Molecular map of the murine S region.

Abstract

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 C4-and 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 M HC (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...

Eucaryotic chromosome transfer: production of a murine-specific cosmid library from a neor-linked fragment of murine chromosome 17.

“…2B) (data not shown). When probed with a C4 probe, which identifies both C4 and the closely related Slp genes (1), all transferents demonstrated hybridization to the Slp-specific restriction fragment, but only the canine transferents contained the C4 gene. Thus the CHMD(D)B1 cell line apparently contains a murine chromosome broken between the Slp and C4 genes.…”

“…DNA probes used in these analyses include an IAp fragment (17), H-2-specific sequence (3), C4-Slp probe (1), and the murine-specific repeat probes (18).…”

“…Highmolecular-weight DNA was prepared for Southern blot analysis by suspending 107 cells in 3 and cloning of this DNA into the cosmid vector pJB-8 was previously described by Chaplin et al (1).…”

Characterization of a Q subregion gene in the murine major histocompatibility complex.

“…A B6 cosmid library was prepared according to Chaplin et al (20). Briefly, high molecular weight B6 liver DNA was partially digested with Mbo I, size-fractionated, and inserted into the BamHI site of the cosmid vector pJB8; 500,000 recombinant colonies were screened with the H-2Ld gene corresponding to most of the C2 domain.…”

Genomic organization of mouse Fc gamma receptor genes.

“…The 1.5-kb cDNA insert of clone b was labeled with 32P by the random priming method (14) and was used to screen two genomic libraries: a mouse BALB/c library in EMBL-3 (Clontech), and a mouse BALB/c library in the cosmid vector pTCF (15). Screening, hybridization, and washing procedures were carried out as described (16)(17)(18). Isolated colonies were rescreened with an Fc,,RIIa-specific probe (a 0.42-kb Bgl I fragment of the cDNA insert of clone a).…”