No abstract

Cells and viruses. CeUs were grown as previously described (8). The SV40-transformed mouse cells (SVA31E7 and VLM) were kindly provided by Y. Ito and S. Tevethia, respectively; the SV40-transformed rat cells (14B), by D. Lane; the SV40-transformed hamster cells (H65 90B), by S. Tevethia; the SV40transformed rabbit cells (TRK54), by P. Black; the SV40-transformed human cells (SV80), by E. Gurney; and the methylcholanthrene-transformed mouse cells (L929), by D. Cox. All viruses were propagated on CV-1 African green monkey kidney cells as described previously (8). The SV-S strain was used as wild-type 861

Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.A number of studies have shown that primary rat cells can be transformed in vitro by cotransfecting an activated ras gene with any of a series of cellular or viral genes (28, 50). Transformed foci have been observed after the cotransfection of an activated ras gene with the gene for the cellular myc protein, polyoma large-T antigen, adenovirus ElA proteins, or MC29 gag-myc fusion protein. Recent studies by three groups demonstrated that cotransfection of the gene that encodes the mouse cellular tumor antigen p53 with an activated human ras gene also yields transformed foci (12,21,42). Results of earlier work had suggested that p53 might play a role in some types of transformation. These suggestions were based not only on comparison of the biochemistry of p53 in normal and transformed cells but also on studies of the immune response of animals to some types of tumors. This work has shown that (i) sera from laboratory animals with tumors or from human patients with some types of neoplasia often contain circulating antibodies specific for p53 (9,11,29,32,49); (ii) transformed cells often have higher levels of p53 than their normal cell counterparts (3,10,11,20,48,53); (iii) in cells transformed by simian virus 40 (SV40) or adenovirus, p53 is found in a stable, highmolecular-weight complex with either the SV40-coded large-T antigen or the adenovirus-coded E1B 57-kilodalton protein (29,32,35,52); (iv) p53 appears to play an important role in the movement of quiescent cells into the S phase after serum stimulation (37, 38); and (v) the synthesis of p53 is temporally regulated after stimulation of cells with mitogens (40,46). These studies suggest that p53 may play an important role in the regulation of cell division in some cell types, but how p53 may be involved in these processes is not known. We present here the isolation, characterization, and nucleic acid sequence of a p53 cDNA clone from human A431 cells that has the coding potential for a full-length p53. * Corresponding author. MATERIALS AND METHODSCells and antibodies. All cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. ...

Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.

We have developed a high-throughput, multiplex reverse transcription PCR (RT-PCR)