Regulation of expression of mouseC4 andSlp genes by non-H-2-linked genes

C4 (the fourth complement component) and Slp (sexlimited protein) are two homologous plasma proteins encoded by genes in theS-region of theH-2 gene complex. We studied the genetic factors influencing the plasma levels of these proteins and their mRNA levels in liver. Considerable differences in both protein and mRNA levels were found between mouse strains carrying the sameS-region allele on different genetic backgrounds, indicating a pretranslational effect of non-H-2-linked genes on the expression of the twoS-region genes. The expression of Slp is androgen-dependent in the strains tested. However, testosterone treatment cannot increase the low levels of Slp caused by non-H-2-linked regulatory genes. In mice with Slp-negativeS-region alleles we found liver mRNA hybridizing with Slp-specific oligonucleotides, indicating expression of theSlp gene in Slp-negative strains. Our data demonstrate the complexity of the regulation of theC4 andSlp genes and pave the way for the analysis of the regulatory factors involved.     

Female expression of the H-2-linked sex-limited protein (Slp) due to non-H-2 genes

Female mice of 15 inbred strains in which males express the H-2-linked sex-limited protein (Slp) were tested for the production of this protein. Four inbred strains (FM, LG/J, NZB, PL/J) were found in which females produce Slp in the absence of hormonal manipulation. Crosses have been made between strains FM, NZB, or PL/J and several Slp-negative strains. Slp-typing of the F₁, F₂, and backcross progeny, as well as of a number of recombinant inbred strains, indicates that production of Slp by normal females of these strains depends upon the concurrent presence of an Slp-positive,H-t2-linked allele and of permissive alleles of one or two non-H-2 autosomal genes. Complementation studies with two of the strains (FM and PL) indicate that an identical genetic mechanism mediates expression of Slp in females of these two strains. FM-derived animals carrying the testicular feminization mutation (tfm) also express Slp, as do castrated NZB mice, indicating that Slp expression in these instances is not dependent upon testosterone as it is in other inbred strains. It is concluded from these results that genes distinct from the putative structural gene for Slp influence the sex-limitation of its expression.     

Three extra copies of a C4-related gene in H-2 w7 mice are C4/Slp hybrid genes generated by multiple recombinational events

Mice bearing the H-2 _w7 haplotype have five C4-related genes and constitutively express the Slp antigen. To understand the structure and evolution of the five C4-related genes of the C3H.W7 mouse, we have determined nucleotide sequences of the 5 end region of these genes. A C4/Slp hybrid nature was confirmed for three of five C4-related genes as predicted previously by restriction enzyme analysis. The nucleotide sequences of the 5 flanking regions of these three hybrid genes showed close similarity to that of the C4 gene, while the 3 side of the ninth exon of the three hybrid genes showed close similarity to that of the Slp gene. In contrast, the regions between the first exon and the middle of the ninth exon of the three hybrid genes showed a mosaic structure of C4-like and Slp-like sequences. Moreover, the boundaries of the C4-like and Slp-like sequences were quite different among the three hybrid genes. The pattern of nucleotide sequence diversity in this region among the five C4-related sequences could be mainly explained not by point mutations but by gene conversions or unequal crossovers. These results suggest that multiple genetic recombinational events between two homologous sequences played an important role in the generation and diversification of the extra copies of the C4/Slp gene in the H-2 ^w7 mouse.The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and have been assigned the accession numbers D90167-71.     

Murine sex-limited protein expression requires androgens and pituitary hormones

Levels of the murine sex-limited protein, (Slp) were measured by an enzyme-linked immunosorbent assay in normal and hypophysectomized female CDF1 (Slp ^a) mice before and after a 15-day treatment with testosterone proprionate. Both groups of mice initially had undetectable levels of circulating Slp. After treatment, Slp serum levels of the nonhypophysectomized group had risen significantly above the Slp serum levels of the hypophysectomized group and the pretreatment controls. This indicates that the pituitary gland is necessary for the androgen-induced expression of Slp.     

A sex-limited serum protein variant in the mouse: Inheritance and association with the H-2 region

An alloantiserum produced in the mouse has been used to detect an antigen which is present only in male serum from certain inbred strains of mice, e.g., DBA/2J, A/J, and BALB/c. Genetic tests reveal that the presence of this antigen is controlled by a dominant autosomal gene which is expressed only in males of the proper genotype. Test crosses and analysis of congenic resistant strains indicate close linkage between the sex-limited protein (Slp) and the histocompatibility-2 (H-2) region of linkage group IX. Analysis of seven intra-H-2 recombinant strains is consistent with the placement of the genetic determinant for Slp within the H-2 region in the same position as the Ss (serum substance) determinant. Immunological evidence suggests that the Slp antigenic sites reflect structural variation in the Ss component of mouse serum.Supported by U.S.P.H.S. Research Grant GM-15419, U.S.P.H.S. Career Development Award K3-HE-24, 980 (D.C.S.), and U.S.P.H.S. Training Grant 2T01-GM-00071 (H.C.P.).     

L3T4 but not LFA-1 participates in antigen presentation by Ak-positive L-Cell transformants

Genomic DNA was isolated from 29 t strains and 4 congenic lines of mice, digested with restriction endonucleases, and hybridized with a probe representing the complement component 4 (C4) gene. All but one of the enzymes revealed restriction fragment length polymorphism in this sample of C4-related genes. Double digestion analysis suggested the presence of three C4 gene copies in some of the t chromosomes and two copies in others. The enzymes distinguished 16 different haplotypes among the 33 strains tested. Based on their restriction fragment length patterns, the t strains could be divided into four groups with strains in each group more closely related to each other with respect to their C4-region genes than strains belonging to different groups. At least three of these four groups represent different branches of the evolutionary tree constructed for the t chromosomes. The C4-related genes of the chromosomes are in strong linkage disequilibrium with the class II genes of the H-2 complex. Typing for the Ss and Slp allotypes of C4 has revealed the presence of the Ss¹ phenotype in two t strains and of the Slp^a phenotype in one strain.     

Genetic Association between H-2 Gene and Testosterone Metabolism in Mice

SEVERAL characters involved in sexual dimorphism or male reproductive performance are influenced by genetic factors that are linked with the histocompatibility-2 (H-2) system of the mouse. These factors influence sperm cell production and function^1–4 interstrain differences in relative weights of vesicular gland and testis^4,5, immune response to the male-specific histocompatibility antigen^6,7 and an androgen-dependent allotypic serum protein designated Slp⁸. Our finding of an H-2 linked gene influencing the size of such male hormone-dependent organs, as is the vesicular gland and testis, suggested that the amount of testosterone in plasma may be influenced by an H-2 linked gene. Whereas the genetic control of some hormonally determined traits is considered to be polygenic^9,10, other data indicate some endocrine variation is due to allelic substitution at a single locus or very few loci^11–14. These genes in the mouse genome have not yet been located.     

Meiotic recombination within the H-2K-H-2D interval: characterization of a panel of congenic mice,including 12 new strains,using DNA markers

Intra-H-2 recombinant congenic strains are widely used to localize traits to specific subregions of the major histocompatibility complex and have provided evidence for the existence of meiotic recombinational hotspots in mammals. Forty-seven intra-H-2 recombinant strains, including 12 not previously reported, have been identified by serological typing in our laboratory. We have extended the analysis of the cross-over sites in these mice using DNA markers for Ab, Aa, Eb, Ea, Cyp21-ps, D17Tu3, Bat7, and Bat5. The recombinant chromosomes of these congenic strains include loci derived from the a, b, f, k, p, q, r, s, u, and v haplotypes of H-2, providing a diverse panel of strains. Although some alleles of Bat7 could not be distinguished from one another, results from the majority of strains indicated a probable gene order of C4Slp/D17Tu3-Bat7-Bat5-H-2D. No recombinants between Cyp21-ps, C4Slp, and D17Tu3 were observed. The crossover sites in 31 of the 47 intra-H-2 recombinants were within the C4Slp/D17Tu3—H-2D interval; of these 31 crossovers, three were bracketed by D17Tu3 and Bat7, ten by Bat7 and Bat5, seven by Bat5 and H-2D, and 11 by D17Tu3 and Bat5. The results from all 47 strains suggest recombinational hotspots within the C4Slp/D17Tu3—H-2D interval and emphasize the influence that specific haplotypes can have on preferred crossover sites. Correspondence to: G. A. Carlson.     

Immune response to the Slp alloantigen in the mouse:H-2-linked qualitative and quantitative control

Immunization of inbred mouse strains lacking the Slp allotype results in the production of Slp antibodies in some strains but elicits no detectable response in other strains. Analysis of standard inbred and congenic resistant strains reveals that both the qualitative and quantitative ability to respond to the Slp allotype is associated with theH-2 haplotype of the recipient. Three different response phenotypes can be identified utilizing complement fixation and quantitative immunodiffusion tests. Strains which carry theH-2 ^q haplotype are high responders,H-2 ^k strains are intermediate in response, whileH-2 ^b andH-2 ^v strains produce no detectable antibody. The characteristic response patterns of high and intermediate responders were manifest by day 35 of immunization and continued as discrete response types after a second booster. Quantitative data in the immune response of the intra-H-2 recombinant B10.A(4R) suggest that the recombination event which established theH-2 ^h4 chromosome disturbs the proper function of the genetic determinant controlling response to Slp.     

Sequence comparison of alleles of the fourth component of complement (C4) and sex-limited protein (Slp).

cDNA clones specific for the fourth component of complement (C4) and its androgen-regulated isotype, sex-limited protein (Slp), have been isolated from two mouse haplotypes (H-2d and H-2w7) that show differential C4 activity and differential regulation of Slp. Clones were first isolated using a cDNA probe enriched by subtractive hybridization. Subsequent screening has resulted in cDNAs spanning the entire C4d mRNA, as well as much of C4w7, Slpw7 and a short region of Slpd. The cDNAs for C4 and Slp show extensive sequence homology, but can be distinguished using oligonucleotide probes synthesized to regions of greatest sequence divergence. Sequence differences between C4 and Slp indicate structurally important features of C4 that have been altered in Slp such that Slp is unable to participate in the complement pathway. Of the few nucleotide differences between C4d and C4w7, a single base change resulting in one less glycosylation site in the C4w7 alpha chain could account for its 4-fold reduced hemolytic efficiency. Sequence comparison of multiple alleles of C4 and Slp indicates that possible gene conversion events occurred in the H-2w7 strain that has multiple Slp genes.     

Recombination of two homologous MHC class III genes of the mouse (C4 and Slp) that accounts for the loss of testosterone dependence of sex-limited protein expression

In most mouse strains, expression of a gene encoding sex-limited protein (Slp), an isotype of the fourth component of complement (C4), is induced by testosterone, or the gene is not expressed at all; however, in some wild-derived strains carrying H-2w7, H-2w16, or H-2w19 haplotype, Slp is expressed constitutively in the same way as C4. To examine the structural basis for the testosterone-independent expression of Slp, 41 overlapping clones together encoding the S region were isolated from C3H.W7 mouse (H-2w7) cosmid library. Five C4-related genes each spanning approximately 16 kb were identified among the cluster of cosmid clones and were isolated for structural study. One of the genes (C4w7) hybridized with the C4-specific oligonucleotide probe but not with the Slp-specific oligonucleotide probe, whereas the other genes (Slpw7a, Slpw7b, Slpw7c, and Slpw7d) hybridized only with the Slp-specific probe. Restriction mapping of these genes and sequencing of the selected regions of 5'-flanking regions of the genes were performed, and the results were compared with the data obtained with the C4 and Slp genes of FM (H-2d) and B10.BR (H-2k). These studies showed that three of the C4-related genes of C3H.W7 (Slpw7b, Slpw7c, and Slpw7d) are C4-Slp recombinant genes comprising a 5'-region derived from C4 gene and a 3'-region derived from Slp gene. It is suggested that 5'-flanking region derived from C4 in these C4-Slp recombinant genes accounts for testosterone-independent expression of Slp in C3H.W7 mouse.     

Mice constitutive for sex-limited protein (SLP) expression contain multiple Slp gene sequences

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins whose structural genes map to the S region of H-2. Serum C4 levels vary as much as 20-fold between C4 high (C4H) and C4 low (C4L) strains, and Slp expression can be null (SlpO), limited to male mice of a subset of C4H strains (Slp+), or "constitutive" (SlpC), in which female as well as male mice express Slp. In this study, we compare, by genomic Southern blot analysis, the C4 and Slp genes from eight congenic inbred mouse strains representative of three distinct phenotypes: C4H, Slp+ (two strains), C4H, SlpO (two strains), C4H, SlpC (three strains), and C4L, SlpO (one strain). By using cDNA probes that recognize both C4 and Slp genes, and are derived from the extreme 5' and 3' ends of the mRNA as well as internal coding sequences, we find no evidence to suggest that strain-specific variations in the expression of C4 and Slp are due to gross deletions of major portions of the structural genes. In most cases, two distinct C4/Slp genes are detected; hybridization with C4- and Slp-specific probes indicate that one of these is C4 and the other is Slp. The three SlpC strains are exceptional: they carry at least four C4/Slp genes; one of these hybridizes to the C4-specific probe whereas the remaining genes hybridize to the Slp-specific probe. Hence, multiple duplication of a gene containing Slp sequences has occurred in certain strains of mice, and this is accompanied by constitutive expression of the Slp protein.     

The C4 and Slp genes of the complement region of the murine H-2 major histocompatibility complex

Recent analyses, at the protein and DNA levels of structure, of the murine complement components C4 and the closely related sex-limited protein, Slp have led to new insights into the H-2/S region-linked C4 and Slp genes and their products. The primary products are 200 000 Da precursors which are cleaved, intracellularly and extracellularly, into the the mature alpha-beta-gamma-subunit molecules of plasma. Precursor order of subunits is beta-alpha-gamma; a complementary DNA clone spanning the alpha-gamma junction has been extensively analysed. The C-terminal of the alpha-chain is of particular interest because of post-secretion processing which differentiates 'secreted' and 'plasma' forms of C4, both apparently functional, and because allelic variants of C4 and the Slp protein, which differ substantially in molecular masses, owe their differences principally to different levels of glycosylation of the alpha-chain. Allelic variations in rate of C4 synthesis (C4-high compared with C4-low) have been analysed in cultures of hepatocytes and macrophages. Three distinct modes of genetic regulation of the expression of the Slp protein have been identified.     

Genetic analysis of the non-H-2-linked Ir genes controlling the cytotoxic T-cell response to H-Y in H-2 d mice

The T-cell mediated immune responses to the male specific minor histocompatibility antigen H-Y in mice have been studied extensively as a model for immune responses to other weak antigens like tumor antigens or autoantigens. In a recent analysis of the strain distribution of the cytotoxic T-cell (Tc-cell) responsiveness to H-Y, it has been found that genes both within and outside the H-2 complex exert an interactive control. Whereas the H-2 ^b strains all are high responders, independent of their non-H-2 background, other H-2 haplotypes (d, k, and s) only allow for a response if they are combined with certain non-H-2 genes. The H-2-linked immune response genes (Ir-genes) have been previously mapped to the I and K or D region of the H-2 complex, but the mapping of the non-H-2 genes has not yet been established. In this study evidence is presented, using recombinant inbred strains and immunoglobulin heavy chain (Igh) congenic strains of mice, to show that there is more than one non-H-2 Ir-gene involved, that the main controlling genes are not linked to the Igh complex, and that at least one non-H-2 Ir-gene is linked to the H-3 region on chromosome 2. This region includes genes for beta-2-microglobulin (2m), the Ly-mllalloantigen a polymorphic cell surface glycoprotein (Pgp-1), a B-cell specific antigen Ly-4, a transplantation antigen H-3, and genes (Ir-2) controlling the immune response to Ea-1 and H-13.     

Tissue-specific variation in C4 and Slp gene regulation.

C4 and Slp are highly homologous mouse genes that differ in function and regulation. Allelic variants exist in quantitative regulation of C4 and in hormonal regulation of Slp. We have examined expression in several tissues, including liver and peritoneal macrophages which are the major sites of synthesis, using a probe that allows direct comparison of C4 and Slp mRNAs. Correctly-sized and initiated RNA, within an order of magnitude of liver levels, is found in mammary gland, lung, spleen, and kidney; lower levels are detectable in testis, brain, heart and submaxillary gland. By comparing expression in congenic mouse strains differing in C4 and Slp loci, regulation of these genes is seen to vary in different tissues. This provides a well-defined genetic system in which to examine cis-acting sequences and trans-acting factors that result in tissue-specific patterns of gene regulation.