Margery O. Nicolson

RD-114 Virus Infectivity Assay by Measurements of DNA Polymerase Activity and Virus Group Specific Antigen

Summary The infectivity of RDV, a non-transforming type-C virus, can be assayed by induction of virus-specific gs antigen and of DNA polymerase activity in infected cell cultures. RD cells are more susceptible to RDV infection than other human cell lines or strains tested. Although RDV infection of the cells can be detected 3 days after exposure of RD cells to large doses of virus, 3-4 wk are required to obtain demonstrable infection of cell cultures infected with small doses of virus. Simpler assays based upon the capacity of RDV to induce syncytia in KB or KC cells 7 to 9 days after infection expectedly yield lower infectivity titers than those obtained by RDP or gs antigen induction assays in RD cells 21-28 days after infection.

Transcription and expression of the Herpes simplex virus tk gene inserted into proviral sequences of feline leukemia virus

Recombinant DNA molecules containing the herpesvirus tk gene inserted near the middle of a cloned feline leukemia virus proviral genome, in the same transcriptional orientation as the long terminal redundancies (LTRs), were used to transform human tk- cells. Analysis of RNA from cloned lines indicates that the 5 LTR promotes a high level of transcription which, as a result of differing RNA splicing and polyadenylation pathways, results in three large, abundant RNAs, two of which contain the entire tk coding region. The tk promoter itself initiates transcription of a smaller, relatively rare tk mRNA, of the same length and abundance as found in cells transformed with the tk gene alone. Assays indicate that there is little if any thymidine kinase (TK) enzymatic activity contributed by the abundant LTR-promoted transcripts. This is presumably due to inefficient initiation of tk translation from the longer LTR-initiated transcripts because of upstream AUG codons in the viral sequences. RNA blots indicate that the viral LTR is stronger as a promoter than the tk promoter. The results also indicate that about one-third of the LTR-initiated transcripts are polyadenylated at the tk poly(A) site, while the rest use the poly(A) site of the 3 LTR.

Liver regeneration: An isolation perfusion system employed to assay hepatic 3-H-thymidine incorporation.

Summary Isolated normal livers perfused with 3H-thymidine containing suspensions which had been previously circulated through isolated livers either (a) regenerating or (b) “sham” operated, showed equal and relatively low levels of both tissue specific activity and nuclear labeling by autoradi-ography. When such blood-simulating perfusates, containing 3H-thymidine, are circulated through whole isolated regenerating livers, nuclear uptake is apparent and specific activity is increased > 100 X over levels obtained when the same perfusate is circulated through nonregenerating livers.