Overproduction of an alpha-amylase/glucoamylase fusion protein in Aspergillus oryzae using a high expression vector.
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Aspergillus oryzae↗
Biomedical subjects
G Tamura
Publications and source records attributed to G Tamura.
Cloning of the alpha-amylase cDNA of Aspergillus shirousamii and its expression in Saccharomyces cerevisiae.
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.
Amino Acid Sequence↗
Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.
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.
Aspergillus oryzae↗
Construction of an alpha-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae.
A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.
Alcohol Dehydrogenase↗
[Bronchial asthma].
In the first study, to clarify whether increased vagal afferent activity contributes to the increase in ventilatory response to CO2 in patients with asthma, we examined the effects of prostaglandin E2 (PGE2) inhalation on the respiratory response to CO2 in seven normal subjects. After PGE2 inhalation, the ventilatory and occlusion pressure responses to CO2 increased significantly compared with those after saline inhalation, with no increase in respiratory resistance. These results suggest that increase in vagal afferent activity may play a role in the increased hypercapnic response during acute exacerbations of asthma. In the second study, to clarify the reduced respiratory chemosensitivity associated with asthma-related deaths, we examined the hypercapnic and hypoxic ventilatory responses in five patients with near-fatal asthma who were given artificial ventilation and/or became unconsciousness during an acute exacerbation of asthma. Hypoxic ventilatory response was significantly lower in these subjects than in patients with uncomplicated asthma and normal subjects, suggesting that lowered hypoxic ventilatory response may be related to death from asthma.
Asthma↗
Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae.
The glucoamylase-encoding gene (glaA) from Aspergillus oryzae was cloned using its cDNA as a probe, which had been isolated previously. From comparison of nucleotide (nt) sequences of genomic clones with its cDNA, the glaA gene was found to contain four short putative introns, 45-56 nt in length. The A. oryzae glaA gene shared 62% homology at the nt level with the A. niger glaA gene with the four introns located at the same position. The 5'-flanking region contained a TATA box at nt-72 from the start codon, and two putative CAAT sequences at nt-87 and -331. Genomic Southern analysis and physical mapping showed that the glaA gene is located on the smallest chromosome (3.4 Mb) of six separated bands of chromosomes. Clones containing the glaA gene, when re-introduced intro A. oryzae, resulted in a three- to eightfold increase in glucoamylase activity.
Amino Acid Sequence↗
Detection of frequent p53 gene mutations in primary gastric cancer by cell sorting and polymerase chain reaction single-strand conformation polymorphism analysis.
Mutations of the p53 gene were investigated after tumor cell enrichment by cell sorting based on differences in DNA content and polymerase chain reaction single-strand conformation polymorphism analysis in 24 surgical specimens of primary gastric cancer. p53 mutations were detected in exons 4-8 in 64% (9 of 14) of aneuploid tumors but in none of 10 diploid tumors examined. Four of five tumors containing two or three aneuploid subpopulations showed the presence of p53 gene mutations. No correlation was found between the presence of p53 mutations and the degree of histological differentiation of tumors. These findings suggest that p53 gene mutations are related to DNA ploidy alterations as relatively late events of carcinogenesis in gastric cancer. The present method is highly sensitive for detection of genetic abnormalities and is applicable even when various kinds of nontumorous cells are present in tumor samples.
Base Sequence↗
Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae.
Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 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 beta-glucuronidase (GUS) down-stream 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.
Aspergillus oryzae↗
Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae.
Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T2 gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T2 revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T2 activity than wild-type strains.
Amino Acid Sequence↗
Do diagnostic procedures other than inhalation challenge predict immediate bronchial responses to inhaled allergen?
To investigate the relationships between allergen inhalation challenge and other diagnostic procedures, inhalation challenge with house dust (HD) allergen, intradermal skin tests with HD allergen, inhalation challenge with methacholine and circulating HD allergen-specific IgE levels were examined in 104 patients with bronchial asthma. Using the single exposure method, allergen inhalation challenge was performed. Forty-three patients had positive bronchial responses to allergen and 61 patients had negative bronchial responses. With serially diluted HD allergen (10(-3) to 10(-6), w/v), skin-test sensitivity was expressed as the highest dilution required to produce a weal of more than 9 x 9 mm. With the continuous exposure method, bronchial responsiveness to methacholine was evaluated as the number of units of inhaled methacholine (PD35-Grs) from the start to the point at which Grs had decreased by 35% from its baseline value. The level of circulating HD allergen-specific IgE was measured with the Phadebas RAST system and the results were assessed as a RAST score. Using discriminant analysis, in which the independent variables were skin-test sensitivity, PD35-Grs and the RAST score, only in 30% of all patients was bronchial responsiveness to inhaled HD allergen predictable. Therefore, we suggest that inhalation challenge with allergen is an essential test for determining the role of a specific allergen in airways at present.
Administration, Inhalation↗
The glucoamylase cDNA from Aspergillus oryzae: its cloning, nucleotide sequence, and expression in Saccharomyces cerevisiae.
A cDNA for Aspergillus oryzae glucoamylase was cloned, using oligodeoxyribonucleotide probes derived from amino sequences of peptide fragments of the enzyme. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae, directed the secretion of active glucoamylase into the culture medium. The complete nucleotide sequence of the cDNA contained an open reading frame encoding 612 amino acid residues. Comparative studies with other fungal glucoamylases showed homologies of 67% with A. niger and 30% with Rhizopus oryzae of the deduced amino acid sequences. In the five conserved regions reported in other fungal glucoamylases, the levels of homologies between those regions of A. oryzae and A. niger enzymes were much higher (78-94%). A. oryzae glucoamylase contained no peptide region abundant in threonine and serine residue (TS-region), like that proposed to adsorb onto raw starch in A. awamori var. kawachii glucoamylase.
Amino Acid Sequence↗
Identification of the promoter region of the Taka-amylase A gene required for starch induction.
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Chromosome Deletion↗
Significance of immunohistochemically detected c-erbB-2 protein expression in stage III breast cancer, with reference to nuclear deformity, DNA content and prognosis.
The significance of c-erbB-2 protein expression was immunohistochemically investigated in 32 patients with stage III breast cancer with regard to area, shape factor and DNA content of cancer cell nuclei. Thirty-one percent of the tumors showed c-erbB-2-positive cell membrane staining (10 out of 32 cases), and the cancer cells of the c-erbB-2-positive tumors had larger and more irregular nuclei with an increased DNA content. All patients with c-erbB-2 protein-positive tumors died of the disease within six years of their mastectomy. It can be concluded that positive immunohistochemical staining of c-erbB-2 protein indicates an aggressive biological behavior of the cancer cells as well as a poor prognosis in patients with stage III breast cancer.
Biomarkers, Tumor↗
[The effect of an oral gold compound, auranofin, on bronchial responsiveness to inhaled methacholine in well-controlled bronchial asthma].
Recently, in Japan, auranofin (6 mg/day) has been demonstrated to be a useful treatment for patients with moderate to severe asthma in a double-blind clinical trial. Therefore, to investigate the mechanism of auranofin on bronchial asthma, we examined pulmonary functions and bronchial responsiveness to inhaled methacholine in well-controlled asthmatics after 12 wks of treatment with auranofin, in a double-blind, placebo-controlled fashion. Twenty-five adult patients with asthma received orally 3 mg of auranofin or inactive placebo twice daily for 12 weeks. Bronchial responsiveness, pulmonary function tests and concentrations of gold in the blood were measured before and 6 and 12 wks after the therapy. Bronchial responsiveness (PD35-Grs) was significantly decreased after 12 wks of treatment with auranofin, compared with that 12 wks after treatment with inactive placebo. We suggest that inhibition of bronchial hyperresponsiveness by auranofin is one of the mechanisms by which auranofin is effective against bronchial asthma.
Adult↗
[Nuclear morphometric and DNA content analyses of cancer cells in superficial esophageal cancer with reference to lymph node metastasis].
In 23 cases of surgically resected superficial esophageal cancer which was defined by Japanese Society for Esophageal Diseases, the nuclear area (NA), nuclear shape factor (NSF) and DNA content (DNA) of cancer cells were measured using an image analysis system, and, in 7 out of the 23 cases, biopsy specimens were examined in the same way as well. The results were analyzed and evaluated with respect to lymph node metastasis. The NA was significantly larger (p less than 0.01) and the DNA content value was significantly higher (p less than 0.05) in the tumors with lymph node metastasis than in those with no lymph node metastasis. By means of a linear discriminant analysis using NA and DNA as variables, z = 0.238 X NA + 0.475 X DNA - 11.9, existence of lymph node metastasis could be correctly presumed at 87%. No false discrimination was found on preoperative biopsy specimens in 7 cases in which the accuracy of the discriminant analysis could be confirmed by microscopic examination of the lymph nodes extirpated at operation for esophagectomy. Conclusively NA and DNA correlate significantly with existence of lymph node metastasis in the patients with superficial esophageal cancer, and might be useful for preoperative predicting whether or not lymph node metastasis exists.
Carcinoma, Squamous Cell↗
Periodic formation of the oriC complex of Escherichia coli.
We examined formation of an oriC-membrane complex through the chromosome replication cycle by dot-blot hybridization using an oriC plasmid as a probe. In a wild-type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event. Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication. Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature. Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture. However, the second periodic peak did not appear when rifampicin or chloramphenicol was added. Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature. We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.
Bacterial Outer Membrane Proteins↗
Eosinophil supernatant causes hyperresponsiveness of airway smooth muscle in guinea pig trachea.
To study the role of eosinophils in airway hyperresponsiveness, we studied the effect of supernatant obtained from activated eosinophils on the responses of isolated guinea pig tracheal smooth muscle segments to histamine. Eosinophils obtained from guinea pig peritoneal fluid were purified and activated with Ca2+ ionophore A23187 using a two-stage reaction. Supernatant obtained from different eosinophil cell numbers (3 x 10(5) to 10(7) cells) did not alter resting tension but potentiated the contractile response to histamine in a cell number-dependent fashion. Thus, at a cell number of 10(7), the supernatant decreased the mean (+/- SE) log histamine concentration producing 50% of maximum contraction significantly from a control value of -5.62 +/- 0.10 to -5.99 +/- 0.07 M (p less than 0.05). The potentiating effect of the supernatant (10(7) cells) was not altered by either removal of tracheal epithelium or by pretreatment with indomethacin when cells were activated. However, pretreatment with AA 861 completely inhibited the supernatant (10(7) cells)-induced potentiating effect associated with inhibition of leukotriene C4, D4, and E4 release in the supernatant. The concentrations of exogenous authentic leukotrienes chosen to match concentrations in the supernatant mimicked the supernatant-induced potentiating response to histamine. These results suggest that leukotrienes released from eosinophils cause hyperresponsiveness of airway smooth muscle in vitro.
Animals↗
Chromosomal transformation in Saccharomyces cerevisiae with DNA isolated by pulse field gel electrophoresis.
We isolated and purified yeast chromosome DNA molecules using pulse field gel electrophoresis (PFG). The isolated DNA had nearly the same size as the native chromosomal DNA on PFG. We could directly transform Saccharomyces cerevisiae yeasts with it, and obtain transformants that were selected by complementation of several markers. They had new chromosome DNA bands observed on PFG. The new chromosome was very stable during mitosis and mating processes, and each of the three homologous chromosomes in the derivative zygotes of transformants was separated equally in daughter cells.
DNA, Fungal↗