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J R Smiley

Publications and source records attributed to J R Smiley.

65 records · Page 4Linked to original sources
PubMed1980

Herpes simplex virus thymidine kinase transcripts are absent from both nucleus and cytoplasm during infection in the presence of cycloheximide.

Nick-translated DNA from a recombinant plasmid containing the herpes simplex virus type 1 thymidine kinase gene was used as a probe for the synthesis of thymidine kinase RNA. The recombinant plasmid was generated by inserting the 3.5-kilobase fragment derived by BamHI digestion of herpes simplex virus type 1 DNA into plasmid pBR322. At 8 h after infection, cytoplasmic and nuclear RNA hybridized to 14% and 19% of the recombinant DNA probe, respectively. However, no significant hybridization was found with either nuclear or cytoplasmic RNA extracted from cells infected and maintained in the presence of cycloheximide. This suggests that no thymidine kinase-related RNA was synthesized in the absence of alpha polypeptides, and supports the hypothesis that the alpha polypeptides effect new thymidine kinase RNA synthesis rather than being involved in processing or transport of thymidine kinase RNA. In cells infected and maintained in the presence of the arginine analog canavanine, about 2 to 3% of the plasmid DNA was found to hybridize with cytoplasmic and nuclear RNA. However, when a recombinant plasmid DNA containing only thymidine kinase coding sequences was used, no significant hybridization was found. The inhibition of thymidine kinase transcription by canavanine suggests that thymidine kinase belongs to the beta 2 kinetic class.

Canavanine↗
PubMed1979

Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus.

We have constructed a hybrid plasmid by insertion of the thymidine kinase (TK) gene of Herpes simplex virus (HSV) type I at the BamHI site on Escherichia coli plasmid pBR322. The restriction endonuclease cleavage site map for the viral DNA fragment was determined for ten nucleases, and the insert in the recombinant plasmid has the same restriction nuclease digestion pattern as bona fide viral DNA. This result indicates that the plasmid contains an accurate copy of the viral DNA. The viral TK gene carried on the plasmid can be introduced into mammalian cells where it is expressed. This source of DNA with a selectable marker should be of considerable practical use in gene-transfer experiments in mammalian cells.

Chromosome Mapping↗
PubMed1978

A herpes simplex virus 1 integration site in the mouse genome defined by somatic cell genetic analysis.

Transfection experiments with HSV 1 in which one uses herpes simplex virus (HSV) thymidine kinase (TK) as a selectable prototrophic marker yield two classes of transformed cells: stable and unstable. In this report, we test the hypothesis that the stability phenotype can be explained by virus genome integration into a recipient cell chromosome. The method of analysis is by means of somatic cell genetics. We have isolated a series of microcell hybrids between a TK- Chinese hamster cell line and a transformed mouse cell line expressing the TK encoded by HSV 1. Several of the hybrid lines contain a single murine chromosome and express only the viral TK. Karyotypic analysis of these hybrids and of TK- derivatives generated by BrdUrd counterselection reveals that the TK+ phenotype is correlated with the presence of the terminal portion of the long arm of a specific murine chromosome. Results of extensive isozyme analyses of the hybrids and their TK- segregants fully corroborate the karyologic data. The results are consistent with the hypothesis that the viral tk gene is covalently integrated into this chromosomal region which itself does not appear to carry the endogenous murine tk locus. Other more complicated models are discussed. Our findings also show that somatic cell genetics can be used to localize viral integration sites in host chromosomes with high resolution.

Animals↗
PubMed1978

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.

Adenoviruses, Human↗
PubMed1976

Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.

Adenoviridae↗
PubMedDate not supplied

Genetic stability of the VP2 hypervariable region of four infectious bursal disease virus isolates after serial passage in specific-pathogen-free chicken embryos.

Infectious bursal disease virus strains U2, 586, L1, and Q2 were isolated from pooled bursal samples collected from commercially reared broilers. These viruses were propagated in specific-pathogen-free (SPF) embryonated chicken eggs for 24 or 25 passages. Nucleotide sequences of a 743-bp reverse transcription (RT)/polymerase chain reaction (PCR) product containing the VP2 hypervariable region were compared before and after passage of the viruses in embryonated chicken eggs. To determine the genetic stability of the viruses, each isolate was compared with its egg-passed ancestor; virus isolates were not compared with each other. When the restriction enzymes BstNI and MboI were used, no differences were observed in the restriction fragment length polymorphism profiles of the RT/ PCR products after embryo passage. After embryo passage, six nucleotide changes were identified in the viruses. Among the four viruses examined, these nucleotide changes resulted in a total of five amino acid changes. The amino acid changes were S-222-L in virus 586, K-249-N in viruses U2, L1, and Q2, and G-281-V in virus Q2. Three of the five amino acid changes occurred at residue 249. The convergent nature of this residue shift in three of four of the chick embryo-passed viruses suggests the occurrence of a functional, as opposed to random, mutation. The original isolates caused typical signs of infectious bursal disease in 3-wk-old SPF chicks. Their embryo-passed ancestors also produced typical signs of infectious bursal disease in 3-wk-old SPF chicks, suggesting the amino acid mutations observed did not affect virulence of the viruses.

Animals↗