The murine Slp gene. Additional evidence that sex-limited protein has no biologic function

Sex-limited protein (Slp) is a mouse serum protein of unknown function that has approximately 95% amino acid sequence identity with murine complement component C4 but is inactive in the complement pathway. The gene for Slp lies in the S region of the murine H-2 complex adjacent to the gene Cyp21 that encodes the Cytochrome P-450 enzyme steroid 21-hydroxylase. We report the sequence of a 26,307 bp long segment of the mouse genome that includes both the Slp and Cyp21 genes. The sequence reported was assembled from the sequences of three overlapping lambda phage genomic clones from mouse strain B10.WR, which carries four tandem pairs of Slp and Cyp21 genes. We also report the sequence of a fourth lambda clone, 12,539 bp in length, carrying parts of a distinct pair of Slp and Cyp21 genes from B10.WR mice. The Slp gene at 14.3 kb in length is about 1 kb shorter than the C4 gene; this difference is due primarily to absences of a simple repetitive sequence and a middle repetitive MT element in the corresponding introns 14 and 15, respectively. The gene sequence reveals an intron/exon organization identical to that of the murine C4 gene, and also that the 9 nucleotide deletion in exon 18, which appears to be directly responsible for the absence of complement activity, is unrelated to differences in intron sequences. Detailed comparisons of C4 and Slp gene sequences indicate that nucleotide substitutions in the Slp gene are occurring at approximately the same rate in both exons and introns. This implies that the murine Slp gene resembles a pseudogene and supports previously reported evidence that the Slp protein has no biologic function.     

Constitutive expression of Slp genes in mouse strain B10.WR directed by C4 regulatory sequences

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins that exhibit very disparate patterns of gene expression: all mice constitutively express C4, whereas only adult male mice from a limited number of standard inbred strains express Slp. Several exceptional strains exhibit constitutive (C4-like) Slp expression, a phenotype that correlates with multiple copies of the Slp gene. To determine the molecular basis for constitutive Slp expression we have isolated genomic clones and compared the sequences of 1.5 kb of 5' flanking DNA from 1 C4 gene and three different Slp genes from the Slp-constitutive strain B10.WR. These sequence comparisons demonstrate C4-like regulatory sequences adjacent to two of the Slp genes. By analysis of cDNA clones isolated from a B10.WR liver library we demonstrate that the constitutive Slp phenotype is due primarily to expression of one of these C4/Slp hybrid genes. It appears likely that Slp gene duplication in strain B10.WR came about via homologous unequal crossover events between C4 and Slp genes; this would accommodate both the gene sequence data and the pattern of C4-like Slp expression in mouse strain B10.WR.     

Multiple C4/Slp genes distinguished by expression after transfection.

The S region of the murine major histocompatibility complex contains two closely related genes: C4, encoding the fourth component of complement, and Slp, encoding sex-limited protein. We cloned these genes from a cosmid library of the B10.W7R strain that does not show androgen regulation of the Slp protein. Restriction site polymorphisms revealed at least four C4-like genes within the Sw7 locus, indicating evolutionary amplification of this region. Transfection of these genes into L cells resulted in expression, processing, and secretion of immunologically correct C4 and Slp proteins. At least two different Slp genes and one C4 gene were capable, after transfection, of expressing C4 and Slp indistinguishable from macrophage-derived protein. A third Slp gene exists within this locus whose recombinant cognate did not express in L cells. Thus, the B10.W7R S region includes one C4 gene and at least three Slp-like genes.     

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.     

Primary structure of the fifth component of murine complement

A cDNA library was constructed by the method of Okayama and Berg, [Okayama, H., & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289] employing size-selected (greater than 28 S) poly(A+) liver RNA from the mouse strain B10.WR. A total of 150,000 recombinants were screened with a partial human C5 cDNA probe; 16 C5-positive clones were identified, 1 of which contained an insert greater than 5.2 kilobase pairs in length. This cDNA insert was fully sequenced by the dideoxy method. The DNA sequence of this insert had an open reading frame of 4920 base pairs specifying a sequence of 1640 amino acid residues. The region corresponding to positions 372-812 exhibited high homology with the previously determined partial structure for human C5 of 438 amino acid residues. A four-residue basic sequence (Arg-Ser-Lys-Arg) was identified upstream of the amino-terminal Asn of C5a, thereby specifying a beta alpha-chain orientation for the promolecule form of murine C5. The 3' end of this clone contained 351 base pairs of untranslated sequence. The presumed polyadenylation recognition site CATAAA was located 17 base pairs upstream of the poly(A) tail. Comparison of the derived murine C5 sequence with previously determined structures for murine C3 and C4 revealed regions of high sequence similarity, including the thiol ester region present in C3 and C4. The cysteine and proximal glutamine which give rise to the intramolecular thiol ester bond in C3 and C4 were absent in C5, having been replaced by serine and alanine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Complete nucleotide and derived amino acid sequences of sex-limited protein (Slp), nonfunctional isotype of the fourth component of mouse complement (C4)

The nucleotide sequence coding for sex-limited protein (Slp), the testosterone-regulated isotype of the fourth component of mouse complement (C4), has been determined from cloned genomic DNA and cDNA fragments. The complete deduced amino acid sequence for the single chain precursor protein of Slp (pro-Slp) consists of 1716 residues. The mature beta, alpha, and gamma subunits contain 654, 763, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted from the beta-chain, five for the alpha-chain, and none for the gamma-chain. From the comparison with the mouse C4 sequences, an extensive overall sequence homology, 96.0% in nucleotides and 94.2% in amino acids, is observed. Only one deletion/insertion event is recognized between C4 and Slp sequences: three residues near the Cls cleavage site are deleted from Slp. The distribution of cysteine residues is completely conserved between pro-Slp and pro-C4.     

An ancient provirus has imposed androgen regulation on the adjacent mouse sex-limited protein gene

The mouse sex-limited protein (Slp) gene is dependent on androgen for expression, unlike its homologous neighbor, which encodes the fourth component of complement (C4). We have found that the extensive identity of Slp and C4 is disrupted by an endogenous provirus inserted 2 kb upstream of Slp. The 5' LTR of this element corresponds to the previously characterized hormone-responsive enhancer associated with Slp regulation, leading to the conclusion that the provirus has conferred androgen response on the adjacent Slp gene. The provirus is extremely old, based on LTR sequence divergence, the accumulation of mutations in former retroviral-like coding regions, and its stability within the mouse genome. The association of this transposable element with Slp regulation thus provides a long-sought example of an insertional mutation that has been maintained in evolution.     

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.     

The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.     

Murine sex-limited protein (Slp) antigenic sites in human complement component C4

Human complement component C4 is encoded by two HLA-linked loci, A and B. In the mouse, the H-2 region contains structural genes for two serum proteins that react with antibodies to human C4, but one of these proteins (Slp) has no C4 hemolytic activity. Because the product of C4-A locus in man has low hemolytic activity, a previous report suggests it may be the homologue of murine Slp. We show here that Slp antigenic determinants are found in human C4. However, they are expressed in the products of both loci A and B, that is, C4A and C4B, since both proteins were specifically immunoprecipitated by the IgG fraction of alloantisera to mouse Slp. Therefore, Slp-associated structural features are preserved in evolution, although they do not seem to be relevant to the hemolytic properties of C4.     

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.     

Characterization of a mouse interferon gene locus I. Isolation of a cluster of four alpha interferon genes.

A BALB/c mouse genomic library was screened with a murine interferon alpha 2 (MuIFN-alpha 2) cDNA coding region fragment. Eight clones were isolated which contain different mouse chromosomal segments related to the MuIFN-alpha 2 probe and a 28 kilobase (kb) region of mouse genomic DNA containing four different MuIFN-alpha genes (alpha 1, alpha 4, alpha 5 and alpha 6) was identified and characterized; an intergenic 1000 nucleotide long conserved sequence was found to be associated with three of these four alpha genes, indicating that this alpha-IFN gene cluster evolved through tandem duplications. Sequence analysis revealed the absence of a polyadenylation site in the 3' untranslated region of MuIFN-alpha 1, and showed that one of the genes (alpha 4) contains an internal deletion of 5 amino acids in the coding region.