Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing.

V79 Chinese hamster cells genetically engineered to express cytochrome P-450IA1 are reported. A full length cDNA encoding rat cytochrome P-450IA1 was obtained from a cDNA library prepared from rat liver mRNA. The cDNA was recombined with the SV40 early promoter and expressed in V79 cells. Three V79-derived P-450IA1-expressing cell lines (XEM1, XEM2, and XEM3) were established. The presence of the rat cytochrome P-450IA1 cDNA in these hamster cells was confirmed by Southern blotting. The transcription of the cDNA into mRNA and translation into the desired cytochrome P-450 protein was detected by Northern and Western blotting. The enzymatic activity was determined by the cytochrome P-450IA1-dependent oxidation of benzo[a]pyrene and 7-ethoxycoumarin. After exposure to benzo[a]pyrene, the mutant frequency increased in XEM1 and XEM2 cells and was higher than in V79 cells in the presence of an exogenous activating system. The mutant frequency was even more increased when XEM1 and XEM2 cells were exposed to the proximate mutagen (trans)-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene.

Isolation and structural analysis of a biologically active chicken c-fos cDNA: identification of evolutionarily conserved domains in fos protein.

To identify functionally important domains in the fos gene product we have studied the evolutionary divergence between chicken and mammalian fos proteins. A cDNA containing the entire chicken c-fos coding region was isolated and its nucleotide sequence determined. The deduced 367-amino acid sequence was compared to that of the mouse and human proteins. This comparison revealed a highly conserved domain (98% homology between mouse and chicken) in the center of the protein (85 amino acids) that coincides with a region known to be indispensible for transforming activity. This highly charged domain presumably contains contact sites for DNA and other proteins as well as a nuclear location signal sequence. Two other regions, that are dispensable for transformation, are also highly conserved and may thus be important for the physiological function of c-fos. These are the N-terminal 88 amino acids (85% homology) and the C-terminal 62 amino acids (92% homology). The C-terminus not only contains a potential DNA-binding Zn-finger structure but is also the least divergent region in the protein at the nucleotide level (92% conservation between chicken and mouse), supporting the hypothesis that mRNA secondary structures in this region may contribute to post-transcriptional regulatory mechanisms. In contrast, the domains between the terminal sequences and the center region of fos protein show considerable divergence (39% and 45% homology, respectively), indicating a minor role, if any, for these sequences. The significance of these conclusions is emphasized by the observation that the chicken c-fos protein, expressed from the cDNA inserted into a retrovirally-derived expression vector, efficiently induces morphological transformation in rat fibroblasts. The chicken c-fos gene product could be identified by immunoprecipitation and in vitro transcription/translation of the isolated cDNA as a protein of Mr approximately 60 K.

Integration of transfected LTR sequences into the c-raf proto-oncogene: activation by promoter insertion.

A malignant cell line (clone S1) isolated after co-transfection of normal NIH3T3 DNA and Moloney leukemia virus long terminal repeat (Mo-LTR) sequences has previously been described to contain an activated c-raf oncogene. Here, we report the isolation by molecular cloning and the structural analysis of the LTR-activated c-raf gene. As shown by Southern blot and nucleotide sequence analyses, the transfected Mo-LTR sequences integrated into the 5th intron of the endogenous c-raf proto-oncogene. This intragenic LTR insertion led to the expression of high levels of LTR-U5-c-raf hybrid transcripts indicating an initiation of transcription from the Mo-LTR promoter. Transcriptional activation of c-raf is accompanied by the synthesis of large amounts of cytoplasmic c-raf protein. Immunoblot analysis suggests that the proteins encoded by the LTR-activated c-raf gene are truncated compared with the normal c-raf gene product(s). Our results indicate a promoter insertion mechanism of c-raf activation.

Biochemical analysis of variant HLA-B27 antigens.

Biochemical analysis of HLA-B27 antigens from individuals that are HLA-ABC identical by serology but distinct in CML typing establishes that two types of HLA-B27 can be defined biochemically. The division of HLA-B27 into a W type and a K type by CML typing correlates perfectly with the biochemical data. HLA-B27 K type possesses a more basic isoelectric point than HLA-B27 W type. Neuraminidase digestions of immunoprecipitated HLA-A,B antigens establish that the difference in isoelectric point between HLA-B27 K and HLA-B27 W is not due to differences in sialic acid content.

Definition of four HLA-A2 subtypes by CML typing and biochemical analysis.

The population of HLA-A2-positive individuals, currently considered serologically homogeneous, can be divided into three subtypes on the basis of antigen recognition by various HLA-A2-specific cytotoxic T lymphocytes (CTLs). When these three types of HLA-A2 antigens were analyzed biochemically, they were found to be distinct. Isoelectric focusing (IEF) of HLA antigens digested with neuraminidase (NANAse) suggested that the difference(s) reside in the polypeptide backbone of the HLA-A2 heavy chain. Biochemical analysis distinguishes three distinct categories of HLA-A2 antigens: (1) a major subtype, designated HLA-A2.I, (2) a minor subtype, designated HLA-A2.II, possessing a more basic isoelectric point (IEP) and (3) a minor HLA-A2 subtype more acidic in its IEP than HLA-A2.I, designated HLA-A2.III. A fourth HLA-A2 subtype could be defined by discordance between cell-mediated lympholysis (CML) typing and biochemical analysis. The latter HLA-A2 antigen was defined as a variant by CTL, but was biochemically indistinguishable from the major subtype HLA-A2.I.

Analysis of human class I antigens by two-dimensional gel electrophoresis. I. Polymorphism, evidence for additional (non-HLA-A, B, C) gene products, and identification of variant HLA-A, B antigens.

Class I antigens were isolated by immunoprecipitation from cell extracts prepared from mitogenically stimulated and internally radiolabeled peripheral blood lymphocytes (PLBs). The precipitating antibodies used are monomorphic and recognize a determinant on the heavy chain of HLA-A, B, C antigens regardless of their allelic specificities when complexed with beta 2m, or determinants on beta 2m itself. Comparison of class I molecules isolated from 25 different homozygous typing cells (HTC) and analyzed by two-dimensional (2-D) gel electrophoresis allowed the identification of those HLA-A, B locus specificities most common in the European Caucasoid population. Class I antigens isolated from HTC that are HLA identical are biochemically indistinguishable also. Evidence was obtained for the expression of additional class I antigens besides the HLA-A, B, C locus products: for some haplotypes, up to six class I genes may be active in mitogenically activated PBLs.No differences in molecular weight and isoelectric point of the class I heavy chains were observed between the antigens recognized by W6/32, the anti-heavy chain reagent, and anti beta 2m reagents. The nature of the mitogenic stimulus, i.e., pokeweed mitogen or phytohemagglutinin, was irrelevant with respect to the class I antigens isolated by this method. Using the HTCs as reference, a panel of HLA-B27 positive heterozygous cells was analyzed. Two types of HLA-B27 antigens, distinct by CML typing were represented. These two forms differed also in their biochemical properties. In addition, we obtained evidence for the existence of an A2 variant. This finding was likewise confirmed by CML typing.