RESUMO
The gene for malic enzyme is expressed at a high level in chick embryo-hepatocytes (CEH) treated with triiodothyronine (T3) and at a low level in the absence of T3. In chick-embryo fibroblasts (CEF), expression of the malic enzyme gene is low and not regulated by T3. Specific nuclear proteins from both CEH and CEF bound to a consensus CCAAT/enhancer binding protein (C/EBP) site at -335 to -327 bp of the malic enzyme gene. The level of binding was much higher in extracts from CEH than in extracts of CEF, and the complexes formed had different mobilities. C/EBPalpha was present in the complex that bound to the C/EBP site in nuclear extracts from CEH but not in those from CEF. The C/EBP element was necessary and sufficient to bestow full T3 responsiveness to 5800 bp of 5'-flanking DNA of the malic enzyme gene in CEH. C/EBPalpha was not detectable in wild-type CEF, and deletion of the C/EBP binding site had no effect on expression of transgenes containing 5800 bp of 5'-flanking DNA of the malic enzyme gene. In CEF, overexpression of C/EBPalpha stimulated promoter activity of constructs that contained the C/EBP site linked to the malic enzyme promoter or a heterologous reporter. The results suggest that C/EBPalpha or a closely related isoform is involved in the tissue-specific expression of the malic enzyme gene.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Malato Desidrogenase/biossíntese , Proteínas Nucleares/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT , Células Cultivadas , Embrião de Galinha , Pegada de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Soros Imunes/metabolismo , Fígado/metabolismo , Malato Desidrogenase/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/fisiologia , Sequências Reguladoras de Ácido Nucleico/efeitos dos fármacos , Deleção de Sequência/genética , Transfecção , Tri-Iodotironina/farmacologiaRESUMO
In vivo, feeding stimulates and starvation inhibits transcription of the malic enzyme gene. In chick-embryo hepatocytes in culture, triiodothyronine (T3) stimulates and glucagon inhibits transcription of this gene. As a first step in the characterization of the involved regulatory mechanisms, fragments of genomic DNA spanning the structural and 5'-flanking regions of the chicken malic enzyme gene were cloned. The coding region of the gene is organized into 14 exons and 13 introns and is greater than 106 kb in length. The size of the gene, the number and lengths of the exons, and positions at which introns are inserted into the coding regions are virtually identical in the chicken and rat genes. When transiently transfected into chick-embryo hepatocytes, 5800 bp of 5'-flanking DNA conferred T3 responsiveness to a linked chloramphenicol acetyltransferase (CAT) reporter gene. Using deletion and site-specific mutations of 5'-flanking DNA, we identified a complex T3 response unit that contains one major T3 response element (T3RE) and several minor ones. The major element contains two degenerate copies of the hexamer, RGGWMA, separated by 4 bp and was a strong repressor in the absence of ligand. Endogenous levels of T3 receptor are sufficient to allow the T3 response elements in the upstream region of the malic enzyme gene to confer responsiveness to T3, suggesting that they are physiologically relevant.
Assuntos
Galinhas/genética , Malato Desidrogenase/biossíntese , Malato Desidrogenase/genética , Sequências Reguladoras de Ácido Nucleico , Tri-Iodotironina/farmacologia , Animais , Sequência de Bases , Células Cultivadas , Embrião de Galinha , Cloranfenicol O-Acetiltransferase/biossíntese , Fígado/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico/efeitos dos fármacos , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase.
