Molecular map of the murine S region ( H - 2 / fourth component of complement / factor B / cosmid cloning / transcriptional control )

Eighteen overlapping cosmid clones spanning 240 kilobases and encoding the gene for factor B and two genes related to the fourth component of complement (C4) were isolated from a murine H-2d genomic library. Cosmid clones were identified by hybridization to human cDNA probes for factor B and C4 and were linked by chromosomal walking procedures. The cluster of clones contains two regions with sequences homologous to the C4 cDNA probe, both in the same orientation, representing a direct duplication of at least 55 kilobases of chromosomal DNA, separated by a shorter (<25 kilobases) segment of nonduplicated DNA. Restriction fragment-length polymorphism seen by using C4 probes maps these sequences to the S region of the major histocompatibility complex. 5' to the two C4-like sequences is an :40-kilobase-long region of -chromosomal DNA remarkable for its lack of restriction fragment-length polymorphism, containing sequences homologous to the human factor B cDNA probe. These experiments demonstrate that the structural gene for factor B is located in the S region of the murine major histocompatibility complex and that this region contains an extensive direct duplication that contains the structural gene for mouse C4 and, we presume, for the sex-limited protein variant, SIp. RNA transfer blot analysis of total liver RNA from high C4and low C4-producing strains showed that steady-state levels of C4-hybridizing RNA were much greater in high C4-producing strains. Regulation of circulating C4 levels in high C4 and low C4 strains is at least partly at the level of mRNA transcription, processing, or degradation. The S region of the murine major histocompatibility complex (MHC), located between the I and D regions, contains the structural genes encoding the fourth component of murine complement (C4) and the sex-limited protein variant, Slp (reviewed in refs. 1 and 2). Slp shares extensive structural homology with C4 but has no demonstrable hemolytic activity and no known function (3). There are two major C4 alleles, C4-high (C4h) and C4-low (C41), which control ==20-fold differences in serum C4 levels. Slp is produced in only some C4h strains and is found only in males (1). In man, the structural genes for the second component of complement (C2) and for factor B of the alternate pathway also map to the MHC (2). Recent studies suggest that the structural gene for mouse factor B maps to the MHC as well and that control of mouse C2 hemolytic levels maps to the S region (4, 5). Together, these proteins make up the class III molecules of the MHC. The class I and class II molecules of the MHC are cell-surface glycoproteins that are involved in many immune reactions (reviewed in refs. 6 and 7). They are notable for the extensive allelic polymorphism that they demonstrate. A great deal has already been learned about the structure and evolution of these genes. Recent experiments have used cosmid cloning to isolate >13 clusters of clones containing sequences encoding class I genes and including >800 kilobases (kb) of genomic DNA (8). Cosmid cloning has also been used to generate a detailed molecular map of 200 kb of DNA containing most of the sequences encoding the class II genes (9). These experiments have disclosed a hot spot for recombination in the I region, demonstrating that, particularly in this region, extrapolating from recombinational map distances to physical map distances may be impossible. The class III molecules are encoded by a much less polymorphic set of genes clustered within the MHC. In the past year, cDNA clones encoding portions of human factor B (10), human C4 (11, 12), and murine C4 (13) have been isolated. In addition, human C4 and factor B genomic clones have recently been described (12, 14). In the present study, we have used the human factor B and C4 cDNAs as probes to recover from a murine H-2d cosmid library overlapping genomic clones that define 240 kb of DNA in the S region, demonstrating that the structural gene for murine factor B is encoded in the S region, tightly linked to the gene encoding C4, and providing evidence for an extensive duplication of chromosomal material in the development of this portion of the MHC. MATERIALS AND METHODS Materials. Restriction enzymes, T4 DNA ligase, and DNA polymerase I were purchased from New England BioLabs or Bethesda Research Laboratories and were used according to the manufacturers' specifications. Calf intestinal phosphatase was from Boehringer Mannheim. Nitrocellulose filters used for plating and screening the cosmid library were Millipore HATF (0.45-,m pore size, 85 mm in diameter). The cosmid vector pJB8 was a gift of R. Flavell. B6.AK1 and B6.K2 liver DNAs were the generous gift of D. Margulies. C57BL/6, BALB/c, and C3H mice were obtained from The Jackson Laboratory. Preparation of the Cosmid Library. High molecular weight DNA was prepared from MOPC-321 tumors (H-2d) according to a modification of the method of Blin and Stafford (15). More than 90% of this DNA was present as fragments larger than 135 kb, as evaluated by gel electrophoresis in 0.25% agarose. Cosmid inserts were prepared by partial Mbo I digestion of 6 mg of MOPC-321 DNA followed by size fractionation by using a Hoeffer Bull's Eye preparative electrophoresis apparatus with a 0.6% agarose gel in 40 mM Tris-HCl/20 mM sodium acetate/ 1 mM EDTA, pH 7.6 (TAE buffer). Fractions containing DNA fragments 35-45 kb in length were extracted with phenol/chloroform, precipitated with ethanol, and dissolved in 10 mM Tris'HCl/1 mM EDTA, pH 7.6, at a concentration of 0.5 mg/ Abbreviations: C2 and C4, second and fourth components of complement, respectively; MHC, major histocompatibility complex; Slp, sexlimited protein; kb, kilobase(s). 6947 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6Proc. Nati. Acad. Sci. USA 80 (1983) ml. Cosmid vector was digested with BamHI, followed by treatment with calf intestinal phosphatase. Ten micrograms of vector and 10 jig of size-fractionated insert (molar ratio of vector to insert, =5: 1) at a total DNA concentration of 0.1 mg/ml were ligated at 13TC for 14 hr. In vitro packaging was performed by a modification of the method of Sternberg et al. (16). Escherichia coli strain 490A (recA-) was transduced at 25TC for 30 min, then diluted 1:5 with Luria-Bertani broth (LB broth), and incubated at 370C for 60 min. Cosmids were plated on nitrocellulose filters at =--5,000 colonies per 85-mm filter, on LB agar containing ampicillin at 25 ,tg/ml. Two sets of replica filters were prepared and processed as described by Hanahan and Meselson (17), one for chloramphenicol amplification (100 Ag of chloramphenicol per ml) and hybridization and the other for storage at -70TC on LB agar plates containing 25% glycerol. This procedure yielded 1.25 x 105 cosmids per /ig of insert DNA; 3 X 105 cosmids were plated per library, distributed on 60 nitrocellulose filters. Screening the Cosmid Library. One microgram of each probe was nick-translated to a specific activity of 2-4 X 108 cpm/Ag as described (18). Filters were divided into groups of 30 and were hybridized for 30 hr at 42TC in 125 ml of 12.5% dextran sulfate/50% formamide/5 mM Tris-HCl, pH 7.6/0.1% NaDodSO4/Denhardt's solution/0.75 M NaCl/0.075 M sodium citrate containing 2 mg of denatured herring sperm DNA and 0.25 Ag (-5 x 107 cpm) of heat-denatured probe. The filters were then washed extensively in 0.3 M NaCl/0.03 :M sodium citrate/0: 1% NaDodSO4 at room temperature, followed by washing with 30 mM NaCl/3 mM sodium citrate/0.1% NaDodSO4 at 500C. Hybridizing colonies were detected by autoradiography overnight by using Kodak XAR film with an intensifying screen. Hybridizing colonies were picked from master filters as described by Grosveld et al. (19), colony purified, and stored frozen in 25% glycerol/LB broth. Cosmid DNA was isolated according to the method of Ish-Horowicz and Burke (20). Southern Blot Hybridization. DNA was digested with the indicated restriction enzymes, separated by agarose gel electrophoresis (0.9% agarose in TAE buffer), and transferred to nitrocellulose filters as described by Southern (21). Hybridization, washing, and autoradiography were as described for cosmid filters, except that hybridization was for 16 hr. Isolation and Analysis of RNA. Freshly obtained liver (2.5 g) from 6to 8-wk-old male mice was minced with scissors, washed in Hanks' balanced salt solution, and homogenized in 20 ml of 4 M guanidine thiocyanate/50 mM sodium acetate, pH 6.5, by using a Polytron at a setting of 6 for 30 sec. The homogenates were cleared by centrifugation at 10,000 X g for 10 min at 40C. RNA was isolated as described by Chirgwin et al. (22). Fifteen micrograms of RNA was incubated for 5 min at 65TC in 30% formamide/6% formaldehyde and then fractionated by electrophoresis in a 0.8% agarose gel (containing 0.6% formaldehyde, and 0.2 jig of ethidium bromide per ml) in 20mM 3-(N-morpholino)propanesulfonic acid/5mM sodium acetate/1 mM EDTA, pH 6.8. After electrophoresis, gels were incubated in 3 M NaCI/0.3 M sodium citrate for 30 min and then RNA was transferred to nitrocellulose by blotting. HyBf C4-X C4-Y I ~~ I_ -Z/ IA '1 Chromosome 17