A technique for replication R- and G-banding of mouse lymphocyte chromosomes was developed, and the replication R-banding pattern was analyzed. Optimal banding patterns were obtained with thymidine- and BrdU-treatment of lymphocytes in the same cell cycle. This produced replication R-band patterns that were the complete reverse of the G-band patterns on all chromosomes. Replication R-banding methods can be used in conjunction with nonisotopic, fluorescence in situ hybridization (FISH) to localize cloned probes to specific chromosomal bands on mouse chromosomes. With these methods the mouse complement factor H gene (cfh) was localized to the terminal portion of the F region of Chromosome 1. Q-banding patterns were also obtained by the replication R-banding method and may be useful for rapid identification of each chromosome.

ABSTRACTcDNA clones specific for the fourth component of mouse complement (C4) and its hormonally regulated isotype, sex-linked protein (Slp), were isolated using as a probe a 20-mer synthetic oligonucleotide corresponding to a known sequence of human C4 cDNA. Two types of clones, one specific for C4 (pFC4/10, with a 3.7 kilobase insert) and one specific for Slp (pFSlp/1, with a 4.7 kilobase insert), were isolated from liver cDNA libraries constructed from the Slp-producing FM mouse strain. The cDNA inserts of these clones shared 70% of the restriction sites determined. Only one type of clone was isolated from the Slp-negative DBA/1 strain; this type showed restriction maps indistinguishable from that of pFC4/10. pFC4/10 and pFSlp/1 displayed extensive homology: 94% nucleotide homology and 89% derived amino acid homology in the C4a region and 92% nucleotide homology and 89% derived amino acid homology in the thiol-ester region. An Arg-Gln-Lys-Arg sequence in the 3-a junction and a Cys-AlaGlu-Gln sequence in the thiol-ester site were identified for both proteins. A remarkable divergency between C4 and Slp sequences was recognized in the region immediately following the C4a sequence.The fourth complement component (C4), the second complement component (C2), and factor B are comprised in the class III antigens of the major histocompatibility complex in higher vertebrates (for review, see refs. 1-3). C4 is a serum glycoprotein composed of three disulfide-linked polypeptide chains (4), but it is translated as a single-chain precursor that is processed to the three-chain structure before it is secreted (5-7). In addition to the hemolytically active C4, a testosterone-regulated isotype, sex-limited protein (Slp), has been identified in the mouse (8, 9). Mouse C4 and Slp display extensive structural similarities, including homology in their partially determined amino acid sequences (10), and both appear to undergo essentially identical intracellular processing (6). Nevertheless, Slp is reported to be hemolytically inactive and insusceptible to enzymatic cleavage by activated Cls (11).The loci coding for mouse C4 and Slp are located in the S region of the murine major histocompatibility complex, H-2 (9, 12-15). In addition to these two structural genes, the S region encompasses regulatory elements for C4 (Ss trait) and Slp expression (8, 9, 16), although it is not clear whether these elements are distinct from their respective structural genes. Two major Ss alleles, C4-high (C4h) and C4-low (C4'), which are found in non-H-2k strains and H-2k strains, respectively, dictate 10-to 20-fold differences in the serum concentration of C4 (9, 16). As for the regulation of Slp, three alleles have been identified which effect (i) testosterone-regulated expression (Slpa), (ii) constitutive expression (Slpw7), or (iii) null expression (Slp°) (8,9,17,18).cDNA clones for mouse C4 and Slp would be extremely useful in studies of the structure and regulation of C4 and Slp genes and in the elucidation of the structure, function, and evol...

SUMMARYThe IgG subclass specificity of Fc receptor(s) induced on cells by infection with human cytomegalovirus (HCMV) was studied in a binding assay by using infected cells and purified iodinated IgG of various subclasses from HCMV seronegative healthy adult donors. All four human IgG subclasses bound to HCMV-infected cells, with the following relative magnitudes: IgG1 t> IgG4 > IgG2 > IgG3. The IgG subclass specificity of the Fc receptor was further analysed in an inhibition assay by using fragments prepared from purified human IgG by papain digestion, and using unlabelled subclass proteins. Fc but not Fab fragments inhibited the binding of 125I-labelled human IgG to HCMV-infected cells. The biological role of the Fc receptor in HCMV infection is discussed.

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A recombinant H-2 haplotype, designated awl8, was produced that underwent meiotic recombination in the Ea (I-E a chain)-Slp (sex-limited protein) interval of the H-2 class III region between B1O.A (H-2a) and wild-derived B1O.MOL-SGR (H-2wm7) strains. It appeared that the H-2awl8 haplotype has a single, recessive, lethal mutation, since homozygotes for H-2aw18 were not detected in progeny generated from the intercross of mice that were heterozygous for this H-2 haplotype. Nine newly established recombinant H-2 haplotypes, which arose from the heterozygous mice that resulted from a cross between common inbred H-2 haplotypes and the awl8 haplotype, allowed us to map the lethal gene to the class III region of the H-2 complex. Southern blot analysis indicated that the awl8 haplotype has a deletion of the C4 gene and a deletion of one of the steroid 21-hydroxylase genes. This result was confirmed by an immunodiffusion test that demonstrated the absence of production of the C4 protein in mice of haplotype H-2aw18. All data that were obtained supported the hypothesis that the meiotic, presumably unequal, recombination between homologous chromosomes of the H-2a and H-2wm7 haplotypes caused the deletion of the C4 and the 21-hydroxylase genes.The class III region of the mouse major histocompatibility complex (MHC), H-2, contains genes that encode components of the early portion of the complement activation pathways, such as C2, factor B (Bf), and C4 (1, 2). The Slp (sex-limited protein) gene, located in the same region, shares marked structural homology with the C4 gene, but Slp has no hemolytic activity. The biological function of Slp is still unknown. In addition to the components of the complement pathway, two structurally homologous steroid 21-hydroxylase (21-OHase) genes, 21-OHase.A and 21-OHase.B, have been mapped adjacent to the 3' ends of the Slp and the C4 genes (3). This enzyme plays a key role in the biosynthesis of adrenal steroids. A similar close linkage of the genes for C4 and 21-OHase and a duplication of a set of these two genes has been also observed in the human class III region (4). Thus, the tandem duplication of sequences spanning, at least and approximately, 55 kilobases (kb) of the DNA segment including the C4, Slp, and 21-OHase genes is a peculiar genetic configuration that is common to both the murine and the human class III region, although it is unclear whether duplication arose prior to the evolutionary differentiation of the two species or whether it arose independently after speciation (5).There are several lines of evidence that further duplication and deletion of genes in this DNA segment take place in the class III region, in both the human and the mouse. For example, Carroll et al. (6) observed human chromosomes with one, two, and three C4 loci. Furthermore, it has been reported (7, 8) that gene deletion occurs fairly frequently in human 21-OHase genes, which, in some cases, causes a congenital adrenal hyperplasia as a result of 21-OHase deficiency. Likewise, variations in the num...