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O Ohara

Publications and source records attributed to O Ohara.

15 recordsLinked to original sources
PubMed1992

A rat brain-derived neurotrophic factor-encoding gene generates multiple transcripts through alternative use of 5' exons and polyadenylation sites.

As a first step toward clarification of the transcriptional controls of the gene encoding brain-derived neurotrophic factor (BDNF), we cloned and sequenced a rat genomic DNA fragment carrying this gene. RNA blotting analysis using a probe derived from the 3'-flanking region of BDNF revealed that alternative use of 3'-polyadenylation sites generates at least two BDNF transcripts that differ in the size of the 3'-noncoding region. Furthermore, sequence analysis of the 5'-end of the BDNF cDNA revealed the presence of at least six different types of transcripts which were probably derived through alternative use of the multiple 5'-exons. Therefore, a single BDNF gene could produce multiple types of transcripts with different noncoding sequences through alternative use of both 5'-exons and 3'-transcription termination sites.

Alternative Splicing↗
PubMed1992

Disorganization of microfilaments is accompanied by downregulation of alpha-smooth muscle actin isoform mRNA level in cultured vascular smooth muscle cells.

In the previous report, we demonstrated that cAMP negatively regulated alpha-smooth muscle actin mRNA levels through destabilization of the mRNA [O. Ohara et al. (1991) J. Biochem. 109, 834-839]. We here report that the decline in the alpha-smooth muscle actin mRNA levels was well correlated with disorganization of microfilaments but not necessarily with the rise in intracellular cAMP levels in rat cultured vascular smooth muscle cells. The decrease in the alpha-smooth muscle actin mRNA induced by microfilament-disorganizing agents also resulted from enhancement of the turnover rate of the mRNA. These results raise the possibility that the post-transcriptional control of the alpha-smooth muscle actin expression is linked to the organization of actin filaments in smooth muscle cells.

Actin Cytoskeleton↗
PubMed1991

cAMP negatively regulates mRNA levels of actin and tropomyosin in rat cultured vascular smooth muscle cells.

The isoform expression patterns of actin and actin-binding proteins have been reported to be good markers for phenotypic modulation of rat vascular smooth muscle cells. In order to elucidate the regulatory mechanism of actin and tropomyosin isoform expression on a molecular basis, we examined the effects of various agents on the isoform expression patterns of actin and tropomyosin at the mRNA level in smooth muscle cells. We found that cAMP-elevating agents induced drastic decreases in the amounts of alpha-smooth muscle type actin and alpha-tropomyosin transcripts, the expression of other actin and tropomyosin isoforms being also repressed, but to a lesser extent. The results of the experiment involving RNA synthesis inhibitors strongly suggest that activation of some mRNA-specific degradation machinery by cAMP might be responsible at least for the rapid disappearance of alpha-smooth muscle type actin and alpha-tropomyosin transcripts in smooth muscle cells.

1-Methyl-3-isobutylxanthine↗
PubMed1990

A simple and sensitive method for determining transcription initiation site: identification of two transcription initiation sites in rat group II phospholipase A2 gene.

We developed a simple and sensitive method for assigning transcriptional initiation sites, and applied it to characterize the transcriptional unit of rat group II phospholipase A2 (PLA2) gene. Our method involves the primer extension reaction followed by detection of its products by hybridization. Using this method, we were able to map two transcriptional initiation sites on the nucleotide sequence of the core promoter region of PLA2 gene with one-base resolution without any difficulties.

Animals↗
PubMed1990

Glucocorticoids suppress group II phospholipase A2 production by blocking mRNA synthesis and post-transcriptional expression.

We investigated the effects of glucocorticoids on group II phospholipase A2 (PLA2) expression in rat cultured smooth muscle cells. Both forskolin-induced and tumor necrosis factor (TNF)-induced PLA2 release responses were almost completely blocked by 10 and 100 nM dexamethasone, respectively, as assayed by protein blotting and PLA2 activity assays. Dexamethasone-mediated inhibition of PLA2 release appeared to be mediated by the glucocorticoid receptor. Dexamethasone at concentrations greater than 10 nM inhibited forskolin-induced elevation of the group II PLA2 mRNA level but not TNF-induced elevation. These data suggest that the mechanism mediating forskolin-induced mRNA accumulation is sensitive to glucocorticoids, but the mechanism mediating the TNF-induced accumulation is not. Inhibition of TNF-induced PLA2 release by glucocorticoids may be explained by the blocking of post-transcriptional synthesis of the group II PLA2.

Animals↗
PubMed1990

Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

Two potent inflammatory mediators, interleukin 1 (IL-1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP-elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL-1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP-elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL-1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.

1-Methyl-3-isobutylxanthine↗
PubMed1989

cDNA cloning and sequence determination of rat membrane-associated phospholipase A2.

Based on the partial amino acid sequences of membrane-associated phospholipase A2 (PLA2M), belonging to group II, purified from rat spleen, the cDNA encoding PLA2M was cloned by a new cloning strategy utilizing enzymatic cDNA amplification. At the N-terminus of the coded 146 residues, which were deduced from the cDNA sequence, the putative signal peptide was found despite the tight adherence of this enzyme to the membrane. The sequence of rat PLA2M exhibits 75% homology with that of human group II PLA2 in the protein-coding region. The result of RNA blot analysis showed that rat ileal mucosa contains the largest amount of the PLA2 transcript among the tissues examined.

Amino Acid Sequence↗
PubMed1989

One-sided polymerase chain reaction: the amplification of cDNA.

We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d(A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle alpha-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydanio rerio) using only 300 ng of a total poly(A)+ preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle alpha-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin.

Animals↗
PubMed1989

Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli.

The genomic sequencing procedure is applied to the direct sequencing of uncharacterized regions of bacterial DNA by a "multiplex walking" approach. Samples of bulk Escherichia coli DNA are cut with various restriction enzymes, subjected to chemical sequencing degradations, run in a sequencing gel, and transferred to nylon membranes. When a labeled oligomer is hybridized to a membrane, a sequence ladder appears wherever the probe lies near a restriction cut. New probes, based on sequence that lies beyond other restriction sites, are then synthesized, and the membranes are reprobed to reveal new sequence. Repeated cycles of oligomer probe synthesis and subsequent reprobing permit rapid sequence walking along the genome. This oligomer walking technique was used to sequence the pyruvate kinase (EC 2.7.1.40) gene in E. coli without resorting to cloning or to library construction. The sequenced region was amplified by the polymerase chain reaction and subsequently transcribed and translated using both in vivo and in vitro systems, and the resultant gene product characterized to show that the gene encodes the type I isoform of pyruvate kinase.

Base Sequence↗
PubMed1987

Anomalous behavior of human leukocyte interferon subtypes on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate.

35S-labeled human leukocyte interferon (IFN) subtypes produced in a cell-free system derived from Escherichia coli were analyzed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Some IFN subtypes anomalously showed lower electrophoretic mobilities than those expected from their formula molecular masses. The results with hybrid IFNs and esterification suggest that this anomaly of IFN subtypes on SDS-PAGE is due to the introduction of one or two negative charges in the middle of the molecule.

Amino Acid Sequence↗
PubMed1986

Purification and characterization of recombinant murine immune interferon.

The recombinant murine immune interferon (rMu-IFN-gamma) was purified to homogeneity from Escherichia coli harboring the expression vector of murine IFN-gamma. The purified rMu-IFN-gamma showed an Mr of 15 000 in SDS-polyacrylamide gel electrophoresis. Results of amino acid analysis, amino- and carboxyl-terminal analyses and peptide mapping of rMu-IFN-gamma suggest that it has the complete protein sequence predicted on the basis of cDNA except for lack of four amino acid residues from the mature carboxyl-terminus.

Amino Acid Sequence↗
PubMed1986

Dog and rat pancreatic phospholipases A2: complete amino acid sequences deduced from complementary DNAs.

We performed molecular cloning of the cDNAs that encode dog and rat pancreatic phospholipases A2 to predict the primary structures of these enzymes. The deduced amino acid sequences exhibited a highly conservative feature which is common to a group of pancreatic phospholipases A2 from various animal species. Furthermore, the structures of the signal sequences of dog and rat pancreatic phospholipases A2 were predicted, although the assignment of the positions cleaved post-translationally is only tentative at the present time.

Amino Acid Sequence↗