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31.
Cloning of the alpha-amylase cDNA of Aspergillus shirousamii and its expression in Saccharomyces cerevisiae. 总被引:2,自引:0,他引:2
I Shibuya G Tamura T Ishikawa S Hara 《Bioscience, biotechnology, and biochemistry》1992,56(2):174-179
alpha-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined. The alpha-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active alpha-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii. 相似文献
32.
K Tsuchiya S Tada K Gomi K Kitamoto C Kumagai G Tamura 《Bioscience, biotechnology, and biochemistry》1992,56(11):1849-1853
The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene. 相似文献
33.
Setsuzo Tada Katsuya Gomi Katsuhiko Kitamoto Kojiro Takahashi Gakuzo Tamura Shodo Hara 《Molecular & general genetics : MGG》1991,229(2):301-306
Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter. 相似文献
34.
Kazuhiro Yoshikawa Masao Seto Ryuzo Ueda Yuichi Obata Kunihiro Notake Takashi Yokochi Toshitada Takahashi 《Immunogenetics》1991,33(5-6):352-360
The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites — 122 base pairs (bp) to — 38 bp from ATG translation initiation site were demonstrated. The promoter region had high G+C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs. 相似文献
35.
The possession of a respiration-dependent primary sodium pump and the requirement of Na for growth were investigated in bacterial isolates from marine environments. The bacteria in which NADH oxidase specifically required Na for maximum activity were believed to possess a primary sodium pump. All bacteria that failed to grow without the addition of NaCl possessed a primary Na pump. All bacteria that had no primary Na pump grew without additional NaCl. The primary Na pump seems to be involved in the Na requirement of marine bacteria, and this can be regarded as a criterion for the definition of marine bacteria. 相似文献
36.
Masahiko Sone Kazuhito Totsune Kazuhiro Takahashi Makoto Ohneda Keiichi Itoi Osamu Murakami Kaoru Yoshinaga Toraichi Mouri 《Peptides》1991,12(6):1333-1335
The presence of immunoreactive porcine brain natriuretic peptide in rat tissues was studied with a specific radioimmunoassay for porcine brain natriuretic peptide-26. The cross-reactivity of the antiserum used was less than 0.001% with rat atrial natriuretic peptide, rat brain natriuretic peptide-32 and rat brain natriuretic peptide-45. Immunoreactive porcine brain natriuretic peptide was detectable in various tissues of the rat, and high concentrations of immunoreactive porcine brain natriuretic peptide were found in the brain and cardiac atrium, with the highest level in the hypothalamus (159±30 fmol/gram wet tissue, mean±SEM, n=4). Reverse phase high performance liquid chromatography showed that the immunoreactive porcine brain natriuretic peptide of the whole brain and heart extracts eluted mainly at an identical position to synthetic porcine brain natriuretic peptide-26. These findings indicate that porcine brain natriuretic peptide-like substance, distinct from rat brain natriuretic peptide, is present in high concentrations in the rat brain and cardiac atrium. 相似文献
37.
A repeating unit of the DYZ1 family on the human Y chromosome consists of segments with partial male-specificity 总被引:1,自引:0,他引:1
Y Nakagome S Nagafuchi S Seki Y Nakahori T Tamura M Yamada M Iwaya 《Cytogenetics and cell genetics》1991,56(2):74-77
An average-sized human Y chromosome contains about 3,000 copies of the repeating DNA family DYZ1. A major repeating unit of the family, pHY10, has been cloned and an entire 3,564-bp sequence has already been determined by Nakahori et al. (1986). In the present study, pHY10 was divided into six consecutive segments, A to F, which were independently amplified by the PCR technique to see if they were strictly male-specific. pHY10 appears to consist of segments of various male-specificity. The B segment was apparently male-specific; however, the use of additional techniques (Southern-blot analysis or second PCR amplification in combination with the standard PCR) revealed homologous sequences in some females. None of the six segments of pHY10 may be male-specific in a strict sense. Different segments appear to be conserved during evolution to different extents. The 323-bp E segment appears to be the least conserved and to be responsible for the generation of most variations within the DYZ1 family. 相似文献
38.
Striking homology of the ''variable'' N-terminal as well as the ''conserved core'' domains of the mouse and human TATA-factors (TFIID). 总被引:18,自引:9,他引:9 下载免费PDF全文
T Tamura K Sumita I Fujino A Aoyama M Horikoshi A Hoffmann R G Roeder M Muramatsu K Mikoshiba 《Nucleic acids research》1991,19(14):3861-3865
39.
M Tamura T Tamura S R Tyagi J D Lambeth 《The Journal of biological chemistry》1988,263(33):17621-17626
The superoxide-generating respiratory burst oxidase is an integral membrane enzyme found in the plasma membrane of polymorphonuclear leukocytes (neutrophils). NADPH-dependent superoxide generation is seen in isolated plasma membranes and in their detergent extracts following activation of the intact cells with phorbol myristate acetate. We have herein examined the effects of phospholipids on the activity of the solubilized oxidase. Solubilization of plasma membranes with 0.5% each of Tween 20 plus deoxycholate resulted in an approximately 2-fold enhancement of activity. Inclusion of phospholipids in the extraction medium resulted in further activation. At 1.0 mg/ml the order of effectiveness was phosphatidylserine (PS) greater than cardiolipin greater than phosphatidylethanolamine greater than phosphatidylinositol; phosphatidylcholine and phosphorylated inositol lipids were not effective. The concentrations required for half-maximal activation by PS and phosphatidylethanolamine were 85 and 200 micrograms/ml, respectively. When PS was used at a maximally activating concentration (0.5 mg/ml), the activity was enhanced 3-5-fold. Detergent solubilization alone elevated the Km of the oxidase for NADPH from 68 microM in intact plasma membranes to 123 microM, but inclusion of PS with detergent restored the Km to near or below that seen in intact membranes. PS also increased the Vmax by a factor of 2-3, but had no effect on the pH optimum. A plot of the activity versus enzyme concentration was linear when membranes were used, but activity showed a quadratic dependence on concentration in solubilized membrane, with lower than expected activity at lower enzyme concentration. PS restored linearity of the concentration-activity plot. The activation by PS was not influenced by the addition of Ca2+, EGTA, or dioctanoylglycerol, indicating that activation was not dependent on protein kinase C. These results implicate phosphatidylserine as a direct effector of the NADPH-oxidase. 相似文献
40.
The adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), is shown to be a potent and specific inhibitor of yeast hexokinase PII. Evidence that the analog binds specifically at the ATP binding site includes the demonstration that glucose binding enhances PLP-AMP binding and that PLP-AMP and ATP bind competitively with an apparent Ki(PLP-AMP) = 23 microM. In addition, from the relationship between the degree of inhibition and extent of modification, it is estimated that the incorporation of 1 mol of PLP-AMP/mol of subunit is required for complete inhibition. Borohydride reduction of the Schiff's base complex formed between hexokinase and [3H]PLP-AMP gives a stable product. The reduced derivative was digested with trypsin and a single radioactive peptide was isolated by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis identified Lys-111 as the modified residue. Taking into account the known structures of the binary complexes (Shoham, M., and Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14), the results suggest that Lys-111, located in the smaller of the two lobes of hexokinase, moves into the active site upon formation of the ternary complex. 相似文献