ajhg00065-0071

Am. J. Hum. Genet. 51:66-80, 1992

A 1.6-Mb Contig of Yeast Artificial Chromosomes around theHuman Factor Vil Gene Reveals Three Regions Homologousto Probes for the DXS I1 5 Locus and Two for the DXYS64 LocusDiha Freije and David SchlessingerDepartment of Molecular Microbiology, Washington University School of Medicine, St. Louis

SummaryTwo yeast artificial chromosome (YAC) libraries were screened for probes in Xq28, around the gene forcoagulation factor VIII (F8). A set of 30 YACs were recovered and assembled into a contig spanning at least1.6 Mb from the DXYS64 locus to the glucose 6-phosphate dehydrogenase gene (G6PD). Overlaps amongthe YACs were determined by several fingerprinting techniques and by additional probes generated fromYAC inserts by using Alu-vector or ligation-mediated PCR. Analysis of more than 30 probes and sequence-tagged sites (STSs) made from the region revealed the presence of several homologous genomic segments.For example, a probe for the DXYS64 locus, which maps less than 500 kb 5' of F8, detects a similar but notidentical locus between F8 and G6PD. Also, a probe for the DXS115 locus detects at least three identicalcopies in this region, one in intron 22 of F8 and at least two more, which are upstream of the 5' end of thegene. Comparisons of genomic and YAC DNA suggest that the multiple loci are not created artifactuallyduring cloning but reflect the structure of uncloned human DNA. On the basis of these data, the most likelyorder for the loci analyzed is tel-DXYS61-DXYS64-(DXS115-3-DXS115-2)-5'F8-(DXS115-1)-3'F8-G6PD.

IntroductionThe subtelomeric region of the long arm of theX chro-mosome has been the subject of intense study, becauseof the linkage of many diseases to polymorphic mark-ers in Xq28 (Mandel et al. 1989). Some of these geneshave been cloned in cosmid or phage clones. Theyinclude the coagulation factor VIII (F8) (Gitschier etal. 1984) and glucose-6-phosphate dehydrogenase(G6PD) genes (Persico et al. 1986). Other genes havenot yet been isolated, including the Emery-Dreifussmuscular dystrophy (Consalez et al. 1991) and adre-noleukodystrophy genes (Aubourg et al. 1987). Be-cause of both the rarity of many of these diseases andthe lack of individuals informative for multiple poly-

Received July 15, 1991; revision received February 13, 1992.Address for correspondence and reprints: Dr. David Schles-

singer, Department of Molecular Microbiology, Washington Uni-versity School of Medicine, 660 South Euclid Avenue, St. LouisMO 63110. 1992 by The American Society of Human Genetics. All rights reserved.0002-9297/92/5101 -0008$02.00

morphisms, linkage analysis has failed to position ac-curately most of the disease genes which map to thisarea. The availability ofDNA from this region wouldallow for a systematic search for additional polymor-phisms and for a more precise ordering of the geneticmarkers.

Another interesting feature of this portion of Xq isthe existence of a region of homology between it andYq. Cooke et al. (1984) have reported that a lambdaphage isolated from a Y chromosome-specific librarycontains DNA identical to a region on the X chromo-some (DXYS61). Arveiler et al. (1989) later isolateda DNA fragment from the X chromosome (DXYS64)and showed that it belongs to the same X-Y homologyregion as does the previously studied phage. DXYS64mapped within 500 kb of the F8 gene (Arveiler et al.1989). Brown et al. (1990) have also noted homologybetween sequences at the telomeres of the long arm ofthe X and Y chromosomes in some individuals andhave suggested that these regions exhibit pseudoau-tosomal behavior.

66

Duplicated Sequences around Factor VIII

Before the question of genetic exchange between thetelomeres of the long arm of these two chromosomescan be studied in detail, it is necessary to define theX-Y-specific region and to isolate additional probes.With the development of the yeast artificial chromo-some (YAC) cloning system (Burke et al. 1987), it hasbecome possible to isolate large fragments of humanDNA which range in size from 50 kb to more than 1Mb. We set out to isolate the XY homology regionin an overlapping set of YACs and to determine itsorientation with respect to other Xq28 markers. Wereport here the isolation of an overlapping collectionof YACs covering 1.6 Mb of Xq28, including G6PD,F8, DXS115, DXYS64, and DXYS61. Analysis of thisregion orders the loci and genes and reveals the exis-tence of a locus homologous to DXYS64 as well asother duplicated DNA segments.

Material and MethodsCell LinesCGM-1 is a lymphoblastoid cell line established

from a male donor and is the source of the CGMYAC library. H/Fra (X3000.11) is a hybrid Chinesehamster ovary (CHO)-human cell line carrying a frag-ment of the human X chromosome (Xq24-q28; Nuss-baum et al. 1986). This cell line was used for thegeneration of the Xq24-q28 specialized library. RJK88is the CHO host strain for the H/Fra cell line. TheX-containing hybrid (GM06318B) and Y-containinghybrid (GM06317B) were obtained from the CoriellInstitute for Medical Research.ProbesProbe 767 for DXS115 (a 1.3-kb HindIII insert in

plasmid pAT153; Hofker et al. 1985) was supplied byDr. G. J. B. van Ommen. Probe st35.239 for locusDXYS64 (a 3-kb EcoRI insert in pBR329; Arveiler etal. 1989) was a gift from Dr. J. L. Mandel. Probe p38for G6PD (a 1.4-kb EcoRI insert in pUC18; Persicoet al. 1986) was provided by Dr. M. D'Urso. Probep114.12 for F8 (a 650-bp EcoRI/HindIII insert inpUC8) was from the American Type Culture Collec-tion.YAC Isolation and PCR PrimersTwo YAC libraries were employed. One, from

Xq24-q28 (Abidi et al. 1990; Wada et al. 1990), con-tains 820 clones which represent about three genomicequivalents of DNA from the region. The other

("CGM") contains another three genomic equivalentsofX-specific clones in a librarymade from total humanDNA (Brownstein et al. 1989). Table 1 lists the YACstrains used in the present study, along with the probeor PCR assay used for the isolation of each strain.Screening for YACs from the Xq24-q28 library wasdone by hybridization essentially according to amethod described by Abidi et al. (1990). Screening ofthe CGM library was done by PCR assays listed intable 2, according to a method described by Green andOlson (1990).Alu PCR AmplificationThe Alu primers were made from conserved regions

of the Alu consensus sequence (Jurka and Smith 1988)(two primers, Alu A and Alu D, for the 5' orientation;and Alu B and Alu C for the 3' orientation). For Alu-Alu amplification, three separate sets of reactions wereset up for each clone. The primers were added in threecombinations, 5' primers (AluA + AluD), 3' primers(AluB + AluC), and all four primers. Thirty-five cyclesof 940C for 1 min, 650C for 2 min, and 72C for 5min were performed in a Perkin-Elmer thermocycler.Sequences of all the primers used are listed below.Most probes generated during the course of this studyare listed in table 1.

Contig AssemblyPCR assays using Alu primers yielded a set ofDNA

fragments, ranging in size from 200 bp to 5 kb, fromthe YAC clones. The number of Alu-Alu fragmentsranged from 1/75 kb to 3/75 kb, depending on theAlu content of the YAC. For the regions rich in Alurepeats, such as the immediate vicinity of F8, primersfor one orientation only were used to obtain well-isolated bands. Alu-Alu bands from each YAC werelettered from "a" to "z" in order of decreasing size.

YAC End-Fragment IsolationTwo methods were used for the isolation of the

human DNA fragments directly adjacent to vector se-quences. Alu-vectorPCR was done as follows: primersfor the left (centromeric) or right arm of the YACvector were used in separate reactions with each Aluprimer, to amplify end fragments. The products ofthe amplification reactions were separated on 1.2%agarose gels, were transferred to Nytran membranes,and were probed with the left or right vector primersto distinguish the appropriate YAC end fragment fromAlu-Alu internal products. Both left and right vector

67

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Iq-kgfe ~~~~~ ~ ~~~~~~~~~~~~~+++0qktH-IdO9Z + + + + + H-hIOSZ t UI-tesSAxU + + + +++ -t,9SAaXUTltl9f + + + + + 9-Itl9f =U-00L, + + + + 'e-09eC-ZZIUI + + +++-ZMuI - U.*-sIIsxU + + +++S-IlsxU 0S-OsZ + + + + ++ s-1osz 3l3-Of9e + + + + + P-iW9v o3-00L, + + + + + Z-9 r z;H-XILf ++++ + + H-Idf.CUH-)Ib98 + + + + + H-Idt98

-

Z-ZwluI + + + z-ZZlUI . Q-z-zzlui 1~~ ~~~~~~~~~0.oz-s51sxU + + + Z-sIsxU B cH-SOL + + + + H-1O5'dtZ6 + + + + -YVZ6 WD-'dtbZ6 + + + + + D-ItZ6G-'dst + + + + + U-N8K t:ve-90V + + + O9-b-tZ6u + + + + r-tZ6EIuOxa-8g, + + + + IuOxa-84q-tZ6v + + + + qj7ZUEIuHOX-81 + + + +

I=c

H-IL + + + + L'-tbZ6L' ++ + + + 3G-UZElUI + + + + U-Z)LZE o

5-VUL6 + + + + :,-toe

I-zzUI + + + + I-Z 3=cT-sJ~sxu + + + PiZ61l -x

" -XP-tZ6u + +PVU'wZ-i9SAXU + + + + + Z-t9SAXG rU.E-088e + + + + + V-088E o -


Table 2Primers and Conditions Used for PCR Assays

PCR Assay and Forward and Reverse Primers

F85-170 exon 1:Forward-GCTTAGTGCTGAGCACATCCAGTG ..............................

Reverse- ACTTATTGCTACAAATGTTCAACT..............................

F8M-270 exon 14:Forward- CTCCCTTATCAGATTGCCTTACGAGGAGTC................Reverse- GAGCCAACCTCTCTTTGATCACCAGTCATC................

924L-220:Forward-ACTACGAATTCTCACATGGTGG ..................................

Reverse-ACAGAATGGGGAGTGACTACT.....................................924R-194:

Reverse-TTGTTTTCAGTTGTGTGAACTT....................................Reverse-ATCTTCCGCGAAACTCTAGA ........................................

Int22-294:Forward-GCTTCTGCGTTTTGTTATTC .......................................

Reverse-AATATGTTAAGAAGTGAGGG.......................................

XYH-165:Forward-TAAATGTCATAGGCCGAAAGAATG..............................Reverse- GTACCTAATCGCAAGAAACACTGT..............................

G6PD-261:Forward-CCTCTATGTGGAGAATGAGAG.....................................Reverse-CACTGCTGGTGGAAGATGTCG.....................................

tel Bam 3.4-335:Forward-TCAGGCCCATGACAAACACCG.....................................

Reverse-AGGGCCATCAGGCACCAAAG.......................................

Conditionsb

940C at 1 min550C at 2 min721C at 1 min

941C at 1 min720C at 3 min







a Denoted by the name of the sequence of origin, followed by the size (in bp) of the amplificationproduct.

b Determined on a Perkin Elmer Cetus DNA thermal cycler. The sequence of the primers is given inthe 5'Y-3' orientation. The composition of the buffer used is given in Material and Methods. All amplifica-tions were done for 35 cycles.

primers amplify a single band from yeast, which servesas an internal control for the amplification reactions.Alu-vector PCR yielded an end fragment for 60% ofthe ends sought. The end fragments isolated by thisprotocol are listed, e.g., as 924L-A, where 924 identi-fies the accession number of the YAC, R or L is for theright or left vector end, and A is for the Alu primer (Athrough D) used. End fragments less than 400 bp longwere not used for this analysis.End fragments used in this study which were not

recovered by Alu-vector PCR were made by ligation-mediated PCR (Riley et al. 1990), with the followingmodifications: Yeast DNA was digested with EcoRV(E), HincIl (H), Hinfl (F), or RsaI (S) and were ligated

with the appropriate "vectorette." After ligation, unli-gated vectorette was removed, by spin dialysis over aSepharose Cl-6B column (Pharmacia). End sequenceswere recovered by two rounds of amplification, onewith primer 224 and either 1089 or 1091 (Riley et al.1990) for 20 cycles and the second with primer 224and the nested primers L or R (from the vector arms;see below) for 35 cycles. The cycling conditions forthe PCR reactions were as follows: 94C for 1 minand 720C for 5 min. End products isolated by thevectorette method are listed by the YAC number fol-lowed by R or L and a letter (E, H, F, or S) representingthe restriction enzyme used in their isolation. Often,more than one enzyme yielded an end product from a

69

Freije and Schlessingergiven YAC. We used, preferentially, DNA fragmentsranging in size between 500 and 1,000 bp.

Both the size of all fragments generated by PCR anda detailed protocol for the generation of all probesreported in this study are available on request. ManyYAC ends could be recovered by more than onemethod. Primers used in various PCR assays in thisstudy included L, TCT CGG TAG CCA AGT TGGTTT AAG G; R, TCG AAC GCC CGA TCT CAAGAT TAC; AluA, AAG TGC TGG GAT TAC AGGCGT GA; AluB, CGA CAG AGC GAG ACT CCGTCT CA; AluC, GGA GGC TGA GGC AGG AGAATC/TG; AluD, GCC TCC CAA AGT GCT GGGATT A; 224, CGA ATC GTA ACC GTT CGT ACGAGA ATC GCT; 1089, CAC CCG TTC TCG GAGCAC TGT CCG ACC GC; 1091, ATA TAG GCGCCA GCA ACC GCA CCT GTG GCG. Buffer com-position was 10 x dNTP (2 mM in each nucleotide[Pharmacia]), 10 x TAQ buffer (500 mM KCl, 100mM Tris [pH 8.4], 15 mM MgCl2, 0.1% gelatin).SequencingDNA for sequencing reactions was generated by

asymmetric PCR, and sequencing was done accordingto a method described by Wilson et al. (1990), byusing a Sequenase kit (USB).DNA PurificationMammalian DNA was isolated according to pub-

lished protocols (DiLella and Woo 1987). Yeast DNAwas isolated according to a method described by Sher-man et al. (1986), with a minor modification: 1 vol ofmethoxyethanol, instead of isopropanol, was used forthe DNA precipitation step.DNA Digests and Southern Blotting

Restriction enzymes were obtained from commer-cial sources and were used according to manufactur-ers' recommendations (New England Biolabs, Boeh-ringer Mannheim, and BRL). DNA was separated byelectrophoresis on either 1% agarose gels, for yeastDNA, or 0.8% agarose, for mammalian DNA. TheDNA was transferred to nylon membranes (Oncor,Nytran, or Hybond) according to the manufacturer'srecommendations. Probes were labeled by randompriming (Feinberg and Vogelstein 1984). Southernblot analysis (Sambrook et al. 1986) of yeast DNAwas done in an aqueous hybridization mix (5 x SSPE,10% dextran sulfate, 1% SDS, 5 x Denhardt's, 100ig salmon sperm DNA/ml) at 65C for 16 h. South-ern analysis of mammalian DNA was done at 420C

for 16 h in 50% formamide, 5 x SSPE, 10% dextransulfate, 1% SDS, 1 x Denhardt's, and 100 gg salmonsperm DNA/ml. After hybridization, the filters wererinsed twice in 2 x SSC, 0. 1% SDS for 30 min at roomtemperature, followed by a third wash at 65C for 30min. A stringent fourth wash was done in 0.1 x SSC,0.1% SDS for 30 min at 65C, unless otherwise notedbelow.

ResultsIsolation of YACs

Screening for YACs around F8 was done by hybrid-ization with four DNA fragments previously mappedin Xq28, identifying DXYS64, F8, DXS115, andG6PD. Three clones (yWXD519, 527, and 588) fromthe Xq24-q28 library hybridized to pl 14.12, theprobe for F8. Two additional YACs, yWXD924 andyWXD932, were recovered from the CGM library byusing a PCR assay for exon 1 of F8 (table 2). Addi-tional clones containing portions of the F8 gene wereisolated by hybridization with probes or PCR productsisolated from this region. These clones includedyWXD585, 695, 705, and 880. (yWXD932 and 695contained a structural rearrangement and were notanalyzed further.)The probe for the DXS115 locus, 767, hybridized

to 14 YACs, and the probe for the DXYS64 locus,st35.239, hybridized to 11 YACs from the Xq24-q28collection; these are higher numbers than are expectedstatistically for single-copy probes in a three-hit li-brary. The DNA sequence identified by 767 was pre-viously shown to be duplicated in Xq28 (Arveiler etal. 1989; Patterson et al. 1989). st35.239 has beenreported as a unique probe in the vicinity of the F8gene (Arveiler et al. 1989). As expected, some of theF8 YACs were also positive for probe 767, since oneof the copies identified by this sequence is present inintron 22 of the F8 gene (Levinson et al. 1990). One40-kb YAC was recovered from the Xq-specific libraryby using a probe for G6PD. One additional YAC (110kb) was isolated from the CGM library by the PCRassay listed in table 2. Tel Bam 3.4, a sequence mappednear the telomere of several human chromosomes(Brown et al. 1990), including X and Y, was presentin two clones, yWXD427 and 2107, as determinedby a PCR assay with oligonucleotide primers derivedfrom the published sequence (table 2).

Because of both the presence of repeated segmentsof DNA in this region and the possibility of errors in

70


contig assembly based on hybridization with a limitednumber of probes, a number of complementary ap-proaches were used to provide independent determi-nation of the physical relationship among the YACs.Contig Assembly from Fingerprinting and Walking Methods

Fingerprinting methods provided confirmation ofthe grouping of YAC clones in the contig shown infigure 1. Two different approaches were used, bothbased on the content of Alu sequences in YAC clones.In one method, as described in Wada et al. (1990),Southern blots of YAC DNA were probed with a ra-dioactively labeled Alu sequence. The occurrence ofbands of the same electrophoretic mobility and rela-tive intensity in two clones is an indication of theirprobable overlap, and the probability of overlap in-creases with the number of bands in common. Theresults were consistent with the map shown in figure1 (data not shown).A second method of fingerprinting is based on prod-

ucts derived from Alu-Alu PCR (Nelson et al. 1989).The Alu-Alu fingerprint for both subcontigs is shownin figure 2. Since the number ofbands perYAC is smalland since band mobilities in gels are often similar,Alu-Alu DNA fragments from a given YAC were hy-bridized to the Xq24-q28 YAC collection to verifyoverlaps detected by fingerprinting methods. An Alu-Alu product which is unique in one oftwo overlappingclones can be used as a hybridization probe to walk tonew clones; and the serial repetition of this methodwas used to verify overlaps as well as to assemblecontigs over the entire region. Furthermore, for anidentified set of overlapping YACs, Alu-Alu finger-prints were expected to be consistent among clones.Any discrepancies or odd bands were indications ofpossible rearrangements or chimeric events in thatclone.End fragments were also isolated from a number of

YACs, to verify overlaps or to extend the contig inregions where overlaps were too small to be detectedby other methods. End fragments were used either ashybridization probes against the collection of Xq24-q28 YACs or for Southern analysis of YAC and ge-nomic DNA; and some were sequenced to developSTSs (Olson et al. 1989). Table 1 summarizes most ofthe data collected, and figure 1 shows the map inferredfrom the data in table 1.The Inferred Contig and Probe LocationsThe 1.6-Mb contig seen in figure 1 was assembled

from 30 overlapping YACs. At least three sequences

(DXS115-1, -2, and -3) homologous to 767 are ap-proximately positioned in relation to the F8 gene. Inaddition, the contig has a locus homologous but notcompletely identical to DXYS64 at the 3' end of theF8 gene. Data are presented below for at least twoclones at every site in the contig, with the exceptionof yWXD527. For simplicity, data for most clonessmaller than 200 kb are not included in this paper.The contig shown in figure 1 is discussed below, in

two portions. The "F8 subcontig" (fig. 1, right) coversabout 850 kb of DNA. It includes all of the clonescontaining the F8 gene and extends from yWXD348or yWXD527, a clone carrying the 3' half of F8 andextending to within 30 kb of the G6PD gene. On thebasis of the organization of the genes in the isolatedYACs, F8 and G6PD are transcribed in the same orien-tation, confirming the order previously determined bypulse-field gel electrophoresis and fluorescent in situhybridization (FISH) (Kenwrick and Gitschier 1989;Trask et al. 1991). If it is assumed that yWXD527 isa faithful copy of genomic DNA, then the 5' end of theG6PD gene is located about 250 kb downstream ofF8, a finding which is in agreement with the in situdata presented by Trask et al. (1991).The "DXYS64 subcontig" (fig. 1, left), covers about

750 kb of DNA. It extends from yWXD630 towardyWXD427 and yWXD2107. The presence of tel Bam3.4 in these two clones indicates that they are at thetelomere, a tentative inference that is being tested fur-ther (R. Nagaraja, personal communication).Verification: Fidelity of YACs and Duplicationsin Genomic DNAThe internal self-consistency of the map of YACs

assembled in figure 1 is a strong indication of both theintegrity of the YACs and the quality of the inferredmap. However, it was critical to try to verify the struc-ture of the YACs and the inferred map by comparisonwith genomic DNA. This was especially important forthis region, where several sequences were apparentlyduplicated (see below).

1. Verification by comparison with other types of clonedDNA from the region. -To verify defined map locationsfor genomic segments cloned in YACs, we took advan-tage of the fact that both independent cloning intolambda phage and sequence analysis had determinedthe structure of portions of more than 175 kb ofDNAin the vicinity of the F8 gene (Gitschier et al. 1984).Consequently, for that region the quality of YACscould be compared with genomic DNA, by STS assayswith oligonucleotide primer pairs derived from pub-

71

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ALU-PCR FingerprintFigure 2 Alu-PCR fingerprint of the YACs across the 1.6-Mb region. For the F8 subcontig (left), an Alu-rich region, two separatePCR amplifications were done for each clone, one with the 5' Alu primers and one with the 3' Alu primers (see Material and Methods).The amplification products from both reactions were pooled after PCR. For the DXYS64 subcontig (right), all four primers were used.One-third of the PCR reactions was separated on a 1.2% agarose gel. The size marker is lambda DNA digested with HindIII and Phi X174 DNA digested with HaeIII. DNA fragments which are present in two different YACs indicate a possible overlap.

lished sequence. All the F8 subcontig clones were ana-lyzed for their content of five STSs (table 2), one eachfor exon 1 and exon 14 of F8, two for the left andright end clones ofyWXD924, and one from the genepresent in intron 22 of F8 (Levinson et al. 1990). OnlyyWXD924 carried the complete F8 gene.

Confirmatory Southern analyses were also donewith several published and newly generated internalprobes (data not shown). In a given clone, any "miss-ing" STSs or probes and any abnormal size restrictionfragment detected by Southern hybridization wouldindicate internal deletions or rearrangements. How-ever, with the exception of yWXD695, which wasmissing a fragment 5' ofthe F8 gene, none of the clonesshowed any rearrangement or deletions. By these crite-ria, the clones that contain F8 in figure 1 appear to bea faithful representation of genomic DNA.

For the DXYS64 subcontig, very little sequence in-formation or very few probes are available. The clonesin this region were analyzed with the two probes forthe respective loci DXS1 15 and DXYS64, and thesedata are discussed below in a separate section. Furtherverification of these clones relied on Alu-Alu and endprobes generated during the course of this study. Theresults were consistent for all probes tested.

2. Verification of duplication of probes. -As anticipatedfrom apparently repeated segments ofDNA in the re-gion, hybridization assays with certain probes identi-fied two sets of nonoverlapping clones. This suggestedthat these probes are duplicated. The term "duplica-tion" is used here very loosely, to indicate the existenceof a homologous but not necessarily identical copy ofa given probe.

In several cases, a given probe hybridized to two

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73

Freije and Schlessinger

sets of clones that, on the basis of their contents ofa number of other probes, had been inferred to benonoverlapping. For example, a630-el hybridized totwo sets of nonoverlapping clones. Southern analysisofYACDNA identified different restriction patterns ineach set of clones, again suggesting that this particularsequence was duplicated. In some cases, an Alu-Alufragment which identified a set of overlapping YACswas duplicated within the YACs. For example, a364-bl is present in two nonidentical copies in each ofyWXD364 and 427, whereas yWXD630 carries onlyone.

3. Verification of the multiple copies of the DXSl 15 lo-cus.-The DXS115 locus detected by the probe 767was previously shown to contain sequences duplicatedwithin an interval of 500 kb (Arveiler et al. 1989;Patterson et al. 1989). The F8 copy can be differenti-

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ated from the upstream copies, since it gives the con-stant bands seen with the BstXi (6.8 kb) and PstI (2.2kb) restriction enzymes used for polymorphism analy-sis (see legend to fig. 3). Southern analysis of humanDNA by PstI and BstXI suggests that on the basis ofthe intensity of the polymorphic bands (1.8 kb and 4.5kb, respectively) when compared with the constantband, theDNA sequences identified by 767 are presentin more than one copy.The analysis of the "DXS1 15 YACs" which carried

the polymorphic copy of this sequence also suggestedthat there are at least two copies of the sequence whichdetects polymorphism. In addition to the constant andthe polymorphic bands, faint bands were seen, re-sulting from a moderately repetitive sequence familypresent in the region and providing a fingerprint forthe clones mapping within this region. Sequence anal-

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Figure 3 Southern analysis of the YACs carrying the DXS1 15 locus with the 767 probe. A 0.5-g.g portion of DNA from each YACwas digested with PstI or BstXI restriction enzyme, was transferred to Oncor membrane, and was hybridized with radioactively labeled767 DNA insert. A posthybridization stringent wash was done in 0.1 x SSC, 0.1% SDS at 65'C for 1 h. The filters were exposed toHyperfilm-MP (Amersham) for 1 h at room temperature. The constant bands seen with this marker (2.2 kb for Pst and 6.8 kb for BstXI)are in the F8 YAC clones yWXD924, 519, 588, and 527. The polymorphic bands (1.8 kb for PstI and 4.4 kb for BstXI) are present inthe YAC clones which map within 500 kb of the F8 promoter region. yWXD705 and 585 did not carry the DXS1 15 locus. The additionalfaint bands which are seen in all clones represent cross-hybridization with a repetitive element which is present in many copies in this region(see text). The size marker is the same as in fig. 2.

74


ysis of a portion of the DXS115 probe showed thatit contained, in addition to the unique sequence, anMstII-like element (Mermer et al. 1987) which couldbe the source of the background hybridization seenhere and on genomic blots (authors' unpublisheddata).

In order to verify the existence of two "DXS115"copies 5' of F8, a series of Southern blots with humanand hybrid DNA were done with various enzymes.The goal was to identify restriction patterns that mightdifferentiate between the polymorphic copies. A seriesof 10 enzymes were used. On the basis of the simplicityofthe hybridization patterns, however, it became clearthat the polymorphic copies ofDXS1 15 are too highlyconserved for this approach to be useful (data notshown). Southern blots of YAC DNA digested withseveral enzymes were also hybridized with the DXS1 15probe, in order to verify the presence of the restrictionfragments carrying the DXS1 15 loci in the YAC clones(data not shown). All YAC clones from the regionupstream of F8 showed the patterns expected from theSouthern analysis with human DNA. This providesfurther evidence that, for this locus, the YACs faith-fully represent the human genomic DNA.The segregation of a duplicated probe in two non-

overlapping YACs provides a different approach toverify the duplication of a given sequence. End frag-ments were isolated from yWXD250 and were shownnot to hybridize with yWXD506 (a clone positive forthe 767 probe) (data not shown). Southern analysis ofYAC and human DNA showed that the yWXD250end clone identifies the same restriction fragment inhuman and YAC DNA. Thus, there are, upstream ofF8, at least two identical copies of sequences homolo-gous to the probe for DXS1 15. The existence of addi-tional DXS1 15-homologous sequences in the humanpopulation cannot be ruled out.The Int 22 gene was previously shown to have two

additional homologous copies 5' of F8 (Levinson et al.1990). On the basis of STS analysis, the Int-22 geneseems to segregate in the YACs, along with the probefor the DXS115 locus. It is very likely that DXS115and the Int 22 gene form a unit which is present inthree copies in this region.

4. Verification of repetition of DXYS64 and other X-Y ho-mologous DNA.-To analyze the location of the X-Yhomology, an STS (XYH-STS) related to the DXYS61locus was generated from the sequence published byCooke et al. (1984). To determine whether this STSdefined a unique sequence, the PCR product was usedas a hybridization probe in Southern analysis of hu-

man DNA. As expected, it identified an 8.6-kb EcoRIrestriction fragment (in addition to several other re-striction fragments which were presumed to be au-tosomal, as suggested in the original report [Cooke etal. 1984]).When the product of the PCR assay was used to

screen the Xq library, it identified several clones whichalso carried the DXYS64 sequence. yWXD364 andyWXD427 showed the strongest signals, whereas therest of the clones showed a weaker signal which couldbe eliminated when filters were washed at higher strin-gency. With XYH-STS, PCR analysis of all the posi-tive clones showed the appropriate amplificationproduct only from yWXD364 and 427. Figure 4shows the result of PCR and hybridization screeningwith this STS. We infer that only two YACs,yWXD364 and 427, contain sequences similar enoughto the primer pairs to give the expected PCR product.(The analysis of clone yWXD2107 will be separatelyreported elsewhere). They define the putative locus ofthe X-Y homology region. The auxiliary sites nearbywould be characteristic of duplicated probes in thisarea of the genome, cross-hybridizing but only par-tially homologous. This inference is in complete agree-ment with all the data available to date.The DXYS64 locus was physically linked to

DXYS61 on a 120-kb XhoI restriction fragment (Ar-veiler et al. 1989). The probe for DXYS64 hybridizedto 11 YACs (see above), a number much higher thanthe expected statistical average for a single-copy probein the Xq24-q28 library. These YACs segregated intotwo sets of nonoverlapping YACs, which suggestedthat this probe is present in at least two copies. FiveYACs (yWXD924, 527, 880, 588, and 585) were alsopositive for the F8 gene. This sequence homologousto st35.239 is positioned between the 3' end of the F8gene and G6PD. The remaining six YACs (yWXD864,630, 601, 427, 364, and 250) mapped 5' of F8. SinceyWXD364 and 427 were positive for the XYH-STSand since yWXD250 was not, the order of loci is in-ferred to be tel Bam 3.4-DXYS61-DXYS64-DXS115.

Because the overlap between yWXD250 andyWXD364 was only determined with probe st35.239,verification that they carried the same DXYS64 locuswas important. End clones from yWXD250 andyWXD364 were used to confirm the overlap, by hy-bridization to the collection of Xq24-q28 YACs andby Southern analysis of the appropriate clones. Theoverlap was confirmed with 364L and 250R, sug-gesting that both YACs carry the same DXYS64 locus.

In addition, a yWXD364L end fragment identified

75

Freije and Schlessinger

Hybridization of XYH Probe to YAC Grid

XYH STS

surt ED F NXo oa0 i) W P-CQ01 *JnnrOI"N wwq-l -XX XXX XZLI -

3!. 90. 3. 3 I)I XV

Figure 4 Analysis of YACs with XYH STS. The left panel shows the results of PCR screening of some of the positive clones withthe XYH STS (for conditions, see Material and Methods). Only yWXD364 and yWXD427 are positive for this STS. Yeast strain AB1380DNA was used as a negative marker for the PCR assay. DNAs from CGM-1 and from hybrid cell lines for the X and Y chromosomes wereused as positive controls. The size marker is Phi X 174 DNA digested with HaeIII. The right panel shows the results of the hybridizationof the 165-bp DNA fragment generated with the XYH STS to the YAC grid representing YAC clones from Xq24-q28. All YACs identifiedby hybridization mapped to the F8 region.

the same 11 clones which hybridized to st35.239 (fig.5). This suggests that yWXD364L falls within the du-plicated region which is around DXYS64 and whichmaps to sites both 5' and 3' of F8.As shown in the right panel of figure 5, Southern

blot analysis gives further evidence about the contentof probe sequences for DXYS64, across the region.yWXD427, 864, and 630 show the expected bandsfor the DXYS64 subcontig. The F8 clones yWXD924and 527 show a different DNA fragment. These twoclones carry a locus which is homologous but not com-pletely identical to DXYS64. Two aberrant restrictionfragments are seen, one each in yWXD364 and 250.These two fragments represent the junction fragmentsbetween the vector and the human sequences, as veri-fied by Southern hybridization with the pYAC4 vectorDNA. From this analysis, it is inferred thatyWXD364

and 250 overlap by a maximum of 25 kb, andDXYS64 maps within this overlap.We infer that the X-Y homology region lies within

theDNA which is in common between yWXD364 and250 and that it extends toward YACs yWXD427 and2107. We expect a discrete boundary for this regionwithin yWXD250. We also infer the existence of alocus homologous to DXYS64 between the 3' end ofF8 and G6PD.

DiscussionRelative Limitations of the Methods Used in ContigAssembly and AnalysisThe limitations of preexisting approaches for ana-

lyzing complex genomic regions, particularly when a

76


A

u. r-N(D sr 0D TN%- N {D I )t C)na do to N 0 to n Lo,XX XXX XX.N 3i 3: B 3 3en >% ;1 >% :1 >% >% -.

I

yWXD364L End Clone

23.1 -9.4-6.5-4.3-

2.3-2.0-

1.3-1.0-.87-.60-

Figure 5 Analysis of YACs with yWXD364L end fragment and st35.239. A, Results of hybridization of yWXD364L to the YACgrid representing all Xq24-q28 YAC clones. The signals can be divided into two categories, strong and weak, after autoradiography for30 min at - 700C. Exposure for 4 h results in indistinguishable signal intensities from all positive clones. Strong signals were obtainedfrom clones in the DXYS64 subcontig, including yWXD364, 427, 630, 250, 864, and 601. The weak signals were obtained from clonesin the F8 subcontig (see text), including yWXD924, 585, 588, 880, and 527. The additional signals identified clones smaller than 150 kband were not included in this analysis. B, Result of Southern analysis, with probe st35.239, of a subset of the clones identified withyWXD364L. A 0.5-jig portion of DNA from each YAC was digested with MspI, was separated on a 1% agarose gel, was transferred toa nylon membrane (Oncor), and was hybridized to a radioactively labeled st35.239 DNA insert. For aberrant fragments, see section onDXYS64, in Results. The size markers are lambda DNA digested with HindIII and Phi X 174 DNA digested with HaeIII.

single approach is used, became obvious during thecourse of this study. The larger size of YACs as com-pared with cosmids and conventional cloning vectorsprovides long-range coverage more readily. However,because of the presence of duplications around F8,difficulties with probe hybridization as a method toassemble overlapping YACs were evident: many YACsshared several sequences but did not seem to overlap.

Hybridization of a given probe to YAC DNA isseveral-fold more sensitive than genomic Southernanalysis of total mammalian DNA. This is because theYAC DNA is present at a relatively high copy numberin a yeast background which has a sequence complex-ity much lower than that of human DNA. Conse-quently, cross-hybridization with homologous copiespresent nearby or elsewhere in the genome can become

a source of miscalled overlaps even when hybridiza-tion is carried out at very high stringency. For thisreason, overlaps must be verified by additional (andpreferably independent) methods. Combining hybrid-ization and PCR-based technology with YAC cloningprovides a way to generate a self-consistent map evenof regions as structurally complex as that reportedhere.The Quality of the Map

Tests for the integrity of YACs and for the qualityof the inferred map (fig. 1) are limited by the availabil-ity of probes in the region. For most regions of thegenome, multiple probes are not readily availablefrom previous studies; but internal Alu-Alu fragmentsand end fragments provide a useful supplementary

77

B

Freije and Schlessingersource. The data generated here indicate (a) that threesegments homologous to the probe for the DXS115locus and two copies homologous to the probe for theDXYS64 locus all have corresponding sequences inthe genome and (b) that the contig truly reflects theorganization of the human DNA.The frequency of chimeric clones in the YAC collec-

tion analyzed during this study was 10% . Two CHO-human chimeric clones were recovered, and, in thecase of yWXD588, the CHO component of the YACwas estimated as being about 20 kb. The other YAC,yWXD868, overlapped with yWXD427 and was notnecessary for this analysis. A third YAC, yWXD748,not presented here, was a human-human chimera.Analyses with a number of probes derived from thisYAC indicate that a DNA fragment from Xq24 wasjoined to a fragment from Xq28 to form a 150-kbYAC. This YAC was not analyzed further.Only one YAC, yWXD695, showed indications of

deletions and a possible rearrangement. For the re-maining YACs, all data collected here were consistent,both between YACs as well as with genomic DNA.The Order of the Loci in Xq28The most likely order for the loci used based on

the basis of this analysis, is tel-DXYS61-DXYS64-DXS1 15-F8-G6PD. Since G6PD mapped between F8and CV by a variety of techniques, the color-vision-gene cluster must be centromeric to G6PD. This orderis in agreement with the pulse-field gel mapping datapresented by Poustka et al. (1991). However, it dis-agrees with the order presented by Kenwrick andGitschier (1989) and with the FISH hybridization datapresented by Trask et al. (1991), both of which re-versed the order of the F8-G6PD-CV cluster withrespect to the telomere. As one possibility, it is notexcluded that, in different individuals, the F8-G6PD-CV cluster exists in inverted orientations with respectto the telomere.

Establishing the genetic order for the Xq28 locicould resolve the discrepancy, but the order has re-mained uncertain. The lack of highly informativemarkers in Xq28 (with the exception of the DXS52locus), as well as the paucity of recombination eventsin the families analyzed so far, have made it difficultto establish a high-resolution genetic map of the distalportion of Xq28. With the cloning of this region inYACs, it is possible to search systematically for poly-morphic markers which might allow the determina-tion of the genetic order of the loci and provide re-sources for further analysis of the many X-linkeddiseases which map in this area (Mandel et al. 1989).

The Occurrence of Scattered Repetitive Sequences

There is ample precedent for sequences that exist ina small number of copies in portions of the genome.On the X chromosome itself, Xq28 DNA containsmultiple copies of the St14 sequence (Oberle et al.1986), as well as copies of sequences such as the STIRprobe (Rouyer et al. 1990) probe and G13.c (Bardoniet al. 1988), which are present elsewhere on the X.Other paucirepetitive sequences are found at a numberof locations in other delimited regions of the X, suchas S232 in Xp22 (Knowlton et al. 1988).The possibility that the DXYS64 region has par-

tially homologous sequences on the X chromosomeraised several questions about the nature and the func-tion of the repeated sequences. It is possible that thisregion arose from a duplication prior to the acquisi-tion of the F8 gene. In this case, many features of thisregion should be conserved evolutionarily. It is alsopossible that the duplicated sequences code for tran-scriptional units and belong to a gene family in Xq28.(Ongoing work is aimed at testing, by isolatingcDNAswhich might map within the duplicated segments, thepossibility that the region contains expressed genes.)The presence of at least three copies of the DXS1 15

locus and three copies of the Int 22 gene in a smallregion of the X chromosome is an example of duplica-tion of sequences that had been considered unique.DXS1 15 and Int22 appear to be near one another,both in intron 22 of the F8 gene and in the YACscarrying the polymorphic DXS1 15 copies. Very likelythe two probes have been duplicated as a unit duringevolution, though the 5' copies are more homologousto each other than they are to the copy in intron 22 ofF8. Preliminary evidence (authors' unpublished data)suggests that the 5' duplicated segments could extendto cover 50 kb or more. As one speculative possibility,it is conceivable that the repeated unit is involved ina large expressed unit and that the physical organiza-tion of elements within it is directly related to genetranscription and regulation in this region.The contig as presented in figure 1 gives the simplest

possible structure consistent with all the availabledata. The possibility that there are additional copiesof DXS1 15 which were not isolated is difficult to ex-clude. In several cases, multiple copies of a tandemlyrepeated sequence seem to be deleted during YACgrowth (Neil et al. 1990). For example, in ongoingwork, yWXD901, a YAC which contains tandem re-peats (Vollrath et al. 1988) of the color-vision genes,is unstable and deletes about 35 kb (the equivalent ofone repeat) before stabilizing. The DXS115 region,

78

Duplicated Sequences around Factor VIII 79

like that of color vision, may be variable in humanpopulations. In fact, all the clones reported here ap-parently contain only one repeat unit, and yWXD695,the only F8 positive clone which extends toward theDXYS64 subcontig, has an internal deletion and wasnot analyzed further. Thus, YACs which carry DXS1 15repeated sequences may lose segments of DNA.How Idiosyncratic Is the F8 Region?On the basis of the data collected so far, other re-

gions in Xq28 might be comparably difficult to ana-lyze. It is suggestive that only small (less than 150 kb)YACs have been recovered for G6PD or the color-vision genes from three different YAC libraries; andYACs containing the color-vision genes, like thosecontaining the more centromeric DXS49 (Abidi et al.1990), are generally unstable. In ongoing work in thislaboratory, the assembly, by hybridization techniques,of an 8-Mb region in Xq26 in overlapping YACs wasrelatively straightforward (Little et al. 1992). On theother hand, YACs mapping to Xq27 have showngreater complexity, and a battery of techniques is nec-essary to elucidate genomic organization. However,very few duplicated sequences were seen in all contigsassembled so far from Xq24 through Xq27 (Schles-singer et al. 1991). It remains to be seen whether thecomplex genomic organization described here is re-lated to the subtelomeric location of these sequences.A Second Pseudoautosomal Region on the Long Armof the Sex Chromosomes?Brown et al. (1990) have suggested the possibility

of a second pseudoautosomal region on the long armoftheX and Y chromosomes. The region ofhomologycovers about 500 kb of DNA, though the borders ofthis region have not been established. However, onthe basis of hybridization analysis of probes fromyWXD364 and 250, it is likely that this homologyhas a well-defined border within yWXD250. We havebegun a search for polymorphic markers from thisregion to attempt to orient the corresponding loci bylinkage analysis and to test for the possibility of geneticexchange between Xq and Yq.

AcknowledgmentsWe thank Drs. J.-L. Mandel, G. J. B. Van Ommen, and

M. D'Urso for providing the DNA probes used for thisstudy. We also thank Drs. Mike Watson, Eric Green, HaroldReithman, and Helen Donis-Keller for helpful discussionsduring the course of this work and for critical reviews of themanuscript. We are also grateful to reviewers, for a careful

review of the manuscript and for providing helpful sugges-tions. Sandy Johnson provided help in the screening ofprobes. D.F. is a graduate student in the division of Biologyand Biomedical Sciences. These studies were supported byNIH grant GM00247 and by the Washington University/Monsanto Biomedical Research Agreement. We also ac-knowledge independent efforts in which Dr. Giuseppe Palm-ieri has also linked F8 to G6PD, using a YAC made fromanother source of DNA.

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