Yutaka Teranishi

Isolation and structural organization of the human preproenkephalin B gene.

Recently, we have elucidated the primary structure of bovine adrenal preproenkephalin by determining the nucleotide sequence of cloned DNA complementary to its mRNA1. The structure of most of this precursor molecule has also been deduced by Gubler et al.2 using cDNA sequencing in conjunction with protein sequencing. Bovine preproenkephalin contains four copies of methionine-enkephalin3 (Met-enkephalin) and one copy each of leucine-enkephalin3 (Leu-enkephalin), Met-enkephalin-Arg6-Phe7 (ref. 4) and Met-enkephalin-Arg6-Gly7-Leu8 (refs 1, 2, 5). The region containing the repeated enkephalin and extended enkephalin sequences, which are each bounded by paired basic amino acid residues, is connected with a cysteine-containing amino-terminal sequence preceded by a signal peptide6. We have now studied the relationship between the repetitive structure of preproenkephalin and the structural organization of its gene by cloning a human genomic DNA segment containing the entire gene. We find that the general organization of the preproenkephalin gene is strikingly similar to that of the gene encoding the common precursor of corticotropin (ACTH) and β-lipotropin (β-LPH)7–9 (alternatively designated preproopiomelanocortin), another multi-hormone precursor. Furthermore, the complete mRNA and amino acid sequences of human preproenkephalin have been deduced from the corresponding gene sequence.

Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae

SummaryA DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn2+, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.

Isolation and structural organization of the human corticotropin-β-lipotropin precursor gene

The common precursor of corticotropin (ACTH) and/3-1ipotropin 03-LPH) [1-8] contains multiple biologically active peptides. We have elucidated the whole primary structure of the bovine ACTH-t3-LPH precursor by determining the nucleotide sequence of cloned DNA complementary to the mRNA coding for the precursor protein [9]. Using this bovine cDNA as a hybridization probe, we have then isolated the entire bovine gene encoding the ACTH-13-LPH precursor as a set of genomic DNA fragments and have determined its structural organization [ 10,11 ]. We have now isolated and characterized a human genomic DNA fragment containing the entire ACTH-t3-LPH precursor gene. The human gene, like the bovine counterpart, consists of 3 exons (mRNA-coding sequences) divided by 2 large introns (intervening sequences). Nucleotide sequence analysis of the whole exons has revealed the complete mRNA and amino acid sequences of the human ACTH-/3-LPH precursor.

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.

Development of microbodies in Candida tropicalis during incubation in a n-alkane medium

Development of microbodies in Candida tropicalis pK 233 was studied mainly by electron microscopical observation. The yeast cells, precultured on malt extract, scarcely contained microbodies and showed very low catalase activity. When the precultured cells were transferred to a n-alkane medium and incubated with shaking, the number of microbodies increased and concomitantly the activity of catalase was enhanced. That is, both the area ratio of microbodies in the cell and the ratio of microbodies to cytoplasm in area increased significantly during the utilization of n-alkanes for 8 hrs. Localization of catalase in the microbodies was demonstrated cytochemically by use of 3,3′-diaminobenzidine, but other organella in the cell, except for vacuoles appearing in the early growth phase and mitochondria, were not stained with this reagent. Microbodies seemed to grow by division. Biogenesis of microbodies in the yeast cells is also discussed.

Ultrastructure ofCandida yeasts grown onn-alkanes

Catalase activities of the cells growing onn-alkanes of various strains ofCandida yeasts wer markedly higher than those of the cells growing on glucose, ethanol or acetate. In connection with this, electron-microscopical studies revealed abundant appearance of specific microbodies having homogeneous matrix surrounded by single unit membrane in the hydrocarbon-growing cells. Localization of catalase activity in the microbodies, in addition to the mitochondria, was confirmed by cytochemical treatment of the cells with 3,3′-diaminobenzidine reagent.

Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae.

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

Tissue-specific expression of a novel GTP-binding protein (smg p25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC-12 cells

We have purified a novel GTP-binding protein, designated as the smg-25A protein (smg p25A), from bovine brain membranes and determined its primary structure. In the present studies, the smg-25A mRNA levels in various tissues have been studied. The 1.6-kilobase smg-25A mRNA is detected in rat brain by Northern blot analysis. This mRNA is not detected in other rat tissues including thymus, lung, heart, liver, small intestine, kidney, and skeletal muscle. The 1.6-kilobase smg-25A mRNA is also detected in bovine adrenal medulla but not in the cortex. Moreover, this mRNA is detected in rat pheochromocytoma PC-12 cells and its level increases after differentiation of the cells into sympathetic neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP. This mRNA level does not increase in response to 12-O-tetradecanoylphorbol-13-acetate incapable of inducing differentiation. These results suggest that the smg-25A gene is specifically expressed in nerve tissues and that smg p25A plays a role in some neuronal functions.

Microbody of n-alkane-grown yeast

Microbodies appearing abundantly in n-alkane-grown cells of Candida tropicalis pK 233 were isolated by means of sucrose density gradient centrifugation. Electron microscopical observation showed that the microbodies isolated were intact. Localization of catalase and d-amino acid oxidase in the isolated microbodies was confirmed. Isocitrate lyase, malate synthase and NADP-linked isocitrate dehydrogenase were also located in the microbody, but malate dehydrogenase, citrate synthase, aconitase and NAD-linked isocitrate dehydrogenase were not. Neither cytochrome P-450 nor NADPH-cytochrome c reductase, the components involved in the n-alkane hydroxylation system of the yeast, were detected in the microbody fraction.

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.