Cloning,characterization and overproduction of nuclease S1 gene (nucS) from Aspergillus oryzae

The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced. It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in lenght. The nucS gene had an open-reading frame of 963 base pairs and coded for a protein of 287 amino acid residues, comprising the signal peptide of 20 amino acids and a mature protein of 267 amino acids. The deduced amino acid sequence agreed well with the published amino acid sequence except for one substitution. Southern hybridization analysis showed that the nucS gene existed as a single copy in the A. oryzae chromosome. When the structural gene of nucS was fused with the promoter of the glaA gene and introduced into A. oryzae, the yield od secreted nuclease S1 increased about 100-fold compared with the recipient strain.     

Lipase modulator protein (LimL) of Pseudomonas sp. strain 109.

Plasmids containing a Pseudomonas sp. strain 109 extracellular lipase gene (lipL) lacking NH2-terminal sequence and a lipase modulator gene (limL) lacking the NH2-terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system. Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein. During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL. The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1. After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex. Activity of the native lipase purified from Pseudomonas sp. strain 109 was also inhibited by rLimL. By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment. LimL was purified from Pseudomonas sp. strain 109, and the NH2-terminal amino acid sequence was determined to be NH2-Leu-Glu-Pro-Ser-Pro-Ala-Pro-. We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.     

Factors necessary for the export process of colicin E1 across cytoplasmic membrane of Escherichia coli

Factors necessary for the export process of colicin E1 across the cytoplasmic membrane of Escherichia coli were investigated. beta-Galactosidase activities from gene fusions between the colicin E1 and lacZ genes were recovered in the inner membrane fraction of E. coli when the region containing the internal signal-like sequence of colicin E1 [M. Yamada et al. (1982) Proc. Natl Acad. Sci. USA 79, 2827-2831] was present, but were found in the soluble fraction when the region was eliminated. The colicin E1 export was reduced upon insertion mutation in a gene that is located downstream from the colicin E1 gene in the same operon and responsible for mitomycin-C-induced killing of the host cell. A frame shift mutation of the colicin E1 plasmid was constructed to direct the protein which had lost the COOH-terminal 13 residues of original colicin E1 and was altered in 6 residues of the new COOH-terminal portion. The aberrant colicin E1 that was inducibly synthesized remained inside the cells. These results indicate that colicin E1 is exported with the aid of a product of the downstream gene and that the COOH-terminal portion is necessary for the export. The binding of colicin E1 to the cytoplasmic membrane through the internal signal-like sequence may be a step in the protein export process.     

Changes in intracellular pH and inorganic phosphate concentration during and after muscle contraction as studied by time-resolved 31P-NMR. Alkalinization by contraction

The morula and the mesenchyme blastula nuclei contained approx. 30 nuclear proteins which were preferentially released by limited digestion with DNase I, but no proteins were released from sperm nuclei. While most of the proteins released by DNase I digestion were common to the two embryonic stages, 2 and 6 proteins were specific or enriched in morulae and mesenchyme blastulae, respectively.     

Isolation and identification of 25-hydroxy-24-oxocholecalciferol: A metabolite of 25-hydroxycholecalciferol

A metabolite of 25-hydroxycholecalciferol has been isolated in pure form from chicken kidney homogenates. It has been identified as 25-hydroxy-24-oxocholecalciferol by means of ultraviolet absorption spectrophotometry, mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and specific chemical reactions.     

Isolation of an actin-binding fragment of fibronectin.

We have identified a specific actin-binding site in the adhesive glycoprotein fibronectin, isolated from chicken fibroblasts. Affinity chromatography of fragments, released from fibronectin by limited proteolysis with trypsin, chymotrypsin and subtilisin, on actin-Sepharose and other protein-Sepharose columns was used to locate the binding site. A 27 000-mol.wt. subtilisin-digest fragment bound efficiently to actin. The results suggest that the actin-binding site is close to, but not identical with, the reported collagen-binding site.     

Disseminated intravascular coagulopathy during experimental pneumococcal sepsis: studies in normal and asplenic rhesus monkeys.

Disseminated intravascular coagulation (DIC) was induced in both normal and asplenic rhesus monkeys by intravenous challenge with Streptococcus pneumoniae. Our observations in the infected monkeys have led us to conclude that (1) pneumococcal capsular polysaccharide (PCP), immune complexes and complement may not have primary roles in the initiation of DIC; (2) intact pneumococci may be catalysts for the development of DIC; (3) the initial event in DIC may be activation of Hageman factor; and (4) evidence of activation of Hageman factor-dependent systems is present regardless of severity of infection.     

Mechanism of the decrease in the major cell surface protein of chick embryo fibroblasts after transformation.

The major cell surface glycoprotein of cultured chick embryo fibroblasts (CSP, a LETS protein) is substantially decreased after neoplastic transformation. We investigated the regulation of this glycoprotein by determining the kinetics of CSP biosynthesis, transit to the cell surface, and degradation before and after transformation by Rous sarcoma virus. CSP synthesis, as measured by immunoprecipitation after pulse-labeling with ¹⁴C-leucine, is decreased 3–6 fold after transformation by the Bryan high titer, Schmidt-Ruppin and temperature-sensitive ts68 and T5 strains of Rous sarcoma virus. Steady state quantities of CSP in intracellular pools are also decreased 4–5 fold after transformation. However, the rate at which newly synthesized CSP is processed and exported to the cell surface is similar before and after transformation.Degradation and release of CSP from cells were measured after labeling for 24 hr. The half-life of CSP on normal cells is 36 hr and is decreased to 16–26 hr after transformation. The absolute amount of intact CSP released into the culture medium is decreased 3 fold after transformation; these amounts, however, represent losses of approximately 20 and 40% of the total CSP synthesized by normal and transformed cells, respectively. These results indicate that the major mechanism for the decrease in CSP after transformation is reduction in its biosynthesis, although increased degradation and loss from the cell surface also contribute significantly. These changes can account for the observed 5–6 fold decreases in cell-associated CSP after transformation of chick embryo fibroblasts.     

Isolation and immunological characterization of a glucose-regulated fibroblast cell surface glycoprotein and its nonglycosylated precursor.

We have isolated and immunochemically characterized a major membrane glycoprotein of mouse 3T3 cells. This GRP (glucose/glycosylation-regulated protein) is labeled by lactoperoxidase-mediated iodination and by ¹⁴C-glucosamine, binds concanavalin A and has an apparent molecular weight in SDS-polyacrylamide gels of 92,000 daltons (or 97,000 daltons in a discontinuous gel system). Glycosylated GRP was isolated from plasma membranes using Triton X-100 extraction, affinity chromatography on concanavalin A-Sepharose and preparative SDS gel electrophoresis.Antibody against this glycosylated GRP stains the external surfaces of mouse cells and induces patches and caps. Immunofluorescence and immunoprecipitation studies indicate that this glycoprotein can exist in the membrane in two molecular forms, either as a glycosylated or as a nonglycosylated protein. The nonglycosylated form is induced under conditions of limited glycosylation or glucose deprivation. This nonglycosylated GRP remains accessible to antibodies on the exterior of cells, but becomes inaccessible to lactoperoxidase.The immunoprecipitation of the 92K GRP with its specific antibody is always associated with the precipitation of a small fraction of the other major GRP of molecular weight 75,000 daltons. We suggest that both GRP (92K and 75K) may function in close association in the membrane.