Matsubara Kenichi

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens

Abstract Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe revealed that the human trypsinogen genes constitute a multigene family of more than ten genes

Lambda phage DNA sequences affecting the packaging process

Abstract Our previous work identified a minimal region of bacteriophage λ DNA that is necessary for packaging into phage particles. It consists of 40 bp of the right arm and 45 bp of the left arm [Miwa and Matsubara, Gene 20 (1982) 267–279]. A part of this region, 22 bp of the right arm and 38 bp of the left arm, is sufficient for cutting at cos λ (the minimal sequence for cos λ cutting). An 84-bp region to its right contains a binding site for λ terminase, a complex of Nu 1 and A gene products. This second region, which we called the enhancing region for packaging, stimulates cutting at cos λ as well as packaging. This region is not active if it is physically separated from the minimal packaging region. The enhancing region has 15-bp inverted repeats. These sequences are conserved in the corresponding region of bacteriophage φ80, which has the same packaging specificity as λ.

Expression of the human salivary α-amylase gene in yeast and characterization of the secreted protein

Abstract Recombinant plasmids were constructed in which the human salivary α-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the α-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary α-amylase. Thus, the human salivary α-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.

Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoteroperator of Escherichia coli

Abstract A novel expression vector using the 236-bp promoter-operator fragment of the recA gene ( recApo ) of Escherichia coli has been constructed. This DNA fragment contains complete signals for the initiation of RNA synthesis, as well as for regulation by the lexA product, but lacks the coding sequence for the RecA protein. The strength of the recA promoter was examined by assaying s-galactosidase activity expressed from a cro-lacZ fused gene placed downstream of the promoter. Under noninducing conditions, the promoter was regulated by the LexA protein, and the fused gene was expressed only weakly. Upon induction by nalidixic acid in a recA + strain, high expression was observed for an extended period. After 5 h under inducing conditions, as much as 11 % of the total cellular protein was cro-lacZ product. The expression level was higher than that from promoters of lac , trp , and λ early genes.

Expression of human salivary α-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence

Abstract A cDNA fragment coding for human salivary α-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast. Yeast cells transformed with this recombinant plasmid synthesized about 5 × 10 5 molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium. The enzyme is stable, and exhibited the same specific activity as α-amylase in human saliva. The amylase-producing yeast grew on starch and produced alcohol.

A human cDNA sequence homologue of bovine phosphatidylethanolamine-binding protein ☆

Abstract Sequencing of about 1000 3′-directed cDNA clones from the human HepG2 cell line revealed that about half of them represent transcripts of abundantly or moderately expressed genes, about 70% of which are novel. We identified one of these clones as encoding the human homologue of bovine phosphatidylethanolamine-binding protein.

Identification of the IL-6-responsive element in an acute-phase-responsive human pancreatic secretory trypsin inhibitor-encoding gene

Abstract Pancreatic secretory trypsin inhibitor (PSTI) has been suggested to be an acute-phase reactant in humans and to be induced by inflammatory cytokines such as the interleukins IL-1 and IL-6. We report that PSTI is synthesized in hepatoma cells and that the gene expression is augmented by IL-6. The start points (tsp) for basal and augmented transcription are exactly the same as the tsp in normal pancreas. Analysis of the PSTI gene revealed that a 40-bp DNA fragment located between kb −3.84 and −3.80 carries the element responsible for both basal transcriptional activity and IL-6-induced gene expression. This 40-bp fragment contains TGTNNGNAAGT, the consensus sequence for the NF-IL6-binding site, which is also known as the IL-6-responsive element that is conserved among various acute-phase genes. The basal activity was augmented by another sequence that lies between kb −4.0 and −3.9.

Expression of human gastrin gene in normal and gastrinoma tissues

Abstract The complete sequence of the human gastrin gene is reported here. This gene consists of three exons. Nine Alu family sequences are found within the gene and in the surrounding region. S1 mapping study showed that the transcription of gastrin gene starts at 60, 57, or 55 bp upstream from the 3′ end of the first exon. The mechanism of mRNA synthesis in a gastrinoma tissue was studied to clarify the ectopic production of gastrin. It was found that mRNA synthesis starts from the same three transcriptional start points. Southern blotting profiles for normal gastric antrum and gastrinoma DNA were indistinguishable from each other within the 18-kb region containing the gastrin gene, showing that no genomic rearrangements are associated with the gastrinoma formation. Thus, the overproduction of gastrin in this tumor is likely to be due to an aberrant expression control system of the cell, rather than a change in the control region of the gastrin gene.

Expression of a synthetic human growth hormone gene in yeast

Abstract A synthetic human growth hormone (hGH) gene was efficiently expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae). More than 106 molecules of hormone were formed per cell despite the fact that the gene was constructed with codon preference for Escherichia coli.

A novel type of human α-amylase produced in lung carcinoid tumor

A novel type of alpha-amylase was detected in a lung carcinoid tissue after surveying the cDNA library constructed from this tumor mRNA. Nucleotide sequence analysis showed that the amylase expressed in this carcinoid tumor has 13 and 6 amino acid substitutions when compared with salivary amylase (Amy1) and pancreatic amylase (Amy2), respectively. The nucleotide sequence homologies of cDNAs between this carcinoid amylase and amy1, amy2 are 97.5% and 98.2%, respectively. The nucleotide sequence comparison strongly suggests that this new amylase is the product of the amy3 gene that has been detected in human genome [Emi et al., Gene 62 (1988) 229-235]