Biomedical subjects

K W Ryan

Publications and source records attributed to K W Ryan.

23 records · Page 2Linked to original sources

PubMed1988

Carboxyl-terminal region of Sendai virus P protein is required for binding to viral nucleocapsids.

The Sendai virus P protein is a component of the viral nucleocapsid, where it participates in RNA synthesis. To identify domains of the protein involved in nucleocapsid recognition, deleted P protein molecules were generated from a cDNA clone of its gene. In vitro transcription of the complete gene and translation of the transcript generated a protein with electrophoretic mobility and immunoreactivity indistinguishable from those of authentic P protein. The in vitro product bound specifically to nucleocapsids when mixed with extracts from infected cells. However, a product lacking only 30 carboxyl-terminal amino acid residues (5% of the molecule) did not bind. Residues within a 195 amino acid region, adjacent to and overlapping by one amino acid with the carboxyl-terminal 30 residues, were also required for binding. No other protein region was required. Therefore, the 224-residue region which includes the carboxyl terminus appears to contain the nucleocapsid attachment site, and the 30 terminal residues either form part of the site or are required to maintain an active conformation.

Animals↗

PubMed1987

Fate and expression of simian virus 40 DNA after introduction into murine cells under nonselective conditions.

When SV40 infects mouse cells, it does not replicate but instead causes neoplastic transformation of a small percentage of the cells. It is unknown, however, what happens to the virus in those cells that do not become transformed. We introduced SV40 into mouse cells by nonselective means, either by cotransfection of SV40 DNA with a selectable marker or by random cloning of SV40-infected cells. We analyzed the fate of viral DNA sequences, expression of T antigens, and transformation properties of these cells. We found that, upon infection, viral DNA integration occurs at a frequency that is at least 10-fold higher than the frequency of transformation. The majority of these cells are not transformed due to lack of expression of T antigen. One cell line which expresses a truncated T antigen is not transformed. We have mapped the viral sequences in the genome of these cells and find that integration in the large T intron is probably responsible for the defect. Lack of transformation can therefore be attributed to both cellular and viral factors, namely, introduction of viral DNA into cells that are resistant to transformation or integration of viral DNA in such a way that T antigen expression is prohibited.

Animals↗

PubMed1986

Functional expression of influenza A viral nucleoprotein in cells transformed with cloned DNA.

Simian cells permissive for influenza A virus infection were stably transformed with a full-length cloned influenza A nucleoprotein gene under the control of an inducible metallothionein promoter and linked to a dihydrofolate reductase gene to facilitate cell selection. Transformed cells synthesized a virus-specific nucleoprotein which was indistinguishable from the nucleoprotein synthesized in virus-infected cells with respect to molecular weight and intracellular localization. It was estimated that transformed cells produced only 1% of the amount of nucleoprotein synthesized in simian cells infected with influenza A virus. Nonetheless, when transformed cells were infected with influenza virus mutants which synthesized temperature-sensitive nucleoprotein, protein expressed by the cloned gene was able to complement the synthesis of plus-strand and minus-strand viral RNA for one mutant and only plus-strand synthesis for another mutant. This indicated that the influenza A nucleoprotein expressed in the transformed cells exhibited functional activity.

Animals↗

PubMed1985

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.

Animals↗

PubMedDate not supplied

Multiple spectral parameter imaging in quantitative fluorescence microscopy. I: Quantitation of bead standards.

Digitized images from fluorescence microscopy may suffer from nonlinearity of camera response to illumination intensity, from uneven spatial response of the camera, and from uneven illumination of the microscopical field. Structureless fluorescence standard slides are used to characterize the camera response. This information permits the images to be corrected for all the above errors, in one operation. The computer algorithms are described, and also those for thresholding and finding objects. The method is illustrated by tests using fluorescent beads.

Algorithms↗