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Y Kaziro

Publications and source records attributed to Y Kaziro.

At least 109 records · Page 6Linked to original sources
PubMed1988

Presence of three distinct molecular species of Gi protein alpha subunit. Structure of rat cDNAs and human genomic DNAs.

We have cloned a new species of rat Gi alpha (Gi3 alpha) cDNA and genomic DNAs for three distinct human Gi alpha proteins (Gi1 alpha, Gi2 alpha, and Gi3 alpha). Gi3 alpha cDNA codes for a protein of 354 amino acids (Mr 40,522) whose sequence is closely related but distinct from that of the previously isolated rat Gi alpha (Gi2 alpha). By screening the human genomic libraries with the two rat Gi alpha cDNAs as probes, clones encoding human Gi1 alpha, Gi2 alpha, and Gi3 alpha were isolated. The human Gi2 alpha and Gi3 alpha genes are composed of eight coding exons and seven introns and possess a completely identical exon-intron organization. Southern blot analysis indicates that a single copy of each Gi alpha gene is present per haploid human genome.

Amino Acid Sequence↗
PubMed1988

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain. Comparison of predicted amino acid sequences from G-protein alpha-subunit genes and cDNAs with partial amino acid sequences from purified proteins.

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2 alpha gene and rat Gi2 alpha cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1 alpha and Go alpha cDNAs, respectively.

Adenosine Diphosphate Ribose↗
PubMed1988

Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants.

Normal (Gly12) and activated (Val12) Ha-ras proteins were produced in Escherichia coli, and purified to an apparent homogeneity without using any protein denaturants. The purified proteins contained an equimolar amount of GDP. They were stable in the presence of 5 mM Mg2+ and 25% (v/v) glycerol when incubated at 60 degrees C for 5 min. The binding of GDP to the protein was greatly stabilized by Mg2+. In the presence of 10 mM Mg2+, the bound GDP hardly exchanged with external guanine nucleotides, even at 30 degrees C. The exchange reaction was markedly enhanced in the presence of 10 mM EDTA or 120 mM ammonium sulfate. The rate-limiting step of the exchange reaction was the dissociation of the bound GDP from the ras protein, and this step was facilitated 40- to 100-fold by the addition of EDTA or ammonium sulfate. The dissociation rate of the normal (Gly12) ras protein was 2- to 3-fold faster than that of the activated (Val12) protein. The dissociation constants (Kd) for GDP of the normal and activated ras proteins were 1.2 X 10(-8) and 3.1 X 10(-9) M, respectively. The overall turnover rate of GTPase activity of the normal ras protein (10.8 mmol.mol-1.min-1) was about 10-fold higher than that of the activated protein (1.1 mmol.mol-1.min-1) in the absence of Mg2+ (less than 10(-8) M).

Base Sequence↗
PubMed1988

Sequence analysis of cDNA and genomic DNA for a putative pertussis toxin-insensitive guanine nucleotide-binding regulatory protein alpha subunit.

We have isolated cDNA clones from rat C6 glioma cells coding for several guanine nucleotide-binding regulatory protein (G protein) alpha subunits (G alpha). The cDNA clones were then used to isolate human chromosomal genes. Among human genomic clones isolated by cross-hybridization with the rat cDNA for the alpha subunit of the inhibitory G protein Gi2, termed Gi2 alpha, a clone designated lambda HGi62 was found to contain a sequence that is highly homologous but distinct from any of the known G alpha sequences, and we have tentatively designated this sequence Gx alpha. We have searched a rat brain cDNA library with the Gx alpha sequence and isolated a cDNA clone containing a rat sequence similar to human Gx alpha. The cDNA contained a single open reading frame of 1065 nucleotides coding for a protein of 355 amino acids with a calculated molecular weight of 40,879. The amino acid sequence of rat Gx alpha shows 66% and 40% similarity with rat Gi2 alpha and rat Gs alpha (the alpha subunit of the stimulatory G protein), respectively. By RNA blot hybridization analysis, mRNA of approximately 3.2 kilobases was detected mainly in brain. Interestingly, the deduced amino acid sequence of Gx alpha predicts that the Gx alpha protein may be refractory to modification by pertussis toxin since the cysteine residue in the fourth position from the C terminus of pertussis toxin-sensitive G alpha is replaced by isoleucine.

Amino Acid Sequence↗
PubMed1988

Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions.

In a previous paper, we demonstrated that a gene coding for a protein homologous to the alpha subunit of mammalian guanine nucleotide-binding regulatory (G) proteins occurs in Saccharomyces cerevisiae. The gene, designated GPA1, encodes a protein (GP1 alpha) of 472 amino acids with a calculated Mr of 54,075. Here we report the isolation of another G-protein-homologous gene, GPA2, which encodes an amino acid sequence of 449 amino acid residues with a Mr of 50,516. The predicted primary structure of the GPA2-encoded protein (GP2 alpha) is homologous to mammalian G proteins [inhibitory and stimulatory G proteins (Gi and Gs, respectively), a G protein of unknown function (Go), and transducins (Gt)] as well as yeast GP1 alpha. When aligned with the alpha subunit of Gi (Gi alpha) to obtain maximal homology, GP2 alpha was found to contain a stretch of 83 additional amino acid residues near the NH2 terminus. The gene was mapped in chromosome V, close to the centromere. Haploid cells carrying a disrupted GPA2 gene are viable. Cells carrying a high copy number of plasmid GPA2 (YEpGPA2) had markedly elevated levels of cAMP and could suppress a temperature-sensitive mutation of RAS2. These results suggest that GPA2 may be involved in the regulation of cAMP levels in S. cerevisiae.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of the human Gs alpha gene.

The gene for Gs alpha (the alpha subunit of the guanine nucleotide-binding protein Gs) was isolated from human genomic libraries using rat Gs alpha cDNA as a probe. Comparison of the nucleotide sequence of the human gene with that of the rat cDNA revealed that the human Gs alpha gene spans approximately equal to 20 kilobases and is composed of 13 exons and 12 introns. Genomic Southern blot analysis suggests that the human haploid genome contains a single Gs alpha gene. Previous reports indicated the presence of multiple species of Gs alpha cDNA. The structure of the human Gs alpha gene suggests that four types of Gs alpha mRNAs may be generated from a single Gs alpha gene by alternate use of exon 3 and/or of two 3' splice sites of intron 3, where an unusual splice junction sequence (TG) instead of the consensus (AG) is used. S1 nuclease mapping analysis of human Gs alpha mRNA identified multiple transcriptional initiation sites. The promoter region of the human Gs alpha gene has extremely high G + C content (85%). It contains 4 "GC" boxes, but no typical "TATA" or "CAAT" box sequence. In the 5' flanking region, there are several blocks of sequences that are similar to the sequences of the 5' flanking region of the human c-Ki-ras2 gene.

Amino Acid Sequence↗
PubMed1988

Suppressors of a gpa1 mutation cause sterility in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae GPA1 gene encodes a protein highly homologous to the alpha subunit of mammalian G proteins and is essential for haploid cell growth. We have selected 77 mutants able to suppress the lethality resulting from disruption of GPA1 (gpa1::HIS3). Two strains bearing either of two recessive mutations, sgp1 and sgp2, in combination with the disruption mutation, showed a cell type nonspecific sterile phenotype, yet expressed the major alpha-factor gene (MF alpha 1) as judged by the ability to express a MF alpha 1-lacZ fusion gene. The sgp1 mutation was closely linked to gpa1::HIS3 and probably occurred at the GPA1 locus. The sgp2 mutation was not linked to GPA1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11 and ste12). sgp2 GPA1 cells showed a fertile phenotype, indicating that the mating defect caused by sgp2 is associated with the loss of GPA1 function. While expression of a FUS1-lacZ fusion gene was induced in wild-type cells by the addition of alpha-factor, mutants bearing sgp1 or sgp2 as well as gpa1::HIS3 constitutively expressed FUS1-lacZ. These observations suggest that GPA1 (SGP1) and SGP2 are involved in mating factor-mediated signal transduction, which causes both cell cycle arrest in the late G1 phase and induction of genes necessary for mating such as FUS1.

Cell Cycle↗
PubMed1988

Induction of neuronal differentiation in PC12 cells by B-cell stimulatory factor 2/interleukin 6.

B-cell stimulatory factor 2 (BSF-2) is a lymphokine which induces the final maturation of B cells. BSF-2 acts on a variety of cells other than B cells, and moreover, expression of BSF-2 mRNA is detected in interleukin-1 beta-stimulated glioblastoma and astrocytoma cell lines. Here, we studied the function of BSF-2 on pheochromocytoma PC12 cells, a model system for induction of neuronal differentiation. PC12 cells possess specific receptors for BSF-2. The BSF-2-stimulated PC12 cells expressed the c-fos proto-oncogene transiently, and they began to change morphologically to neurite-extending cells after several days. The number of voltage-dependent Na+ channels was also increased.

Adrenal Gland Neoplasms↗
PubMed1988

Role of STE genes in the mating factor signaling pathway mediated by GPA1 in Saccharomyces cerevisiae.

The ste mutants (ste2, ste4, ste5, ste7, ste11, and ste12) are insensitive to mating factors and are, therefore, sterile. Roles of the STE gene products in the GPA1-mediated mating factor signaling pathway were studied by using ste gpa1 double mutants. Mating efficiency of a ste2 mutant defective in the alpha-factor receptor increased 1,000-fold in a gpa1 background, while G1 arrest and aberrant morphology (shmoo) caused by gpa1 were not suppressed by ste2. Furthermore, the steady-state level of the FUS1 transcript, which normally increases in response to mating factors, was also elevated when the GPA1 function was impaired. These results suggest that the GPA1 protein functions downstream of the STE2 receptor. Conversely, the sterility of ste4, ste5, ste7, ste11, and ste12 mutants was not suppressed by gpa1, but the lethal phenotype of gpa1 was suppressed by these ste mutations. Northern (RNA) blotting analysis revealed that the ste7, ste11, and ste12 mutations caused reductions of 50 to 70% in the steady-state levels of the GPA1 transcript, while ste4 had a slight effect and ste5 had no effect. This implies that the suppression by ste7, ste11, and ste12 could be due to reduced syntheses of additional components, including an effector, and that suppression by ste4 and ste5 may result from direct effects on the signaling pathway. The STE4, STE5, STE7, STE11, and STE12 products, therefore, appear to specify components of the signal transduction machinery, directly or indirectly, which function together with or downstream of GPA1.

Crosses, Genetic↗
PubMed1987

Chromosomal gene structure of human myeloperoxidase and regulation of its expression by granulocyte colony-stimulating factor.

By using the full-length cDNA as a probe, the chromosomal gene for human myeloperoxidase was isolated from a human gene library. Comparison of the nucleotide sequence of the chromosomal gene with that of the cDNA has revealed that the human myeloperoxidase gene is composed of 12 exons and 11 introns. S1 mapping analysis of human myeloperoxidase mRNA identified the single transcription initiation site at 180 base pairs upstream of the ATG initiation codon. In the 5'-flanking region of the human myeloperoxidase gene, there are several blocks of sequences which are homologous to the sequences found on the 5'-promoter region of the human c-myc proto-oncogene. The myeloperoxidase gene is expressed in mouse myeloid leukemia WEHI-3B D+ and NFS-60 cells. When these cells were treated with human recombinant granulocyte colony-stimulating factor, the steady-state level of myeloperoxidase mRNA declined to zero within 36 h in WEHI-3B D+ cells, but not in NFS-60 cells.

Amino Acid Sequence↗
PubMed1987

GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction.

GPA1 protein of Saccharomyces cerevisiae is homologous to the alpha subunit of mammalian G protein. GPA1 transcript was found in haploid cells but was not detected in diploid cells. Disruption of GPA1 resulted in a haploid-specific lethal phenotype, indicating that GPA1 is a haploid-specific essential gene for cell growth. Upon regulation of expression of GPA1 by the galactose-inducible GAL1 promoter, the loss of GPA1 function was found to lead to cell-cycle arrest at the late G1 phase. Mutants that suppress the lethality of the gpa1::HIS3 mutation showed a sterile phenotype that was not cell-type-specific. These results suggest that GPA1 protein may control the signal for mating-factor-mediated cell-cycle arrest.

Cell Cycle↗
PubMed1987

The chromosomal gene structure for murine granulocyte colony-stimulating factor.

The chromosomal gene for murine granulocyte colony-stimulating factor (G-CSF) has been isolated from a mouse genomic library. The nucleotide sequence analysis of the gene and its flanking region has revealed that the murine G-CSF gene is composed of four introns and five exons, which is similar to the human G-CSF gene. The S1 mapping analysis of murine G-CSF mRNA has identified the major transcription initiation site for the murine G-CSF gene at 36 base pairs upstream of the ATG initiation codon. In the flanking region, there are sequences of about 300 base pairs which are highly conserved between human and murine G-CSF genes. The decanucleotide GAGRTTCCAC, which was found at the 5'-flanking region of genes for other hemopoietic growth factors, was also present within the corresponding regions of human and murine G-CSF gene. Unlike the human G-CSF gene, no alternative splicing was observed for the murine G-CSF gene.

Amino Acid Sequence↗
PubMed1987

Molecular cloning and characterization of cDNA for human myeloperoxidase.

Partial amino acid sequence of human myeloperoxidase was determined, and a 41-base oligonucleotide containing deoxyinosines at four positions was chemically synthesized. By using the oligonucleotide as a probe, cDNA clones for human myeloperoxidase were isolated from a cDNA library constructed with mRNA from human promyelocytic leukemia HL-60 cells. One of the clones containing a 2.6-kilobase insert was subjected to nucleotide sequence analysis. The sequence was found to contain an open reading frame, 2,235 nucleotides coding for a protein of 745 amino acids with a calculated Mr of 83,868. The heavy chain of myeloperoxidase, consisting of 467 amino acids, was located on the COOH terminus half of the protein. The RNA specified by the cDNA was prepared using SP6 RNA polymerase and translated in rabbit reticulocyte lysates, and the product was identified as human myeloperoxidase by immunoprecipitation with rabbit anti-human myeloperoxidase antibody. By Northern hybridization analysis of RNA from leukemic cells, it was shown that myeloperoxidase mRNA is abundantly expressed in human promyelocytic HL-60 and mouse myeloid leukemia NFS-60 cells. Furthermore, the results of Southern hybridization analysis of human genomic DNA suggest that there are one or two genes for myeloperoxidase in the human haploid genome.

Amino Acid Sequence↗
PubMed1987

Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. Large scale purification, subunit functions, and subcellular localization.

Messenger RNA capping enzyme (GTP:mRNA guanylyltransferase) purified from yeast Saccharomyces cerevisiae consisted of two polypeptides (45 and 39 kDa) and possessed two enzymatic activities, i.e. mRNA guanylyltransferase and RNA 5'-triphosphatase (Itoh, N., Mizumoto, K., and Kaziro, Y. (1984) J. Biol. Chem. 259, 13923-13929). In this paper, we describe an improved procedure suitable for the large scale purification of the enzyme. The steps include glass beads disruption of the cells and several ion-exchange and affinity column chromatographies. The enzyme was purified from kilogram quantities of yeast cells to apparent homogeneity. The purified enzyme had an approximate Mr of 180,000 and consisted of two heterosubunits of 80 and 52 kDa and had the same two enzymatic activities as above. We consider that this is the more intact form of the enzyme. Using the in situ assays on sodium dodecyl sulfate-polyacrylamide gels, RNA 5'-triphosphatase, and mRNA guanylyltransferase activities were located on the 80- and 52-kDa chains, respectively. In agreement with this, the 52-kDa enzyme-[32P]GMP complex was formed on incubation of the enzyme with [alpha-32P]GTP. Guinea pig antisera against purified yeast capping enzyme recognized both 80- and 52-kDa chains in Western blot analysis. The antibody did not cross-react with the enzymes from rat liver. Artemia salina, or vaccinia virus. Nuclear localization of the enzyme was demonstrated by immunofluorescence microscopy.

Acid Anhydride Hydrolases↗
PubMed1987

Characterization of recombinant human granulocyte-colony-stimulating factor produced in mouse cells.

Mouse C127I cells were transformed with a chimeric plasmid consisting of bovine papillomavirus DNA and human granulocyte-colony-stimulating factor (G-CSF) cDNA placed under the control of the SV40 early promoter. The transformed cells secreted constitutively a high level of human G-CSF, 10-20 micrograms/ml in a low-serum medium. The secreted G-CSF has been purified to homogeneity by a two-step procedure including gel filtration and hydrophobic column chromatography. The purified recombinant G-CSF runs as a single band with an apparent Mr of 19,000 on a polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This value corresponds to that of the native human G-CSF purified from the medium conditioned by human carcinoma CHU-2 cells. The recombinant human G-CSF was as active as native G-CSF in vitro in supporting proliferation of mouse NFS-60 cells and stimulating colony formation from human as well as mouse bone marrow cells. When the recombinant human G-CSF was subcutaneously administrated into mice, a remarkable stimulation of granulopoiesis and splenomegaly was observed.

Animals↗
PubMed1987

Human gene coding for granulocyte-colony stimulating factor is assigned to the q21-q22 region of chromosome 17.

Granulocyte-colony stimulating factor (G-CSF) is a member of colony stimulating factors which regulate the proliferation and differentiation of hematopoietic progenitor cells. A full-length cDNA clone coding human G-CSF was used as a hybridization probe to detect homologous sequence in human-mouse somatic cell hybrids, flow-sorted human chromosomes, and in situ human metaphase chromosomes. The results indicate that the gene encoding human G-CSF is on the q21-q22 region of chromosome 17, which is involved in translocation of t(15;17) (q23;21) in human acute promyelocytic leukemia.

Animals↗