Cloning and sequencing of CATR1.3, a human gene associated with tumorigenic conversion.

The human squamous cell carcinoma cell line SCC83-01-82 (SCC) contains mutations in both the H-ras and p53 genes, but it exhibits a nontumorigenic phenotype in nude mice. This cell line can be converted into a cell line with a tumorigenic phenotype, SCC83-01-82CA (CA), by treatment with the mutagen methyl methanesulfonate (MMS). This indicates that additional genetic events leading to expression of a cooperating tumor susceptibility gene(s) may be required for tumorigenicity. To identify the cooperating gene(s), an expression cDNA library was made from tumorigenic Ca cells. The library DNA was transfected into nontumorigenic SCC cells and the transfected SCC cells were then injected into nude mice for the selection of a tumorigenic phenotype. Tumors developed in 3 of the 18 mice after injection. Several new cell lines were established from these transfected cell-induced tumors and designated as CATR cells. Tumor histology and karyotype analysis of these cells indicated that they were of human epithelial cell origin. All the CATR cells have the library vector sequence integrated in their genome. Cell line CATR1 expressed a single message from the integrated library representing a 1.3-kb cDNA insert that was absent from untransfected SCC cells or MMS-converted CA cells. This 1.3-kb cDNA insert was cloned by PCR amplification of reverse-transcribed CATR1 total RNA and was designated CATR1.3. The nucleotide sequence of CATR1.3 encodes a peptide of 79 amino acids, has a long 3' untranslated region, and represents an unknown gene product that was associated with the tumorigenic conversion due to the transfected expression library.

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.