Restriction fragment length polymorphism at the HLA-C locus

We have used the HLA-C-specific DNA probe pC250 to investigate restriction fragment length polymorphism (RFLP) at the HLA-C locus. Genomic Southern blot hybridization included DNA prepared from a panel of homozygous typing cells representing serological specificities Cw1 to Cw8 and also from samples representing Cw blanks. Although many restriction nucleases failed to reveal any polymorphism, RFLPs were evident with Taq I, Pvu II, Bst XI, Nde 1, and Nci I in addition to the previously reported Eco RI. In the case of Bst XI, a unique RFLP defined a subset of serologically defined Cw blanks. Comparison of RFLP sizes with restriction fragment lengths obtained from the known HLA-Cw3 gene sequence permitted the localization of intragenic C locus RFLLs and the identification of a variable Taq I site in the second intron, a variable Nci I site near the end of the fourth exon, and a variable Pvu lI site in the fifth intron.     

Effect of dietary copper sulfate, Aureo SP250, or clinoptilolite on ureolytic bacteria found in the pig large intestine

The predominant ureolytic bacteria in the pig large intestine were determined while growing pigs were fed a basal diet or basal diet plus copper sulfate, Aureo SP250, or clinoptilolite. Fecal samples were collected from four pigs fed each diet at 3, 9, and 14 weeks and analyzed for total colony counts and percent ureolytic bacteria. Fecal urease activity, ammonia nitrogen, and identity of the ureolytic bacteria were determined at 14 weeks. Copper sulfate and Aureo SP250 reduced the number of ureolytic organisms, with a marked decrease occurring in the Streptococcus spp., which made up 74% of the ureolytic isolates from the pigs on the basal diet. Other ureolytic species detected at lower concentrations were Staphylococcus spp., Selenomonas ruminantium, Bacteroides multiacidus, and Eubacterium limosum. Copper sulfate also reduced fecal urease activity (P less than 0.10). Fecal ammonia concentrations were not different between pigs fed the various diets. These data suggest that the streptococci are the most numerous ureolytic species in the pig intestinal tract and are significantly reduced by copper sulfate and Aureo SP250; however, only copper sulfate reduced intestinal urease activity.     

Characterization of a class of small auxin-inducible soybean polyadenylated RNAs

Four new auxin-responsive RNAs from soybean (Glycine max (L.) Merr., var. Wayne) are described. The RNAs were identified by hybridization to three cDNA probes obtained from a library enriched for sequences which increase in abundance within 60 min after 2,4-D (2,4-dichlorophenoxyacetic acid) treatment. These RNAs appear to define a new class of small (i.e. approximately 550 nucleotides) RNAs that respond extremely rapidly to application of exogenous auxin. In excised elongating hypocotyl sections, an increase in the abundance of these RNAs can be detected 2 to 5 min after treatment with 50 M 2,4-D. This response is half maximal after 10 min and reaches steady state in 60 min. RNA blot analysis shows that these RNAs are expressed differentially in various parts of the seedling. The degree of inducibility by auxin is also organ-specific, with the elongating hypocotyl being the most responsive of the organs tested. The RNAs display identical response specificities with one exception. Accumulation of one RNA, designated 10A, is completely abolished by simultaneous addition of cycloheximide and 2,4-D. This RNA also displays a different 2,4-D dose response than other RNAs examined. These results suggest that more than one mechanism is involved in rapid modulation of gene expression by auxin.     

Interleukin-1 (IL-1) depresses cytochrome P450 levels and activities in mice

Endotoxin depresses cytochrome P450 levels when injected into animals. The purpose of this study was to determine whether endotoxin itself, or monokine(s) released in response to endotoxin administration are responsible for this effect. Cytochrome P450 levels and drug metabolizing activities were measured in endotoxin resistant C3H/HeJ mice 24h after single intraperitoneal injections of either lipopolysaccharide (LPS), a semipurified murine monokine preparation containing interleukin-1 (IL-1), or murine recombinant IL-1. In endotoxin sensitive C3H/HeN mice, LPS (0.5 mg/Kg) decreased total cytochrome P450 levels, benzphetamine demethylase activities, and ethoxyresorufin-0-deethylase activities. This dose of LPS did not alter cytochrome P450 levels or activities in the C3H/HeJ mice. However, after injection of the semipurified monokine preparation or the recombinant IL-1, there were significant decreases in cytochrome P450 levels and activities similar to the decreases observed with LPS in the C3H/HeN mice. These findings suggest that the alterations in hepatic cytochrome P450 seen with endotoxin injection are mediated, at least in part, by IL-1.     

Dsi-1, a region with frequent proviral insertions in Moloney murine leukemia virus-induced rat thymomas.

Dsi-1 is a region of chromosomal DNA that underwent proviral insertion in 3 of 24 Moloney murine leukemia virus-induced rat thymomas. In one of these tumors, a provirus is also integrated adjacent to the proto-oncogene c-myc. The proviruses in Dsi-1 have been characterized and appear to be complete. The proviruses were located within a 2-kilobase region that contained four prominent DNase I-hypersensitive sites. These hypersensitive sites were observed in Moloney murine leukemia virus-induced thymomas but not in NRK cells. The region of Dsi-1 immediately 3' to the insertions cross-hybridized with human and chicken DNA, indicating that it contains highly conserved sequences. No evidence could be found for the expression of this highly conserved region. Dsi-1 was mapped to mouse chromosome 4. This location demonstrates that Dsi-1 is different from 16 of the known proto-oncogenes (c-abl, c-erbA c-erbB, c-ets-1, c-ets-2, c-fes, c-fos, c-myb, c-myc, c-raf, A-raf, c-Ha-ras, c-Ki-ras, N-ras, c-sis, and c-src) and 12 cellular regions of tumor-associated integrations in retrovirus-induced tumors (c-erbB, Fis-1, int-1, int-2, Mis-1/pvt-1, Mlvi-1, Mlvi-2, c-mos, c-myb, c-myc, Pim-1, and c-Ha-ras). Hybridization experiments indicated that Dsi-1 is probably different from five additional proto-oncogenes (c-fgr, c-fms, c-mos, neu, and c-yes) and from two additional frequent integration regions (lck and Mlvi-3).     

Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane.

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the 'cold' synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.     

Sequence of the sites phosphorylated by protein kinase C in the smooth muscle myosin light chain

We have determined the sequence of the sites phosphorylated by protein kinase C in the turkey gizzard smooth muscle myosin light chain. In contrast to previous work (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072), two-dimensional tryptic peptide maps of both heavy meromyosin and the isolated myosin light chain showed two major phosphopeptides, one containing phosphoserine and the other phosphothreonine. We have purified the succinylated tryptic phosphopeptides using reverse phase and DEAE high pressure liquid chromatography. The serine-containing peptide, residues 1-4 (Ac-SSKR), is the NH2-terminal peptide. The phosphorylated serine residue may be either serine 1 or serine 2. The threonine-containing peptide, residues 5-16, yielded the sequence AKAKTTKKRPQR. Analysis of the yields and radioactivity of the products from automated Edman degradation showed that threonine 9 is the phosphorylation site.