon and the muc I umu opeons howeve is the pesence of a thid gene, designated impc, coding fo a small potein of MW 9,491, fo which no homologue exists in the othe two opeons. The biological function of this potein is not yet clea, although ou initial expeiments indicate that it may be involved in the estoation of the induced imp opeon to the epessed state, once epai of DNA damage is completed. It does not howeve shae homology with the plasmid-coded/wib function, which is epoted to have a simila ole (22), no have we been able to detect any significant homology to poteins of known function in the SwissPot data base. We have howeve demonstated by the ceation of deletions mat impc is not absolutely equied fo the UV potection and mutation effects (Lodwick and Stike, manuscipt in pepaation). In tems of evolution of the UV potection / mutation functions, it is not possible to say whethe the imp opeon has acquied this exta sequence, o whedie the umu and muc opeons have lost it. It is pehaps elevant howeve that imp like sequences ae widely distibuted amongst most Incl, IncB and IncFTV plasmids (Lodwick and Stike, manuscipt in pepaation), both in plasmids deived fom ecent clinical infections, and fom plasmids isolated befoe the geneal use of antibiotics (11). In each of these cases, sequences equivalent to impc ae pesent and have not been lost. The ImpC potein does appea to be toxic to the cell when poduced constitutively in lage quantities, and also appeas to intefee with the SOS induction pocess (Lodwick and Stike, manuscipt in pepaation). Expeiments ae cuendy in pogess to futhe chaacteise its biological popeties. EFEENCES 1. Walke.G.C. (1984) Micobiol. ev., 48, Bidges, B.A. and Woodgate,. (1984) Mol. Gen. Genet., 196, Tessman, I. (1985) Poc. Nail. Acad. Sci. USA, 83, Chistensen, J.., Le Clec, J.E., Tata, P.V., Chnstensen,.B. and Lawence, C.W. (1988) J. Mol. Biol., 203, Sedgwick, S.G. and Goodwin, P.A. (1985) Poc. Nail. Acad. Sci. USA, 82, McCann, J.,Spingam, N.E.,Koboi,J. and Ames, B.N (1975) Poc. Natl. Acad. Sci. USA, 72, Pey.K.L., Elledge, S.J., Mitchell, B.B., Mash, L. and Walke, G.C. (1985) Poc. Nail. Acad. Sci. USA, 82, Kitagawa, Y., Akaboshi, E., Shinagawa, H., Hoii, T., Ogawa, H. and Kato, T (1985) Poc. Nail. Acad. Sci. USA, 82, Glazebook, J.A., Gewal, K.K. and Stike, P. (1986) J. 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