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N Tsuchida

Publications and source records attributed to N Tsuchida.

At least 91 records · Page 5Linked to original sources
PubMed1984

New shuttle vectors for Escherichia coli and Bacillus subtilis. I. Construction and characterization of plasmid pHY460 with twelve unique cloning sites.

We have constructed chimeric plasmid vectors, pHY460 and pHY310, from the streptococcal tetracycline resistance (TcR) plasmid pAM alpha 1 (9.2 kb) and the Escherichia coli vector pACYC177 (3.7 kb). These bifunctional plasmids can replicate and express the TcR gene in both E. coli and Bacillus subtilis. Plasmids pHY460 (7.0 kb) and pHY310 (4.8 kb) contain the TcR gene of pAM alpha 1 and the ampicillin resistance (ApR) gene of pACYC177. Both plasmids showed high transformation efficiency in both host cells. pHY460 was maintained stably in B. subtilis and, thus, is a useful shuttle vector functioning in E. coli and B. subtilis. The PvuI, PstI, BglI and BanI sites in the ApR gene and the HpaI, BalI and EcoRV sites in the TcR gene can be used for selection of recombinant plasmids by insertional inactivation. In addition, plasmid pHY460 has unique sites for SacII, BstEII, XbaI, AvaI and BamHI.

Bacillus subtilis↗
PubMed1984

Physical mapping of the virion and the prophage DNAs of a temperate Lactobacillus phage phi FSW.

We analysed the physical structure of the DNA of phi FSW, which is a temperate phage of Lactobacillus casei S-1. A circular restriction map of the virion DNA has been constructed with three restriction endonucleases, BamHI, SalI and XhoI. Other data indicated that the phage genome was circularly permuted. In lysogens, the DNA of the prophage was found to be linearized at a specific site and integrated into a specific locus of the host genome, with the same orientation in each case, as evidenced by Southern filter hybridization. We compared the physical structure of phi FSW with its three virulent mutants. One of them had a restriction map indistinguishable from that of phi FSW and two of them contained host-derived DNA sequence(s) in a specific region of the phi FSW genome (V-region). The prophage integration site was mapped on a different segment of the phage genome to the V-region. Derivation of virulent mutants from phi FSW is discussed in relation to the physical structure of the phage genome.

Bacteriophages↗
PubMed1983

Nucleotide sequence of the staphylokinase gene from Staphylococcus aureus.

We have determined the entire nucleotide sequence of a 1,4-kilobase segment containing the staphylokinase gene, sak, molecularly cloned from the bacteriophage S phi-C genome of Staphylococcus aureus. The probable coding region is 489 base pairs long and these base pairs are translated into a polypeptide of 163 amino acid residues (Mr = 18,490) with a presumed signal sequence of 27 amino acid residues at the NH2-terminal end. In regions adjacent to the sak structural gene a possible promoter sequence and three possible terminator sequences for transcription were found about 100 base pairs upstream from the initiation codon and about 300, 400, and 500 base pairs downstream from the termination codon, respectively; they are active in an in vitro transcription system using Escherichia coli RNA polymerase. The immunoactive 18,500-dalton and 15,500-dalton proteins corresponding to a precursor form before secretion and a mature form after secretion of the sak gene products, respectively, were identified by the E. coli maxicell system.

Base Composition↗
PubMed1983

Prophage Origin of a Virulent Phage Appearing on Fermentations of Lactobacillus casei S-1.

For protection from the abnormal fermentation of Lactobacillus casei S-1 caused by contamination of a virulent phage, phiFSV, the origin of this phage was studied. Morphologies, viral structural proteins, and DNA structures of three independent isolates of phiFSV were compared with those of phiFSW, which is lysogenized in strain S-1. The results showed (i) that the morphology of phiFSV phages is indistinguishable from that of phiFSW and (ii) that all viral structural components found in phiFSW are present in the particles of phiFSV's. In addition, restriction endonuclease analyses of viral DNA showed that the HindIII-digested fragments of phiFSW DNA, the sum of which covered at least 94.7% of this phage genome, were conserved in the phiFSV DNA digests. Results of Southern filter hybridization of the S-1 and prophage-cured cell (C239) DNAs with phiFSV DNA as a probe revealed that C239 had lost most of the phiFSV DNA sequence, whereas S-1 had about one copy of the phiFSV DNA sequence. These results indicate that virulent phage phiFSV is derived from the lysogenized phage phiFSW. Therefore, the appearance of phiFSV can be eliminated by using the prophage-cured derivative of S-1.

Journal Article↗
PubMed1983

Dissociation between transformed and differentiated phenotype in rat thyroid epithelial cells after transformation with a temperature-sensitive mutant of the Kirsten murine sarcoma virus.

Differentiated rat thyroid epithelial cells, infected in vitro with a temperature-sensitive mutant of the Kirsten murine sarcoma virus, expressed at the permissive temperature (33 degrees C) some phenotypic properties typical of transformed cells, including morphological features, colony formation in agar, and induction of tumors in newborn animals. Specific functional markers of these differentiated cells, i.e., synthesis/secretion of thyroglobulin, synthesis of thyroglobulin mRNA and iodide uptake, were blocked during growth at 33 degrees C. Normal morphology, failure to grow in agar, and the requirement of hormones for optimal growth were all restored after shifting to the temperature nonpermissive for transformation (39 degrees C), though the typical differentiated functions remained blocked. Infection with a leukemia helper virus clone (Moloney or Kirsten murine leukemia virus) did not lead to the loss of the differentiated phenotype of rat epithelial thyroid cells, thus demonstrating that the loss of the differentiated phenotype is caused by the sarcoma virus component. These results indicate that the expression of some of the phenotypic properties of transformed differentiated rat thyroid epithelial cells is under the direct control of the p21 thermosensitive activity, whereas the block in the expression of two typical differentiation markers of thyroid epithelial cells is irreversible and probably controlled by different mechanisms.

Animals↗
PubMed1982

Nucleotide sequence of the oncogene encoding the p21 transforming protein of Kirsten murine sarcoma virus.

The transforming protein of Kirsten murine sarcoma virus (Ki-MuSV) is a virally encoded 21-kilodalton protein called p21 kis. The sequences encoding p21 kis were genetically localized to a 1.3-kilobase segment near the 5' end of the viral genome by assaying the capacity of a series of defined deletion mutants of molecularly cloned Ki-MuSV DNA to induce focal transformation of mouse cells. Nucleotide sequencing of a portion of this region has led to the identification of an open reading frame of 567 nucleotides coding for p21 kis protein.

Animals↗
PubMed1982

Block in the expression of differentiation markers of rat thyroid epithelial cells by transformation with Kirsten murine sarcoma virus.

Well-differentiated epithelial cells, derived from primary cultures of normal rat thyroid glands (T-79 cells), as well as a cloned cell line also derived from normal rat thyroid glands (FRT-L cells) were infected with Kirsten murine sarcoma virus carrying outer coat of the helper Kirsten murine leukemia virus. Infected T-79 and FRT-L cells changed morphologically and began to proliferate rapidly, suggesting malignant transformation by the virus. Both cell lines can support the replication of both transformation-competent and transformation-incompetent viruses such as murine or rat leukemia viruses. Infected T-79 and FRT-L cells had a high colony-forming efficiency (68 and 64%, respectively) when grown in agar and formed tumors when transplanted s.c. into syngeneic rats. These tumors morphologically resemble undifferentiated adenocarcinomas, thus showing that Kirsten sarcoma virus carrying the outer coat of the helper Kirsten murine leukemia virus is able to transform differentiated epithelial cells. Transformed T-79 and FRT-L cells, in contrast to uninfected cells, neither secrete thyroglobulin concentrate iodide, two biochemical markers of differentiated thyroid function. Thus, expression of the differentiated phenotype is blocked as a consequence of cell transformation. The system described may be useful in studying epithelial cell carcinogenesis in terms of regulated expression of differentiated functions.

Animals↗
PubMed1981

Transformation of rat thyroid epithelial cells by Kirsten murine sarcoma virus.

Fischer rat thyroid epithelial cella (FRT) growing continuously in culture were infected with the Kirsten murine sarcoma virus KiMSV(KiMuLV) and found to produce this virus constitutively. Although the morphology of the FRT cells did not change appreciably, the cells became malignant after infection with KiMSV(KiMuLV) as shown by the growth of infected cels in semi-solid media (uninfected FRT cells did not grow) and by the tumorigenicity of infected when injected into syngeneic animals (uninfected FRT cells or FRT cells infected with non-transforming retro-viruses were not tumorigenic). The induced tumors morphologically resembled moderately differentiated carcinomas. Two markers of thyroid epithelial differentiation were absent in the original FRT clone and remained unexpressed after transformation. Fully differentiated rat thyroid epithelial cells (FRT-L cells) infected with another strain of the Kirsten murine sarcoma virus, the KiMSV(MolMuLV), were also transformed as demonstrated by the ability also of these cells to grow as carcinomas (after in vitro transformation) in syngeneic animals. Our results clearly demonstrate that the Kirsten murine sarcoma virus can transform in vitro cells of epithelial as well as of fibroblastic origin.

Animals↗
PubMed1981

The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes.

The Harvey and Kirsten strains of murine sarcoma virus encode enzymatically and serologically related p21 src proteins which are required for virally mediated cellular transformation. The genes in each virus encoding p21 show such extensive divergence from each other that cloned probes from these genes detect distinct sets of cellular genes in the DNA from several vertebrate species. These data suggest that cellular p21 sarc genes constitute a divergent family of vertebrate genes that can regulate the growth of cells.

Animals↗
PubMed1981

Structure and functions of the Kirsten murine sarcoma virus genome: molecular cloning of biologically active Kirsten murine sarcoma virus DNA.

The unintegrated closed circular form of viral DNA prepared from NIH3T3 cells infected with Kirsten murine sarcoma virus was cloned into bacterial plasmid pBR322. The closed circular DNA, which consisted of two different-sized populations, was enriched from the virus-infected cells, linearized with BamHI, and inserted into pBR322 DNA. Four different recombinant DNAs (clones 2, 4, 6, and 7) were obtained, and a physical map of each was constructed by using various restriction enzymes. Clone 4 DNA had the largest insertion, corresponding to a complete copy of the linear DNA. This suggested that this insertion contained two copies of the 0.55-kilobase pair long terminal redundant sequence. Clone 2 and clone 6 insertion DNAs had deletions of 0.2 and 0.5 kilobase pair, respectively, which mapped near the right end (3' side of viral RNA) of the linear DNA. Clone 7 DNA appeared to have a deletion of a single copy of the large terminal redundant sequence. Transfection of BALB3T3 cells with the clone 4 DNA insertion showed that this DNA had transforming activity. The efficiency of transfection with clone 4 Kirsten murine sarcoma virus DNA was enhanced eightfold by inserting EcoRI-cleaved viral DNA into the EcoRI site of pBR322. The EcoRI-inserted DNA produced foci with single-hit kinetics, suggesting that a single molecule of Kirsten murine sarcoma virus DNA can induce transformation. Results of transfections with EcoRI-inserted Kirsten murine sarcoma virus DNA cleaved with various restriction enzymes suggested that the first 3.3-kilobase pair region at the left end of the linear DNA is important for the initiation of transformation or maintenance of transformation or both.

Animals↗
PubMed1981

Identification of unintegrated forms of Kirsten murine sarcoma viral DNA and restriction endonuclease cleavage map of linear DNA.

We detected unintegrated linear 7.0-kilobase pair DNA and covalently closed circular DNA species in NIH3T3 cells recently infected with Kirsten murine sarcoma virus. Using the linear DNA, we constructed a restriction endonuclease cleavage map and compared it with the map of Harvey murine sarcoma virus. The restriction endonuclease maps of two segments, one 1.2 kilobase pairs (SmaI site) to 3.7 kilobase pairs (HindIII site) from the right end (corresponding to the viral 3' side) and the other 0.5 kilobase pair (SmaI and KpnI sites) to 0.9 kilobase pair (KpnI site) from the left end, were identical in the two virus types.

Animals↗
PubMed1979

A murine teratocarcinoma stem cell line carries suppressed oncogenic virus genomes.

Murine teratocarcinoma stem cells are nonpermissive for productive infection by a variety of DNA (polyoma and SV40 virus) and RNA (murine leukemia and sarcoma virus) tumor viruses whereas differentiated murine cells derived from the stem cells are permissive for productive (or abortive in the case of SV40) infection by these same viruses. The block to productive infection by these oncogenic viruses is at a postpenetration step in the replication cycle of these viruses but the precise level of the block has not been established for any of these viruses. In this report we describe teratocarcinoma-derived stem and differentiated cell lines which should be especially useful in determining the level of the block to replication of ecotropic murine leukemia virus in murine teratocarcinoma stem cells. The stem cell line, OTT6050AF1 BrdU, which is completely nonpermissive to productive infection by Moloney murine leukemia virus and consists of 97% pluripotent stem cells, contains DNA copies of an RNA tumor virus which is indistinguishable from the N-tropic murine leukemia virus of AKR mice. The stem cells are negative for expression of viral reverse transcriptase, p30 and gp69/71 and no virus is found by XC plaque assay or other biological tests. Differentiated cells established from the same teratocarcinoma tumor are 100% positive for viral gp69/71, p30, and produce large amounts of reverse transcriptase activity and N-tropic virus as detected by biological assay. The virus isolated from the differentiated cells is closely related, if not identical to AKR N-tropic virus by nucleic acid hybridization studies and is thus not an endogenous virus of the 129 strain of mice. The teratocarcinoma tumor from which the cell lines were established had been carried in 129 mice and perhaps at some time in the mouse passage history the tumors were infected (nonproductively) with the N-tropic virus. Regardless of the origin of this viral DNA, the OTT6050A derived stem and differentiated cell lines should be extremely useful in defining in stem cells the step at which ecotropic murine leukemia virus replication is blocked.

Animals↗
PubMed1978

A temperature sensitive mutant of Escherichia coli which does not allow replication of RNA phage at a high temperature.

A conditional mutant, referred to as RepR43, was isolated from Escherichia coli W2252 by N-methyl-N'-nitro-N-nitroso-guanidine mutagenesis. Although RepR43 does not permit growth of RNA phage beta at the restrictive temperature, 43 degrees C, cell growth and synthesis of macromolecules such as RNA and protein continue at a somewhat reduced rate. Several lines of evidence indicate that a RepR43 function is indispensable for normal phage RNA replication. In addition, this function appears to be involved in the maintenance of the perpetuated phage genome. The addition of 10% sucrose to the medium at the restrictive temperature resulted in the production of the phage, suggesting that the mutant cell might have an altered membrane organization which interferes with normal viral replication.

Coliphages↗
PubMed1978

Transmission electron microscopy surveillance of retroviruses in tissue culture cells prepared by the critical-point drying method.

Tissue culture cells grown on grids were processed by the critical-point drying whole-cell method. With the use of a conventional transmission electron microscope operating at 100 kV, this technique permitted visualization of intracytoplasmic organelles of unsectioned whole cells. The morphology of type C virus in the process of budding and also in extracellular locations closely resembled that revealed in thin sections. Prelimininary results of virus surveillance of tissue culture cells prepared by this technique was corroborated by the levels of reverse transcriptase activity in culture media and by immunofluorescence staining of viral antigens on the cell surface.

Antigens, Viral↗
PubMed1977

Analysis of DNA from adenovirus 12-transformed cells for virus-specific DNA sequence with viral DNA fragments cleaved with restriction endonuclease.

32P-Labeled adenovirus-12 (Ad-12) DNA was treated with restriction enzyme (EndoR-Hin dIII) isolated from Haemphilus influenzae (Rd strain) and the resulting 16 specific fragments were separated through gel electrophoresis. This kinetics of renaturation of each of the fragments was measured in the presence of unlabeled Ad-12-transformed hamster embryo cell, clone 9 (Ad-12HE-C19). more than 77% of the viral genome nucleotide sequence was present in Ad-12he-c19 cell DNA with 5 to 10 copies per haploid quantity of cell DNA of each of the sequences of 11 fragments examined; A, C, D, E, F, G, H, I, J (J1+J2), K, and L. However, it is suggested that only a part of the nucleotide sequence in B fragment may b present in the cell DNA.

Adenoviruses, Human↗
PubMed1975

Type-C virus-specific nucleic acid sequences in cultured rat cells.

Single-stranded DNA transcripts of rat type-C viruses prepared in the presence of actinomycin D, hybridized specifically to DNA of several rat cell cultures with no obvious qualitative or quantitative differences. Similar products prepared from a pseudo-type sarcoma virus with contributions from rat and mouse type-C viruses hybridized to both rat and mouse cellular DNA, while mouse viral transcripts did not hybridize to rat cell DNA. Viral RNA was detected in all rat cells by means of the rat viral DNA transcripts, with some differences between untreated low-passage cells and sister cultures treated with bromodeoxyuridine or bromodeoxyuridine and methylcholanthrene. Cells treated with both compounds were previously shown to be transformed and turorigenic, and these were distinguishable by kinetic analysis from the control cells.

Amino Acid Sequence↗
PubMed1975

Size of virus-specific RNA in B-34, a hamster tumor cell producing nucleic acids of type C viruses from three species.

B-34 is the designation of a hamster tumor-derived cell line induced by the Harvey sarcoma virus. This cell line produces virions which contain structural proteins common to edogenous hamster viruses and nucleic acid sequences of hamster, mouse, and rat origin. The sedimentation characteristics of the intracellular virus-specific RNA was determined in sucrose gradients after treatment with dimethylsulfoxide by molecular hybridization using complementary DNA of strict virus specificity. Hamster virus-specific RNA sedimented at 35S (major peak) as is characteristic of productive infection by type C leukemia viruses of other species. Rat virus-specific RNA sedimented at 30S which is characteristic of the sarcoma virus-related genome found in nonproducer cells transformed by Kirsten sarcoma virus. Both Harvey and Kirsten sarcoma viruses contain a related but not necessarily identical 30S rat-specific component which is also found in normal cultured rat cells. Mouse cells producing Harvey sarcoma virus also contain a rat-specific 30S RNA. Mouse virus-derived sequences also sedimented at 30S in B-34 cells and in a similar size range in Harvey virus-infected mouse cells. The possibility that the mouse and rat-derived sequences are present on a single 30S RNA species which would then be related to sarcomagenic potential is one attractive hypothesis suggested by these data.

Animals↗