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R C Condit

Publications and source records attributed to R C Condit.

13 recordsLinked to original sources
PubMed1991

Genetic and molecular biological characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component.

The gene affected by five previously isolated temperature-sensitive (ts) mutants (ts 10, ts 18, ts 38, ts 39, ts 44) of vaccinia virus strain WR constituting a single "normal" complementation group has been characterized. Marker rescue and DNA sequence analysis show that the five members of the complementation group map in an open reading frame, ORF 18R, which spans the HindIII I-G junction and has the capacity to encode a 77.6-kDa protein. The nucleotide sequence change responsible for temperature sensitivity in each of the five mutants was determined. Two of the mutants, ts 38 and ts 44, have the identical nucleotide change and may therefore be sisters. Northern blot analysis demonstrates that ORF 18R is transcribed at both early and late times during infection. Two distinct early transcripts have been observed which are 5' coterminal and which contain a 518 nucleotide 5' untranslated region. The long early transcript spans the entire 18R gene while the 3' end of the shorter early transcript maps to an early transcription termination signal contained within the 18R coding sequence. The 5' ends of the late transcripts have been mapped to a family of AUG proximal sites using both S1 nuclease and primer extension analysis. Primer extension analysis also identifies additional late 5' ends which map between nucleotides -500 and -1000 relative to the ORF 18R AUG.

Amino Acid Sequence↗
PubMed1991

Phenotypic characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component.

Genetic and biochemical evidence is presented which shows that the product of the vaccinia virus gene 18R is a virion protein. Western blot analysis of virion proteins using anti-18R serum detects a 78,000-Da protein, localized in the virus core. Of five ts mutants which map to gene 18R, two mutants, ts 10 and ts 44, possess thermolabile virions. Temperature shifts performed during single-step growth of ts 44 suggest that precursors required for virion maturation accumulate during nonpermissive infections with ORF 18R mutants and that protein synthesis is required for recovery from nonpermissive condition.

Animals↗
PubMed1991

A vaccinia virus isatin-beta-thiosemicarbazone resistance mutation maps in the viral gene encoding the 132-kDa subunit of RNA polymerase.

The mutation in a vaccinia virus mutant resistant to inhibition by isatin-beta-thiosemicarbazone was mapped by marker rescue. DNA from the resistant mutant was cloned into cosmid and plasmid vectors and tested for its ability to convert wild-type vaccinia virus to IBT resistant virus in a helper-mediated marker rescue protocol. Resistance was mapped in this way to a 0.9-kb DNA fragment derived from the HindIII A fragment of vaccinia genome. Southern blot hybridization using this DNA as a probe demonstrated that the 0.9-kb fragment is contained within the DNA sequence encoding the second largest subunit of vaccinia RNA polymerase, rpo132. Thus, mutation of rpo132 can cause resistance to IBT in vaccinia virus.

Blotting, Southern↗
PubMed1991

Genetic and molecular biological characterization of a vaccinia virus gene which renders the virus dependent on isatin-beta-thiosemicarbazone (IBT).

We have sequenced and analyzed the transcription of a gene capable of rendering vaccinia virus (VV) dependent upon isatin-beta-thiosemicarbazone (IBT) for growth. Marker rescue analysis of an IBT-dependent mutant of VV, IBTd-1, and a temperature-sensitive mutant of VV, ts56, both of which require IBT to grow at 40 degrees, showed that both lesions mapped to gene G2R. VV mutants with G2R deletions were constructed and shown to also be dependent upon IBT for growth. The nucleotide sequence changes responsible for IBTd-1, ts56, and the gene G2R deletion mutants were determined, and taken together show that IBT dependence results from inactivation of the orf G2R gene product. Gene G2R, which has the capacity to encode a 26-kDa protein, is transcribed solely early during infection. The 1.3-kb mRNA contains a 5' untranslated region of almost 600 nucleotides, and terminates about 20 nucleotides downstream from an early transcription termination signal. Transcription analyses of three flanking genes, as well as the map positions of the VV mutants ts11 and ts60 are also presented.

Adenine Nucleotides↗
PubMed1991

Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus.

The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.

Cell Line↗
PubMed1990

Orthopoxvirus genetics.

Genetic analysis of orthopoxviruses has contributed substantially to our understanding of the functional organization of the poxvirus genome, and individual mutants provide invaluable tools for future studies of poxvirus biology. Deletion and transposition mutants, localized primarily in the termini of the genome, may be particularly useful for studying virus host range and pathogenicity. Numerous drug resistant and dependent mutants provide keys to understanding a wide variety of virus genes. A large number of well-characterized ts mutants, clustered in the center of the virus genome, are taking on an increasingly important role in research on the function of essential poxvirus genes. Genetic characterization of orthopoxviruses has progressed rapidly during the past decade, and one can reasonably anticipate a time when mutants will be available for the study of any poxvirus gene. Considerable progress toward this goal can be achieved through organized attempts to integrate and further characterize existing mutant collections and through the continued isolation and characterization of deletion, drug resistant, and ts mutants using established techniques. The most exciting possibility is that soon techniques will be available for directed mutagenesis to conditional lethality of any essential poxvirus gene.

Poxviridae↗
PubMed1990

Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase.

We have used plasmid clones spanning the region encoding the 132-kDa subunit of the cowpox virus RNA polymerase (CPV rpo 132) to marker rescue each of five vaccinia virus (VV) temperature sensitive (ts) mutants, ts 27, ts 29, ts 32, ts 47, and ts 62, which together constitute a single complementation group. The experiments fine-map the vaccinia mutations to a 1.3-kb region containing the 3' end of the CPV rpo 132 gene. Phenotypic characterization shows that all five mutants are affected to varying extents in their ability to synthesize late viral proteins at the nonpermissive temperature, similar to other ts mutants with lesions in the 22- and the 147-kDa subunits of the VV RNA polymerase. Two mutants, ts 27 and ts 32, exhibit a delay in the synthesis of late viral proteins at both the permissive and the nonpermissive temperatures. We conclude that the five VV mutants affect the 132-kDa subunit of the VV RNA polymerase. Additional genetic experiments demonstrate intragenic complementation between ts 62 and three other members of this complementation group, ts 27, ts 29, and ts 32.

Animals↗
PubMed1990

Extrachromosomal recombination in vaccinia-infected cells requires a functional DNA polymerase participating at a level other than DNA replication.

Homologous recombination was measured in vaccinia-infected cells cotransfected with two plasmid recombination substrates. One plasmid contains a vaccinia protein lacZ coding region bearing a 1.1 kb 3' terminal deletion while the other plasmid contains a non-promoted lacZ coding region bearing a 1.1 kb 5' terminal deletion. Homologous recombination occurring between the 825 bp of lacZ common to both plasmids regenerates a functional lacZ gene from which B-galactosidase expression was measured. The entire 3 kb lacZ gene was used as a positive control. A panel of thermosensitive mutants was screened in cells either transfected with the positive control plasmid or cotransfected with the recombination substrates. A DNA - mutant, ts42, known to map to the viral DNA polymerase gene was found to be defective in recombination. Significantly, other DNA - mutants, ts17 or ts25, or other DNA polymerase mutants did not exhibit a defect in recombination similar to ts42. Inhibitors of viral DNA synthesis did not uniformly affect recombination. Cytosine arabinoside and aphidicolin inhibited B-galactosidase expression from the recombination substrates but not from the positive control plasmid, whereas hydroxyurea enhanced expression from both. Marker rescue with the cloned wildtype DNA polymerase gene repaired the defect in ts42. Southern and western analyses demonstrated that B-galactosidase activity was consistent with a recombined lacZ gene and unit size 116 kDa protein. Measurement of plasmid and viral DNA replication in cells infected with the different DNA - mutants indicated that recombination was independent of plasmid and viral DNA replication. Together these results suggest that the vaccinia DNA polymerase participates in homologous recombination at a level other than that of DNA replication.

Animals↗
PubMed1990

Structure and expression of the vaccinia virus gene which prevents virus-induced breakdown of RNA.

Three noncomplementing vaccinia virus temperature-sensitive mutants, ts4, ts22, and ts23, exhibit an abortive late phenotype characterized by the simultaneous cessation of protein synthesis, the breakdown of rRNA and viral mRNA, and an increase in intracellular concentrations of 2'-5'-linked oligoadenylates late during infection at the nonpermissive temperature (R.F. Pacha and R.C. Condit, J. Virol. 56:395-403, 1985; R.J. Cohrs, R.C. Condit, R.F. Pacha, C.L. Thompson, and O.K. Sharma, J. Virol. 63:948-951, 1989). We have identified the virus gene affected by the abortive late mutants, determined its DNA sequence, and analyzed its transcription. The gene resides in the HindIII A DNA fragment, it has a predicted coding capacity of 57 kilodaltons, and it is transcribed both early and late during infection. The early transcript of the abortive late gene is unusual; it contains a 426-nucleotide 5' untranslated region, and it must be synthesized by transcription through an early transcription termination signal which is located in the middle of the gene in a hairpin loop structure. DNA sequence and transcription analysis of two flanking genes is also presented.

Amino Acid Sequence↗