The Generation and Regional Localization of 303 New Chromosome 5 Sequence-Tagged Sites

GENOMICS

32, 91–96 (1996) 0080

ARTICLE NO.

The Generation and Regional Localization of 303 New Chromosome 5 Sequence-Tagged Sites DEBORAH L. GRADY,*,1 DONNA L. ROBINSON,* MERYL GERSH,† ELIZABETH NICKERSON,‡ JOHN MCPHERSON,‡ JOHN J. WASMUTH,‡ JOAN OVERHAUSER,† LARRY L. DEAVEN,* AND ROBERT K. MOYZIS* *Center for Human Genome Studies and Life Sciences Division, Los Alamos National Laboratory, University of California, Los Alamos, New Mexico 87545; †Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and ‡Department of Biochemistry, University of California Irvine, Irvine, California 92717 Received September 18, 1995; accepted November 21, 1995

(Botstein et al., 1990), facilitating the identification of disease genes and accelerating our understanding of many aspects of cellular and molecular biology (Olson, 1993). Various approaches have proven successful in generating robust PCR-based physical and genetic markers (Green et al., 1991; Murray et al., 1994), and the worldwide human genome effort is likely to produce thousands of STSs in the foreseeable future [for example, see the Whitehead Institute/MIT Center for Genome Research, Human Physical Mapping Project, Data Release 8 (September 1995)]. Nevertheless, many additional STSs will be needed to produce the desired 0.1-Mb resolution required for wide dissemination of the genome map to the world’s scientific community. It has been demonstrated previously that flow-sorted human chromosomes can be an efficient source of DNA for both recombinant DNA library construction and the systematic generation of chromosome-specific STSs (Deaven, 1986; Green et al., 1991; McCormick et al., 1993). The usefulness of chromosome-specific STSs is enhanced if further regional localization can be obtained. Hybrid cell panels consisting of deletions or translocations of specific human chromosomes allow the assignment of STSs to small chromosome regions (Vollrath et al., 1992; Overhauser et al., 1994; Doggett et al., 1995). Following this initial low-resolution (approximately 2–5 Mb) ‘‘binning’’ of STSs, further regional resolution and ordering of 0.1–0.5 Mb may be achieved by direct YAC-content mapping (Green et al., 1990; Foote et al., 1992; Doggett et al., 1995) or radiation hybrid mapping (Cox et al., 1990). Ultimately a ‘‘sequence-ready’’ map of much higher resolution can be generated in Escherichia coli-based vectors (Stallings et al., 1990, 1992; Riethman et al., 1993; Nizetic et al., 1994; Doggett et al., 1995). In this paper, we describe the use of flow-sorted human chromosome 5 DNA to generate 303 new regionally assigned STS markers. This marker density (approximately 1 STS/640 kb), in addition to the numerous

With the ultimate goal of creating a sequence-ready physical map of all of chromosome 5, 303 new human chromosome 5-specific STS markers have been systematically generated and regionally ordered. Chromosome 5 DNA prepared from flow-sorted chromosomes was digested with restriction enzymes BamHI and HindIII and cloned in bacteriophage M13mp18. Random clones were sequenced, and appropriate PCR deoxyoligomers were synthesized. An acceptable sequence-tagged site (STS)-PCR assay yielded the appropriate size amplification product from both total human DNA and hybrid cell line DNA containing only human chromosome 5. Each STS has been regionally localized by breakpoint analysis using a set of hybrid cell panels consisting of natural deletions or translocations of human chromosome 5. This hybrid cell panel was able to localize the STSs to 1 of 51 bins on the short arm and 1 of 15 bins on the long arm. The STS markers appear to be randomly distributed along the length of this 194-Mb chromosome. The current overall density of these markers (approximately 1 STS/640 kb), combined with the numerous PCR-based physical and genetic markers generated by other groups, will provide sufficient ‘‘nucleation points’’ for YAC contig assembly and verification in any region of human chromosome 5. q 1996 Academic Press, Inc.

INTRODUCTION

The generation of sequence-tagged sites (STSs) (Olson et al., 1989) is an integral part of strategies designed to produce high-quality, low-resolution physical maps of human chromosomes (Green and Olson, 1990; Foote et al., 1992; Chumakov et al., 1992; Cohen et al., 1993; Doggett et al., 1995). STS-based maps, with an average resolution of approximately 0.1 Mb, are one of the initial goals of the Human Genome Project 1 To whom correspondence should be addressed at Los Alamos National Laboratory, MS M888, Los Alamos, NM 87545.

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0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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PCR-based physical and genetic markers generated by other groups (Murray et al., 1994), will provide sufficient ‘‘nucleation points’’ for YAC contig assembly in all regions of this 194-Mb chromosome (Morton et al., 1991; Goodart et al., 1994). MATERIALS AND METHODS Chromosome sorting. Complete digest chromosome 5-specific recombinant DNA libraries were prepared from chromosomes derived from two sources. Chromosomes 5 were sorted either from diploid human fibroblasts (GM130A) or from a Chinese hamster–human hybrid cell line (Q826–20) (M. Sinensky, Eleanor Roosevelt Institute, Denver, CO). The cells were grown using conventional techniques, and chromosome isolation utilized the polyamine method (Deaven et al., 1986). The purity of sorted chromosomes is critical to the construction of chromosome-specific DNA libraries. In general, sorting purity was analyzed by both quantitative flow-sorting estimates and in situ hybridization following sorting (Deaven et al., 1986). For library construction, sort purity was greater than 90–95%. This represents a 20- to 50-fold enrichment of the desired chromosome. Library construction. Approximately 200,000 chromosome 5 molecules (approximately 70 ng in 400 ml) sorted from cell line GM130A or Q826–20 were stored in microfuge tubes at 0207C. After thawing, TE (10 mM Tris–HCl, pH 8.0/1 mM EDTA, pH 8.0) was added to a total volume of 1.5 ml. Sodium dodecyl sulfate and proteinase K (International Biotechnologies Inc.) were added to final concentrations of 0.5% and 100 mg/ml, respectively. Following incubation at 377C overnight, the sample was extracted with an equal volume of phenol:chloroform (1:1) and then chloroform:isoamyl alcohol (24:1). The extracted aqueous phase was dialyzed (3 1 15 min) against TE in a collodion bag. Approximately 50 ng of DNA from this extraction was digested to completion with the restriction enzymes BamHI and HindIII (NEB). After heating to 707C for 10 min, phenol:chloroform and chloroform:isoamyl alcohol extraction (as above), and dialysis against TE (3 1 15 min), the DNA was concentrated to a total volume of 40 ml with secbutanol and redialized (TE, 3 1 15 min). The restriction enzyme-digested DNA was ligated to a twofold excess of M13mp18 replicative form DNA that had been cleaved with BamHI and HindIII and then treated with alkaline phosphatase. The ligation mixture was used to transform competent cells prepared from Escherichia coli strain DH5aF (BRL). Approximate cloning efficiencies of 103 recombinants per nanogram of insert DNA were obtained. Propagation of individual M13 clones and isolation of single-stranded DNA were carried out using standard protocols, substituting Strataclean (Stratagene) for phenol:chloroform extractions. DNA sequencing was performed using the dideoxy chain termination method (Sanger et al., 1977) and universal M13 sequencing primers, with a Dupont Genesis 2000 automated sequencer. M13 clones were chosen at random, and the sequence of 0.25–0.45 kb of insert DNA sequence immediately adjacent to the vector cloning site was determined. Development of STS assays—Sequence analysis The chromosome 5-enriched DNA sequences were subjected to computer-based analysis. Each sequence was scrutinized for similarity to DNA segments known to occur frequently in the human genome, specifically, Alu, L1, alphoid (Green et al., 1991), THE (Fields et al., 1992), satellites I, II, and III (Grady et al., 1992), and telomere (Moyzis et al., 1988) motifs as well as M13 vector sequence. Sequences found to contain large sequence identities to any of these repetitive elements were eliminated from further analysis. Regions of minimum sequence identity to any of these repetitive elements were noted on the remaining sequences. These regions of minimum sequence identity were avoided when choosing oligonucleotide primers. Even minimum identity to repetitive elements can affect PCR yield, due to the large number of pseudosites for primer annealing in human DNA (Moyzis et al., 1989). Each sequence was analyzed using the grail program (Uberbacher et al., 1991). We have found that primers synthesized in putative coding regions often yield poor results, giving equivalentsized bands in rodent DNA or multiple bands. Such results are ex-

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pected for true coding regions. PCR primers were chosen manually from the remaining DNA sequence using stringent criteria. Primers chosen were (1) a minimum of 20 nucleotides in length; (2) without an identity greater than 7 nucleotides with a repetitive sequence; (3) without internal secondary structure; (4) without complementarity between primers; (5) with 40–50% G/C content (same for both primers); and (6) with G or C at the 3* elongation end. The predicted Tm of the chosen oligomer was computed, using a program based on nearest neighbor frequencies (Gray et al., 1981; Doggett et al., 1995), that for short oligomers yields a more accurate Tm than other approaches. PCR assays. Oligodeoxynucleotide primers were synthesized on Applied Biosystems Model 391 or 394 DNA synthesizers and subsequently purified on Nensorb (NEN) reverse-phase columns. Purified oligonucleotide yields averaged 15–20 A260 (approximately 495–660 mg). PCR, as described below, was performed using slight modifications of the approach described previously (Green and Olson, 1990; Green et al., 1991). PCR assays were carried out in a 20-ml volume containing 50 ng DNA template, 22.5 mM each primer, 1.5 mM MgCl2 , 200 mM dNTPs, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 6.25 U Taq DNA polymerase (AmpliTaq; Perkin–Elmer/Cetus), and 0.001% (w/v) gelatin. Amplification was performed with a Perkin– Elmer 9600 thermocycler as follows: denaturation at 927C for 15 s, renaturation at 107 below the Tm of the primer set for 35 s, and elongation at 727C for 35 s. Thirty-five amplification cycles were performed. PCR products were analyzed by agarose gel electrophoresis on 3% agarose gels (FMC NuSieve, 3:1) and detected by ethidium bromide staining as described (Green et al., 1991). Standard analysis of each STS involved synthesis from both human DNA (GM130) and cell line HHW105, which contains only human chromosome 5. A successful PCR assay amplified a single DNA product of the appropriate molecular weight in these two reactions and no product when amplification was attempted from hamster DNA, the rodent component of cell line Q82620. Regional localization of STSs. A description of the somatic cell hybrids used for mapping STSs to 5p and 5q has been given previously (Warrington and Wasmuth, 1993; Overhauser et al., 1994). Genomic DNA was isolated using standard procedures (Moyzis et al., 1981, 1989). PCR was performed in a 25-ml reaction volume in PCR buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl, 0.01% w/v gelatin) containing 200 mM dNTPs, 1.2% deionized formamide, 0.5 U Taq polymerase, 100 ng genomic DNA, and 75 ng each primer. Optimal Mg concentrations (1.0–2.5 mM) and annealing conditions (50– 607C) for human-specific amplification were determined for each STS. A two-step 35-cycle PCR program of 947C for 30 s and the optimal annealing temperature for 30 s was performed. This was followed by a final extension cycle at 727C for 10 min. The amplified products were run on a 1.5% agarose gel at 75 V for 1 h.

RESULTS

DNA Sequencing and PCR Analysis Chromosome 5 DNA prepared from flow-sorted chromosomes was digested with restriction enzymes BamHI and HindIII and cloned in bacteriophage M13mp8. Seven hundred sixty-one clones were chosen at random and sequenced. Of these 761 sequences, 122 (16.0%) showed significant sequence identity to human repetitive sequences, 45 (5.9%) were identical to the M13 vector, 36 (4.7%) were unsequenceable, 11 (1.4%) were under 50 nucleotides in length, and 2 (0.3%) were duplicates. The remaining 546 (71.7%) sequences were used to generate STSs. Each STS was screened against (1) total human genomic DNA (GM130A), (2) total hamster genomic DNA (CHO), and (3) DNA from a hamster somatic cell hybrid

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CHROMOSOME 5 STSs

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TABLE 1 LANL STS Nos. and GSDB Accession Nos. for Each of the 303 Chromosome 5-Specific STSs

containing only human chromosome 5 (HHW105). Because of the sources of human chromosome 5 DNA used in the construction of the M13 libraries, namely, total human chromosomes (GM130A) and a hamster/human chromosome 5-containing hybrid (Q826–20), two forms of DNA contamination were detectable by PCR. Contaminants from the total human chromosome preparations produced an appropriate size band with PCR from total genomic DNA but not with PCR from DNA derived from human chromosome 5. Using hybrid cell panel DNA, these STSs could be assigned to other human chromosomes. Contaminants from the hamster/ human cell hybrid amplified a strong PCR signal from hamster DNA. When these 46 human contaminants (8.4%) and 62 hamster contaminants (11.3%) were subtracted from the total possible number of sequences capable of producing chromosome 5 STSs (546–108), 438 sequences remained. Thirty-two sequences (7.3%) gave ambiguous results in PCR assays. This category refers to those reactions that yielded multiple bands, bands of inappropriate product size, or the same size bands in both human and hamster DNA. A total of 23% (101) of the primer pairs gave no amplification product.

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The remaining 305 (69.6%) primer sets generated robust, chromosome 5-specific PCR products when tested under the standardized conditions. Unsuccessful primer sets have not been retested under customized MgCl2 and temperature conditions (Green et al., 1991). These 305 STSs were regionally mapped along chromosome 5 using somatic cell hybrid panels (see below). Two STSs on the short arm were unmappable on these panels. The remaining 303 STSs and their genome sequence database (GSDB) Accession Nos. are shown in Table 1. Regional Localization of STSs STSs were first mapped to a specific arm of chromosome 5 using the somatic cell hybrids HHW661 and HHW693. HHW661 contains chromosome 5 DNA from 5p14–qter. HHW693 is derived from HHW661 and contains chromosome 5-specific DNA from 5p14–cen (Wasmuth et al., 1986). If the STS mapped to the q arm, the amplification results would be positive for HHW661, but negative for HHW693. For subsequent mapping to 5p, PCR was performed

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proximal to JH132. Sixty-four (21.1%) of the chromosome 5-specific STSs mapped to the short arm. Regional assignment of q arm markers was carried out using a similar PCR-based assay on an expanded set of human–Chinese hamster cell hybrids, each of which retains naturally occurring deleted or translocated chromosome 5 (Warrington and Wasmuth, 1993). Two hundred seventeen long arm STSs were localized to one of 16 bins, with the exception of bins B and D (Fig. 2). With the present hybrid cell panel, markers originating in these 2 bins cannot be distinguished.

FIG. 1. Idiogram of the short arm of human chromosome 5. Bin assignments were derived from a natural deletion/translocation somatic cell hybrid panel established from patient material. Localization of each STS into 1 of 51 bins is indicated.

on an initial set of six somatic cell hybrids to determine the chromosomal band location (5p15.3, 5p15.2, etc.) of the STS. The somatic cell hybrids that had chromosomal breakpoints within the chromosomal band were then used in PCR experiments for more precise mapping (Overhauser et al., 1994). The results are shown in Fig. 1. In several cases, the mapped STS allowed for the further ordering of the deletion breakpoints. For example, the order of HHW786 and JH132 could not be initially determined; however, LANL550 and LANL221 were found to map distal to HHW786, but

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FIG. 2. Idiogram of the long arm of human chromosome 5. Bin assignments were derived from a natural deletion/translocation somatic cell hybrid panel. Localization of each STS into 1 of 15 bins is indicated.

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CHROMOSOME 5 STSs

Additionally, three other STSs not shown in Fig. 2 were localized to multiple bins. STS 203 was localized to bins B, D, and E. STS 152 was placed in bins F, G, and H. STS 331 was localized to bins K, L, and M. Whether this represents multiple locations detected by these STSs or other ambiguities remains to be determined. An additional 19 STSs that, to date, have only a q arm localization are included in Table 1. Given the current uncertainties of the hybrid breakpoint map, where the indicated bin size is known only approximately, the distribution of STS markers shown in Figs. 1 and 2 is essentially random. For example, the fraction of the STSs mapping to the short arm and the long arm are 21.1 and 78.9%, respectively, consistent with their observed cytological lengths (Morton, 1991). A similar analysis between any other two arbitrarily defined regions of chromosome 5 yields observed numbers of STSs proportional to the designated bin size. DISCUSSION

The generation of 303 new STSs from flow-sorted human chromosome 5 DNA and their regional localization have been accomplished. These STSs were all developed from experimentally generated DNA sequence data. None were derived from previously reported genes or sequenced regions on human chromosome 5. A comparison with the efficiency of STS production from other flow-sorted chromosome sources, namely, the chromosome 4 work (Goold et al., 1993), the Y chromosome work (Foote et al., 1992), and the chromosome 11 work (Smith et al., 1993), revealed similar results. From the initial number of sequenced clones, 71.9% for chromosome 5, 53% for the Y chromosome, 86% for chromosome 11, and 68% for chromosome 4 were suitable for PCR analysis. When primer sets from these sequences were tested by PCR for STS generation, 69.6% for chromosome 5 (this work), 77% for chromosome 11 (Smith et al., 1993), 95% for the Y chromosome (Vollrath et al., 1992), and 88% for chromosome 4 (Goold et al., 1993) were reported to be successful. These numbers translate into 303 STSs for chromosome 5, 182 STSs for the Y chromosome, 212 STSs for chromosome 11, and 822 STSs for chromosome 4. Clearly, if sorting purities greater than 90% can be achieved, the generation of STSs from flow-sorted DNA is an efficient procedure. REFERENCES Botstein, D., Cantor, C., Carbonell, J., Carrano, A., Caskey, T., Collins, F., Francke, U., Lander, E., Lerman, L., Moyzis, R., Olson, M., Pardue, M., Pearson, M., Tilghman, S., Wexler, N., White, R., Wolff, S., and Zinder, N. (1990). ‘‘Understanding Our Genetic Inheritance. The U.S. Human Genome Project: The First Five Years. FY 1991–1995,’’ No. DOE/ER-0452P, NIH No. 90–1590. Chumakov, I., Rigault, P., Guillou, S., Ougen, P., Billaut, A., Guasconi, G., Gervy, P., LeGall, I., Soularue, P., Grinas, L., Bougueleret, L., Bellanne-Chantclot, C., Lacroix, B., Barillot, E., Gesrouin, P.,

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