Search PubMed⌕ Search

Biomedical subjects

K Peden

Publications and source records attributed to K Peden.

23 records · Page 2Linked to original sources
PubMed1983

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.

Adenosine Triphosphatases↗
PubMed1982

Homology between mammalian cell DNA sequences and human herpesvirus genomes detected by a hybridization procedure with high-complexity probe.

Mouse and human DNA used as in vitro-labeled "high-complexity" probes revealed hybridization between specific herpesvirus DNA fragments on Southern transfers and repetitive sequences present at 10(3) to 10(5) copies per mammalian cell genome. Several different sites of major cell-virus sequence homology have been detected in both the herpes simplex virus type 1 and type 2 genomes, and these are located predominantly within the L and S inverted repeat regions and near the center of the L unique region. The hybrids persisted even in relatively stringent conditions, and appear to correlate closely with some of the previously recognized regions of size heterogeneity in the viral genome. Cloned viral DNA fragments from each site hybridized to different sets of discrete bands and dispersed elements within restriction-endonuclease-digested genomic DNA from a variety of vertebrate species. Localized cell-virus homology was also detected in both the human Epstein-Barr virus and cytomegalovirus genomes.

Animals↗
PubMed1979

A study of the organisation of the ribosomal ribonucleic acid gene cluster of Neurospora crassa by means of restriction endonuclease analysis and cloning in bacteriophage lambda.

1. Total Neurospora crassa DNA was restricted with endonucleases and fragments carrying rRNA coding sequences were identified by hybridization with Xenopus laevis ribosomal DNA probes. 2. The repeating unit of the rRNA gene cluster was found to be 8.6 kbp, arranged in a head-to-tail fashion. 3. Digestion with Hind III yielded fragments of 3.4 kbp and 5.2 kbp and both were cloned. 4. Digestion with Eco RI yielded fragments of 2.2 kbp, 3.0 kbp and 3.4 kbp; the 3.0 kbp fragment was cloned. 5. Sequences coding for RNA (S-rRNA)1 of the smaller subribosomal particle were found (at least 90%) in the 2.2 kbp EcoRI subfragment of the 5.2 kbp Hind III fragment. 6. The coding sequences for the major RNA species (L-rRNA) of the larger subribosomal particle were located mainly (at least 95%) in the 3.4 kbp Hind III fragment. 7. For comparison, a Hind III digest of total yeast DNA was cloned and recombinants containing a 6.4 kbp rDNA fragment were isolated.

Animals↗
PubMedDate not supplied

Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing.

Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/mL) were substantially above the detection limit of the TM-PERT assay ( approximately 10(6) pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.

Animals↗