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Developmental regulation of the dictyostelium ras gene
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DIFFERENTIAL GENE EXPRESSION DURING LECTIN INDUCED TRANSFORMATION OF HUMAN LYMPHOCYTES. G. Muscat, A. Caputo , E. McCairns and P.B. Rowe. Children's Medical Research Foundation, P.O. Box 61, Camperdown, N.S.W., 2050, Australia. A cDNA library in Xgtl0 was constructed from the cytoplasmic poly(A)+RNA of human lymphocytes after 72h of phytohemagglutinin (PHA) stimulation in which maximum cell transformation is achieved. Clones coding for mRNAs preferentially expressed in stimulated as opposed to resting lymphocytes were isolated by hydroxylapatite depletion and differential screening techniques. Four recombinants were isolated which coded for mRNAs (0.72.4 kb) which were preferentially induced 5- to 20-fold. The mRNAs corresponding to two of the clones were maximally elevated after 24h exposure to PHA, coincident with the onset of DNA replication, while the other two were elevated at 6h prior to overall transcriptional increases. The relative contribution of transcription, processing and stability to the observed changes in mRNA levels is being investigated. Ten additional clones were isolated which contained ~lu repetitive sequences and were transiently induced with the onset of G1 phase. 11
EXPRESSION OF EXOGENOUS GENES IN TRANSFORMED XENOPUS LAEVIS EMBRYOS L. Etkin, B. Pearman, S. Ba--aTc-eTls, Dept. of Genetics, M. D. Anderson Hospital and Tumor I n s t i t u t e , Houston, TX 77030. Transformed Xenopus laevis were used to examine the developmental and tissue specific expression of several genes. Genes for chloramphenicol acetyl transferase (CAT), Drosophila alcohol dehydrogenase (ADH), and sea urchin histones are all expressed at the mid-blastula t r a n s i t i o n stage in i n i t i a l t r a n s f o r mants. We feel that most of the expression at MBT is from the extrachromosomal plasmids. The presence of strong promoters (SV 40 early gene promoter) or r e l a t i v e l y weak promoters (adenovirus E3 promoter-E3CAT) does not affect the time of expression of these genes. However, they do a f f e c t the expression of the genes q u a n t i t a t i v e l y . EIA protein coinjected with E3CAT affects expression q u a n t i t a t i v e l y but not qualitatively. The injected DNAs are integrated into the Xenopus genome and exhibit a random pattern of expression in individual tissues of larvae and young f r o g l e t s . Supported by NIH. 12 GLUCOCORTiCOID REGULATION OF MOUSE MAMMARY TUMOR VIRUS SEQUENCES IN TRANSGENIC MICEo S.R. Ross, D.H. Henrard, and J.J. Michon. We have introduced chimeric genes containing the promoter and glucocorticoid receptor binding element into the germ line of mice by microinjection. These chimeric genes are expressed in the lactating mammary gland and in the testes of males containing these genes. This transcription is under the control of glucocortiooid hormones° We are further dissecting those sequences responsible for the tissue-specific expression and examining their role in the hormone regulated transcription.
DEVELOPMENTAL REGULATION OF THE DICTYOSTELIUM ras GENE. C.D~ Reymond, R.H. Gomer, W. Nellen, and R.A. Firtel. Department of Biology, University of California San Diego, La Jolla CA 92093. We have cloned and sequenced a Dictvostelium gene encoding a protein h i g h l y homologous to the mammalian ras proteins. We are using DNA probes and a polyclonal antibody to examine the differential accumulation of ras RNA and protein through development. The Dd-ras gene is expressed in vegetative cells-and later during development in prestalk cells. The level of the Dd-ras protein remains constant until early culmination and then decreases. Expression of the Dd-ras gene can be modulated by extracellular-cAMP and cell-cell contact. Using DNAmediated transformation, we have introduced Dd-ras genes, truncated at different positions, into Dictyostelium cells. A construct, encoding the first 59 amino acids, blocks the DictyosteZium development. These studies suggest that the Dd-ras gene plays a role both in vegetative cell growth and during development.
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DIFFERENTIAL GENE EXPRESSION DURING LECTIN INDUCED TRANSFORMATION OF HUMAN LYMPHOCYTES. G. Muscat, A. Caputo , E. McCairns and P.B. Rowe. Children's Medical Research Foundation, P.O. Box 61, Camperdown, N.S.W., 2050, Australia. A cDNA library in Xgtl0 was constructed from the cytoplasmic poly(A)+RNA of human lymphocytes after 72h of phytohemagglutinin (PHA) stimulation in which maximum cell transformation is achieved. Clones coding for mRNAs preferentially expressed in stimulated as opposed to resting lymphocytes were isolated by hydroxylapatite depletion and differential screening techniques. Four recombinants were isolated which coded for mRNAs (0.72.4 kb) which were preferentially induced 5- to 20-fold. The mRNAs corresponding to two of the clones were maximally elevated after 24h exposure to PHA, coincident with the onset of DNA replication, while the other two were elevated at 6h prior to overall transcriptional increases. The relative contribution of transcription, processing and stability to the observed changes in mRNA levels is being investigated. Ten additional clones were isolated which contained ~lu repetitive sequences and were transiently induced with the onset of G1 phase. 11
EXPRESSION OF EXOGENOUS GENES IN TRANSFORMED XENOPUS LAEVIS EMBRYOS L. Etkin, B. Pearman, S. Ba--aTc-eTls, Dept. of Genetics, M. D. Anderson Hospital and Tumor I n s t i t u t e , Houston, TX 77030. Transformed Xenopus laevis were used to examine the developmental and tissue specific expression of several genes. Genes for chloramphenicol acetyl transferase (CAT), Drosophila alcohol dehydrogenase (ADH), and sea urchin histones are all expressed at the mid-blastula t r a n s i t i o n stage in i n i t i a l t r a n s f o r mants. We feel that most of the expression at MBT is from the extrachromosomal plasmids. The presence of strong promoters (SV 40 early gene promoter) or r e l a t i v e l y weak promoters (adenovirus E3 promoter-E3CAT) does not affect the time of expression of these genes. However, they do a f f e c t the expression of the genes q u a n t i t a t i v e l y . EIA protein coinjected with E3CAT affects expression q u a n t i t a t i v e l y but not qualitatively. The injected DNAs are integrated into the Xenopus genome and exhibit a random pattern of expression in individual tissues of larvae and young f r o g l e t s . Supported by NIH. 12 GLUCOCORTiCOID REGULATION OF MOUSE MAMMARY TUMOR VIRUS SEQUENCES IN TRANSGENIC MICEo S.R. Ross, D.H. Henrard, and J.J. Michon. We have introduced chimeric genes containing the promoter and glucocorticoid receptor binding element into the germ line of mice by microinjection. These chimeric genes are expressed in the lactating mammary gland and in the testes of males containing these genes. This transcription is under the control of glucocortiooid hormones° We are further dissecting those sequences responsible for the tissue-specific expression and examining their role in the hormone regulated transcription.
DEVELOPMENTAL REGULATION OF THE DICTYOSTELIUM ras GENE. C.D~ Reymond, R.H. Gomer, W. Nellen, and R.A. Firtel. Department of Biology, University of California San Diego, La Jolla CA 92093. We have cloned and sequenced a Dictvostelium gene encoding a protein h i g h l y homologous to the mammalian ras proteins. We are using DNA probes and a polyclonal antibody to examine the differential accumulation of ras RNA and protein through development. The Dd-ras gene is expressed in vegetative cells-and later during development in prestalk cells. The level of the Dd-ras protein remains constant until early culmination and then decreases. Expression of the Dd-ras gene can be modulated by extracellular-cAMP and cell-cell contact. Using DNAmediated transformation, we have introduced Dd-ras genes, truncated at different positions, into Dictyostelium cells. A construct, encoding the first 59 amino acids, blocks the DictyosteZium development. These studies suggest that the Dd-ras gene plays a role both in vegetative cell growth and during development.
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