We have subjected the viral mos oncogene (v‐mos), the activated human H‐ras oncogene [H‐ras (A)] and the normal human H‐ras protooncogene [H‐ras (N)] to the transcriptional regulation of glucocorticoid hormones by in vitro recombination with the promoter region of the mouse mammary tumor virus long terminal repeat (MMTV LTR) and transfection into NIH 3T3 cells. Cell clones were selected which exhibit a transformed phenotype strictly dependent on the presence of hormone in the growth medium. The expression of the chimeric genes as a function of time after hormone stimulation was studied at the level of transcriptional rate, mRNA and protein accumulation. Oncogene expression was stimulated rapidly to high levels, after hormone addition, but declined in the continuous presence of hormone. Measurements of the transcriptional rates in nuclei from LTR v‐mos and LTR H‐ras (A) transfected cells showed a repression of LTR v‐mos and LTR H‐ras (A) transcription after the initial stimulation by hormone. LTR H‐ras (N) transcription was not affected. An independently transfected LTR H‐2Ld construct in LTR v‐mos or LTR H‐ras (A) containing cells is also transcriptionally repressed. These experiments demonstrated a transcriptional repression effect of the oncogene products on the glucocorticoid hormone‐dependent MMTV LTR transcription.

Previous attempts to relate the structure and function of hepatocytic membranes have compared biochemical data of fractions to morphological data derived from either intact tissue or fractions. The effects of the original homogenization aside, biochemical recoveries comparing membrane marker enzymes of the homogenate to subsequent fractions suggest a general conservation of activity. A stereological study was undertaken to estimate membrane surface areas in the intact tissue, homogenate, and fractions of the same livers and then to test the comparability of these data with membrane marker enzymes by calculating both morphological and biochemical recoveries. The stereological data were corrected for errors due to section thickness and compression.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

Cloning of vitellogenin cDNA of Xenopus laevis revealed that vitellogenin is encoded in a small family of genes representing two distantly related main groups A and B, each comprising two more closely related subgroups A1, Az, and B1, BZ respectively. To characterize the proteins derived from these genes we have isolated the corresponding mRNAs by hybridizing, under stringent conditions, cytoplasmic poly(A)-containing RNA from the liver of estrogen-stimulated Xenopus to filter-bound cDNA clones containing sequences specific for all four vitellogenin genes.Hybridization of the isolated mRNAs with nick-translated cDNA clones revealed that contamination of the mRNAs by those of the other main group was less than 0.1 %. Melting curves of the hybrids prepared with the isolated mRNAs and cDNA clones specific for the four vitellogenin genes showed that the isolated vitellogenin mRNAs are also specific for the four subgroups. Analysis of R loops formed between isolated mRNAs and cDNA clones representing the corresponding subgroup further indicated about 10 % cross-contamination between the more closely related mRNAs. In a reticulocyte lysate each of the four mRNAs coded for a 200000-M, protein immunoprecipitable by monospecific vitellogenin antibody.From these results we conclude that the four different mRNAs A1, A2, B1 and Bz, which all can be isolated efficiently, code for vitellogenin and are expressed simultaneously in response to estrogen stimulation.Estrogen-dependent synthesis of vitellogenin, the precursor of the major egg-yolk proteins in the liver of Xenopus represents an attractive system of gene regulation by hormones, allowing reversible activation of a normally silent gene in male animals to produce a female protein (see review [l -31). Isolation and characterization of the vitellogenin mRNA as well as its accumulation in response to estrogen was reported by various laboratories [4,5]. Recently we demonstrated by restriction analysis and R-loop mapping of various clones of Xenopus vitellogenin cDNA that Xenopus vitellogenin mRNA is composed of four related RNAs [6]. Restriction analysis of genomic DNA demonstrated that all these RNAs are coded for by different genes [6].In our previous experiments we assumed that all these related RNAs are mRNAs and that all code for vitellogenin proteins. In this paper we now report on the isolation of the four vitellogenin mRNAs from poly(A)-containing RNA of estrogen-stimulated Xenopus and it is shown that each of these poly(A)-containing RNAs directs synthesis of a polypeptide in vitro which has about the same size as vitellogenin and is immunoprecipitable with vitellogenin antibody.Abbreviations. cDNA, complementary DNA; Hepes, 4-(2-hydroxyethy1)-1 -piperazineethanesulfonic acid; 1 x NaCl/Cit, 150 mM NaC1/15 mM trisodium citrate; pXlvc, cDNA clone designation : plasmid, Xenopus laevis, vitellogenin, cDNA.Definition. rot or cot product of [RNA] or [DNA] and time of hybridisation (M.s).

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