10.1101/gr.7.3.210Access the most recent version at doi: 1997 7: 210-222Genome Res.

 R Nagaraja, S MacMillan, J Kere, et al. recombination and GC content.X chromosome map at 75-kb STS resolution, revealing extremes of  

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RESEARCH

X Chromosome Map at 75-kb STS Resolution, Revealing Extremes of Recombination and

GC Content Ramaiah Nagaraja, Sandra MacMillan, Juha Kere, Carmela Jones,

Stephanie Griffin, Matthew Schmatz, Jennifer Terrell, Michael Shomaker, Christopher Jermak, Christian Hott,

Mochubeloa Masisi, Steven Mumm, Anand Srivastava, Giuseppe Pilia, Terence Featherstone, Richard Mazzarella, Sheila Kesterson,

Brigid McCauley, Brian Railey, Frank Burough, Volker Nowotny, Michele D'Urso, David States, Bernard Brownstein, and

David Schlessinger ~

Center for Genetics in Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

A YAC/STS map of the X chromosome has reached an inter-STS resolution of 75 kb. The map density is sufficient to provide YACs or other large-insert clones that are cross-validated as sequencing substrates across the chromosome. Marker density also permits estimates of regional gene content and a detailed comparison of genetic and physical map distances. Five regions are detected with relatively high G + C, correlated with gene richness; and a 17-Mb region with very low recombination is revealed between the Xq13.3 {XISTJ and Xq21.3 XY homology loci.

[The STSs and YACs can be accessed at http'//genome.wustl.edu/cgm/cgm.html/.]

In addition to sustaining an exponentially increas- ing rate of gene finding (Collins 1995), yeast artifi- cial chromosome/sequence-tagged site (STS/YAC)- based maps (Burke et al. 1987; Olson et al. 1989) have begun to reveal additional features of chromo- some structure and dynamics. For example, during the development of maps for subportions of the X chromosome, the existence of a second "pseudoau- tosomal" region at the Xq terminus of the chromo- some was demonstra ted (Freije and Schlessinger 1992; Freije et al. 1992; Li and Hamer 1995), fol- lowed by the discovery that the region shows a unique phenomenon of gene inactivation on both the X and Y homologs (D'Esposito et al. 1996). In another instance, it was shown that a cluster of genes in a delimited segment of XpI 1 escape X in- activation (Miller et al. 1995). As the density of markers across the chromosome has increased be- yond the 100-kb resolution goal suggested for the

1Corresponding author. E-MAIL davids@sequencer.wustl.edu; FAX (314) 362-3203.

"genome init iative," addit ional features are re- vealed, as described here.

The average inter-STS distance o f - 7 5 kb has been achieved by the placement of 2091 STSs on cognate YACs across the 160 Mb of the chromo- some. Collectively, the STSs sample -1% of X- specific sequences. About half of the STSs (962) are made from YAC insert ends (Kere et al. 1992), and another 592 are from randomly derived unique X- chromosomal sequences. However, the STSs also in- clude 97 expressed sequence tags (ESTs) and 190 gene-specific STSs from known genes, as well as 192 dinucleotide and 38 tri- and tetranucleotide repeat markers that detect polymorphism. As a result, the YAC/STS map can be integrated with transcriptional and genetic maps.

RESULTS

Mapping Strategy and Performance

We used a modified "all-walking" form of STS con- tent mapping (Kere et al. 1992) in which STSs were

210 ~GENOME RESEARCH 7:210-222 01997 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/97 $5.00

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derived systematically from YAC insert ends at the edges of contigs to ensure that subsequent screening of YAC libraries would move progressively into new territory. From the screening results, SEGMAP (see Methods) efficiently generates the most probable orders of clones and STSs, and the likely size of cur- rent contigs.

Based on simulations, we expected that an essen- tially complete map should have been assembled after -600 screenings; instead, >1500 were required. One of the unexpected factors that increased the effort was the smaller size and fourfold higher chimera fre- quency than had been estimated previously for some YAC collections (for review, see Nagaraja et al. 1994). Also, the amount of insert ends that yielded STSs was lower than anticipated, because >60% were either co- cloned sequences from other chromosomes, con- tained repetitive sequence elements, or had a nucleo- tide composition too high in A and T for STS devel- opment. Nevertheless, the intuitive strength of STS content mapping to achieve map closure was realized in the final year of mapping, with a drop in the num- ber of contigs from 120 to 13.

Map Features

The 2091 STSs are aligned and ordered in 5423 YACs (see Methods) across the X chromosome (excluding the 3-Mb centromeric region, which remains un- mapped). The DNA contents of the YACs have all been estimated to be + 10% by pulsed-field gel elec- trophoresis (PFGE) compared to the size of yeast chromosomes (Goffeau et al. 1996), and provide an average of 10-fold clone coverage of each STS on the chromosome.

In Figure 1, which is a photograph of a WEB representation, parallel vertical components show the following. (1) An ideogram of the chromosome with approximate cytogenetic band positions. (2) The Centre d'Etude du Polymorphisme Humain (CEPH)/Genethon 198.1-cM genetic linkage map (Dib et al. 1996) normalized to the same overall length as the 160-Mb physical map (with its own index scale farther right). (3) Lines joining the cor- responding positions of dinucleotide repeat markers that are placed on both the physical and genetic linkage maps; crossed lines indicate discordances, which are usually minor. (4) Boxed cohorts of or- dered STSs in 10-Mb intervals from Xpter to Xqter; polymorphic (CA)n markers, genes, and ESTs are color-coded (see Methods and Fig. 1 legend).

The current map is based on a composite inter- active assembly of contigs made completely in the Center for Genetics in Medicine or developed in col-

X CHROMOSOME YAC/STS MAP

laboration with other groups (displayed on a World Wide Web page and published in some cases; see Methods and Acknowledgments). The 13 remaining YAC contigs range from 1.5 to 52 Mb in size, with an average of 12 Mb (the contigs are listed with their marker limits in Methods).

During the construction of the map, YAC sizes and STS content were used in SEGMAP to estimate the minimum, maximum, and average size of na- scent contigs. As STS content increased, the three size estimates converged; and by the time 100-kb inter-STS distances were approached, the sizes of contigs were in close agreement with sizes from other sources. For example, the extent of contigs was the same when assessed by rare-cutter restric- tion enzyme mapping in PFGE for regions of up to 8 Mb in Xp22 (Trump et al. 1996), Xq26 (Pilia et al. 1996), Xq27 (Zucchi et al. 1996), and Xq28 (Palm- ieri et al. 1994). Also, the total sum of successive contigs is within 5% of the 160-Mb content of the X chromosome estimated from cell-sorting experi- ments (Trask et al. 1990); and the contigs in Xq24- qter add up to about one-third the total, or about the cytogenetic fraction of the X included in the X3000.11 cell line containing that region (Nagaraja et al. 1994 and references therein). Thus, the index positions assigned to STSs along the chromosome provide a reasonable first approximation of position and can guide the allocation of regions of known size for long-range sequencing projects (Seventh X Chromosome Workshop 1996, unpubl, report).

Fewer than 20 of 2400 STSs tested thus far fail to find cognate YACs in the screening of 15 genomic DNA equivalents in YACs, suggesting that the map covers >99% of the chromosome. Some of the re- maining apparent gaps are still being filled by on- going screening. Furthermore, in two cases analyzed in Xq28 (Palmieri et al. 1994; Rogner et al. 1994; M. D'Urso, in prep.), DNA gaps in contigs were only on the order of 10-20 kb. Thus, any small amount of uncloned DNA may well be recoverable by long- range PCR amplification from chromosomal DNA preparations.

DISCUSSION

Universality of the Map

The internal consistency of the YAC/STS map in clones derived from seven different libraries of hu- man DNA is in accord with the anticipated general- ity of the structure of the X chromosome in human populations. As expected, the cohort of STSs present in a region proximal or distal to a translocation

GENOME RESEARCH J 21 1

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214 @ GENOME RESEARCH

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X CHROMOSOME YAC/STS MAP

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Figure 1 (Continued)

GENOME RESEARCH @ 21 5

Cold Spring Harbor Laboratory Press on July 15, 2011 - Published by genome.cshlp.orgDownloaded from

NAGARAJA ET AL.

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Cold Spring Harbor Laboratory Press on July 15, 2011 - Published by genome.cshlp.orgDownloaded from

X CHROMOSOME YAC/STS MAP

breakpoint or missing in particular deletions (e.g., in Xp22; Ferrero et al. 1995) are consistently found in a contiguous group in the inferred ordered list of STSs of Figure 1. As another example, YACs yWXD1 to yWXD829 in Xq24-qter, derived from an African American, show the same intrinsic content of STSs seen in the region in other YACs, derived from Cau- casians.

Overall, the results also support the fidelity of human DNA sequences cloned into YACs. For ex- ample, in a contig in Xp22.1 (Trump et al. 1996), 54 STSs were ordered across 3.5 Mb by their content in 58 YACs from five collections. The resulting map showed that an STS was negative at a site between two others in only 4 of 400 instances, consistent with 99% faithful recovery of X unique sequences.

At the average 75-kb marker density, the essen- tially universal set of STSs are sufficient not only to evaluate clone quality, but also to screen for alter- native large-insert clones, such as bacterial artificial chromosomes (BACs; Shizuya et al. 1992), as se- quencing substrates. In initial pilot work, for ex- ample, STS-based screening easily recovered mini- mal t i l ing paths of over lapping BACS for five projects of at least 1 Mb.

Recombination Indices Compared to Location

Overlapping sets of STSs and clones are sufficient to permit comparisons to other maps. These include partial physical maps assembled at lower resolution and with less than one-third the number of markers, using only mega-YACs, somatic cell hybrid panels, or hybridization or radiation hybrid-based methods (e.g., Chumakov et al. 1995; Roest Crollius et al. 1996). Figure 2 gives an example of possible special interest to human geneticists. Based on the parallel map representations in Figure 1 of the YAC/STS scale and the complete genetic linkage map of Dib et al. (1996), the ratio of genetic to physical distance is plotted in Figure 2 as a cumulative curve showing the integrated sum of cM/Mb starting from the Xp terminus. The overall average of 1.3 cM/Mb (Dib et al. 1996) is clear, but the ratio varies considerably. An extensive region of very low recombination (B in Fig. 2) is especially striking. Between DXS8046 and DXSl196, a distance o f -16 .8 Mb, only 2.7 cM in-

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Figure 2 Cumulative cM vs. Mb distances across the X chromosome. Distances in cM between successive markers that detect polymorphism are summed and plotted against the corresponding distances in Mb (ab- scissa) from Xpter to Xqter. The broken line is drawn with the average (overall) slope of 1.3 cM/Mb. A and C delimit two regions of possible hot spots for recombi- nation; B is a region that shows very low recombination (see text).

tervene, or a ratio of 0.16 cM/Mb. Unexpectedly, this region does not fall at the centromere, where an anticipated but slightly less marked inhibition of recombination is observed (a t -62 Mb on the index scale). Instead, this zone is distinctly more distal, roughly at the center of mass of the chromosome. It begins at or very near the X-inactivation center of the chromosome, the XIST locus (Lyon 1992) and extends distally to another distinctive region that has been delineated by the map, -4 Mb of DNA homologous on the X and Y chromosomes (Sargent et al. 1996).

The conjunction of the XIST locus, with one edge of an extensive region of low recombination, is suggestive. Possibly the involvement of this portion of the chromosome in Barr body formation and glo- bal transcriptional regulation may inhibit local syn- apsis.

The overall average requires that there be re- gions of higher recombination to compensate for the "cold" region. Two such possible regions are A in Xp and C near the Xq26-27 border (Pilia et al.

Figure 1 The STS map compared to the genetic map. Shown is the order of 2091 STSs along the X chromosome, with positions indexed to an approximate scale in megabases. The ideogram of the cytogenetic band pattern is also shown, and the comparative genetic distances (from Dib et al. 1996) are indicated with the 198.1-cM total scaled to the same overall length as the physical map of 160 Mb. Mapped markers that detect polymorphism are shown in bright pink between these scales; (Right) Ordered STSs in 10-Mb cohorts also show ESTs in blue, genes in green, and GDB DXS numbers (or sWXD Center designations for those in the submission process) in black. Further updates of marker lists will be posted on the World Wide Web site (see Methods).

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NAGARAJA ET AL.

1996; Zucchi et al. 1996), with apparent local values of recombinat ion as high as 5 cM/Mb. (A third pos- sible region of high recombinat ion is seen at 115- 120 Mb but falls in a region with relatively few ge- netic markers.)

Regional recombinat ion values can have a con- siderable impact on searches for disease genes. For example, a translocation breakpoint associated with premature ovarian failure was localized between PGK1 and DXS447 (Powell et al. 1994), a distance of 0.1 cM; however, those markers fall in the region with very low recombinat ion values and are >1 Mb apart on the chromosome. In general, the physical map and recombinat ion-based map agree in marker order where distances are appreciable on both maps; and the physical map provides further ordering in- formation for genetic markers in the regions where recombinat ion values are very low.

Estimate of Gene Concentration by GC Levels in STSs

The STSs also provide a measure of local gene con-

centration. STSs made from mRNA sequences are direct indicators of the location of genes, but at pre- sent o n l y - 3 0 0 have been mapped on the X. An alternative measure of regional gene concentrat ion is provided by the relative content of GC in DNA segments . As suggested or ig ina l ly by Bernardi (1993) and confirmed for Xq26-qter (Pilia et al. 1993) and 22q (Gardiner et al. 1993), segments of chromosomal DNA spanning up to 1 Mb or more have characteristic GC contents, and the concentra- t ion of the genes is a function of GC. Because the nucleot ide composi t ions of the STSs themselves provide a sampling of GC content, their GC content can be plotted to obtain a profile of GC levels and corresponding putative relative gene concentrat ion across the X. Figure 3 shows the resultant distribu- tion. The middle line and its associated dots show the average GC content of STS sequences in a mov- ing window of groups of 80. The th inner lines above and below give the 95% and 5% confidence limits, respectively, of the estimated GC levels.

Five regions of relatively high ambient GC are seen: one at -20 Mb, two on either side of the cen-

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Figure 3 GC content across the chromosome estimated by % (G + C) in the sequences of sets of STSs derived from corresponding locations in the YAC/STS contig map. The curve showing the experimental points found by a sliding window of 80 STSs is accompanied by higher and lower curves representing 95% and 5% confidence intervals (see text). (Inset) An enlarged view of the curve between 125 and 160 Mb compared to the GC content of regions estimated with probes on Southern blots of DNA fractionated by density (Pilia et al. 1993).

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X CHROMOSOME YAC/STS MAP

t romere , one in Xq26.1, and one, especial ly marked , r ising sharply in sub te lomer ic Xq28. Two lines of ev idence suppor t t h e va l id i ty of these es t imates . First, t he prof i le in Xq26 to Xq te r s t r o n g l y re- sembles t ha t f o u n d by an i n d e p e n d e n t assay of GC levels. The inset in Figure 3 compares an en la rged

s e g m e n t of the GC curve f rom Figure 3 wi th assays based on Sou the rn analyses wi th specific probes as- sayed agains t DNA f rac t iona ted accord ing to its GC c o n t e n t by cen t r i fuga t ion to equ i l ib r ium in dens i ty gradien ts (Pilia et al. 1993). The direct assays were d o n e wi th on ly a l imi ted n u m b e r of probes and wi th local izat ion less precise t h a n tha t of Fig. 2; bu t the two inferred d is t r ibut ions are genera l ly concor- dant . Especially clear is the very h igh GC level in the Xq sub te lomer ic region, w h i c h has also b e e n con- f i rmed direct ly by long- range s equenc ing and o the r m e t h o d s (see, for example , C h e n et al. 1996).

A second i n d e p e n d e n t l ine of ev idence comes f rom f luorescent in situ hybr id iza t ion (FISH) of the

h ighes t GC fract ion of h u m a n DNA (the "H3" iso- chores; Bernardi 1993) to m e t a p h a s e c h r o m o s o m e s . Such expe r imen t s have de tec ted four of the p e a k s m one in t e lomer ic Xp and th ree in Xq. High GC seg- m e n t s in the one region u n d e t e c t e d by FISH, near the c e n t r o m e r e in Xp, have also b e e n d e m o n s t r a t e d direct ly by long- range s equenc ing (G. Nyakatura , M. Platzer, B. Drescher, H. Hel lebrand, D. Schindel- hauer , A. Meindl , and A. Rosenthal , pers. comm. ) . Fur the rmore , a very similar d i s t r ibu t ion has b e g u n

to emerge f rom the p l a c e m e n t of ESTs a long the X c h r o m o s o m e (Schuler et al. 1996). Thus, groups of STSs provide a reliable sample of the GC c o n t e n t of u n i q u e sequences in di f ferent po r t ions of the X.

In the s equenc ing phase of g e n o m e analysis, strategies m a y be adapted especially for regions of h igh GC, which tend to show compress ions in se- quenc ing and of ten con ta in h igh levels of Alu se- quences (Chen et al. 1996), or for regions of low GC, which m a y conta in a n u m b e r of long, nearly identical LINE sequences (Hood et al. 1995). Similarly, the analysis of regions of h igh or low GC requires software wi th differential sensitivity to detect CpG islands or putat ive exons, and a region tha t shows low recombi- na t ion m a y cue an analysis to look for any correlated

"suppressing" sequences. It will be of interest to com- pare the locations of ho t or cold spots of recombina- t ion and h igh or low GC levels on au tosomes for which maps reach comparable STS density.

METHODS

STSs and YACs

STSs and YACs are described in detail both in the Genome

DataBase (GDB) and on the World Wide Web site at http:// genome.wustl.edu/cgm/cgm.html; STSs that are not yet as- signed DXS numbers are identified by their Washington Uni- versity Genome Center accession (sWXD) numbers. All YACs have been deposited at the American Type Culture Collection (ATCC). Contigs include 5423 YACs from seven libraries (Na- garaja et al. 1994), supplemented with 569 cosmids from the X-specific Lawrence Livermore Laboratory collection and a growing number of BACs, currently totaling 250. The YACs comprise 1360 and 829 clones from two X-specific libraries [F and X3000 (Nagaraja et al. 1994), with an average size of 240 kb]; 993 and 1153 clones from two collections made from cell lines containing multiple X chromosomes (E and I, with an average size of 330 kb); 290 clones from the CEPH mega-YAC library [(Albertsen et al. 1990), with an average size of 1014 kb], and some additional clones from the Imperial Cancer Research Fund (ICRF) (Larin et al. 1991) and original St. Louis (Nagaraja et al. 1994) YAC libraries.

Strategy and Contigs from STS Content Mapping

The original formulation of STS content mapping (Green and Olson 1990; Green and Green 1991) was based on initial PCR- based screening of YAC libraries with a series of randomly generated STSs to yield substantial contigs across most of the area of a chromosome. As coverage becomes extensive and new random STSs increasingly fall within already mapped re- gions, directed walking is initiated with STSs (pseudoends) that map nearest the ends of nascent contigs. In this project, a modified all-walking approach was adopted in which STSs were made from YAC insert ends, and a series of walking steps at many points was then instituted and reiterated (Kere et al. 1992) until contigs merged across the entire X chromo- some. This strategy was theoretically more efficient (Palazzolo et al. 1991; Kere et al. 1992) and was facilitated by the avail- ability of two collections of X-specific YACs to initiate STS development and the merger of seed contigs (Nagaraja et al. 1994).

Contigs were aligned and oriented by a combination of FISH (Srivastava et al. 1995) and published marker orders and deletion panels. The somatic cell hybrid panel used hamster cell lines containing an entire X (GM06218C) or defined frag- ments of X chromosomes [Xp21.2-qter (GMl11713); Xp11.2-qter (GM10501); Xq13-qter (GM10095); Xq21.3-qter (GMl1172); and Xq24-qter (X3000.1)].

The current 13 contigs, with their bracketing markers, index positions roughly in megabases, and relevant references to some of the significant partial published versions (in addi- tion to Schlessinger et al. 1991 and Nelson et al. 1995), are as follows, from Xpter to Xqter: Xpter-DXS6667 (0-52 Mb; Cof- fey et al. 1992; Monaco et al. 1992; Alitalo et al. 1995; Black et al. 1995; Ferrero et al. 1995; Ried et al. 1995; Trump et al. 1996; Boycott et al. 1996); OATL2-centromere (52-58.5 Mb; Miller et al. 1995); centromere; DXS62-DXS1 (62-63 Mb); DXS1-DXS1225 (63-79 Mb; Villard et al. 1995); DXS447- DXS26 (79-84 Mb; van der Maarel et al. 1995); DXS995-TBG (84-106 Mb; Vetrie et al. 1994; Forbes et al. 1996, Sargent et al. 1996); DXS7667-COL4A5 (107-109.5 Mb; Srivastava et al. 1995); DXS1210-DXS287 (109.5-113 Mb); DXS1059- DXS8088 (113-118.5 Mb); DXS7308-DXS7872 (118.5-120 Mb); ANT2-DXS7317 (120-124 Mb); DXS8081-DXS1206 (124-131 Mb); and DXS1339-Xqter (131-160 Mb; Palmieri et al. 1994; Rogner et al. 1994; Pilia et al. 1996; Zucchi et al. 1996).

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NAGARAJA ET AL.

Use of SEGMAP

STS content mapping was sustained and directed by the ap- plication of the SEGMAP program (developed by C. Magness and P. Green, available from Chuck@chimera.biotech.wash- ington.edu). The most probable marker order was inferred from YAC/STS content (Green and Green 1991) with the pro- gram. In the middle game of mapping, contig formation and software representations of maps were very sensitive to false positives, which often joined distant portions of the X chro- mosome; but these were later sorted out by additional clones and cross-testing of STS content.

SEGMAP also facilitated the integration of outside data, based on the screening of published STSs to identify their location in relation to existing contigs. The comparative lo- calization was extended to infer relative genetic and physical distances, as in Figures 2 and 3.

ACKNOWLEDGMENTS

We are indebted to many collaborators in the X community for cooperative mapping interactions, including the provision of auxiliary STSs, YACs, and cosmids. Our major collaborators include G. Rappold (XpPAR); G.B. Ferraro, A. Ballabio, D. Nel- son, T. Alitalo, and D. Trump (Xp22); K. Boycott and T. Bech- Hansen (Xp11.3-p11.4); A. Miller and H.F. Willard (the peri- centromeric region); P. Stanier and F. Cremers (Xq21.3); and especially I. Zucchi, G. Palmieri, G. Romano, and the group of M. D'urso (Xq24-qter); Jean Weissenbach and Jeff Murray (simple sequence repeat markers and genetic map informa- tion prior to publication); and GDB (additional STS informa- tion). This work was supported by GESTEC (grant HG00201) from the National Center for Genome Research.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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Received October 1, 1996; accepted in revised form January 17, 1997.

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