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D C Shreffler

Publications and source records attributed to D C Shreffler.

At least 37 records · Page 2Linked to original sources
PubMed1985

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.

Animals↗
PubMed1984

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.

Alleles↗
PubMed1983

H-2-linked murine factor B phenotypes.

A hemolytic assay has been developed which is specific for Factor B (B) activity in murine EDTA-plasma. Three discrete levels of B activity were observed among B10-congenic strains. Mice with standard H-2 haplotypes, b, d, k, r, f, q, s, and u, all exhibited the same mean level of activity. However, plasma from H-2v (B10.SM) mice contained only 0.25 of that level, and those with standard haplotype H-2ja (B10.WB) or wild haplotype H-2wr7 (B10.WR) exhibited 2.5 times the H-2b (B10) basal level of activity. These differences among B10 congenic lines suggested that the activity is H-2 controlled; further tentative mapping with intra-H-2 recombinants indicated that the gene is located in the S region. A fourth phenotype was found among progeny of backcross generations between B10.BR (H-2k) and mice of subspecies Mus musculus molossinus and M. m. bactrianus. This ultra-high activity was found also to be governed by a gene very closely linked to Ss, the primary S region marker. F1 generations between disparate phenotypes yielded progeny with activity levels intermediate between the parents; progeny of parents of different strains with the same phenotype expressed B hemolytic titres equal to those of the parental strains. No differences in antigenic levels of the protein among the strains of different phenotypes could be detected by radial immunodiffusion. In mixing experiments, resultant activity levels were intermediate between the higher and the lower phenotype, ruling out independent inhibitors or activators of the reaction. These studies indicate that an H-2-linked S region-located single gene governs structural differences in allelic B molecules that lead to differences in specific activities.

Alleles↗
PubMed1983

cDNA clone spanning the alpha-gamma subunit junction in the precursor of the murine fourth complement component (C4).

cDNA clones carrying parts of murine fourth complement component (C4, serum substance protein) mRNA sequences have been identified by differential hybridization to mRNA from a high C4-producing strain, B10.WR, and a congeneic low C4 strain, B10.BR, followed by hybrid-selected translation and DNA sequence analysis. One clone, pMLC4/w7-2, encodes an open amino acid reading frame that includes four tandem arginine residues immediately preceding a sequence 85% homologous with the NH2-terminal sequence of the human C4 gamma-chain. The amino acid composition of the predicted sequence upstream of the tandem arginines matches quite closely with the composition of a similar sized peptide at the COOH terminus of the human C4 alpha chain. The latter result raises questions regarding the nature and extent of plasma-mediated postsynthetic processing of the C4 alpha-chain COOH terminus. The results also demonstrate that strain differences in plasma C4 levels (low C4 vs. high C4) reflect differences in steady-state levels of liver C4 mRNA in these strains.

Amino Acid Sequence↗
PubMed1983

Cultured hepatocytes from mouse strains expressing high and low levels of the fourth component of complement differ in rate of synthesis of the protein.

Primary hepatocyte cultures prepared from the livers of inbred strains of mice show different rates of synthesis for the fourth component of complement (C4). The relative amounts of C4 synthesized by hepatocyte cultures correspond to the relative plasma levels of C4 among strains, a trait determined by the S region of the H-2 major histocompatibility complex. There is no evidence for increased intracellular catabolism of C4 in hepatocyte cultures from a strain with a low level of plasma C4. Processing and secretion of C4 proceed very slowly in vitro relative to other hepatocyte proteins.

Animals↗
PubMed1983

Expression of cell surface antigens by suppressor T cell hybridomas. I. Comparison of phenotype and function.

The phenotypic expression of cell surface markers by T cell hybridomas that elaborate suppressor factors specific for the polymers L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) or L-glutamic acid50-L-tyrosine50 (GT) has been analyzed. We found that determinants encoded by the I-J subregion of the H-2 complex were borne on the surface of these hybrid cells and on the factors they secrete, whereas I-J determinants were not expressed by the AKR thymoma fusion parent, BW5147. The level of expression of I-J determinants fluctuated widely depending upon culture conditions, but I-J products and other cell surface markers of normal T cells could be quantitatively increased, or induced to appear, by treatment of the hybridomas with chemical agents, such as dimethyl sulfoxide (DMSO) or phorbol myristate acetate (PMA). In contrast, the surface expression of the viral product gp70 was decreased by the same treatment. Using chemical induction, we typed BW5147, a group of antigen-specific suppressor T cell hybridomas, and two control hybridomas for expression of I-J, Thy-1, Lyt, and H-2K alloantigens. Also, a haplotype-specific hybridoma that produces an antigen-nonspecific factor was analyzed. The results demonstrated that BW5147 failed to express I-J or Lyt alloantigens but expressed Thy-1.1 and H-2Kk gene products. The pattern of expression of these antigens by T cell hybridomas was very complex, but three conclusions could be drawn: 1) Good correlation exists between the expression of certain I-J determinants and the ability of T cell hybridomas to produce suppressor factor. 2) The expression of Thy-1, Lyt, or H-2Kk determinants is variable, and no correlation was found between expression of these antigens and the ability to produce active suppressor factors. 3) I-Jk products contributed by the AKR thymoma fusion partner are expressed by T cell hybridomas.

Absorption↗
PubMed1982

Genetic variation in glycosylation of the fourth component of murine complement. Association with hemolytic activity.

The molecular basis for variation in the Mr of the C4 alpha-chain of the fourth component of murine complement from several strains was investigated. All strains were capable of incorporating radiolabeled mannose into the alpha-chain of C4 from peritoneal macrophage cultures. In most cases, carbohydrate was found on both alpha-chain autolytic fragments produced by denaturation of the native C4. However, mice bearing the H-2 haplotypes w7, w16, and w19 produce C4 with no carbohydrate on the larger (COOH-terminal) autolytic fragment. This lack of carbohydrate is sufficient to cause the difference in Mr seen in the C4 alpha-chain from these mice. The chemical removal of all carbohydrate abrogated this difference. The levels of C4 antigen in the plasma of mice from these three strains are normal while the levels of hemolytic activity are reduced 60-80%. This suggests a strong role for carbohydrate in the functional properties of C4.

Animals↗
PubMed1982

Amino acid sequence homologies and glycosylation differences between the fourth component of murine complement and sex-limited protein.

Limited primary sequence data have been obtained for all three subunits of the fourth component of murine complement (C4) and its related homologue, the sex-limited protein (Slp). These data show a high degree of NH2-terminal homology between C4 and Slp: four of the six residues identified for the alpha chain, seven of eight for the beta chain, and four of four for the gamma chain. This suggests that apparent molecular weight differences between C4 and Slp subunits are not, as previously suggested, due to a shift in the proteolytic processing sites in the pro-Slp polypeptide molecule. Chemical deglycosylation (apparently complete) of the C4 and Slp alpha chains with trifluoromethanesulfonic acid removes the molecular weight difference between them, suggesting that acquisition of extra glycosylation sites in the latter is responsible for this difference.

Amino Acid Sequence↗
PubMed1982

Characterization of the Mr difference between secreted murine fourth component of complement and the major plasma form: evidence for carboxyl-terminal cleavage of the alpha chain.

The alpha-chain of murine fourth component of complement (C4) secreted by cells in vitro and in vivo has a Mr that is larger by approximately equal to 4,000 than that of the alpha-chain of the principal form of C4 in plasma. By using in vivo labeling of C4 with [35S]methionine, C4 was shown to be first synthesized with the higher Mr ("secreted") alpha-chain, which was then quickly processed (t1/2 approximately equal to 1 hr) extracellularly to the mature ("plasma") C4 possessing the lower Mr alpha-chain. Both forms of C4 were functional as assayed by the ability of their alpha-chains to be cleaved by the protease C1, to bind methylamine, and to undergo denaturation-dependent autolysis. When secreted C4 and plasma C4 were activated to C4b, the Mr difference of 4,000 was maintained in the alpha'-chains. The Mr difference was localized to the carboxyl-terminal autolytic fragment of the alpha-chain and was unaffected by the removal of carbohydrate. C4 from resident peritoneal macrophage cultures could be converted to the plasma form by incubation with heparin/plasma. This conversion could be blocked by EDTA or 1,10-phenanthroline. These data suggest that an enzyme, presumably a neutral proteinase present in mouse plasma, cleaves the carboxyl terminus of newly synthesized C4 alpha-chains, thereby creating the major form of C4 in plasma.

Animals↗
PubMed1982

Biosynthesis of C4 by mouse peritoneal macrophages. I. Characterization of an in vitro culture system and comparison of C4 synthesis by "low" vs "high" C4 strains.

C4 biosynthesis and secretion in mouse resident peritoneal macrophage cultures was examined by functional and antigenic analysis. The rate of secretion of functional and antigenic C4 decreased in parallel in short-term culture despite constant total protein secretion and increasing factor B secretion. Feedback inhibition and protease degradation did not account for the decreasing rate of C4 secretion. Despite a 10-fold difference in plasma C4 levels between high C4 (H-2d haplotype) and low C4 (H-2k haplotype) mouse strains, resident peritoneal macrophages from both strains synthesized and secreted similar amounts of antigenic and functional C4 in short-term culture. The C4 secreted by H-2d and H-2k macrophages was relatively stable, because it exhibited only approximately 2% loss of hemolytic activity per hour in culture. These data suggest that an unidentified environmental signal leads to the specific decrease in C4 synthesis and secretion by peritoneal macrophages and that a regulatory defect may not be present, at least in macrophages, to account for the low C4 levels in the H-2k haplotype.

Animals↗
PubMed1982

Differences in expression of MHC products between several H-2-restricted CTL clones.

Five cytotoxic T lymphocyte (CTL) clones, all specific for influenza virus A/JAP/57 and all H-2Kd-restricted, were typed for expression of MHC products. All express H-2K and H-2D encoded antigens, but they differ in their expression of I region products. One clone, 14-2, is positive with antisera directed against serologically detectable products apparently related to three different I regions, namely I-A, I-J, and I-E. Two clones, 11-1 and 14-1, lack I-E-associated antigens but express I-A and I-J products, and two other clones, 14-7 and 14-13, lack detectable I region products. These observations show that although discrete CTL clones may be homogeneous in their function, they differ in their expression of I region products.

Animals↗
PubMed1982

Structural and functional characterization of an incompletely processed form of murine C4 and Slp.

The properties of a 128,000-dalton polypeptide, corresponding to the uncleaved alpha- and gamma- chains of the fourth component of murine complement (C4), were studied. A fragment of similar size (137,000 daltons) from the structurally related sex-limited protein (Slp) was also found. These polypeptides have methylamine and iodoacetamide binding sites. suggesting that they, like the alpha-chains of C4 and Slp, possess an internal thiolester. In tryptic peptide map analysis, extensive homology is seen between the 128,000-dalton fragment and native C4 alpha- and gamma-chains. N-terminal sequences for this fragment and C4 alpha are identical, as are those for the 137,000-dalton Slp fragment and Slp alpha. Although the alpha-gamma fragment, like C4 alpha, undergoes denaturation-dependent autolytic cleavage, unlike C4 alpha, it cannot be cleaved by C1 This indicate that some of the properties of C4 do not require the native three-chain structure, whereas others do require it. These findings suggest that the alpha-gamma fragments represent an intermediate step in the processing of the C4 and Slp precursor polypeptides.

Animals↗