ISL1: a new transposable element in Lactobacillus casei.

The genome structures of a temperate Lactobacillus phage, phi FSW, and its virulent mutants, phi FSVs, were examined by restriction, heteroduplex and nucleotide-sequence analyses. The results showed that two out of three phi FSVs had the same 1.3 kbp insertion (designated as ISL1) at different positions in the phi FSW sequence.

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).

A novel combination of K-ras and myc amplification accompanied by point mutational activation of K-ras in a human lung cancer.

Amplifications of two oncogenes, c-K-ras-2 and c-myc, were found in a human lung giant cell carcinoma (LGCC) Lu-65, which is maintained in nude mice. The extent of c-K-ras-2 and myc amplifications were estimated to be 10- and 8-fold, respectively, by means of the Southern hybridization procedure. In addition, NIH3T3 cells were transformed by transfection of Lu-65 DNA and the transforming gene was identified as c-K-ras-2.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Sarcoma-virus-related RNA sequences in normal rat cells.

A rat type C virus spontaneously activated from the NRK (normal rat kidney) cell line was found to have two major size classes of viral RNA subunits sedimenting at 35 and 30 S. Virus-producing cells contained both RNA species, while normal "virus-free" rat cells contained primarily virus-specific 30S RNA species. A DNA transcript, specific for Kirsten sarcoma virus, prepared from virus activated in nonproducer BALB/c cells originally transformed by Kirsten sarcoma virus and rendered specific for the virus by absorption of sequences related to mouse helper virus hybridized only with the 30S RNA species of virus-producing rat cells and normal rat cells.

Sarcoma and helper-specific RNA tumor virus subunits in transformed nonproducer mouse cells activated to produce virus by treatment with bromodeoxyuridine.

A tumor line (58-2T) was established from a slowly growing tumor in a BALB/c mouse inoculated with M58-2 cells. The latter clonal cell line was isolated after bromodeoxyuridine treatment as a flat variant from nonproducer BALB/3T3 cells transformed by the Kirsten sarcoma virus. The 58-2T cells produced type C virus with two discrete virus-specific RNA species.

Intracellular viral RNA species in mouse cells nonproductively transformed by the murine sarcoma virus.

The size and quantity of virus-specific RNA in five non-virus-producing mouse cells transformed by the Moloney isolate of murine sarcoma virus (MSV) was determined. Hybridization of RNA from transformed cells with the [(3)H]DNA product of the RNA-directed DNA polymerase of the murine sarcoma-leukemia virus was used to detect and quantitate virus-specific RNA. The amount of virus-specific RNA in non-virus-producing cells was less than one-sixth of that found in virus-producing cells.

Mutants of nonproducer cell lines transformed by murine sarcoma virus. 3. Detection and characterization of RNA specific for helper and sarcoma viruses.

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells ( approximately 2.5 x 10(3)/cell) whether infected with RLV or activated to produce virus with BrdU.