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F Yamao

Publications and source records attributed to F Yamao.

At least 19 recordsLinked to original sources
PubMed1995

Abnormal integrity of the nucleolus associated with cell cycle arrest owing to the temperature-sensitive ubiquitin-activating enzyme E1.

A mouse cell mutant, ts85, containing the temperature-sensitive ubiquitin-activating enzyme was arrested in G2 phase at the non-permissive temperature. In the arrested cells, azure C, a nucleolus-specific stain, revealed a U-shaped or ring-shaped arrangement of nucleolar lobes with an unstained region in the center. Silver staining of the nucleolar organizer region (NOR) and fluorescence in situ hybridization (FISH) with rDNA both gave signals in azure C-positive regions. Electron microscopic examination revealed a cloud of unidentified electron-dense particles (diameter approximately 70 nm) in the azure C-negative center space. When the arrested cells were released into M-phase, we observed the association of NOR-bearing chromosomes with a pulverization-like abnormality. FISH with rDNA and NOR silver staining demonstrated that the pulverization-like abnormality was restricted to NORs. The frequent occurrence of persistent nucleolar material in prophase and prometaphase of the stressed cells after release indicated a delayed dissociation of the nucleolus that brought about the abnormal chromosomes in M-phase. ts85 cells transfected with the mouse E1 cDNA recovered growth at the non-permissive temperature and no longer showed abnormal nucleolar morphology. It seems that the ubiquitin system plays a role in the dissolution of the nucleolus, possibly involving the NOR-bearing chromosomes.

Animals↗
PubMed1995

Ubiquitin-activating enzyme, E1, is phosphorylated in mammalian cells by the protein kinase Cdc2.

The ubiquitin-activating enzyme (E1) is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 was found to be phosphorylated in cells of a mouse mammary carcinoma cell line, FM3A. Peptide mapping of trypsin digests of labeled E1 indicated that two oligopeptides were mainly phosphorylated in vivo. The same oligopeptides were also labeled in vitro on Cdc2 kinase-mediated phosphorylation of E1, affinity-purified from the same cell line. The Cdc2 kinase is a key enzyme playing a pivotal role in G2/M transition in the cell cycle. The phosphorylation of one of the two oligopeptides was prominent at the G2/M phase of the cell cycle, and dependent upon the Cdc2 kinase activity in vivo since it was significantly reduced in tsFT210, a mutant cell line deficient in Cdc2 kinase. Mutation analysis indicated that the serine residue at the fourth position of the E1 enzyme was a phosphorylation site of Cdc2 kinase. These findings suggest that E1 is a target of Cdc2 kinase in the cell, implying that the ubiquitin system may be dynamically involved in cell cycle control through phosphorylation of this key enzyme.

Amino Acid Sequence↗
PubMed1993

Conditional resistance to thymineless death predominantly selects DNA synthesis-deficient mutants of mammalian cells.

Temperature-sensitive growth mutants of the mouse mammary carcinoma cell line FM3A were isolated by selecting survivors of thymidylate starvation for a limited time at the restrictive temperature (39.5 degrees C). Nineteen lines of independent isolates were established and all were found to be deficient in DNA synthesis. Cell-cell hybridization with authentic mutant lines of FM3A demonstrated that the mutants fell into three complementation groups, which were deficient in DNA polymerase alpha or ubiquitin-activating enzyme E1 or both.

Animals↗
PubMed1992

tsBN75 and tsBN423, temperature-sensitive x-linked mutants of the BHK21 cell line, can be complemented by the ubiquitin-activating enzyme E1 cDNA.

tsBN75 and tsBN423 are independently isolated temperature-sensitive (ts) mutants of the BHK21 cell line for cell growth. Both tsBN75 and tsBN423 belong to the same complementation group and show G2 block at the nonpermissive temperature. Both were efficiently transformed to ts+ cells with the mouse and human cDNA encoding the ubiquitin-activating enzyme, E1. While no transformants of tsBN423 cells had a DNA content greater than the parental 2C, several ts+ transformants of tsBN75 cells acquired a multiploid DNA content. These data thus demonstrate the function of the human and mouse E1 cDNAs and further suggest that E1 functions in more than one step in cell cycle progression.

Animals↗
PubMed1991

Levels of tRNAs in bacterial cells as affected by amino acid usage in proteins.

Transfer RNAs of Mycoplasma capricolum were separated by two-dimensional polyacrylamide gel electrophoresis, and the relative abundance of each of the 28 known tRNA species was measured. There existed a correlation between the relative amount of isoacceptor tRNAs and the frequency in choosing synonymous codons that could be translated by the isoacceptors. Furthermore, it was observed that the total amount of tRNAs for a particular amino acid was paralleled by the composition of the amino acid in ribosomal proteins. A similar relationship was obtained from reexamination of the previous data on Escherichia coli tRNAs, suggesting that the amount of tRNAs for an amino acid is affected by the usage of the amino acid in proteins.

Amino Acid Sequence↗
PubMed1990

Prokaryotic genetic code.

The prokaryotic genetic code has been influenced by directional mutation pressure (GC/AT pressure) that has been exerted on the entire genome. This pressure affects the synonymous codon choice, the amino acid composition of proteins and tRNA anticodons. Unassigned codons would have been produced in bacteria with extremely high GC or AT genomes by deleting certain codons and the corresponding tRNAs. A high AT pressure together with genomic economization led to a change in assignment of the UGA codon, from stop to tryptophan, in Mycoplasma.

Anticodon↗
PubMed1990

The organization and evolution of transfer RNA genes in Mycoplasma capricolum.

The genes for presumably all the tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been cloned and sequenced. There are 30 genes encoding 29 tRNA species. This number is the smallest in all the known genetic systems except for mitochondria. The sequences of 9 tRNA genes of them have been previously reported (1-3). Twenty-two genes are organized in 5 clusters consisting of nine, five, four and two genes (2 sets), respectively. The other eight genes exist as a single transcription unit. All the tRNAs are encoded each by a single gene, except for the occurrence of two tRNA(Lys)(TTT) genes. The arrangement of tRNA genes in the 9-gene cluster, the 5-gene cluster, the 4-gene cluster and one of the 2-gene clusters reveals extensive similarity with a part of the 21-tRNA gene cluster and/or the 16-tRNA gene cluster in Bacillus subtilis, respectively. The results suggest that the present M. capricolum tRNA genes have evolved from large tRNA gene clusters in the ancestral Gram-positive bacterial genome common to M. capricolum and B. subtilis, by discarding genes for redundant as well as non-obligate tRNAs, so that all the codons may be translated by as small a number of tRNAs as possible.

Base Sequence↗
PubMed1989

Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria.

The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.

Amino Acids↗
PubMed1988

Evolutionary dynamics of tryptophan tRNAs in Mycoplasma capricolum.

Mycoplasma capricolum uses two tryptophan codons, the "universal" nonsense codon UGA and the universal codon UGG. The bacterium contains two tryptophan tRNAs, one with anticodon UCA, (U: 2'-O-methyl U derivative), and the other with CCA (5'-C: partially 2'-O-methylated). tRNAUCA would translate codons UGA and probably UGG by wobbling. tRNACCA is much less charged by tryptophan in the cells than tRNAUCA, and the intracellular amount of tRNACCA is 5-10 times lower than that of tRNAUCA. The genes for these two tRNAs are separated by a terminator-like structure in a single operon. In vitro transcription experiments suggest that the predominance of tRNAUCA over tRNACCA results from the attenuation of transcription by this terminator-like structure.

Base Sequence↗
PubMed1988

Directional mutation pressure and transfer RNA in choice of the third nucleotide of synonymous two-codon sets.

Bacterial species have diverged into a series of families, some with high G + C content in their DNA, and other with high A + T content, resulting, respectively, from G.C- and A.T-directional mutation pressures. Such mutation pressure (G.C/A.T pressure) may be an important determinant for codon usage. It has also been suggested that tRNA acts as a selective constraint for determining codon usage. We have studied the relation between G.C/A.T pressure and tRNA constraints in determining choice of the third nucleotide of eight two-codon sets, using codon usage data obtained from protein genes in four bacterial species, Mycoplasma capricolum, Bacillus subtilis, Escherichia coli, and Micrococcus luteus, and in liverwort (Marchantia polymorpha) chloroplasts. The genomic G + C contents of these range from 25% to 74%. The results demonstrate that tRNA levels act additively to A.T and G.C pressure in affecting contents of A (pairing with *UNN anticodons, in which *U indicates a 2-thiouridine derivative) and C (pairing with GNN anticodons) or G (pairing with CNN anticodons), respectively, in third nucleotide positions of codons.

Chloroplasts↗
PubMed1988

Mischarging mutants of Su+2 glutamine tRNA in E. coli. I. Mutations near the anticodon cause mischarging.

In order to select the mischarging mutants of Su+2 glutamine tRNA, auxotrophic amber mutants of E. coli K12 which cannot be suppressed particularly by Su+2 were screened. By utilizing these mutants, cysam235 and metam3, several tens of mischarging mutants of Su+2 were isolated, as those conferring altered suppression patterns for a set of tester amber mutants of bacteria and phages. Nucleotide sequence analysis revealed that the mutation sites were found to be exclusively at psi 37 residue located at the 3'-end of anticodon loop, changing it to either A37 or C37. These mutants were obtained as those suppressing cysam235, and not metam3. From these, secondary mutants were selected. In these mutants suppression patterns were further altered by the additional base substitutions, capable of suppressing metam3. Such mutants were obtained exclusively from A37 and not from C37 mutant tRNA. Additional mutations to A37 were found to be either A29 or C38, which are located at the lowermost two base pairs in anticodon stem. The mischarging sites in Su+2 glutamine tRNA locate in the newly detected region of tRNA, differing from the previous case of Su+3 tyrosine or Su+7 tryptophan tRNAs. Implication of this finding is discussed on L-shaped tRNA molecule in relation to aminoacyl-tRNA synthetase recognition. Suppression patterns given by the double-mutants, A37A29 and A37C38, were consistent with the observation that the mutant tRNAs interact with tryptophanyl-tRNA synthetase.

Anticodon↗
PubMed1988

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs.

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.

Base Sequence↗
PubMed1988

Escherichia coli glutaminyl-tRNA synthetase: a single amino acid replacement relaxes rRNA specificity.

We described previously the isolation, by genetic selection, of a mutant of Escherichia coli glutaminyl-tRNA synthetase that misaminoacylates supFtRNATyr with glutamine. The single amino acid change responsible for the mischarging phenotype was identified at amino acid 235 in the translated glnS gene. The mutant, called glnS7, has an Asp----Asn change, and studies with the purified glnS7 gene product show it may mischarge a number of presumably different tRNAs. We have carried out extensive homology searches that show E. coli glutaminyl-tRNA synthetase shares regions of homology with other aminoacyl-tRNA synthetases, although little apparent similarity at the site of the glnS7 mutation. In addition to the conserved 'HIGH' motif implicated in aminoacyl adenylate formation, there are regions of homology of glutaminyl-tRNA synthetase with other synthetases which may be involved in tRNA binding. These include short stretches of homology in sequences acting as a tRNA 'anchor' as well as homology of some aminoacyl-tRNA synthetases to a recently identified motif in ribonucleoproteins. Therefore, our results show that E. coli glutaminyl-tRNA synthetase may share with other aminoacyl-tRNA synthetases regions responsible for tRNA binding, while other regions of the protein, of which the glnS7 mutation may be a component, are responsible for tRNA discrimination.

Acylation↗
PubMed1987

The ribosomal protein gene cluster of Mycoplasma capricolum.

The DNA sequence of the part of the Mycoplasma capricolum genome that contains the genes for 20 ribosomal proteins and two other proteins has been determined. The organization of the gene cluster is essentially the same as that in the S10 and spc operons of Escherichia coli. The deduced amino acid sequence of each protein is also well conserved in the two bacteria. The G + C content of the M. capricolum genes is 29%, which is much lower than that of E. coli (51%). The codon usage pattern of M. capricolum is different from that of E. coli and extremely biased to use of A and U(T): about 91% of codons have A or U in the third position. UGA, which is a stop codon in the "universal" code, is used more abundantly than UGG to dictate tryptophan.

Amino Acid Sequence↗
PubMed1987

Occurrence of unmodified adenine and uracil at the first position of anticodon in threonine tRNAs in Mycoplasma capricolum.

Codon usage pattern in the threonine four-codon (ACN) box in Mycoplasma capricolum is strongly biased towards adenine and uracil for the third base of codons. Codons ending in uracil or adenine, especially ACU, predominate over ACC and ACG. This bacterium contains two isoacceptor threonine tRNAs having anticodon sequences AGU and UGU, both with unmodified first nucleotides. It would thus appear that ACN codons are translated in an unusual way; tRNA(Thr)(AGU) would translate the most abundantly used codon ACU exclusively, because adenine at the first anticodon position can, according to the wobble rule, pair only with uracil of the third codon position. The tRNA(Thr)(UGU) would mainly be responsible for translation of three other codons, ACA, ACG, and ACC. Anticodon UGU would also be used for reading codon ACU as a redundancy of tRNA(Thr)-(AGU), as deduced from the mitochondrial code where unmodified uracil at the first anticodon position can pair with adenine, cytosine, guanine, and uracil by four-way wobble. The tRNA(Thr)(AGU) has much higher sequence homology to tRNA(Thr)(UGU) from M. capricolum (88%), Bacillus subtilis (77%) and Escherichia coli (86%) than to tRNA(Thr)(GGU) from B. subtilis (66%) and E. coli (63%), suggesting that tRNA(Thr)-(AGU) has been derived from tRNA(Thr)(UGU), but not from tRNA(Thr)(GGU).

Adenine↗