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.     

Restriction fragment length polymorphism of the murine C4 and Slp genes: two C4 groups.

We have examined the related H-2 genes coding for the fourth component of complement (C4) and the sex-limited protein (Slp) from 30 inbred mouse strains by Southern blot analysis. With four restriction enzymes, 11 RFLP patterns distributed among 26 different H-2 haplotypes have been identified. Strains of the same serologic H-2 haplotype were found to have identical RFLP patterns. It was confirmed that the number of C4-related genes in most haplotypes is two, Slp and C4; but H-2SWI6 (SWI6) and SWI9, which have the same RFLP pattern, have four and Sw7 has five. Although C4 and Slp have many similarities, they also were found to contain distinctive features: relative to Slp, each C4 allele examined has two insertions totaling 1.1 kb located in introns 14 and 15; and each Slp allele examined, excluding hybrids, has a provirus insertion upstream. No other large deletions or insertions were detected. The RFLP patterns are also due to 10 polymorphic restriction sites, which have been placed on standard maps; two are associated with Slp and eight are associated with C4.Sk strains, the only strains that express low serum levels of C4, have the same RFLP phenotype as Sw14, Sw18, and Swx; Sk may have arisen from a recent common ancestor of these strains. Homologous recombination has been important in the formation of existing C4 alleles. However, based on complete linkage disequilibrium between three RFLP internal to C4, the haplotypes have been divided into two groups that may have functional significance.     

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.     

Constitutive expression and induction of CYP1B1 mRNA in the mouse.

Previous studies appeared to indicate that CYP1B1 was not constitutively expressed in mouse liver. In our laboratory, we demonstrated using aromatic hydrocarbon-responsive receptor knock-out (AHR-(-)/-) mice that both piperonyl butoxide (PBO) and acenaphtyhlene (ACN) are AHR-independent inducers of murine CYP1A2 and CYP1B1 mRNA. In the current study, we demonstrate both constitutive levels and induction of CYP1B1 in mouse liver. The induction of CYP1B1 mRNA by PBO or ACN was higher in DBA/2 (Ahrd) than in C57BL/6 (Ahrb-1) mice, while 3-methylcholanthrene induced CYP1B1 more in C57BL/6 than in DBA/2 mice. These results suggest that CYP1B1 may also be induced by more than one mechanism. In addition, constitutive expression of CYP1B1 was detected in liver, kidney, and lung of untreated C57BL/6 mice. There was no gender difference in CYP1B1 expression; however, in C57BL/6 mice, the kidney contained less CYP1B1 than either liver or lung.     

C lambda 2 and C lambda 4 immunoglobulin light chain genes in a wild-derived inbred mouse strain

A genomic clone containing the lambda constant (C) region genes (C lambda 2S and C lambda 4S) has been isolated from a genomic library from the mouse strain SPE. SPE is an inbred strain derived from progenitors trapped near Grenada in Spain and has been classified as mus 3 or mus spretus. The sequence of the C lambda 2S gene is virtually identical to that of BALB/c both in the coding region and in flanking sequences, suggesting that it is an expressed gene in the SPE strain. By contrast, the C lambda 4S gene on the same cluster has diverged in sequence from that of BALB/c and contains a large deletion that precludes its normal expression. Whereas BALB/c J lambda 4 region contains substitutions that probably preclude its usage, the SPE J lambda 4 gene includes all sequences required for a functional J gene. Comparison of the C lambda 2S and C lambda 4S gene sequences with those available for BALB/c C lambda 3 and C lambda 1 confirms the close relationship between the C lambda 1-C lambda 4 and C lambda 2-C lambda 3 gene pairs. The C lambda 3 gene of BALB/c is more closely related to C lambda 2S than is C lambda 1 of BALB/c to C lambda 4S. If it is assumed that C lambda 1 and C lambda 2 are respective duplicates of C lambda 4 and C lambda 3 and that these duplications occurred at the same time, then the C lambda 2 gene has been under stronger selective pressure than C lambda 4.     

Analysis of regulatory sequences in androgen-responsive genes

We have analysed the effect of androgens on the activity of promoters from MMTV, and the rat prostate C3(1) and mouse secretory protease inhibitor genes. MMTV promoter activity was stimulated by testosterone as well as progesterone and dexamethasone but not by oestradiol. Deletion analysis indicated that the three steroids acted through DNA sequences between nucleotides -201 and -69 upstream of the MMTV cap site. In contrast, the promoters for the C3(1) gene and the protease inhibitor gene were unaffected by testosterone in a number of cell types, including prostate cells, despite the fact that the MMTV promoter was stimulated in such cells.     

异源表达afsRScla全局性调控基因提高工业菌株纳他霉素产量

前期研究表明棒状链霉菌的全局性调控基因afsRScla异源表达可以激活变铅青链霉菌中两种抗生素的合成。本研究将包含afsRScla基因的质粒pHL851整合到纳他霉素工业生产菌株褐黄孢链霉菌TZ1401的基因组中,使纳他霉素产量提高38%,达到3.56g/L。qRT-PCR检测6个纳他霉素生物合成基因的转录情况,发现其转录水平提高1.9-2.7倍,表明afsRScla通过正调控纳他霉素生物合成基因的转录,从而提高纳他霉素的产量。本研究结果对afsRScla在抗生素工业生产菌株的高产育种应用具有借鉴意义。     

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.     

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.     

Degradation of o-toluate by Pseudomonas sp. strain WR401

Abstract A Pseudomonas sp. strain WR401 was isolated for growth on 3-, 4-, and 5-methylsalicylate. The organism was capable of growth on o -toluate. The data on enzyme activities in cell-free extracts, DHB dehydrogenase and catechol 2,3-dioxygenase, as well as the cooxidation of the substrate analog 2-chlorobenzoate yielding 3-chlorocatechol indicated a pathway for o -toluate degradation through 6-methyldihydrodihydroxybenzoate, 3-methylcatechol and further through the meta -pathway. In contrast to other toluate dioxygenating enzymes found in m - and p -toluate degrading organisms, strain WR401 was able to dioxygenate a wider range of chlorobenzoates including 2-chlorobenzoate.     

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.     

High frequency expression of S107 VH genes by peritoneal B cells of B10.H-2aH-4bP/WTS mice

Our previous analyses of peritoneal Ly-1 B cells indicate that a high percentage express VH genes of the VH11 and VH12 families, and that this bias is due to clonal selection. The antibodies encoded by these genes bind the same hapten, phosphatidyl choline (PtC). Twenty-one of 73 hybridomas generated from fusions with peritoneal Ly-1 and Ly-1 sister population B cells of B10.H-2aH-4bp/Wts mice produce anti-PtC specific antibodies. We show here that 19 of these express VH11 and VH12 family genes and two express VH36-60 family genes. To assess whether there is a bias in VH gene use among non-PtC-specific hybridomas we analyzed the remaining 52 hybridomas for VH family expression by using VH family-specific probes in an RNA dot blot assay and by Ig mRNA sequencing. We find a seven-fold increase in the expression of the VHS107 family genes, and only slight differences in the expression of VH genes of other families relative to splenic B cells. We attribute the increase in VHS107 gene expression to clonal selection inasmuch as five of the seven VHS107+ hybridomas express the same VH gene (V11) and VL association is nonrandom. The bias in VH gene use among the entire panel of 73 peritoneal hybridomas is to the extent that approximately one-third express one of three genes: the V11 gene of the S107 family, the CH34 gene of the VH11 family, and the VH12 family gene.     

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.