Sequence and gene expression analyses of plasmid pHPM8 from Helicobacter pylori reveal the presence of two operons with putative roles in plasmid replication and antibiotic activity.

The DNA sequence of a 7.8-kb Helicobacter pylori plasmid, pHPM8, was determined. Six open reading frames (ORFs) were present. Ribonuclease protection studies showed that ORF1/ORF2 and ORF3/ORF4 genes are organized in operons possibly involved in plasmid replication and in production of a peptide with antibiotic activity, respectively. Finding areas of pHPM8 with a high level of identity to H. pylori chromosomal DNA supported the hypothesis that recombination occurs between plasmids and the chromosome of H. pylori.

Molecular cloning and functional expression of the gene for a cotton Delta-12 fatty acid desaturase (FAD2).

Two overlapping genomic clones spanning 16.5 kb of cotton DNA were found to encompass a Delta-12 fatty acid desaturase (FAD2-3) gene. A partial FAD2-3 cDNA clone was also analyzed. The FAD2-3 gene has one large intron of 2967 bp entirely within its 5'-untranslated region, only 12 bp upstream from the ATG initiation codon. Several potential promoter elements, including several light-responsive motifs, occur in the 5'-flanking region. The continuous FAD2-3 coding region is 1155 bp and would encode a protein of 384 amino acids. The polypeptide has four putative membrane-spanning helices, indicative of an integral membrane protein, and is most likely localized in the endoplasmic reticulum. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.

Characterization of a 3.5-kbp plasmid from Helicobacter pylori.

A 3.5-kbp plasmid (pHPM180) was isolated from Helicobacter pylori (HPM180) and the DNA sequence was determined. Two open reading frames (ORF1 and ORF2) were identified which could encode polypeptides of 54,517 and 27,629 Da, respectively. Ribosome binding and promoter consensus sequences were identified, as well as two 232-bp direct repeats and four 22-bp direct repeats. DNA sequence homology was found between pHPM180 ORF1 and a 684-base pair HindIII fragment from a 5.6-kbp H. pylori plasmid. ORF1 showed amino acid homology with six replication proteins from bacterial plasmids with theta-type replicons. Additional sequence identity was found between pHPM180 noncoding DNA and a segment of H. pylori pHPK255, a plasmid that replicates via a rolling circle type mechanism. A ribonuclease protection assay determined that ORF1 was transcribed in H. pylori HPM180.

Localization of 3H-thymidine-labeled HSV-1 in latently infected rabbit trigeminal ganglion cells.

Although current data favor conservation of virus in a nonreplicating form during latency, the actual host cell-virus relationship during this quiescent period remains an enigma. The purpose of this study was to develop a highly specific probe for direct localization of the HSV type 1 (HSV-1) genome in an animal model that closely mimics human disease. Tritium-labeled HSV-1 was inoculated onto trigeminal ganglion (TG) neurons in vitro and onto New Zealand white rabbit corneas in vivo. During acute infection in vivo and after establishment of latency in vivo or suppressed infection in vitro the TG neurons were processed for autoradiography. Silver grains were localized over nuclei of 8-10% of TG neuron cell bodies during suppressed infection in vitro. Acute infection in vivo resulted in the localization of label over 5-10% of neuron cell bodies and satellite cells per section. During latency the label appeared over nuclei of 1-10% of TG bodies per section. This study shows that directly labeled HSV-1 can be found in TG neuron nuclei both in vivo and in vitro. It also suggests that HSV genetic material is lost from certain neurons when latency is established.

Expression of endogenous murine leukemia virus in transplantable tumor cells and DNA methylation of the viral sequences.

A cell line derived from early passage of a transplantable tumor in athymic mice that initially had been injected with human breast adenocarcinoma cells produced a substantial amount of RNA sequences related to the Kirsten murine leukemia virus. The cell line derived from later passage of transplantation of these tumors diminished viral RNA production. Expression of the viral RNA sequences in these cell lines is inversely correlated with the level of viral DNA methylation in the mouse genome.

Sequence relationships between Kirsten retrovirus genomes and the genomes of other murine retroviruses.

RNA sequence relationships between the genomes of the Kirsten murine sarcoma virus (MSV-K) complex, the Kirsten murine leukemia virus (MuLV-K) complex, the Gross murine leukemia virus (MuLV-G), and the Moloney murine leukemia virus (MuLV-M) were investigated. Sedimentation analyses revealed the expected 30 and 34 S RNA subunits in the MSV-K complex and a previously undetected 30 S RNA subunit accompanying the 34 S RNA subunit in the MuLV-K complex. Nucleic acid hybridization data indicated that each Kirsten virus 30 S RNA subunit had about 40% sequence homology with the RNA genome of MuLV-G, although these sequences were only partially homologous between the two 30 S subunits. In contrast, the MuLV-K 34 S RNA subunit had 96% sequence homology with the MuLV-G genome, whereas the MSV-K 34 S RNA subunit displayed only 71% sequence homology with the MuLV-G genome. Similar relationships were indicated by oligonucleotide fingerprinting. The oligonucleotide data, taken with published sequence data on the MuLV-G and MuLV-M genomes, enabled us to construct partial sequence maps of the MuLV-K 34 S RNA subunit and the MSV-K 34 and 30 S RNA subunits. The sequence arrangements indicated that (1) the MuLV-K 34 S RNA subunit is a variant of the MuLV-G genome; (2) the MSV-K 34 S RNA subunit is a recombinant molecule, which maintains the length of its leukemia virus parent; and (3) the MSV-K 30 S RNA subunit may have been generated from the MuLV-K 34 S genome by a two-stage process, culminating in the retention of parental sequences only within the U5 and U3 noncoding segments and within several amino-terminal coding segments. Further examination of published retrovirus genome sequences revealed several strategically situated sets of potential recognition signals for transcription and translation and suggested a model for genetic recombination based on mRNA splicing signals and areas of limited sequence homology. This model may explain how foreign gene elements can be inserted into retrovirus genomes to generate either functional or defective recombinant retroviruses.

Inhibition of the release of virion-associated murine leukemia virus genomic RNA by vesicular stomatitis virus.

The genomic RNAs of murine leukemia virus (MuLV) and vesicular stomatitis virus (VSV) were distinguishable based on a difference in sedimentation coefficient, polyadenylic acid content, and nucleotide sequence homology. Applying these biochemical characteristics, we found that the release of virion-associated MuLV genomic RNA from virus-producing cells was inhibited by VSV as early as 2 h after superinfection. This finding offers an explanation to our previous inability to detect MuLV(VSV) pseudotype formation during mixed infection with VSV and MuLV.

Quantitative measurement of intracellular RNA genomes of Rauscher murine leukemia virus by competition hybridization in DNA excess.

The technique of competition hybridization in DNA excess was used to determine the intracellular distribution of RNA genomes of Rauscher murine leukemia virus. An examination of subcellular RNA fractions revealed that 59% of intracellular viral RNA genomes were associated with the nuclear-enriched fraction, 41% with the cytoplasmic fraction, and 18% with the polysomal-enriched fraction. Also, an analysis of total cellular RNA disclosed that 20% of intracellular viral RNA genomes were polyadenylated and 80% were not polyadenylated.

Search for oncogenic viruses in human prostate cancer.

Electron microscopic, immunologic, and biochemical methods have been used in an attempt to detect and characterize oncornaviruses in human prostatic carcinoma (PCa) and benign prostatic hyperplasia (BPH), and in prostates of mice of high and low mammary cancer or leukemia strains. Ultrastructural examination of 37 PCa and nine BPH specimens has revealed the presence of particles resembling type C virus in five cases of PCa and one of BPH, and also two different types of intracisternal virus-like particles in seven other cases of PCa. Type B virus particles have been observed in prostate of old mice of high mammary cancer strains, while type C virus particles have been found in the prostates of most mice of all the ten strains examined. Immunofluorescence tests with sera from patients with PCa and BPH and with cells derived in vitro from PCa have shown that sera of patients with PCa contained antibodies directed mainly against Forssman-like and tumor-related antigens. In immunofluorescence tests of antisera to major proteins of oncornaviruses with cells of PCa and BPH tissues grown in vitro, positive reactions have been obtained with antisera to p30 protein of murine, feline, and simian type C viruses. Fixed immunofluorescence (FIF) tests of sera of PCa (38%) and BPH (25%) and of some normal donors (27%) gave positive cytoplasmic reaction with mouse prostate cells infected with Soehner-Dmochowski murine sarcoma virus (SD-MSV). Immunoferritin tests of 11 sera positive by FIF gave ferritin labeling of type C virus particles in the SD-MSV-infected mouse prostate cells...

Alteration of the syncytium-forming property of XC cells by productive Moloney leukemia virus infection.

The effect of productive murine leukemia virus (MuLV) infection of the syncytium-forming property of XC cells was studied MuLV-Moloney-infected XC cells, designated XC(M), initially went through a period of spontaneous syncytium formation. The syncytia then disappeared, and XC(M) cells continued to propagate and produce both infectious MuLV and MuLV group-specific antigens. However, XC(M) cells became refractory to syncytium formation that was induced by Kirsten, Friend, Rauscher, and Moloney strains of murine oncornaviruses. These data suggest that XC(M) cells lost their syncytium-forming ability as a result of productive MuLV infection.

Quantitative nucleotide sequence relationships of mammalian RNA tumor viruses.

A molecular hybridization technique has been used to quantitatively measure the nucleotide sequence relationships of selected mammalian RNA tumor viruses. Reciprocal cross-hybridization tests were done in which a given radioactively labeled, viral genomic RNA species was annealed with an excess of unlabeled, complementary DNA product synthesized in endogenously instructed reverse transcriptase reactions. Hybrid formation was measured with pancreatic RNase A. Three representative mammalian RNA tumor virus groups were examined: murine viruses, simian viruses, and feline viruses. The results of reciprocal cross-hybridization testing have revealed that the murine viruses consist of four distinctly related subgroups: (i) the Friend leukemia virus/Rauscher leukemia virus subgroup, (ii) the Gross leukemia virus subgroup, (iii) the Moloney sarcoma virus subgroup, and (iv) the Kirsten sarcoma virus subgroup. Simian sarcoma virus, the only simian virus examined, appeared to share limited interspecies sequence relationships with members of the other virus groups and in particular with Kirsten sarcoma virus. Of the two members of the feline virus group tested, Rickard feline sarcoma virus and RD-114, each was placed in a separate, unrelated subgroup. Rickard feline sarcoma virus exhibited limited sequence relatedness with members of the other virus groups, whereas RD-114 exhibited none.

Strandedness and complementarity of DNA from long-term RNA-dependent DNA polymerase reactions of Soehner-Dmochowski murine sarcoma virus.

The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA.

Structural characteristics and nucleotide sequence analysis of genomic RNA from RD-114 virus and feline RNA tumor viruses.

The results of molecular hybridization experiments have demonstrated that the RNA genome of RD-114 virus has extensive nucleotide sequence homology with the RNA genome of Crandell virus, an endogenous type C virus of cats, but only limited homology with the RNA genomes of feline sarcoma virus and feline leukemia virus. The genomic RNAs of RD-114 virus and Crandell virus also had identical sedimentation coefficients of 50S. A structural rearrangement of genomic RNA did not exist within released RD-114 virions, whereas a structural rearrangement of genomic RNA did occur within feline sarcoma virions and feline leukemia virions after release from virus-producing cells.

Structural rearrangement and subunit composition of RNA from released Soehner-Dmochowski murine sarcoma virions.

Two types of genomic, high-molecular-weight RNA species were found in Soehner-Dmochowski murine sarcoma virions released from virus-induced rat tumor cells grown in tissue culture. The type of RNA species observed depended on the length of exposure of the tumor cells to radioactive precursor. Early RNA of virions labeled up to 4 h with radioactive uridine had a sedimentation coefficient of 50S, and late RNA of virions labeled for 24 h had a sedimentation coefficient of 58S. Thermal transitions of early and late RNA indicated a difference in the configuration or structure of these two types of RNA. The late RNA may represent either a different configurational state of the early RNA or an aggregate molecule of two early RNA components joined together. Heat dissociation revealed that the major subunit of both RNA types was a 28S species, which was not susceptible to degradation by the addition of micrococcal nuclease to virions. A transitional, intermediate RNA species with a sedimentation coefficient of 37 to 40S was detected when early RNA was dissociated by dimethyl sulfoxide or heat at temperatures suboptimal for complete conversion. No free RNA subunit components were detected in virions harvested at intervals as short as 30 s or 5 min. A model for the assembly of genomic RNA from 28S RNA subunits is proposed.