RESUMO
A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.
Assuntos
Dimetilaliltranstransferase/genética , Testículo/fisiologia , Transcrição Gênica , Transferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Expressão Gênica , Fígado/fisiologia , Masculino , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/genética , Ratos , Ratos EndogâmicosRESUMO
The isolation and characterization of the rat genomic clone encoding the cholesterogenic enzyme farnesyl diphosphate (FPP) synthase is reported. The gene is localized on a 15-kilobase (kb) genomic fragment, spans approximately 12 kb and contains eight exons. Sequences containing from 3.9 kb to 132 base pairs (bp) of the putative promoter were joined to the coding region of the bacterial reporter gene chloramphenicol acetyltransferase (CAT). The CAT activities or CAT mRNA levels of the hybrid genes were determined following either transient transfections into human hepatoma HepG2 cells or stable transfections into Chinese hamster ovary cells. The transient transfections identified a 319-bp fragment that was required for a 4-fold induction in the absence of sterols. Sequence analysis of this region showed it contained five potential copies of the sterol regulatory element (SRE-1) (Smith, J.R., Osborne, T.F., Brown, M.S., Goldstein, J.L., and Gil, G. (1988) J. Biol. Chem. 263, 18480-18487) previously identified in the promoters of the 3-hydroxy-3-methyl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein receptor genes. Further mutational and deletion analysis of the FPP synthase promoter-CAT constructs followed by stable transfection and primer extension of the CAT mRNA levels indicated that these potential SRE-1 regulatory elements were not involved in the sterol-mediated transcriptional regulation of the gene. Our analyses have identified a 115-bp region that is required for the transcriptional induction of FPP synthase in the absence of sterols. These results suggest that the FPP synthase gene may be regulated at the transcriptional level by a different mechanism than other sterol regulated genes.
Assuntos
Alquil e Aril Transferases , Fígado/enzimologia , Regiões Promotoras Genéticas , Transferases/genética , Animais , Sequência de Bases , Células CHO , Carcinoma Hepatocelular , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular/métodos , Cricetinae , DNA/genética , DNA/isolamento & purificação , Éxons , Genes Reguladores , Biblioteca Genômica , Geraniltranstransferase , Humanos , Íntrons , Neoplasias Hepáticas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Transcrição Gênica , Transfecção , Transferases/metabolismoRESUMO
Previous studies identified a 115-base pair (bp) region of the farnesyl diphosphate (FPP) synthase promoter which is involved in the transcriptional regulation of this gene by sterols (Spear, D. H., Kutsunai, S. Y., Correll, C. C., and Edwards, P. A. (1992) J. Biol. Chem. 267, 14462-14469). In the current study we fused a 117-bp fragment, containing this region of interest, upstream of the heterologous minimal promoter of the herpes simplex virus thymidine kinase gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Chinese hamster ovary (CHO) cells were stably transfected with this fusion gene and incubated in the absence or presence of sterols. Analysis of CAT mRNA by primer extension indicated that transcription of the fusion gene was under sterol-mediated control. Thus, when cellular sterols were present, the CAT mRNA levels were reduced 2-4-fold. To further localize the FPP synthase sterol-responsive element(s), additional promoter-reporter gene constructs containing either deletions or mutations were constructed and transfected into CHO or CV-1 cells. These studies localized a 6-bp region (ATTGGC) that is required for both transcriptional induction in the absence of sterols and transcriptional repression in the presence of sterols. Gel shift and footprinting analyses demonstrated that nuclear proteins isolated from CHO cells bound to six distinct regions of the promoter between nucleotides -293 to -47. Taken together, these results further define both the cis-acting elements controlling normal transcription of the FPP synthase gene and identify a novel sequence involved in sterol regulation.