Biomedical subjects

F R Cross

Publications and source records attributed to F R Cross.

59 records · Page 4Linked to original sources

PubMed1984

Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts.

The transforming activity of the cellular src (c-src) gene as well as of hybrid genes between viral and cellular src was tested by constructing derivatives of Rous sarcoma virus DNA in which all or part of the viral src gene (v-src) was replaced by the corresponding portion of the c-src gene. After these derivatives were introduced into chicken embryo fibroblasts by transfection, replication-competent virus was recovered, which induced the expression of p60src at a level equivalent to p60v-src expression in cells infected with Rous sarcoma virus wild type. Replacement of the portion of the v-src gene, either upstream or downstream of the Bgl I site, with the homologous portion of the c-src gene resulted in fully transforming viruses. On the other hand, the virus stock obtained from cells transfected with Rous sarcoma virus DNA containing the entire c-src gene had a very low titer of focus-forming virus, while it contained a high titer of infectious virus. We present evidence that the rare small foci are formed by mutant viruses generated from the original c-src-containing virus. These results indicate that overproduction of the c-src gene product does not cause cell transformation, and that this proto-oncogene is subject to a relatively high rate of mutation when incorporated in a retrovirus genome, resulting in the acquisition of transforming capacity.

Animals↗

PubMed1984

A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation.

We have constructed mutants by using linker insertion followed by deletion in the region of cloned Rous sarcoma virus DNA coding for the N-terminal 9 kilodaltons of the src protein. Previous work implicated this region in the membrane association of the protein. The mutations had little effect on src tyrosine kinase activity. Substitution of a tri- or tetrapeptide for amino acids 15 to 27, 15 to 49, or 15 to 81 had little effect on the in vitro transforming capacity of the virus. Like wild-type p60src, the src proteins of these mutants associated with plasma membranes and were labeled with [3H]myristic acid. In contrast, a mutant whose src protein had the dipeptide Asp-Leu substituted for amino acids 2 to 81 and a mutant with the tripeptide Asp-Leu-Gly substituted for amino acids 2 to 15 were transformation defective, and the mutant proteins did not associate with membranes and were not labeled with [3H]myristic acid. These results suggest that amino acids 2 to 15 serve as an attachment site for myristic acid and as a membrane anchor. Since deletions including this region prevent transformation, and since tyrosine kinase activity is not diminished by the deletions, these results imply that target recognition is impaired by mutations altering the very N terminus, perhaps through their effect on membrane association.

Amino Acid Sequence↗

PubMed1983

Local mutagenesis of Rous sarcoma virus: the major sites of tyrosine and serine phosphorylation of pp60src are dispensable for transformation.

We have constructed mutants of Rous sarcoma virus expressing p60srcs that are underphosphorylated on serine or tyrosine, by linker insertion or insertion/deletion into cloned Rous sarcoma virus DNA, and recovery of mutant virus by transfection of chicken embryo fibroblasts. Cells infected with mutants whose p60srcs lack the major site of either serine or tyrosine phosphorylation were morphologically transformed and formed colonies in soft agar. The tyrosine kinase activities of the mutant p60srcs measured in vivo and in vitro were close to the wild type activity. Peptide mapping showed that phosphorylation on tyrosine and serine of p60src is independent: the major phosphorylated tyrosine and the major phosphorylated serine can each be phosphorylated in the absence of phosphorylation of the other.

Animals↗

PubMed1983

Cycloheximide stimulates early adenovirus transcription if early gene expression is allowed before treatment.

The inhibition of protein synthesis by cycloheximide markedly stimulated adenovirus 2 early transcription (assayed in vivo and in vitro in nuclei) when the drug was added 3 h after infection of HeLa cells. The stimulation was not uniform but ranged from three- to eightfold for region 1A to 10- to 30-fold for region 2 of the adenovirus genome. The increase was complete by about 60 min after treatment. The stimulation reversed rapidly after cycloheximide was removed. Treatment with cycloheximide either before infection with wild-type adenovirus type 5 or at 3 h after infection with dl312, a mutant which fails to express early gene products in HeLa cells, both resulted in levels of transcription at or substantially below the control (infection with wild-type adenovirus without treatment). Therefore we conclude that cycloheximide treatment and previous early gene expression interact to yield the maximum levels of transcription.

Adenoviruses, Human↗

PubMedDate not supplied

An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins.

The src gene product, p60src, of Rous sarcoma virus (RSV) is a tyrosine-specific protein kinase which is associated with the plasma membrane of infected cells. Myristic acid is bound in an amide linkage to glycine 2 of p60src. Of the N-terminal 30 kilodaltons of p60src, only amino acids 1-14 are required for myristylation, and myristylation of p60src may be required for its membrane association, and for cell transformation. To test the hypothesis that the first 14 amino acids of p60src contain a recognition sequence for myristylation, we have fused the DNA sequence coding for these amino acids to either the fps gene of the F36 derivative of Fujinami sarcoma virus (FSV), or to the chimpanzee alpha-globin gene. We report here that although the fusion proteins were myristylated, the parental proteins were not, and unlike the non-myristylated F36 p91fps which was not bound to the plasma membrane, the myristylated fusion protein was bound, like p60src. We conclude that the first 14 amino acids of p60src contain a sequence which is sufficient for myristylation, and which may direct proteins to the plasma membrane.

Amino Acid Sequence↗