Search PubMed⌕ Search

Biomedical subjects

H Iba

Publications and source records attributed to H Iba.

At least 73 records · Page 4Linked to original sources
PubMed1985

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.

Animals↗
PubMed1984

Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts.

The transforming activity of the cellular src (c-src) gene as well as of hybrid genes between viral and cellular src was tested by constructing derivatives of Rous sarcoma virus DNA in which all or part of the viral src gene (v-src) was replaced by the corresponding portion of the c-src gene. After these derivatives were introduced into chicken embryo fibroblasts by transfection, replication-competent virus was recovered, which induced the expression of p60src at a level equivalent to p60v-src expression in cells infected with Rous sarcoma virus wild type. Replacement of the portion of the v-src gene, either upstream or downstream of the Bgl I site, with the homologous portion of the c-src gene resulted in fully transforming viruses. On the other hand, the virus stock obtained from cells transfected with Rous sarcoma virus DNA containing the entire c-src gene had a very low titer of focus-forming virus, while it contained a high titer of infectious virus. We present evidence that the rare small foci are formed by mutant viruses generated from the original c-src-containing virus. These results indicate that overproduction of the c-src gene product does not cause cell transformation, and that this proto-oncogene is subject to a relatively high rate of mutation when incorporated in a retrovirus genome, resulting in the acquisition of transforming capacity.

Animals↗
PubMed1983

Transcriptional regulation of three double-stranded RNA segments of bacteriophage phi 6 in vitro.

Three double-stranded RNA segments of bacteriophage phi 6 (L, M, and S) were transcribed in vitro by a virion-associated RNA polymerase. Regulation of L transcription was distinct from regulation of M and S transcription. Transcription of the L segment, which codes for early proteins, required manganous ion and high concentrations of all four ribonucleoside triphosphates and was inhibited by polyamines such as spermine. Transcription of the M and S segments, which code for late proteins, required manganous or magnesium ion and relatively low concentrations of all ribonucleoside triphosphates except GTP and was enhanced by polyamines. Optimal conditions for L transcription were more stringent than those for M and S transcription. These two apparently different patterns produced in in vitro transcription presumably reflect the two distinct in vivo transcription patterns; i.e., (i) similar amounts of three single-stranded RNA species were transcribed from the three corresponding segments of double-stranded RNA (early pattern) and (ii) a much larger amount of single-stranded RNA species was transcribed from M and S segments than from the L segment (late pattern). The early transcription pattern may be changed into the late pattern by a change of environment, such as substrate concentration. This suggests that the different enzymatic properties under the different environmental conditions of the virion-associated transcriptase are responsible for the transcriptional regulation throughout the infection cycle of bacteriophage phi 6.

Cations, Divalent↗
PubMed1982

Virion-associated RNA polymerase of bacteriophage phi 6 synthesizes three complete transcripts of double-stranded RNA genome in vitro.

Three single-stranded RNA transcripts synthesized in vitro by a virion-associated RNA polymerase of bacteriophage phi 6 were sequenced at their 5'- and 3'-termini. The sequences agreed with those of the + strands of the 3 double-stranded RNA segments [FEBS Lett. (1982) 141,111-115]. The results show that the transcription by phi 6 RNA polymerase initiates exactly at the 3'-ends of the template RNAs (-strands of the genomic RNA) and terminates exactly at the 5'-ends.

Coliphages↗
PubMed1981

The chromosome structure and the cell cycle of Caulobacter crescentus. Isolation and analysis of envelope-free nucleoids.

Envelope-free nucleoids were isolated from an asymmetrically dividing bacterium, Caulobacter crescentus. In the nucleoid fraction, most of the DNA and nascent RNA in the cell and about 2% of the total cellular proteins were recovered. The sedimentation coefficient of the nucleoid was constant (1260 S) during the G1 period of the swarmer cell cycle and increased to 1940 S during the S period. Since both replicating (S period) and non-replicating (G1 period) chromosomes had a similar superhelical concentration, the increase in the sedimentation coefficient was simply explained by duplication of the nucleoid structure. The duplicated nucleoid was shown to segregate prior to the cell division. The pulse-labeled proteins recovered in the nucleoid fraction contained several stage-specific species, most of which were detected at the beginning of S period.

Bacterial Proteins↗
PubMed1981

Nucleotide sequence of a cloned cDNA copy of TMV (cowpea strain) RNA, including the assembly origin, the coat protein cistron, and the 3' non-coding region.

The cloned cDNA derived from the 3' end of cowpea strain (Cc) RNA of tobacco mosaic virus (TMV) has been sequenced. Substantial sequence information of 1,060 nucleotides from the 3' end of the RNA reveals some interesting features: (1) the coat protein cistron corresponds to residues 210-701 from the 3' end. Some errors in the amino acid sequence previously reported have been corrected and the revised total length of the coat protein is 162 amino acid residues. The capping site of the coat protein mRNA is at residue 711 from the 3' end of genome RNA. (2) The assembly origin of reconstitution is positioned within the coat protein cistron at residue 369-461 which can be formed into a highly base-paired hairpin loop structure. The sequence, GAXGUUG, in the loop region and a triplet-repeated purine base tract surrounding the loop are found. These structural features are common to assembly origins of both Cc and vulgare strains. (3) We find the sequence highly homologous to, but distinct from, the genuine assembly origin. It will be called the pseudo-assembly origin, which is located in the corresponding region to the assembly origin of the vulgare strain, outside the coat protein cistron. There is also the sequence, GAXGUUG, in the middle of the region. (4) In the 5' flanking region of the coat protein cistron, a long reading frame, probably of 30 K protein, is found. The coding region is terminated in the coat protein cistron and thus the 30 K protein and the coat protein cistrons overlap. (5) The 3' non-coding region is 209 residues long and can be folded into a possible tRNA-like structure. Surprisingly, we find that the 3' terminal sequence of Cc RNA is not very similar to that of vulgare RNA but extensively homologous to that of turnip yellow mosaic virus (TYMV) RNA.

Amino Acid Sequence↗
PubMed1981

A lytic enzyme in the bacteriophage phi 6 virion.

The results of our biochemical studies on the early step of phi 6 infection were schematically summarized in Fig. 6. To our knowledge, no other bacteriophage was reported to synthesize its mRNA in the parental subviral particle. The unique feature of the life cycle of this phage seems to be supported by two enzymes associated with the virion; the lytic enzyme and the RNA polymerase. The study of the RNA polymerase reaction observed in vitro showed the mode of the phi 6 RNA synthesis clearly and seems to be very useful to know the mechanism of the initial viral RNA synthesis in vivo.

Bacteriolysis↗
PubMed1980

Assignment of viral proteins to the three double-stranded RNA segments of bacteriophage phi 6 genome: translation of phi 6 messenger RNAs transcribed in vitro.

Pseudomonas phaseolicola bacteriophage phi 6 has a double-stranded (ds) RNA genome in three segments (L, M, S) which can serve as templates for in vitro transcription by phi 6 nucleocapsid. Single-stranded (ss) RNA (l, m, s) synthesized in vitro functioned as messenger RNA of viral proteins in an Escherichia coli cell-free protein-synthesizing system. Each of the three ssRNA species was isolated in virtually pure form and translated, providing a means of determining the polypeptides encoded by each segment. From the analysis of polypeptide products by SDS-polyacrylamide gel electrophoresis, coding assignments of three dsRNA segments were established. The major structural proteins P8 and P9 were shown to be encoded by the S segment transcript (s). The membrane proteins P3, P6, and P10 are encoded by the M segment transcript (m). The nucleocapsid proteins P1, P4, and P7 are probably synthesized by the L-segment transcript (l), but there remained a possibility that P4 and P7 are encoded also by the M-segment transcript (m). The nucleocapsid protein P2 was not synthesized in detectable amounts by transcripts of any segments in our experiments. This protein is known to be synthesized in small amounts in vivo. The lytic enzyme P5 could not be identified owing to the difficulty in separating P5 from products of the endogenous protein-synthesizing activity of E. coli extracts.

Bacteriophages↗
PubMed1980

Semi-conservative transcription of double-stranded RNA catalyzed by bacteriophage phi 6 RNA polymerase.

Treatment of Pseudomonas phaseolicola double-stranded RNA bacteriophage phi 6 with sodium deoxycholate converted the virions to nucleocapsids, which had in vitro RNA polymerase activity. The incorporation of [3H]UMP continued for at least 7 h. The initial incorporation was detected as intermediate RNA. Radioactivity was chased first into three segments of double-stranded RNA, and then into small, medium, and large species of single-stranded RNA successively via the intermediate RNA. Several copies of single-stranded RNA at least were synthesized from a template. The RNA synthesis clearly took place by a semi-conservative mechanism with respect to templates. That is, 5-bromo UTP was incorporated into one strand of double-stranded RNA to make a hybrid RNA of brominated and unbrominated strands. Furthermore, one strand of the 3H-labeled parental double-stranded RNA was shown to be released as single-stranded RNA.

Bacteriophages↗
PubMed1978

Rate of major protein synthesis during the cell cycle of Caulobacter crescentus.

The rate of major protein synthesis was examined during the synchronous differentiation of Caulobacter crescentus. Total cell proteins were pulse-labeled with [35S]methionine at different times in the swarmer cell cycle and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The rates of synthesis of total cell proteins and of about one-half of the individual major proteins examined increased through G1 and S periods but remained nearly constant during G2 period. The rates of synthesis of the other half of the individual major proteins either increased continuously throughout the swarmer cell cycle or doubled during S period. One stage-specific protein was also detected in late S period. For most of the major proteins examined, the rate of synthesis in the swarmer cell was less than that in the stalked cell. It seemed that, before the onset of G2 period, the Caulobacter cell was already able to synthesize each major protein at the additive rate of the two progeny cells. Compared to the stability of cellular proteins, the functional degradation rate of mRNA coding for individual major proteins was rapid, with half-lives of 0.4 to 5.8 min. It thus seems that the rate of major protein synthesis mainly reflects the transcriptional control of gene expression.

Bacteria↗
PubMed1977

Chromosome replication in Caulobacter crescentus growing in a nutrient broth.

The pattern of chromosome replication in the Caulobacter crescentus cell cycle was studied by examining the rate of deoxyribonucleic acid (DNA) synthesis during synchronous growth in a fast-growth nutrient broth. As reported previously for the cell cycle in a slow-growth minimal medium (Degnen and Newton, 1972), the Caulobacter cell cycle (at the fastest available growth rate) in nutrient broth consisted of three distinct periods in terms of DNA synthetic activity. The swarmer-cell cycle consisted of a presynthetic period (G1), synthetic period (S), and postsynthetic period (G2) of 30, 50, and 35 min, respectively, whereas the stalked-cell cycle consisted of S and G2 periods of 50 and 35 min, respectively. Synchronously growing cells in the nutrient broth were stained to visualize nuclear bodies. Two nuclear bodies could be discerned in both swarmer and stalked cells, and four could be discerned in predivisional cells. DNA content per cell was determined chemically and found to be about the same in swarmer and stalked cells; it was equivalent to roughly twice the value expected from the kinetic complexity reported previously (Wood et al., 1976) for Caulobacter DNA.

Bacteria↗
PubMed1977

Stalkless mutants of Caulobacter crescentus.

A stalk, a single falgellum, several pili, and deoxyribonucleic acid (DNA) phage receptors are polar surface structures expressed at a defined time in the Caulobacter crescentus cell cycle. When mutants were isolated as DNA phage phiCbK-resistant or ribonucleic acid (RNA) phage phiCp2-resistant, as well as nonmotile, strains, 5 out of 30 such mutant isolates were found not to possess stalks, but did possess inactive flagella. These stalkless mutants were resistant simultaneously to both DNA and RNA phages and did not possess pili and DNA pendent stalkless mutants. All motile revertants simultaneously regained the capacity to form stalks and susceptibility to DNA and RNA phages. It is suggested that a single mutation pleiotropically affects stalk formation, flagella motility, and coordinate polar morphogenesis of pili and DNA phage receptors. The stalkless mutants grew at a generation time similar to that of the wild-type strain at 30 degrees C. Cell size and morphology of a stalkless mutant, C. crescentus CB13 pdr-819, were also similar to those of the wild-type strain, except for the absence of a stalk. In addition, the CB13 pdr-819 predivisional cells were partitioned into smaller and larger portions, indicating asymmetrical cell division, as in the wild-type strain. From these results, it is suggested that swarmer cells undergo transition to cells of a stalked-cell nature without stalk formation and that the cell cycle of the stalkless mutant proceeds in an ordered sequence similar to that defining the wild-type cell cycle.

Bacteria↗