Atypical regions in large genomic DNA sequences (DNA sequence analysis/markov chain/chromosome srcture)

Transcription

1 Proc. Nti. cd. Sci. US Vol. 91, pp , July 1994 Genetics typicl regions in lrge genomic DN sequences (DN sequence nlysis/mrkov chin/chromosome srcture) STEWRT SCHERER*t*, MRY SR MCPEEK, ND TERENCE P. SPEED *Humn Genome Center, Lwrence Berkeley Lortory , Berkeley, C 94720; tdeprtment of Microiology, Box 196, University of Minnesot School of Medicine, Minnepolis, MN 55455; nd Deprtment of Sttistics, University of Cliforni, Berkeley, C Communicted y Ronld W. Dvis, Mrch 30, 1994 BSTRCT Lre genomic DN sequences contin regions with disnctive ptterns of sequence orgniztion. We descrie method using logrithms of proilities sed on seventh-order Mrkov chins to rpidly identify genomic sequences tht do not resemle models of genome orgnztion uilt from compiltion of octnucleotde usge. Dt ses hve een constructed from Escherichi coli nd Scchromyces cerevisie DN sequences of >1000 nt nd humn sequences of >10,000 nt. typicl genes nd clusters of genes hve een locted in cteriophge, yest, nd primte DN sequences. We consider criteri for tisl s nce of the results, offer possile expntons for the oserved vrition in genome orgniztion, nd give dditionl pplictions of these methods in DN sequence nlysis. Lrge contiguous genomic DN sequences hve een determined for numer of species. The Humn Genome Project nd similr efforts for importnt model systems will e producing such sequences t n incresing rte. The most widely used computtionl technique for the nlysis of new sequence informtion is the comprison of the sequence with ll other known sequences for regions of similrity. With the growth of the pulic dt ses, this pproch hs ecome incresingly successful, with out one-third of ll lrge open reding frmes in new genomic sequences or new cdn sequences eing relted to known genes (1, 2). The genome projects will hve mjor impct on this pproch ecuse decresing frction of dtse entries will e sequences of known function with the welth of iologicl informtion ssocited with them. Existing computtionl tools for the nlysis of DN sequences were generlly developed for the nlysis of individul genes. Most sequence nlysis softwre reports tles of regions of interest sed on scoring system. This tulr presenttion ecomes unwieldy when fced with sequences of tens or hundreds of kiloses. Sttisticl nlyses of DN sequences hve een used since the origins of moleculr iology. t ws first noted tht nerest-neighor frequences of different species vried gretly nd tht these differences could not e explined y the known vrition in se composition (3). With the dvent of efficient DN sequencing techniques, it ecme cler tht there were wide vritions in codon usge (4), tetrnucleotide (5), nd hexnucleotide (6) frequencies mong species. The increse in ville sequence dt s result of the genome projects will permit the sttisticl evlution of frequencies of longer oligonucleotides. Building on these oservtions, we hve developed flexile pproch to the nlysis of lrge genomic DN sequences sed on Mrkov chin models. dt se is uilt from lrge collection of sequences nd the frequency of occurrence, termed usge, of ll possile sequences of The puliction costs of this rticle were defryed in prt y pge chrge pyment. This rticle must therefore e herey mrked "dvertisement" in ccordnce with 18 U.S.C solely to indicte this fct. length 8 is tulted. This length ws chosen for severl resons. First, it is the lrgest size for which effective sttistics could e developed when the complete genomes of the importnt model microorgnisms ecome ville. Second, it is out the smllest size where protein-dn interctions might e reflected. Finlly, this size represents 16 its of informtion nd offers certin computtionl economies. Proilities re clculted for window of constnt length t every possile position long query sequence nd the results re presented grphiclly. The query cn e tested ginst vriety of models constructed y the selection of different sets of sequences for the underlying dt se. The disply indictes how well regions of the sequence resemle the model. Our sttisticl model ssumes sttionry ehvior of the query sequence ut mkes no iologicl ssumptions out the nture of tht sequence or those used to uild the dt se. For exmple, coding nd noncoding regions re treted equivlently. For long genomic sequences, one would expect sustntil locl sttisticl vritions. We discuss the vlidity of these ssumptions nd criteri for the determintion of significnce of the extreme vlues tht re oserved. By uilding dt se from ll known sequences from single species, we hve found regions in lrge query sequences with typicl orgniztion in oth prokryotes nd in eukryotes. These results gree well with our understnding of DN orgniztion of these regions nd highlight res for future iologicl investigtions. We discuss possile explntions for the presence of genes nd gene clusters with unusul orgniztion nd other pplictions of this pproch to DN sequence nlysis. METHODS DN Sequence Dt. ll DN sequences used re derived from those in GenBnk relese 75. Compiltions of sequences from prticulr species used the nme in the orgnism field of the entry. Sequences less thn 1000 ses in length (10,000 for humn DN) were not included nd the reverse complement of ech sequence ws dded to compiltions. Octmer Usge Sttistics. The usge of ll octmersjl1, 2,..., g) (where is ny se ndfis the tulted count) nd totl usge N = Xll svmersfwere determined. From these dt, conditionl proilities were clculted: P(gll,..., 7) = [f(1, 2,..., g) + k]/>8[f(1, 2,..., %) + k] where k = 0.01 N/48. This flttening constnt k is pproximtely unity for the dt ses used elow tht were uilt from lrge collections of genomic sequences. See ref. 7 for discussion of this topic. Trnslted se 2 logrithms of these vlues were determined s Zi = log2 P(iji-7,..., i-1) + 2. Dinucleotide nd trinucleotide frequencies were-determined tto whom reprint requests should e ddressed. Present ddress: Deprtment of Sttistics, University of Chicgo, Chicgo, L the 7134

2 Genetics: Scherer et l. from the octmer usge. These vlues were similrly zero djusted nd used to determine vlues of Z. Confidence Limits. DN sequences of length equl to tht of the query sequence were generted rndomly y using the usge of heptmers in the dt se to provide n initil point nd using the conditionl proilities descried ove. One hundred such sequences were used to estlish 95% confidence limits for the upper limit of the plots. Grphicl Presenttion. Scores for sequence locks of length j + 1 were clculted for ll vlues of i where i + j is less thn the sequence length; Yij = -k+jizk ws plotted t x = i + (j/2). The y xis ws divided y the men gj = [(j + 1)/N]Xll 8-mersZf, so tht the vlues presented re multiples of u. The entire sequence is plotted in single frme with the x xis eing the position on the sequence. Becuse of vrition in the lengths of the query sequences, widths of the peks for similr sized structures vry etween figures. Note tht the plots re -10g2 P. Therefore, sequences tht do not resemle the underlying model will point upwrds nd those overrepresented in the dt se will point downwrds. Three horizontl lines re shown. The upper line is the position where ll vlues would plot if the dt se hd uniform distriution of ll sequences. The centrl line is the men of the distriution, 1.t. f the sequence resemled the underlying model, one would expect noise nd centered on this position. The third line is twice the men. RESULTS ll figures in this work present the comprison of DN sequence with n octmer usge dt se constructed from genomic DN sequences from prticulr species. Generlly ll known sequences from given species were used s descried in Methods. n some cses, the counts were tulted from single DN sequence. Proilities were determined for ll susequences of given length (512 or 1024 ses) in the query sequence nd vlues for ech position long the sequence were plotted s descried in Methods. Bcteriophge. mong lrge cteril DN sequences, phge is one of the most intensively chrcterized (8). n prticulr, it exhiits modulr orgniztion with djcent genes often hving relted functions (9). Fig. 1 presents n nlysis with the entire phge genome s query nd Escherici coli sequences used to construct the model. Fig. 1 uses seventh-order Mrkov chin nlysis, wheres Fig. l uses first-order Mrkov sttistics (i.e., dinucleotide frequencies). While the overll pttern is similr, it is cler tht the first-order chin, which is uilt y using nerest-neighor frequencies, does not effectively revel the lock structure present in the phge genome. Severl fetures stnd out in the plot. First, the lte genes -J clerly re representtive of typicl E. coli sequences. The first significnt pek ner the end of the lte trnscript is the loin gene (nt 18,695-19,582; ref. 10). This gene is trnscried during lytic growth ut is not essentil for plque formtion. Other regions tht produce the downwrd peks in the right hlf of the genome include the recomintion, repliction, nd lysis genes. The regions not essentil for lytic growth, most notly the 2 region (leftwrd from 27,731), devite most from the model. Scchromyces Chromosome 3. This sequence ws the first complete eukryotic chromosome to e determined (2). Becuse of the extreme length of the yest sequences eing tested, the size of the window ws douled to ssure sttisticl significnce. This lso mkes the remining lrger fetures esier to detect given the compression of the x-xis. Fig. 2 shows the pttern produced using the entire chromosome s query with the model uilt from ll other yest sequences of >1 k. While the extent of vrition from the model is not s extensive t tht oserved with phge, there is lrge region centered out coordinte 210,000 tht does not Proc. Ntl. cd. Sci. US 91 (1994) 7135 J 2 OP SR ; Di il. h im1111 ll J 2 OP SR i _ ~~~ ~~~~~~~~ FG. 1. Seventh-order () nd first-order () Mrkov chin nlysis of the cteriophge sequence. The lock size ws 512 nt. n, the 95% confidence limit for significnt devition from the model is Loctions of mrkers re given t the top of ech pnel. strongly resemle yest sequences. Severl other sttisticlly significnt vritions re oserved. Notle is the lowest point in the grph, which is t the loction of Ty element. This is expected for sequences tht re highly represented in the dt se. When yest chromosome 3 is tested ginst n E. coli model, no prt of the sequence strongly resemles cteril DN. The men of the noise nd is t nd none of the low points on this plot (not shown) lign with the peks in Fig. 2 or. Fig. 2 is plot where the dt se is uilt solely from the ctul sequence of yest chromosome 3. Most of the sequence shows no significnt vrition from other sequences on the chromosome. Severl fetures re of interest. The lowest point on the plot is gin the Ty element. The other three pronounced downwrd peks re HML (12,000), MT (199,000), nd HMR (292,000). This indictes tht ny sequence representing out 1% of dt se will e redily detected without resorting to conventionl homology serch. The two highest points re the region round 210,000 which ws found not to resemle yest sequences in generl nd the glucokinse gene (50,000).

3 7136 Genetics: Scherer et l. Proc. Ndl. cd. Sci. US 91 (1994) HML Ty MT HMR TSM1 - THR _ _-1. 1.i 1. lu~ _ HML Ty MT 9.L.g ll,. EL v,+1.0 ^ f r = _ HMR T. T F Tl.-- WF F _ FG. 2. nlysis of yest chromosome 3 with the yest model () or yest chromosome 3 model (). The lock size ws 1024 nt. The 95% confidence limit for the upper ound in is The loctions of severl chromosome 3 lndmrks re shown t the top of ech pnel. Fig. 3 shows n enlrgement of the region ner coordinte 210,000 on chromosome 3. The window size hs een reduced to show smller fetures of the plot. Note tht the most typicl regions lign well with the lrgest open reding frmes of the region; however, mny lrge open reding frmes nery, such s TSM (201, ,000) nd THR4 (216,000), show more typicl orgniztion. Scchromyces Chromosome 1. mong severl lrge yest genomic sequences exmined y this pproch, including totl ofout 100 k from chromosome 5, the most significnt vrition from the generl yest model ws noted for this sequence from chromosome 1 (Fig. 4). The highest pek in the plot gin ligns with n open reding frme. While the function of this gene in the region of this pek is not completely understood, it is homologous to the hmster RCCJ locus (11). Given the role of this gene in the cell cycle, one would expect it to hve some type of yest counterprt; however, it is uncler why such gene would require typicl orgniztion. * F FG. 3. n enlrgement of prt of yest chromosome 3 with the lock size reduced to 512 nt. Open reding frmes (>100 mino cids strting with methionine) re shown t top with rrows indicting direction of trnsltion. Note the lignment of the peks with the lrgest open reding frme in those regions. P-Gloin Genes. Fig. 5 nd show seventh-order nd second-order nlyses of the humn 3-gloin gene cluster (12) using ll other humn sequences >10 k to uild the dt se. This higher cutoff is necessry ecuse high proportion of the dt se is cdn sequences. The presence of highly repeted DN sequences such s lu repets clerly influence the seventh-order results ut not the second-order plot. ll of the strong downwrd peks re t the position of lu elements. When the model is uilt solely with relted sequence such s the 40-k Glgo crssicudtus (ush y),-gloin gene cluster (13), the only significnt downwrd peks re t the regions of homology encoding the (-gloin-like genes (dt not shown). Fig. 6 shows the results of seventh-order plot with the ush y (3-gloin cluster s query nd humn model uilt from ll sequences >10 k. Note the lrge region round position 7000 tht differs gretly from the humn model. portion of this region ws known to e highly homologous to < ( = 4. ', _ FG. 4. nlysis of region ofyest chromosome 1 with the yest model. The query sequence is GenBnk entry YSCLTESPO. The lock size nd confidence limits re s descried in Fig. 2. Open reding frmes re shown t top.

4 Genetics: Scherer et l. Proc. Ntl. cd. Sci. US 91 (1994) 7137 _ N E 1! _ H 11 N iiiiiiiii _ -1.0 'T To C = n FG. 5. is HUMHBB with lock size of 512 nt. presents seventh-order plot; presents second-order plot. The strong downwrd peks in re ll t the loction of lu elements in the sequence. Humn P-gloin gene cluster. The query sequence used the E. coli insertion sequence S186. Our sttisticl nlysis detected much lrger region of unusul orgniztion. The lrger region is highly homologous to the E. coli entd gene (14) with the left end homologous to S421. Fig 6 shows the sme sequence plotted with n E. coli model. While the entd homology fits well with the E. coli model, the other pek in Fig. 6 does not. This region (ner 32,000) contins segment homologous to the E. coli lc operon. DSCUSSON We hve developed simple method, using seventh-order Mrkov chins, to locte genes nd clusters of genes in cteril nd yest DN sequences tht re quite dissimilr from other sequences in those species. Our pproch mkes no ssumptions out the nture of the sequences eing tested nd cn scn tens of kiloses per second. Similr nlyses cn lso revel repeted elements nd rpidly locte regions of homology etween two sequences. This pproch should permit the rpid loction of interesting regions in lrge DN sequences for susequent computtionl nlyses nd iologicl experimenttion. f sttionry seventh-order Mrkov model were pproprite for the query sequences, then for most susequences if FG. 6. GCRHBEGEB with lock size of 512 nt. seventh-order Mrkov chin nlysis is shown using the humn () ore. coli () model. Both of the upwrd peks in contin sequences homologous to E. coli DN sequences. Bush y P-gloin gene cluster. The query sequence is of length j + 1 the vlue -log2 P(i, i+1,.., i+j) should e pproximtely (j + 1)H, where H is the entropy of the Mrkov chin. Our nlysis differs in tht our -log2 P vlues re derived from dt se of other sequences, zero djusted, nd conditionl on The evidence we hve ccumulted rgues strongly tht the seventh-order Mrkov model does not hold glolly nd forms the sis for identifying iologiclly interesting regions in lrge sequences. We hve estlished our confidence limits y using simultion procedure (see Methods). Other efforts to determine the proility of the plots crossing ny vlue ssumed norml distriution for the vlues eing plotted ecuse ech would represent the sum of mny identiclly distriuted rndom vriles. Q-Q plots (not shown) of these vlues for lrge sequences clerly indicte tht this ssumption is not correct nd tht the distriutions of the plotted vlues lck the tils tht would expnd the rnge of vlues tht might e expected. The vrince determined y fitting line to these plots ws used in efforts to clculte the proility of extreme vlues rising in Mrkov chins of the length under study. These ltter vlues gve 95% confidence limits fr outside the rnge of the plots shown. While it is importnt not to equte iologicl nd sttisticl significnce (15), the

5 7138 Genetics: Scherer et l. confidence limits determined y the simultion procedure re quite effective in identifying interesting regions in the plots. Mrkov chins hve een used extensively to study DN sequences (16, 17), prticulrly with shorter sequences. While some fetures mentioned ove would hve een reveled through nlysis of se composition nd nerestneighor frequencies, much dditionl useful informtion is present in the higher-order Mrkov chins. With cteril sequences, the generl outline of the plot emerges in second-order chin, reflecting the high frction of coding sequence. This is not true with humn genomic sequences, s coding sequences do not produce the predominnt fetures of the plot. Fifth-order plots egin to indicte the presence of highly repeted elements such s lu sequences. The sequence under study is not included when the dt se is constructed; however, multiple entries or similr sequences my e present for susets of the sequence. t present, there is no systemtic wy to resolve these two possiilities for prticulr regions of lrge DN sequence. This type of overrepresenttion does not significntly ffect our conclusions. By use of dt ses the size of known yest nd E. coli sequences, n extr copy of sequence will lower its position in the plot y 0.1 j (dt not shown). There re mny possile explntions for why portion of chromosome might hve drsticlly different sequence composition from the reminder of the chromosome. Some vrition would e expected y chnce lone in the sttionry model. Unknown selective pressures or structurl fetures of chromosomes my e operting. dditionl possiilities include horizontl trnsmission from n unrelted orgnism, virl infection, nd incorportion of sequences from n orgnelle into the nucler genome. The demonstrtion of trnsfer of plsmids from cteri into yest is one exmple of the first possiility. Mny viruses re known to integrte their DN into humn chromosomes, nd frctions of the yest mitochondril genome hve een detected in nucler DN. The methods presented here will redily detect heptitis B virus in the ckground of humn genomic DN sequences (dt not shown). The modulr structure of cteril genomes is uilt on the clustering of genes with relted functions nd the genetic exchnges tht cn tke plce etween species. The centrl (2) region of phge hs long een known to hve se composition tht is mrkedly different from the composition of other prts of the phge genome. This region is the prt sustituted in mny trnsducing phges with E. coli DN, mking it surprising tht this is the prt of the genome lest like E. coldi DN. Similrly, the loin gene nd the sequences t the extreme right end of re expressed in lysogens (10). ll of the regions we find different from typicl E. coli orgniztion re known to e dispensle to the phge. Such regions of the phge pper to e under different selective pressures thn the rest of the genome nd might e optimized for function in nother host. The sequence of yest chromosome 3 hs een extensively nlyzed y vriety of sttisticl tests. The open reding frmes ner the glucokinse gene nd to the right of MT hve een noted to hve unusul se compositions in the third position of their codons (18). This grees well with our findings. While there is much evidence for movement of locks of genes t the telomeres, much less is known out internl insertions or sustitutions in yest chromosomes. The results in Fig. 6 indicte tht this pproch will permit the rpid scnning of lrge sequences for the presence of vector or other cteril contminnts tht might hve een included s result of some step ofthe cloning or sequencing. Other reports hve indicted the presence of E. coli S elements in eukryotic dtse entries s the result of Proc. Ntl. cd. Sci. US 91 (1994) homology serches (19, 20). The pproch tken here does not rely on homology to known sequences to detect suspect regions. The usge tles need not e constructed from ll sequences of n orgnism. ny suset of dequte size could e used such s trnscried or trnslted regions. Our results indicte tht cution must e exercised in the construction of dt sets for use in gene-finding lgorithms. t is cler tht severl uthentic E. coli nd yest genes differ significntly from the norm using our sttisticl mesure. Using known genes to uild the trining set will significntly is the set of reding frmes selected y computer progrms towrd finding more genes like those we lredy know. The highly nonrndom chrcter of DN sequences gretly complictes efforts t sttisticlly modeling lrge genomic DN sequences (21). Our studies provide simple method to visully disply this ehvior. Grphicl presenttion of the results of sequence nlysis offers severl dditionl dvntges over fmilir tulr presenttions. For exmple, the growth in size in pulic dt ses nd query sequences cuses n increse in the score required for sttisticl significnce in vriety of tests. Even with n incomplete understnding of the sttisticl ehvior of DN sequences, the presence of scores ll long lrge query rpidly gives the investigtor feel for ckground scores. We expect tht this pproch to the presenttion of DN sequence nlysis results will hve wide ppliction. We thnk John Mullign for providing yest genomic DN sequences prior to puliction. 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