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A Class I Jumping Clone Places the HLA-G Gene Approximately 100 Kilobases from Hfa-H Within the MA-A Subregion of the Human MHC

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A Class I Jumping Clone Places the HLA-G Gene Approximately 100 Kilobases from Hfa-H Within the MA-A Subregion of the Human MHC GENOMICS lo,%%914 (19%) A Class I Jumping Clone Places the HLA-G Gene Approximately 100 Kilobases from HfA-H within the MA-A Subregion of the Human MHC HRIDAYABHIRANJAN SHUKLA,* GERALD A. GILLESPIE, t RAKESH SRIVASTAVA,$ FRANCIS COLLINS,~ AND MICHAEL J. CHORNEY~~ Departments of *Human Genetics and tlnternal Medicine, Yale University School of Medicine, New Haven, Connecticut 06570; *Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037; §Departments of Internal Medicine and Human Genetics, University of Michigan, Ann Arbor, Michigan 48109; and )IDepartments of Microbiology and Immunology and Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Received August 3, 1990; revised February 18, 1991 endonucleases (most of which are sensitive to C meth- By the combination of cosmid cloning, ehromosomal ylation) and pulsed-field gel electrophoresis (PFGE) jumping, and pulsed-field gel electrophoresis (PFGE), we technology, have identified the major megabase frag- have fine-mapped the HLA-A subregion of the human ma- ments upon which reside the genes encoding the jor histocompatibility complex (MIX). Through the isola- transplantation antigens, HLA-A, -B, and -C. The tion of a class I jumping clone, the Qa-like HLA-G class I positions of additional members of the class I gene gene has been placed within 100 kb of HLA-H. The tight family, such as HLA-E (Srivastava et aZ., 1987) and physical linkage of these class I genes has been further sup- cdal2 (Ragoussis et aZ., 1989), have recently been ported by hybridizing PFGE blots with locus-specific added to the molecular map. However, the large num- probes. It has been found that both of the above class I genes ber of highly homologous class I sequences as well as are linked to HLA-A, with HLA-H residing no more than the large size of this stretch of DNA have impeded the 200 kb from the HLA-A gene. These data support the possi- ble existence of a Qu-like subregion composed of nonclassi- map’s completion. cal HLA class I genes within the human MHC linked telo- Weissman (1987) has outlined alternative cloning merically to the HLA-A locus. o 1~~1 Academic press, IDC. and mapping approaches that already have proven useful in the generation of mapping data. These tech- nologies include the use of jumping and linking li- INTRODUCTION braries (Collins and Weissman, 1984; Poustka and Lehrach, 1986) as well as yeast artificial chromosome The class I region of the human major histocompati- (YAC) vectors capable of cloning megabase DNA bility complex (MHC) is composed of about 2000 kb fragments (Burke et a& 1987). The former technology of DNA (Lawrance et al., 1987) and comprises approx- has proven instrumental in the successful identifica- imately 20 class I genes, pseudogenes, and gene frag- tion of the gene responsible for cystic fibrosis (Rom- ments (Srivastava et al., 1985; Koller et al., 1987; mens et al., 1989). Using YACs as cloning vectors, Lawlor et al., 1990). The functional members of the Little et al. (1989) have succeeded in isolating HLA class I gene family are among the most polymorphic and immunoglobulin clones. The continued applica- genes in nature (Parham et al., 1989); the mechanisms tion of these and other technologies will undoubtedly involved in the maintenance of this diversity are aid in the completion of the physical map not only of largely unknown. the MHC but of the human genome as well. The construction of a molecular map of the class I From deletion analysis studies (Orr and Demars, region, situated on the short arm of chromosome 6 1983) and data obtained in our own laboratory (M. (6p21.3), is under way (Lawrance et al., 1987; Ragous- Chorney, G. Gillespie, and S. Weissman, unpub- sis et al., 1986,1989; Carroll et aZ., 1987); this has been lished), it appears that many of the class I genes, aided in part by the isolation of lambda and cosmid pseudogenes, and gene segments are linked telomeri- clones containing unique and low-copy-number tally to the HLA-A locus. In the present report, we probes that flank HLA class I sequences. These have applied cosmid cloning, PFGE, and chromo- probes, in conjunction with rare-cutting restriction somal jumping technologies to the mapping of several 905 o&x&3-7543/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved. 906 SHUKLA ET AL. genes and pseudogenes within the human HLA-A isolated in the Weissman laboratory at Yale Univer- class I subregion of the MHC and discuss the possible sity can be found in Srivastava and Lambert (1989). occurrence of a human Qa region analogous to that found on chromosome 17 of the mouse (Weiss, 1987). Locus-Specific Probes We also discuss the possibility of a recombinational hot spot occurring in this region of the human MHC. HLA-G (6.0)-specific probe. On the basis of nu- cleotide sequencing (Srivastava and Lambert, 1989), one of the EcoRI class I+ fragments contained within MATERIALS AND METHODS CosRS27 was found to contain an allele of the HLA- 6.0 gene described previously by Geraghty et al. Cells (1987). This gene has been subsequently redesignated 3.1.0 (HLA-A2, HLA-B27, HLA-Cwl) is an EBV- HLA-G (Koller et al., 1989). A 700-bp low-copy EcoRI transformed B-cell line that is hemizygous with re- fragment located approximately 17 kb upstream of spect to its MHC (Levine et al., 1985). JY is an EBV- the HLA-G gene was subcloned into pUC19 and transformed line that is serologically HLA-A2 and termed pMC27.7 (see Fig. 3). The probe resides -B7; whether this line is an MHC homozygote as origi- within a nonpolymorphic 2-kb Hind111 fragment but nally suggested has been questioned (van Seventer et weakly cross-hybridizes to a series of four polymor- al., 1988). Both cell lines were maintained in RPM1 phic Hind111 fragments, each of which is linked to a 1640 containing 10% fetal calf serum and 1% penicil- specific HLA-A gene (Chorney et al., 1991). The locus lin-streptomycin. defined by this probe has been designated D6SllO (Chorney et al., 1991). HLA-H-specific probe. A 700-bp EcoRI fragment Jumping Library Production located approximately 21 kb 5’ of the HLA-H gene was The construction of the lOO-kb jumping library is subcloned into pUC19 (subsequently termed pMC6.7; described in Collins et al. (1987). Briefly, 3.1.0 DNA Chorney et al., 1990). This fragment was derived from was partially digested with Mb01 and size-fraction- CosRS6, the map of which is presented in Fig. 3. On ated by pulsed-field gel electrophoresis. DNA in the Southern genomic blots, pMC6.7 hybridized most size range of 80-130 kb was removed from the gel and strongly to a nonpolymorphic HindIII fragment of 10 ligated under dilute conditions in the presence of an kb but cross-reacted with several bands, two of which excess of BamHI-ended supF gene. Conditions that displayed polymorphism. Among the clones in our promoted the formation of DNA circles were chosen. class I+ collection, the probe hybridized only to After ligation, the circularized genomic DNA was di- CosRS6. The specificity of this fragment in hybridiz- gested with EcoRI and ligated to the lambda vector ing only to CosRS6 was previously noted (Srivastava Ch3AAlac. This lambda vector, which is Aam Bum, et al., 1986). accepts DNA in the size range of O-12 kb. The ligation HLA-A-specific probe. A 490-bp PvuII-MspI frag- was packaged in vitro and plated onto the supF- host ment derived from the 3’ untranslated region of the strain, MC1061. Approximately 2 X lo6 plaques were HLA-A2 gene was subcloned into pUC9 by Koller et obtained. These were divided into four groups and al. (1984) and termed pHLA-2A.l. The probe hybrid- amplified for future screening. ized most strongly to fragments carrying HLA-A genes but was also observed in our experiments to 3.1.0 Class I-Containing Cosmids cross-hybridize weakly to fragments carrying an HLA-A-linked pseudogene (Chorney et al., 1990) and The 3.1.0 cosmid libraries were made according to HLA-H. Lawrance et al. (1985). EcoRI and Sau3AI partially digested 3.1.0 genomic DNA was size-fractionated on Pulsed-Field Gel Electrophoresis a sucrose density gradient. The approximately 40-kb genomic DNA fragments were then ligated into either 3.1.0 genomic DNA, digested in agarose plugs, was EcoRI-digested pJB8 or BamHI-digested pcos- resolved on OFAGE (orthogonal field alteration gel BEMBL vectors. Packaged cosmids were then used to electrophoresis) gels using an LKB Pulsaphor gel ap- infect the recA- Escherichia coli strain HBlOl. paratus. Gels contained 1% agarose, 0.5X TBE (1X Unamplified cosmid libraries were screened using TBE equals 0.089 M Tris borate, 0.089 M boric acid, either HLA-B7 cDNA (Sood et al., 1981) or the class I 0.002 M EDTA, pH 8.0) and were run at 14°C for 48 h gene HLA-H (Chorney et al., 1990) as a probe. The at 250 V with a pulse time of 2 min. After electrophore- HLA-H gene is carried on a 6.6-kb EcoRI fragment sis, the gels were depurinated with 0.2 N HCI for 15 and lacks repetitive sequences. The compilation and min and then denatured with 0.5 N NaOH. The gels further description of the class I-containing cosmids were then blotted against 0.5 N NaOH and 1.5 M A CLASS I JUMPING CLONE FROM THE HLA-A SUBREGION 907 I I WI1 Eco RI Eco RI ms.PA PSI 1 SW I i pHS.1 1 kb FIG.
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