Comparative analysis of the gene-dense ACHE/TFR2 region on

14 Pages·2001·1.25 MB·English

1352–1365 NucleicAcidsResearch,2001,Vol.29,No.6 ©2001OxfordUniversityPress
Comparative analysis of the gene-dense ACHE/TFR2
region on human chromosome 7q22 with the
orthologous region on mouse chromosome 5
MichaelD.Wilson,CathyRiemer1,DuaneW.Martindale,PamelaSchnupf,AndrewP.Boright2,
Tony L. Cheung3, Daniel M. Hardy3, Scott Schwartz1, Stephen W. Scherer2, Lap-Chee Tsui2,
Webb Miller1 and Ben F. Koop*
DepartmentofBiology,CentreforEnvironmentalHealth,POBox3020,UniversityofVictoria,Victoria,BritishColumbia
V8W3N5,Canada,1DepartmentofComputerScienceandEngineering,PennsylvaniaStateUniversity,UniversityPark,
PA16802,USA,2DepartmentofGenetics,TheHospitalforSickChildren,Toronto,OntarioM5G1X8,Canadaand
3DepartmentofCellBiologyandBiochemistry,TexasTechUniversityHealthSciencesCenter,3601FourthStreet,
Lubbock,TX79430,USA
ReceivedOctober13,2000;RevisedandAcceptedJanuary26,2001 DDBJ/EMBL/GenBankaccessionnosAF312032,AF312033
ABSTRACT canbeviewedathttp://web.uvic.ca/~bioweb/laj.html.
Laj is available at http://bio.cse.psu.edu/, along with
Chromosome7q22hasbeenthefocusofmanycyto-
online documentation and additional examples of
genetic and molecular studies aimed at delineating
annotatedgenomicregions.
regionscommonlydeletedinmyeloidleukemiasand
myelodysplastic syndromes. We have compared a
gene-dense, GC-rich sub-region of 7q22 with the INTRODUCTION
orthologous region on mouse chromosome 5. A
TheGiemsanegativebandq22ofhumanchromosome7isan
physicalmapof640kbofgenomicDNAfrommouse
area known to be gene-rich and prone to chromosomal
chromosome 5 was derived from a series of over-
breakage. Aberrations at 7q22 are commonly observed in
lapping bacterial artificial chromosomes. A 296 kb myeloid leukemias and myelodysplastic syndromes, with
segment from the physical map, spanning Acheto criticaldeletedregionsbeingmappedusingfluorescentinsitu
Tfr2,wascomparedwith267kbofhumansequence. hybridization (1–4) and polymorphic microsatellite markers
We identified a conserved linkage of 12 genes (5). Specifically, the segment between CYP3A4 and CUTL1
includinganopenreadingframeflankedbyAcheand containsaregiondeletedintwopatientswithchronicmyeloid
leukemia (3). Recent evidence for the association of schizo-
Asr2, a novel cation-chloride cotransporter inter-
phrenia with 7q22 further establishes the need for a detailed
acting protein Cip1, Ephb4, Zan and Perq1. While
characterizationofthisregion(6).
some of these genes have been previously
The fact that the mouse is the leading model for studying
described,ineachcasewepresentnewdataderived
disease processes in mammals makes the emerging mouse
from our comparative sequence analysis. Adjacent
genomic data a valuable resource for functional studies (7).
unfinished sequence data from the mouse contains Mouse–humancomparative analyseswillfacilitate thedesign
an orthologous block of 10 additional genes and interpretation of mouse knockout studies. Extensive
including three novel cDNA sequences that we refinements to the cytogenetically-based Mouse Genome
subsequently mapped to human 7q22. Methods for Informatics database map and the Davis Human/Mouse
displaying comparative genomic information, homology map have been made by identifying human
including unfinished sequence data, are becoming orthologstomappedmousegenes(8).
Detailed characterizations of disease regions are increasingly
increasingly important. We supplement our printed
being done through mouse–human genomic comparisons
comparative analysis with a new, Web-based
aimedatidentifyingnovelgenesandregulatoryelements.For
program called Laj (local alignments with java). Laj
example,acomparisonof7q11.23withitsorthologousregion
provides interactive access to archived pairwise
onmousechromosome5hasidentifiedonenewgeneandruled
sequence alignments via the WWW. It displays
outtheexistenceoftwopreviouslyproposedgeneswithinthe
synchronized views of a dot-plot, a percent identity region commonly deleted in patients with Williams–Beuren
plot,anucleotide-levellocalalignmentandavarietyof syndrome(9).Thisgrowingnumberofmouse–humancompar-
relevantannotations.Ourmouse–humancomparison isonsofincreasingsizecreatesaneedfornewwaystodisplay
*Towhomcorrespondenceshouldbeaddressed.Tel:[phone];Fax:[phone];Email:[email protected]
NucleicAcidsResearch,2001,Vol.29,No.6 1353
comparative genomic information. While several workbench H_DJ0138m12, the 3 kb gap clone and the 6357 nt XbaI
programs such as Genotator (10), RUMMAGE (11), Power- fragment from BAC clone 183H5. Clones were isolated with
BLAST/SEQUIN (12,13) and GESTALT (14) address the NucleoBond Plasmid Maxi Kits (Clontech) and randomly
demandforeffectiveanalysis,annotationanddisplayoflarge sheared by nebulization. Blunt ends were assured and frag-
singlegenomicsequences,newtoolsfordisplayingcomparative ments from 1.5 to 3 kb were size-fractionated by agarose gel
sequence analysis are just beginning to emerge. These electrophoresis. Fragments were ligated into SmaI-cut
programs include CGAT (15), Intronerator (16,17) and M13mp19vectorandsingle-strandedtemplateswere purified
Alfresco(18).InthispaperweintroduceaprogramcalledLaj withQiagenM13plates.Randomclonesfromeachsub-library
(local alignments with java), which can be used as an inter- werethenrunonABI373or377automatedDNAsequencers
active WWW-based tool for displaying pairwise sequence using fluorescently labeled primers (Amersham). Clones
alignmentsandassociatedannotations. 423o3, 558e17, cos159d9.L and H_DJ0138m12 were
In addition to the proven value of comparing complete sequencedto7-foldredundancy.GapswerefilledusingaPCR
genomic sequences, analysis of the rapidly-generated data approach and dye-terminator sequencing with unique primers
from lower redundancy sequencing may also be of use to the (AmershamandABI).Atthetimeofanalysis,BAC139n8had
biological community (13,19). The PipMaker Web server been sequenced to 3.75-fold and 493b1 to 1.57-fold redun-
supportsanalysisofworkingdraftsequencesbypermittingone dancy.
ofthetwosequencestoconsistofmultipleunorientedcontigs
(20). Laj provides a graphical way to organize, label and Sequenceanalysis
display the results from PipMaker, thereby facilitating the
DNAStar software was used for gel trace analysis, contig
analysisofunfinishedsequence.
assembly and DNA and protein alignments. Repeat elements
We extend the sequence of the gene-dense, GC-rich EPO were characterized using RepeatMasker2 (A.F.A.Smit and
contigonhuman7q22,previouslydescribed(11)andcompare P.Green, unpublished results; http://ftp.genome.washington.edu/
it with ∼380 kb of the corresponding mouse genomic DNA.
cgi-bin/RepeatMasker). Comparative sequence alignments
This genomic comparison involves both complete and partial
displayed in this paper were generated using PipMaker (20;
mouse sequences, which together reveal extensive conserva-
http://bio.cse.psu.edu/). DNA and protein sequences were
tion of gene content and order. Novel transcripts and new
compared with available public databases using the various
splice variants are identified, and the mouse–human map for
BLAST programs available through the networkserver atthe
thisregionisfurtherrefined.OurWebsite,http://web.uvic.ca/
National Center for Biotechnology Information (http://
~bioweb/laj.html, provides an electronic supplement to the
www.ncbi.nlm.nih.gov/). Putative exons were identified using a
resultspresentedinthispaper.
variety of programs GENSCAN (21; http://bioweb.pasteur.fr/
seqanal/interfaces/genscan-simple.html), FGENES, FGENESH
MATERIALSANDMETHODS and FGENES-M from the BCM Gene-Finder (V.V.Solovyev,
unpublished results; http://dot.imgen.bcm.tmc.edu:9331/
Isolationandcharacterizationofclones gene-finder/gfb.html). Compiled, overlapping sets of
expressedsequencetags(ESTs)weredownloadedasUniGene
The P1-derived artificial chromosome (PAC) clone
clusters (22) and the individual ESTs were assembled using
H_DJ0138m12 was from the RPCI PAC whole genome
DNAStar software. Additional, overlapping cDNA and EST
library. The cosmid clones cos159d9.L and cos8a5.L were
sequences were then obtained by submitting the consensus
obtainedfromthechromosome7-specificcosmidlibraryofthe
sequence of the assembled ESTs to BLAST. This facilitated
Lawrence Livermore National Laboratory (LL07NCC01).
Human bacterial artificial chromosome (BAC) 183H5 was theverificationofsplicesites,establishedthelocationofnovel
obtained from the BAC library of Genome Systems, Inc. genes and allowed the extension of previously published
(StLouis, MO) using human probes for zonadhesin. The cDNA sequences. Differences, including SNPs, alternative
mouse genomic DNA BAC clones (423o3 and 558e17) were splicing events and predictions not supported by EST data,
obtained from the Research Genetics CITB-CJ7-B mouse werefurtherinvestigatedbyinspectingthetracedatafromthe
BAC library using PCR probes for the mouse Ache and Epo originalgenomicsequenceassembly.
genes,respectively.A3kbgapbetweenBACs423o3and558e17
Expressionstudies,cloningandpartialcDNAs
was bridged by PCR using primers (423sp6-F: 5′-CTGA-
GAGAGACTGACACCAGAAGG-3′,and558T7-R:5′-TGA- MouseandhumanexpressionstudiesofASR2andCIP1were
CTCGGGTCAATTCTAAGTT-3′)andmousegenomicDNA. carried out on Clontech multiple tissue panels using
The resulting DNA was T-A cloned into pGEM-T vector HotstarTaq (Qiagen).A 50µlvolwasused for allexpression
(Promega).AdditionalmouseBACcloneswereobtainedfrom studies,andthecyclingparameterswereasfollows:15minat
theResearchGeneticsCITB-CJ7-BmouseBAClibraryusing 95°C for Taq activation, and eight cycles of 94°C for 30 s;
gene-orBACend-specificprimers.ClonesfromtheRPCI-11 65°Cfor1min;72°Cfor1min;followedby30–33cyclesof
Human Male BAC Library were identified by hybridization 94°C for 30 s; 58°C for 1 min; 72°C for 1 min; and a final
using BAC end sequence from NH452F23, NH0044M06, extensionof5minat72°C.Primersusedforexpressionstudies
NH0264N05andNH0126L15. are available from the authors upon request. Partial mouse
cDNAsforAsr2,Cip1andPerq1wereamplifiedfrommouse
DNAsequencing
brain cDNA (Clontech). CIP1 was amplified from human
A shotgun sequencing strategy was employed for clones brain cDNA (Clontech). The PCR products from the cDNA
423o3, 558e17, 139n8, 493b1, cos159d9.L, cos8a5.L, were T-A cloned into pGEM-T vector (Promega). Asr2 and
1354 NucleicAcidsResearch,2001,Vol.29,No.6
Figure1.Overviewoftherefinedchromosome7q22physicalmapfortheregionspanningthegenesPAI1andCYP3A4,withadirectcomparisonwith640kbof
orthologous genomic sequence from mouse chromosome 5. Black rectangles represent completely sequenced regions, gray rectangles represent partially
sequencedorPCR-mappedclonesandgrayhatchesrepresentpartsofcloneswhoseoverlappingdistancesarenotknown.(A)Representativegenesandclones
providefurtherrefinementtothehumangenemappreviouslydescribed(11).Aminimaltilingpathdeterminedbyhybridization,PCRandinsilicomethodsclearly
establishesMUC3onthetelomericsideofACHEandlinkstheHRBLendofAF053356totheCYP3A4locus(seehttp://www.genet.sickkids.on.ca/chromosome7/for
a more detailed physical map including additional genes, further clones and experimental details). (B) Comparative human–mouse genomic map showing
conservedsyntenyofatleast22genes.Thecompositegenemapshowshumangenes(uppercaseletters),allofwhichhaveamouseortholog,aswellasthemouse
geneCyp3a13,whosehumanorthologisyettobedetermined.TheorientationofthemouseBACclonesinrelationtochromosome5wasinferredfromthelocation
ofthemouseCyp3agenelocusonarefinedchromosome5map(http://genome.nhgri.nih.gov/chr7/comparative;8).PCRmappingdeterminedthataTSC-22-like
gene,THG-1,isnexttoHRBLinbothhumanandmousefollowedbyZE03F02andFLJ20257(Ze03f02andD5Wsu46e)whoseorderhasnotyetbeendetermined.None
ofthegenesshownbetweenFLJ20257andCYP3A4hasbeenidentifiedinourpartialmousesequencedataforBACs139n8and493b1(D5Wsu46etoCyp3a13).
CIP1 were completely sequenced by primer walking. Perq1 sequencefrompositions38341to45778ofAF053356butdid
andCip1cloneswereendsequenced. match positions 1–38340 and 45779–48328 of AF053356.
Position38340ofAF053356correspondstotheonesequence
gap in the EPO contig that could not be filled using a PCR
RESULTS
approach (11). Furthermore, alignments with our complete
WeobtainedaminimaltilingpathofBACsrepresenting∼640kb sequence of the human zonadhesin (ZAN) cDNA (T.Cheung,
ofmousechromosome5orthologoustoagene-dense,GC-rich M.Wassler,G.CornwallandD.Hardy,manuscriptinpreparation)
isochorefromthe EPOregionof7q22(Fig.1).MouseBACs revealed that ZAN exon 7 was missing and exons 1–6 were
423o3 and 558e17 were completely sequenced along with a downstream of exons 8–11 in AF053356. The positioning of
3kb segment that links the two clones, producing a gap-free exons 1–6 (including the putative promoter region) down-
contig of 296 kb. Two overlapping human clones (cosmid streamofexons8–11alterstheconserveddomainorderofthe
159d9 and PAC 138m12) were also sequenced completely, zonadhesin protein observed in mouse, human and pig,
yieldinga118kbcontig.Thiscontigwasusedtofillina94kb strongly suggesting that the ZAN locus on AF053356 was
gap linking the ACHE-containing genomic clone AF002993 incorrectlyassembledanddoesnotrepresentanaturalvariant
(23)tothe228kbEPOcontigAF053356previouslydescribed of the gene. To resolve this we obtained human BAC 183H5
(11;Fig.1). fromGenomeSystemsandutilizedunfinishedhighthroughput
We detected a sequence transposition within the previously genomic (HTG) data from The Washington University
reported human genomic sequence AF053356 (11) when we sequencing project (AC009488; Fig. 1). The sequence of a
compareditwithourmousegenomicsequenceinthe5′region 1253 bp PCR product amplified from a human placental
ofthezonadhesin(Zan)locusandwiththemousezonadhesin genomic DNA template confirmed the accuracy of
cDNA (MMU97068) (24). Inspecting our partial human AC009488_3ctg3frompositions24030–25292(senseprimer:
sequencefromcosmid8a5(Fig.1)reinforcedthepossibilityof CTCTATCCGCCGGGGCTCCTGTA; antisense primer:
sequence transposition, as cosmid 8a5 did not contain any CACGCCTGGCTTTCCTGATGACC). In addition, the
NucleicAcidsResearch,2001,Vol.29,No.6 1355
sequence of a 2268 bp PCR product bridged a gap between cited (29–31), the most abundant Ache mRNA species are
exons 11 and 12 in AF053356 (sense primer: ACACTGG- believedtorangefrom2to2.4kb(29).Whileitisstillpossible
GCCCTCGGACATAAAA; antisense primer: GACTGGC- that this novel ORF is part of an uncharacterized variant of
CTGCGTGGGAGAAC). We also identified a 6357 bp XbaI acetylcholinesterase, or thatthe two genes are co-transcribed,
fragment from BAC clone 183H5 that spans exons 7–8 by there is currently no sequence data supporting this. Taken
Southernblottingusingpositions809–1103ofthezonadhesin together, our preliminary evidence suggests that a new gene
cDNAasa probe. The sequenceofthisfragmentmergedtwo separatefromAcheexistsbetweenAcheandAsr2(Fig.2B).
contigsofAC009488,and,togetherwiththesequencesofthe
PCR products, properly ordered exons 1–6 and 8–11, which GNB2,CGGrepeatsandarecombinederythroleukemic
are transposed in AF053356. Thus, collectively these cellline
sequences resolved discrepancies between AC009488 and A deletion on mouse chromosome 5 resulting in a rearrange-
AF053356 and corrected the sequence transposition in mentofthe5′UTRofGnb2withpartofexon1ofEpocauses
AF053356. the abnormal erythropoietin gene expression found in murine
Wedirectlycompared267kbofthecorrected365kbhuman erythroleukemic cell line IW32 (see GenBank: MUSERPB)
contigto296kbofthecontiguousmousesequence,revealing (32). We find that this deletion removes a 45 kb region
thegenomicstructureofaconservedlinkageof12genes(Ache containing Rpp20, Perq1 and Gnb2 (Fig. 2B). The region
to Tfr2; Fig. 2). The data are shown as a traditional dot-plot extending2.5kbfromthedeletionbreakpointontheGnb2side
(Fig. 2A) andpercentidentityplot(PIP;Fig.2B).New infor- (positions 251000–253567 on Fig. 2B) is 79.4% similar
mationregardingspecificgenesisprovidedinTables1and2. betweenhumanandmouseandisveryGC-richinbothspecies
We also compared adjacent incomplete sequence data from (68.6and59.8%,respectively).Inthe5′UTRpartofthissame
BAC139n8totheorthologousregiononAF053356(PCOLCE 2.5kbregion,10consecutiveCGGrepeatswereidentifiedby
to HRBL; Fig. 1). This revealed that the mouse orthologs to screeningabraincDNAlibraryforCGGrepeatsinasearchfor
PCOLCE,FBXO24,LRN,IRS3LandHRBLareallpresenton neurologicaldiseasecandidategenes(33).Elevenconsecutive
BAC 139n8 (Fig. 1). In addition to the above tables and CGG repeats are seen in AF053356 (11), suggesting that this
figures,weutilizeournewprogramLajtointeractivelydisplay region may be polymorphic. Recent findings demonstrate a
both our complete and partial genomic alignments (http:// strong correlation between chromosome deletion breakpoints
web.uvic.ca/~bioweb/laj.html). This electronic supplement and CGG repeats in patients with Jacobsen (11q-) syndrome
displays the alignments themselves, hyperlinks to reference (34,35), which raises interesting questions about a human
cDNA sequences, UniGene clusters (22), GeneCards (25), breakpointanalogoustotherearrangementeventcharacterized
LocusLinks(26)andESTsnotlinkedtoanycDNA,alongwith inthemouseerythroleukemiccelllineIW32.
PubMedlinkstorelevantliterature(Fig.3).
DefiningtheboundariesofAcheandapotentialnovelgene
A recent search for genes that are expressed during mouse
development revealed the expression of a novel cDNA frag-
ment, D5Ertd655e, in a 2-day-old mouse embryo (27).
D5Ertd655e is part of a mouse UniGene cluster (Mm.21836)
that is homologous to clusters observed in human and rat
(Hs.157779 and Rn.13571, respectively). This 3′ cDNA
sequenceisidenticaltothe3′endofourpredictedgenelying
between Ache and Asr2 and contains the polyadenylation
signalpreviouslyattributedtoanalternativeformofAche(28).
We searched the data banks and found no mRNA or EST
sequencethatcontainedAche exon6andthe second(alterna-
tive)polyadenylationsite.WedidfindadditionalmurineESTs
thatoverlapwiththeD5Ertd655e,providingevidencethatthe
entire predicted open reading frame (ORF) is transcribed in
mouse and rat (see Table 1 and http://web.uvic.ca/~bioweb/
laj.htmlforfurtherdetails).
Initially it was assumed that two major mRNA species of
mouseAche(2.5and3.7kb)seeninnorthernblotexperiments
differed only in the 3′ UTR region and thatthe larger mRNA
species was due to the use of a second polyadenylation site
(28). The only published northern blot evidence supporting
Figure2.(A)Dot-plotdisplayofmouse(x-axis)andhuman(y-axis).(B;overleaf)
alternate polyadenylation usage comes from an experiment
PIP.Nucleotidepositionsareshownforthemousesequenceonthex-axis,and
utilizing a 1.5 kb probe that begins 200 bp 3′ of the first percentsequenceidentitywiththecorrespondinghumansequence(50–100%)
polyadenylation signal and extends through the second poly- isshownonthey-axis.Thepurpleandorangeshadingontheupperandlowerhalves
adenylationsignal(28).Thisprobewouldalsocontainthelast ofthePIPrepresentthelocationsofexonsonthe+and–strands,respectively,while
thepinkandlightorangeareasrepresentUTRs.Redstripesindicateregulatory
two exons of the ubiquitously expressed Asr2, which would
regionsthathavebeenfunctionallycharacterized.Genesarelabeledbasedon
complicatetheinterpretationofthesenorthernblots.Although cDNA,ESTandcomparativegenomicinformation.RepeatMasker2(A.F.A.Smit
theexistenceofalternatepolyadenylationusageinAcheisstill andP.Green,unpublishedresults)wasusedtolocaterepeatelements.
1356 NucleicAcidsResearch,2001,Vol.29,No.6
NucleicAcidsResearch,2001,Vol.29,No.6 1357
Extendingthemouseandhumanphysicalmaps these orthologs has proven highly successful in providing
sequence-ready maps for mouse (8). We also find that in
Identifyingmouseorthologsforhumangenesfoundonhuman difficult-to-map areas of chromosome 7 not represented by
chromosome 7 and then probing mouse BAC libraries with contiguous YAC sequence, human orthologs of mouse genes
1358 NucleicAcidsResearch,2001,Vol.29,No.6
Table1.MouseandhumangenesintheACHE/TFR2region
ACHE AChE(acetylcholinesterase)hasmultiplemolecularforms(28)(Fig.2BdisplaysthedominantE4-E6hydrophilicvariant).Ahighlyconserved
Ache regionjust5′ofthetranscriptionstartsite,highlightedbyaredstripeinFigure2B(positions7445–7516),containstranscriptionfactorbinding
sitespreviouslydescribed(55).Asecondregioncontaininganalternativepromoterthatactssynergisticallywiththeproximalpromoterin
enhancingAcheexpressioninneuroblastomacells(29)isalsohighlyconserved,andhighlightedbyaredstripeinFigure2B(positions
6374–7139).Theconservedregionsfoundupstreamofthealternativepromoterhaveyettobeexploredforregulatoryelements.
Hs.157779 Usingourcomparativegenomicdatawepredicta217aminoacidproductinmousejustdownstreamofAche.Inhumanwecanpredicta
Mm.21836 227aminoacidproductfromAC002993thatis79.2%identicaltoaputative217aminoacidproductinmouse(Table2).Theseproductsshow
similaritytotheC-terminalendoftheputativeproteinsCG16979(Drosophila),CAB41160(Arabidopsis),F38A5.1(C.elegans)and
BAA92064(human).However,onourhumancosmid159d9andcloneNH0126L15sequencedbyWashingtonUniversityinStLouis
(AC011895),thestartcodonhasaCinsteadofanA,makingthelargestORF146aminoacids.
ASR2 Asr2(arseniteresistanceprotein2)wasnamedforitsabilitytoconferarseniteresistancetoanarsenite-sensitiveChinesehamsterovarycell
Asr2(Also line(56).WecharacterizedthecompletegenomicstructureofASR2(Table2andFig.2B)andfoundittranscribedinalltissuestestedinboth
knownas:Ars2) humanandmouse(datanotshown).The876aminoacidhumanproductdiffersinbothsizeandaminoacidcontentfromthatpredictedbya
full-lengthcDNAcloneAL096723.Thiscanbeexplainedbytwoseparate,singlenucleotidedifferencesinAL096723thatcauseaframeshift
error.ThesedifferencesareresolvedbyourhumangenomicsequenceandwereprobablyduetosequencecompressionsinAL096723.
AlternativesplicingoftheAsr2mRNAwasrevealedbysequencingtwodifferentPCR-derivedAsr2cDNAclonesthatcontainthefullcoding
regionofthemouseAsr2cDNA(lessthefirstmethionineandthelasteightcodons).BycomparingthesecDNAcloneswithgenomicandEST
sequenceswefoundthreealternativespliceeventsthatdecreasedthecodingsizeby12,21or33(12plus21)bp(Table2).Anexaminationof
humanESTsrevealedonlythe12bpalternativespliceeventwhich,asinmouse,usesanon-canonicalsplicesite(TGinsteadofAGinthe
AG–GTsplicerule).
CIP1 Weidentifiedandcharacterizedanovelcation-chloridecotransporter-likegeneinbothhumanandmouse.Recently,Caronetal.(57)
Cip1 independentlycharacterizeda3.2kbtranscriptthattheynamedCIP1(cation-chloridecotransporter-interactingprotein)asit
coimmunoprecipitateswithendogenousNKCC(Na-K-Clcotransporter)andinXenopuslaevisoocytes,itinhibitsNKCC1-mediatedcation
transport.CIP1is100%identicaltoourputativecation-chloridecotransporter-likeproteinandispredictedbyPSORT(58)tohave12
transmembranehelices.ToconfirmourgenepredictionsweclonedandsequencedapartialcDNA,whichspansall13exon/intronboundaries,
fromahumanbraincDNApool.ThiscDNA,combinedwithhumanandmousegenomicsequencecomparisons,allowedustopredictthe
mouseexonstructure,ofwhichexons7–13havesubsequentlybeenconfirmedbysequencingclonedmousebraincDNA.Wescreened
ClontechmouseandhumanmultipletissuecDNApanelsusingPCR,andfoundthatthisgeneisubiquitouslytranscribedinalltissuestested
(datanotshown).Alternativesplicevariantswereapparentinallmousetissuestested,whilehumanexpressionstudiesusingprimersdesigned
forthecorrespondingpositionsonhumancDNAshowednoevidenceofalternativespliceproducts.However,humanESTAW675796reveals
analternativesplicingeventthatresultsina627aminoacidisoformofCIP1thatispredictedtohaveatruncatedC-terminus,yetisstill
predictedtocontain12transmembranespanningregions.CIP1showshighersimilaritytoDrosophila(60%;CG10413),C.elegans(54%;
T04B8.5)andputativeyeast(49%;NP009794.1)proteins,thanitdoestoothermembersofthetwoestablishedbranches(Na+-K+-Cl–and
K+-Cl–)ofthecation-chloridecotransporterfamily.ThisevolutionaryconservationcombinedwithpreliminaryfunctionalstudiesplaceCIP1in
athirdbranchofthecation-chloridecotransporterfamilyandsuggestthatitmaybepartofanewfamilyofproteinsthatmodifytheactivityor
kineticsofothercation-chloridetransporters(57).
TRIP6 TRIP6(thyroidreceptorinteractingprotein6)isahumanLIMdomain-containingproteinpreviouslymappedto7q22(59)whoseproposed
Trip6 functionistotransmitsignalsfromthecellsurfacetothenucleus(60).Wehavecharacterizedthegenomicstructureofthemouseandhuman
Trip6genes(Table2).
EPHB4 EPHB4(ephrintype-Breceptor4precursor),originallynamedHTK(hepatomatransmembranekinase),isareceptortyrosinekinaseexpressed
Ephb4 inavarietyoftissues,withitshematopoieticexpressionlocalizedtothemonocyticlineage(61,62).EPHB4ispreferentiallyexpressedinveins
(63)andrecentstudieshaveclearlylinkedEphb4toangiogenesisinX.laevis(64).Themouseortholog,Ephb4,wasoriginallydesignatedMyk1
(mousetyrosinekinase;65)andlatercalledMdk2(mousedevelopmentalkinase2;66).TheESTsequenceAI049017extendsthe5′UTRofthe
Ephb4cDNAandsuggeststhatanalternativesplicingeventoccursinthisregionofthegene.Whileourgenomicdataconfirmboththecoding
regionandtheoveralltranscriptsizepreviouslydescribed(66),wedonotfindamatchforthe3′end(positions
3756–4473)ofthatsequence(Z49085);however,thissequencematchesperfectlytopositions781–1496oftheCctggene(Z31556),whichhas
beenmappedtomousechromosome3(67).WethusconcludethatZ49085containssequencefromtwoseparategenes.
ZAN ZAN(Zonadhesin)codesforaspermmembraneproteinthatbindstothezonapellucidaoftheegginaspecies-specificmanner(68).Inmice,
Zan theZanproductisalarge(5374aminoacid)testes-specificproteinwithacomplexmosaicstructureconsistingofMAM(meprin,A5receptor,
proteintyrosinephosphatasemu),mucin,VWD(vonWillebrandfactortypeD),EGF(epidermalgrowthfactor),transmembraneand
intracellulardomains(24,69).BycomparingourmousegenomicsequencewiththemouseZanmRNAMMU97068(16.1kb)wefindthatthe
mouseZangenecontains88exonsandspans98.2kbofgenomicDNA.Incontrast,thehumanZANgenecontains48exons,spans61kband
codesforaproteinof2724aminoacids(T.Cheung,M.Wassler,G.CornwallandD.Hardy,manscriptinpreparation).Theexpansionofthe
mouseZangenestandsoutinthedot-plot(Fig.2A)asa39.5kbelongationalongthex-axis(mouse;positions108788–147614onFig.2B),
andisprimarilyduetomultipleduplicationofthepartialD3domainsequence.Eachofthe20partialD3domains(exons38–77)consistsofa
segmentcontainingtwoexonsof∼218and142bpinlength.
NucleicAcidsResearch,2001,Vol.29,No.6 1359
Table1.Continued.
EPO Erythropoietin(EPO)regulatestheproductionofredbloodcellsbypromotingtheproliferationanddifferentiationoferythroidprecursorcells
Epo (reviewedin70).ThemouseandhumangenomicstructuresforEPOhavepreviouslybeendescribedindetail,withconsiderableprogressbeing
madeincharacterizingtheregulatoryelements(reviewedin49).ThemajorproximalregulatoryelementsareapromoteratPIPpositions
205140–205195inthemousesequenceanda3′enhancerat201210–201252(71).Theseelements,markedbyredstripesinFigure2B,are
substantiallymoreconservedthanthesurroundingsequence.Whileinitialcluesfortheirlocationwereprovidedbycarefulinspectionof
human–mousealignmentsbetweenmuchshortersequences,Figure2Bverifiesthattheyalsostandoutclearlyinacompletelyautomated
comparisonoflonggenomicregions.Inparticular,thepromotercorrespondsverycloselytoa48bpgap-freesegmentwith94%nucleotide
identity(PIPpositions205144–205191),andtheenhanceriscontainedwithina149bpgap-freesegmentwith79%identity(PIPpositions
201141–201289).TwoadditionalelementsregulatingEPOtranscriptionhavebeenmappedtotheregion14kbupstreamofthetranscription
startsiteinhumansbyinsertinglargeDNAfragmentsintotransgenicmice(summarizedin49).Theseresultssuggestthattheconserved
regionsaroundpositions209–214kbmaybeworthyofinvestigation.
RPP20 RPP20codesfora20kDaproteinthatco-purifieswithhumanribonucleaseP(RnaseP),atRNAprocessingribonucleoprotein(72).TheRpp20
Rpp20 geneconsistsofatleasttwoexons,oneofwhichaccountsfortheentire140aminoacidproduct,whichis94.3%identicalbetweenhumanand
mouse(Table2).
PERQ1 PERQ1codesforanovelproteinrichintheaminoacidsproline,glutamate,arginineandglutamine(P,E,RandQ),withaglycine-tyrosine-
Perq1 phenylalanine(GYF)domain.TheGYFdomainispartofauniquehelix–bulge–helixdomainthatbindstoproline-richmotifs(73).Our
mouse–humancomparison,ESTdataandpartialcDNAsequencingpredictaputativePerq1mouseproteinandanorthologousPERQ1human
proteinthatdiffersomewhatinstructurefromtheputativeORF2(11).Inordertopredictthe1004aminoacidproductinhuman,wehadto
assumethatarunofninecytosinebases(position88127ofAF053356)actuallycontainedeightorten,asthereadingframeshiftproduceddid
notcorrespondtoourmousecDNAsequenceorgenomicdata.WhilethecDNAsarestillnotfullycharacterized,wepredictaminimum3kb
transcriptwith24exonsinbothspecies(Table2).Consideringthemouse–humansequencesimilarityandthepresenceofaCpGislandfound
withintheregion8.8kb5′oftheannotatedPerq1gene(Fig.2B;PIPpositions229–231kb),itispossiblethatpartoftheupstream5′UTRand
promoterregionhasnotbeencharacterized.Atthe3′endofthegene,severalESTsequencesfromavarietyofspeciesmatchsignificantlywith
theregionbetweenPerq1andthe3′endofGnb2.PERQ1shows58%identitytotwoputativehumanproteins(AK001739andAB014542)and
55%identitytoaputativechickenprotein(U90567).ThesethreeproteinsarealsorichinP,E,RandQ,andcontainconservedGYFdomains.
GNB2 GNB2(guaninenucleotidebindingprotein,βpolypeptide2)encodesaβsubunitofguaninenucleotide-bindingproteins(G-proteins).
Gnb2 G-proteinsareheterotrimeric,containingα,βandγsubunits,andareinvolvedinamultitudeofcellularsignalingprocesses(forarecentreview
see74).WecharacterizedthemousegenomicstructureforGnb2.Partofthehighlyconservedregionfoundinthe5′UTR(PIPpositions
250459–252757)appearstobetranscribedinvariousmouseandhumantissues(seehttp://web.uvic.ca/~bioweb/laj.html).
BAF53B ThehumanACTL6-likegeneismorecloselyrelatedtoactingenesoflowerorganismsthantothoseofvertebrates(11).Sinceitsdiscovery,
Baf53b ACTL6-likehasconfusinglybeencalledACTL6(BAF53A).However,itisclearfromcomparingourmousegenomicsequencetoAF053356
thattheACTL6-likegeneisactuallyBAF53B.ThemouseorthologBaf53bencodesa426aminoacidputativeproductthatis84.1%identicalto
theputative475aminoacidhumanBAF53B(Table2).Baf53bis84%identicaltomouseBaf53a,whoseroleinchromosomeremodelinghas
previouslybeendescribed(75).
TFR2 Transferrinreceptorsmediateironuptakebybindingandinternalizingthecarrierproteintransferrin.Thetransferrinreceptor2gene(TFR2)
Tfr2 contains18exons,is2.9kbinlengthandisprimarilyexpressedintheliver(11,76).Wehavedeterminedthegenomicstructureofthefirstsix
exonsofmouseTfr2andidentifiedanalternativesplicevariantoccurringinthe5′UTRregionofthegene(ESTAA537969).
Anexpandedversionofthistableandlinkstospecificsequencescanbefoundathttp://web.uvic.ca/~bioweb/laj.html.
identifiedinorthologousregionscanhelptoorientanddefine UsingLajtodisplaycomparativegenomicinformation
human physical maps. With PCR primers designed from
Laj gives the reader an immediately accessible interactive
human orthologs for Hrbl, Thg-1, Ze03f02, D5Wsu46e and
supplementtothetext,providingconsiderablymorepowerfor
Cyp3a13, we ordered seven BACs previously identified by
viewingandinterpretingdatathancanbeprovidedonaprinted
probingwiththeendsequenceofNH0452F23(AZ254552;see
page. It can easily be set up as a Web-based applet, allowing
Fig.1forarepresentativepair).FurtherPCRandhybridization scientists to curate their sequence data and display the infor-
experiments join the EPO contig (AF053356) to a nearly mationtothepublicfromtheirownWebsites.Furthermore,this
completed sequence contig that extends all the way up to electronicsupplementcanbeupdatedasresearchprogresses.The
AZGP1andCYP3A4(Fig.1).OntheACHEsideofour365kb applet can be viewed from any computerwithInternetaccess
contig, BAC end sequence from clone NH0126L15 andaWebbrowserthatsupportsJava 1.2 (e.g.,aPCrunning
(AC011895) identifies several clones that were previously Windows95/98/NT or Linux,or aSparc workstation running
mapped near MUC3. Specifically, end sequence from clone Solaris),thusenablingawideaudiencetoreadilyexaminethe
NH1001A20 lines up with clone CIT-HSP-306C13 whose information.Lajcandisplayadot-plot,aPIPandanucleotide-
other end aligns to the locus designated MUC3 on Figure 1, levellocalalignmentsimultaneously.Italsosupportsavariety
whichcontainstwogenes,MUC3AandMUC3B(36). of annotations, including locations of exons and repeat
1360 NucleicAcidsResearch,2001,Vol.29,No.6
Table2.Comparativegeneinformation
Genes,UniGene Genestructure Protein Conservation
cluster(s) mRNAlengtha Exons Representativealternativesplicedform Length %Aminoacid %nt(ORF)
(aminoacids) identity
Ache mouse 2089 6 614 88.4 84.8
human 2218 6 NM000665,NM015831 614
Mm.21836 mouse 1001 1 217 79.2 80.9
Hs.157779 human 809 1 227
Asr2 mouse 2901 20 AA562274,AA929503,AW488339 875 97.8 88.7
human 2896 20 AA317461,AW572565 876
Trip-6 mouse 1754 9 480 86.8 83.4
human 1735 9 476
Cip1 mouse 3302 14 observedb 914 93.5 85.3
human 3310 14 AW675796 914
Ephb4 mouse 4271 17 AI049017 987 91.9 90.5
human 3945 17 987
Zan mouse 16125 88 5374 61.2 71.1
human 8852 48 2724
Epo mouse 1385 5 192 78.2 79.6
human 1367 5 193
Rpp20 mouse 784 2 140 94.3 87.5
human 893 2 AA405580,AA779813,AA324940 140
Perq1 mouse 3033 24 1011 89.1 87.8
human 3312 24 1004
Gnb2 mouse 1860 10 340 99.7 92.6
human 1661 10 340
Baf53b mouse 1472 14 426 83.7 84.1
human 1495 14 429
Tfr2 mouse 5′partial 1–6 AA537969
human 5′partial 1–6
aDeterminedusingEST,cDNAandcomparativegenomicdata.
bEvidenceforalternativesplicevariantsseenonCLONTECHmultipletissuecDNApanels.
elements, color underlays that can be used to represent Cards(25),LocusLinks(26)andPubMedreferences.Notethat
GENSCANpredictionsoranyotherregionsoftheannotator’s when provided with exon locations in the first sequence, Laj
choice and hyperlinks to relevant Web resources such as displaysthemintwoways:astallblackboxesinthediagram
sequences from GenBank (37), UniGene clusters (22), Gene- abovethePIP,andasshadedregionsinthetoprowofthetext
NucleicAcidsResearch,2001,Vol.29,No.6 1361
Figure 3. Using Laj to view complete and partial comparative genomic
sequencesfromhumanchromosome7q22(y-axis)andtheorthologousregion
inthemouse(x-axis).(A)ViewoftheentireACHE/TFR2genomicregion.The
overallscalebar,dot-plot,relevantannotations,PIP(labeledasinFig.2B)and
localalignmenttextarealldisplayedinthesamewindow.Thefirstmessagebox
belowthemenubardisplaysinformationabouttheobjectthemouseispointing
at,inthiscaseahyperlinktotheUniGeneclusterMm.21836.Alocalalignment
intheACHE/ASR2regionhasbeenselectedbyclickingonit,whichhascaused
the local alignment to be highlighted in red and the clicked position to be
marked with a red circle. Information about these is provided in the second
messagebox.Thescrollablepanelatthebottomofthewindowdisplaysthe
selectedlocalalignmentinanucleotide-leveltextformat,withthewhitecursor
correspondingtothemarkedposition.(B)‘Zoomingin’ontheregionselected
in (A). Themarkedpositionliesina geneflanked by ACHE and ASR2that
containsUniGeneclusterMm.21836,andthemousepointeriscurrentlyresting
onaB1elementfoundinthe3′UTRofthisgene.Thepurpleandorangeshading
ontheupperandlowerhalvesofthePIPrepresentthelocationsofexonsonthe
+and–strands,respectively,whilethepinkandlightorangeareasrepresent
UTRs.SimilarshadingoftheexonsandUTRsappearsinthetoprow(mouse
sequence)ofthetextpanel.HyperlinkedannotationsincludeUniGenecluster
Mm.21836(bluebarabovemousepointer)flankedbyAcheandAsr2UniGene
clusters,cDNAs,alternativelysplicedESTs(allbluebars),LocusLinks(pink
bars)andaGeneCardforACHE(orangebar).(C)Displayofunorderedpartial
mousedata(y-axis)alignedtocompletehumansequence(x-axis).Eightmouse
contigsfromBAC139n8orthologoustothePCOLCE/HRBLregiononhuman
clone AF053356 (11) are displayed, separated by horizontal gray lines. An
alignment(highlightedinred)thatcontainsthe5′endoftheIRS3Lgenehas
beenselected,andthemousepointeriscurrentlyrestingonahyperlink(purple
bar) to the PubMed entry for a paper about the mouse Irs3 gene (54). The
orange-shadedtexthighlightsthebeginningoftheIRS3LORFandUTR.(See
http://web.uvic.ca/~bioweb/laj.htmlforadisplayoftheorderedcontigdata.)
graphicalandtextrepresentationsofthealignmentsandfacili-
tatesthevisualizationofintron–exonjunctions.
Lajisavailablefordownloadathttp://bio.cse.psu.edu/andis
well documented, so interested researchers can (i) use it as a
stand-alone tool to display and annotate their sequences, or
(ii)set up their comparative sequence analyses for display on
the Internet. Once Laj is installed, the user need only supply
the data and annotationsin specific formats.PipMaker, along
with some small programs available from the PipMaker site,
can rapidly transform input files obtained from various other
bioinformatic sources (e.g., GENSCAN and RepeatMasker2)
intoaformatsuitableforLaj,thusincreasingLaj’sutilityasa
comparative workbench. For more information about Laj,
includingonlinedocumentation,asoftwaretourdescribingthe
program itself and additional examples of annotated genomic
regions,pleaseseehttp://bio.cse.psu.edu/.
DISCUSSION
Comparative sequence analysis is a useful supplement to
traditionalgene-findingmethodsforcompletingthecatalogof
allmammaliangenes.Wedescribe640kbofmousegenomic
sequenceorthologoustoagene-dense,GC-richisochoreinthe
EPOregiononhumanchromosome7q22.Ourdirectcomparison
ofthecontiguousportionofthissequencefromACHEtoZAN
allowedustocharacterizeanovelcation-chloridecotransporter-
interactingproteinCIP1,provideevidenceofthepossibilityof
a gene flanked by ACHE and ASR2, and characterize the
genomic structure of ASR2, TRIP6, EPHB4 and ZAN. In
addition, our comparative and physical mapping data have
determined the orientation of the AF053356 contig on the
physical map of chromosome 7q22. Even in regions where
alignments (purple for the forward strand, orange for the highqualitysequenceanalysishasbeendoneinonespecies,as
reverse strand). This serves as a visual link between the wasthecasefortheZAN/TFR2region(11),wefindthatadual-