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F Imamoto

Publications and source records attributed to F Imamoto.

At least 37 records · Page 2Linked to original sources
PubMed1992

Construction and characterization of an Escherichia coli mutant deficient in the metY gene encoding tRNA(f2Met): either tRNA(f1Met) or tRNA(f2Met) is required for cell growth.

The Escherichia coli metY gene, encoding tRNA(f2Met), was split by the kanamycin-resistance-encoding gene. The resulting mutant exhibited the same growth rate as the wild type, indicating that tRNA(f2Met) is not indispensable as is the case with the metZ gene encoding tRNA(f1Met) [Kenri et al., Gene 103 (191) 31-36]. beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or a trpA'::lac'Z fusion gene, in the metY mutant. The lac repressor from the lacI gene and the chimeric protein from a hupB'::lac'Z fusion gene, whose start codons are GUG, were also synthesized efficiently in the insertion mutant. These results provide evidence that tRNA(f2Met) is not essential for growth of E. coli and that the start codons, AUG and GUG, are both recognized by tRNA(f1Met), a major N-formyl methionine-specific tRNA, in the tRNA(f2Met)-depleted cells. We were unable to construct mutants deficient in both tRNA(f1Met) and tRNA(f2Met) by P1 phage-mediated transduction with the metY and metZ mutations. Moreover, the ampicillin-resistance marker of the pUC9 plasmid carrying metZ+ was not cured at 42 degrees C in host cells with the polAts and metY-metZ double mutations. These results indicate that either tRNA(f1Met) or tRNA(f2Met) is required for the growth of E. coli.

Blotting, Southern↗
PubMed1992

Preferential binding of E.coli histone-like protein HU alpha to negatively supercoiled DNA.

Binding specificity of histone-like HU alpha protein to supercoiled DNA was examined by gel retardation assay and chemical probing with OsO4. The latter method was proved to be a unique means for detecting torsional tension restrained in supercoiled plasmid in the presence of HU alpha. It was shown that HU alpha protein has preferential affinity to negatively supercoiled DNA relative to relaxed, nicked and linearized DNAs. There were two modes for binding of HU alpha to the supercoiled DNA: one was the binding associated with topological changes in DNA and the other was relatively strong binding, probably specific to certain particular structures of DNA. It was suggested that HU in vivo interacts preferentially with the regions deformed under torsional stress or with the metabolically active regions along DNA.

Bacterial Proteins↗
PubMed1991

Participation of the histone-like protein HU and of IHF in minichromosomal maintenance in Escherichia coli.

The closely related Escherichia coli genes, hupA, hupB, himA and himD (hip), encode the bacterial histone-like protein subunits, HU-2, HU-1, IHF chi and IHF beta, respectively. We report here that E. coli minichromosomes [plasmids (2.7-12.2 kb) with oriC] carrying the intact mioC region were unable to transform mutants deficient in both HU and integration host factor (IHF), whereas they could transform mutants deficient in either HU or IHF as efficiently as the wild-type strain. Minichromosomes carrying a deletion of the proximal part of mioC or a DnaA box just upstream from mioC could not transform cells deficient in IHF, but could transform cells deficient in HU. These results suggested that HU and IHF participate in minichromosomal replication from oriC in E. coli.

Bacterial Proteins↗
PubMed1991

Construction and characterization of an Escherichia coli mutant with a deletion of the metZ gene encoding tRNA (f1Met).

The Escherichia coli metZ gene encoding tRNA (f1Met) was replaced by the chloramphenicol-resistance-encoding gene. The resulting mutant exhibited slightly lower growth rates as compared to the wild type at 37 degrees C or 42 degrees C, but grew apparently slower than the latter at 30 degrees C, indicating a slight cold sensitivity of growth. beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or trpA'::lac' Z fusion gene, in the metZ deletion mutant. The lac repressor from the lacI gene and the chimeric protein from a hupB' ::lac'Z fusion gene, whose start codons are GUG, were also synthesised in the deletion mutant. These results provide evidence that tRNA (f1Met) is not essential for growth of E. coli and that the start codons, AUG and GUG, are both recognized by tRNA (f1Met), a minor N-formyl methionine-specific tRNA, in the tRNA (f1Met)-depleted cells.

Blotting, Southern↗
PubMed1991

Presence of circulating anti-c-myb oncogene product antibodies in human sera.

Bacterially expressed mouse c-myb protein was purified from E. coli extracts and used as antigen to screen human sera for circulating anti-c-myb oncogene product antibodies. Using Western blotting, we have found that human sera contain IgG antibodies to the c-myb gene product. The percentage of positive sera in cancer patients appears to be dependent upon cancer type and is significantly higher in breast-cancer patients than in normal donors: 43% of sera from breast-cancer patients are positive, whereas in neuroblastoma cancer patients the production of IgG anti-c-myb appears to be rare. No significant correlation was observed between the presence of circulating anti-c-myb antibodies and the expression of the c-myb gene in breast tumors.

Antibodies↗
PubMed1991

Effects of prokaryotic termination signals on RNA polymerase II transcription in HeLa cells.

Three prokaryotic termination signals, the Escherichia coli trp attenuator, lambda 4S RNA terminator, and lambda tR1 terminator, were examined as to their effects on transcription in vivo in HeLa cells. The trp attenuator inhibited the expression of downstream genes in an orientation-dependent manner, but both the lambda 4S RNA and lambda tR1 terminators did not. Furthermore, a point mutation of the attenuator that disrupted the secondary structure of mRNA abolished this inhibitory effect. These results suggest that an attenuator-like secondary structure is effective in inhibiting transcriptional elongation by RNA polymerase II.

Bacteriophage lambda↗
PubMed1990

HU-1 mutants of Escherichia coli deficient in DNA binding.

We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.

Amino Acid Sequence↗
PubMed1990

Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial F1F0-ATPase and construction of mutant yeast lacking the protein.

A gene coding for yeast 15-kDa protein, a regulatory factor of mitochondrial F1F0-ATPase, was isolated. The cloned gene was disrupted in vitro and mutant strains that did not contain the 15-kDa protein were constructed by transformation of yeast cells with the disrupted gene. The ATP-synthesizing activity of the mutant mitochondria was the same as that of wild-type cells, suggesting that the 15-kDa protein is not required for mitochondrial oxidative phosphorylation. Collapse of the membrane potential induced ATP-hydrolyzing activity of F1F0-ATPase of the mutant mitochondria but not of normal mitochondria. Activation of the enzyme was also observed during incubation of submitochondrial particles from mutant cells, but not of those from wild-type cells. Thus, it is inferred that the 15-kDa protein supports the action of an intrinsic ATPase inhibitor of the ATP-hydrolyzing activity of the enzyme upon de-energization of mitochondrial membranes.

Amino Acid Sequence↗
PubMed1990

Promoters and autogenous control of the Escherichia coli hupA and hupB genes.

Three start sites and a single start site for transcription of the hupB and hupA genes, respectively, have been identified in Escherichia coli. Preceding the RNA start sites are DNA sequences that conform to canonical promoter consensus sequences. The two most upstream promoters of the hupB gene function in vivo at comparable efficiency, while the third is not expressed significantly. Both hupB and hupA genes possess a DNA sequence with a rho-independent transcriptional terminator in their respective regions downstream from the coding regions. The hup genes are both transcribed in vivo into monocistronic mRNA molecules. Upon introduction of an HU-overproducing plasmid carrying either the hupB or the hupA gene into the wild-type and hup single deletion mutants, the intracellular levels of mRNA from the chromosomal hup genes are reduced. The HU-1 and HU-2 proteins both repress both hup genes, repression of the hupB gene being less efficient. The HU protein selectively represses mRNA synthesis starting at the hup promoters in the hupB promoter-CmR and hupA promoter-KmR fusion genes, but does not have a negative regulatory effect on mRNA synthesis from the true CmR and KmR promoters. These findings suggest that the signals for the actions of HU proteins are located in the DNA regions upstream from the sites near the 5' extremities of the coding regions of the hupB and hupA genes.

Bacterial Proteins↗
PubMed1990

Requirement of integration host factor (IHF) for growth of Escherichia coli deficient in HU protein.

Escherichia coli mutants deficient in histone-like protein, HU, and integration host factor (IHF) were constructed and their growth characteristics were examined. Mutants deficient in both HU and IHF grew slowly and were filamentous at both 30 degrees C and 37 degrees C. These mutants scarcely grew in LB broth at 42 degrees C. They formed minute colonies on LB plates at 42 degrees C and no colonies at 46 degrees C, indicating temperature-sensitive (ts) growth. On the contrary, mutants deficient in either HU or IHF were not ts for growth. These results indicate that IHF compensates for the absence of HU and permits normal cell growth; this suggests functional similarity between HU and IHF.

Bacterial Proteins↗
PubMed1990

Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast.

An intrinsic ATPase inhibitor and 9-kDa protein are regulatory factors of mitochondrial ATP synthase in Saccharomyces cerevisiae. A gene encoding the ATPase inhibitor was isolated from a yeast genomic library with synthetic oligonucleotides as hybridization probes and was sequenced. The deduced amino acid sequence showed that the precursor protein contains an amino-terminal presequence of 22 amino acid residues. Mutant strains that did not contain the inhibitor and/or the 9-kDa protein were constructed by transformation of cells with their in vitro disrupted genes. The disruption of the chromosomal copy in recombinant cells was verified by Southern blot analysis, and the absence of the proteins in the mutant cells was confirmed by Western blot analysis. All the mutants could grow on a nonfermentable carbon source and the oxidative phosphorylation activities of their isolated mitochondria were the same as that of normal mitochondria. However, an uncoupler, carbonylcyanide-m-chlorophenylhydrazone, induced marked ATP hydrolysis in the inhibitor-deficient mitochondria, but not in normal mitochondria. These observations suggest that the ATPase inhibitor inhibits ATP hydrolysis by F1F0-ATPase only when the membrane potential is lost.

Adenosine Triphosphate↗
PubMed1990

Maintenance of plasmids in HU and IHF mutants of Escherichia coli.

Complementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al. 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1. Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants). Mini-P1 plasmids and mini-F plasmids could not be introduced into the delta hupA-delta hupB double deletion mutant. Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the delta hupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the delta hupA-delta hupB double deletion mutant. The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable.

Amino Acid Sequence↗
PubMed1990

Characterization of HU-like protein from Bifidobacterium longum.

A HU-like protein (HBl) of Bifidobacterium longum was purified and characterized. HBl is heat-stable and acid-resistant, and has a molecular weight of about 9.1 kDa as estimated by its mobility on electrophoresis. HBl is intermediate in basicity (pI 9.8) between the HU-1 and HU-2 proteins of Escherichia coli, and is dissociated from a calf thymus DNA-cellulose column at 300-400 mM NaCl. Its amino acid composition shows many similarities with that of E coli HU. The NH2-terminal amino acid sequence of HBl also shows significant similarities to the consensus sequence deduced from the sequences of eleven HU-like proteins from prokaryotic sources. Chemical crosslinking analysis indicated that the HBl protein predominantly forms a homotypic dimer.

Amino Acid Sequence↗
PubMed1990

Genetic transformation of Lactobacillus casei by electroporation.

Lactobacillus casei IAM1045 was transformed with a plasmid pAM beta 1-1, a tra deleted derivative of pAM beta 1, by electroporation. Effective transformation was achieved in electroporation buffers of a wide range of pH values, and in all phases of cell growth tested, with highest frequency in the early log phase. Polyethylene glycol increased the transformation frequency, whereas divalent cations such as Mg2+, Ca2+ and Mn2+ at 0.25 mM decreased the frequency by 2 to 3 orders. Highly efficient transformation of approximately 10(-4)/viable cell was achieved under optimal conditions. A plasmid harboring the trpD, C, F, B and A genes from L casei RNL7 was introduced by electroporation into tryptophan auxotrophic L casei JCM1053. The resulting transformant was found to express the trp genes introduced.

Calcium↗
PubMed1990

Nucleotide sequences and genomic constitution of five tryptophan genes of Lactobacillus casei.

Five trp genes, trpD, trpC, trpF, trpB, and trpA, of Lactobacillus casei were cloned by transformation of tryptophan auxotrophic mutants of the respective trp genes in Escherichia coli. These trp genes appear to constitute an operon and are located in the above order in a segment of DNA of 6,468 base pairs. The entire nucleotide sequence of this DNA segment was determined. Five contiguous open reading frames in this segment can encode proteins consisting of 341, 260, 199, 406, and 266 amino acids, respectively, in the same direction. The amino acid sequences of these proteins exhibit 25.5-50.2% homology with the amino acid sequences of the corresponding trp enzymes of E. coli. Two trp genes, trpC and trpF, from L. casei can complement mutant alleles of the corresponding genes of E. coli. However, neither the trpA gene nor the trpB gene of L. casei can complement mutations in the E. coli trpA gene and the trpB gene, respectively, suggesting that the protein products of the L. casei and E. coli trpA and trpB genes, respectively, cannot form heterodimers of tryptophan synthetase with activity. Other features of the coding and flanking regions of the trp genes are also described.

Amino Acid Sequence↗
PubMed1989

Negative regulation of human insulin gene expression by the 5'-flanking region in non-pancreatic cells.

We have examined the effect of the 5'-flanking region of the human insulin gene on its expression in non-pancreatic cells. The presence of the region containing the insulin gene enhancer (-339 to -169 bp) markedly repressed the promoter activity of the insulin gene. This suppressive phenomenon was restored by the addition of forskolin or dibutyryl cAMP, suggesting that this region alone is not sufficient to repress completely insulin gene expression in the presence of extracellular stimuli which increase the intracellular cAMP level. The hypervariable region (HVR) located at -365 bp also repressed the promoter activity. These results show negative regulation of human insulin gene expression in non-pancreatic cells by these regions.

Animals↗
PubMed1989

Cooperative function of two separate enhancers of the human epidermal growth factor receptor proto-oncogene.

Two separate enhancers were identified both upstream and downstream of the promoter of the human epidermal growth factor receptor gene. Transcriptional enhancer activity was found to be associated with a region 1172 and 852 base pairs upstream of the most upstream RNA start site, and within a 530-base pair fragment located in the first intron about 2000 base pairs downstream of the most upstream RNA start site. These two separate enhancers stimulated promoter activity cooperatively in HeLa cells synthesizing the epidermal growth factor receptor. The enhancer downstream of the promoter functioned only in the presence of the other upstream enhancer. Several areas of sequences homology with viral and cellular enhancers were noted in both the upstream and downstream enhancers. The region essential for the downstream enhancer activity has 10 nuclear factor-binding sites.

Base Sequence↗