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Biomedical subjects

A F Purchio

Publications and source records attributed to A F Purchio.

77 records · Page 5Linked to original sources

Transformation of human embryonic kidney cells by human papovarirus BK.

Infection of secondary human embryonic kidney (HEK) cells with human papovavirus BK (BKV) resulted in cellular lysis and degeneration within 7 days. After 30 days, multilayered colonies of transformed cells were found and subcultured for analyses. These BK-HEK cells uniformly expressed the BKV T-antigen but were only 1% V-antigen positive. They produced infectious virus and were resistant to superinfection by BKV. They reached a saturation density of 1.3 x 10(5) cells per cm2 in medium with 5% fetal calf serum, were able to grow in medium containing 2% serum, and did not form colonies in soft agar or tumors in nude mice. Nonintegrated, superhelical BKV DNA was detected in the noncloned cells as expected because they were persistently infected and contained RNA transcripts complementary to both early and late regions of the BKV genome. Analysis of T-antigen-positive clonal isolates of these BK-HEK cells by the Southern technique revealed an absence of free viral DNA and the presencce of integrated BKV DNA sequences corresponding to the early region of the BKV genome. These studies demonstrate the stable transformation of human cells by BKV. However, the transformed human cells which retain and express part of the BKV genome do not fully manifest the growth properties of other papovarirus-transformed cells.

Antigens, Neoplasm↗

Identification of a polypeptide encoded by the avian sarcoma virus src gene.

Two techniques were used to search for the polypeptide encoded by the avian sarcoma virus (ASV) src gene. First, antiserum from rabbits bearing ASV-induced fibrosarcomas was used to immunoprecipitate a transformation-specific antigen from ASV-transformed chick embryo fibroblasts. This antigen has an apparent molecular weight (Mr) of 60,000. Second, the 3' one-third of the ASV genome, selected by oligo(dT)-cellulose chromatography and sucrose gradient sedimentation, was translated in a mRNA-dependent reticulocyte cell-free lysate. This RNA species programmed the synthesis of a polypeptide that comigrated with the transformation-specific antigen of Mr 60,000 immunoprecipitated from transformed cells. The methionine-containing tryptic peptides from the polypeptides of Mr 60,000 obtained from translation in vitro and from immunoprecipitation were found to be identical upon two-dimensional fractionation.

Alpharetrovirus↗

Translation of 35S and of subgenomic regions of avian sarcoma virus RNA.

Rabbit antiserum monospecific for an internal structural protein, p27, of avian sarcoma viruses (ASV) was found to immunoprecipitate polypeptides with molecular weights (Mr) of 180,000 and 76,000 from cell-free reticulocyte lysates programmed by ASV 35S RNA and also from lysates of ASV-infected cells. In addition, the Mr 180,000 protein was also precipitated by antiserum raised against virion DNA polymerase, suggesting that is a product of the two genes nearest the 5' end of virion 35S RNA. We have also investigated the ability of subgenomic portions of virion RNA to program cell-free protein synthesis. A 10-12S poly(A)-containing fragment of RNA from both nondefective and transformation-defective ASV directed the synthesis of a polypeptide of Mr 29,000 immunologically unrelated to the gs antigens; 20-24S poly(A)-containing RNA from nondefective ASV directed the synthesis of a polypeptide of Mr 60,000 not found when a similar RNA preparation from transformation-defective ASV was translated, suggesting that it is the product of the ASV src gene. These results indicate that internal initiation sites for protein synthesis exist on the 35S RNA genome.

Avian Sarcoma Viruses↗

Transforming growth factor-beta 2: cDNA cloning and sequence analysis.

We have obtained a cDNA clone coding for human transforming growth factor (TGF)-beta 2. The clone was isolated from a tamoxifen-treated human prostatic adenocarcinoma cell line (PC-3) using oligonucleotide probes based on the partial amino acid sequence of purified TGF-beta 2. The cDNA sequence predicts that TGF-beta 2 is synthesized as a 442-amino-acid polypeptide precursor from which the mature 112-amino-acid TGF-beta 2 subunit is derived by proteolytic cleavage. The proteins coded for by the human TGF-beta 1 and TGF-beta 2 cDNAs show an overall homology of 41%. The mature and amino-terminal precursor regions show 71% and 31% homology, respectively. Northern blot analysis identified TGF-beta 2 transcripts of 4.1, 5.1, and 6.5 kb using mRNA from several different sources. Analysis of polyadenylated RNA from tamoxifen-treated PC-3 cells showed that these cells contain higher numbers of transcripts for TGF-beta 1 than for TGF-beta 2, although they produce more TGF-beta 2 protein than TGF-beta 1. This suggests that there is a post-transcriptional level of regulation for the production of these proteins.

Amino Acid Sequence↗

Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells.

A stable Chinese hamster ovary (CHO) cell line producing high levels of human oncostatin M (OM) was generated by transfecting a heterologous gene coding for the protein. This novel construct was comprised of the gene for the transforming growth factor-beta 1 (TGF-beta 1) signal peptide fused to the gene for mature human OM. Amplification with methotrexate produced milligram quantities of this recombinant OM, which was processed correctly, glycosylated, and found to have biological functions similar to those of natural OM.

Animals↗