RESUMO
The asteroid gene of Drosophila was found to lie within 189 bp of Star. Asteroid cDNA clones were isolated and sequenced and a single putative open reading frame was identified that encodes a novel protein of 815 amino acids with a calculated molecular mass of 93 kilodaltons. Using cDNA probes, asteroid transcripts were localized to the proliferative tissues of embryos and to the mitotically active tissue anterior to the morphogenetic furrow in eye imaginal discs. Ribonuclease protection assays identified a mutation of asteroid that acts as a dominant enhancer of Star mutations and also enhances the Ellipse mutation, EgfrE1. Based on these data, a model for asteroid gene function in EGF receptor signaling is presented.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Dosagem de Genes , Genes erbB-1 , Fosfoproteínas/genética , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta , Mapeamento por Restrição , Transdução de Sinais/genéticaRESUMO
The lanosterol demethylation intermediate 3 beta-hydroxylanost-8-en-32-al is a known suppressor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthesis. Studies on the mechanism of action of this compound have been hampered by its rapid metabolism. As one approach to this problem, the effects of 3 beta-hydroxy-lanost-8-en-32-al on HMGR gene expression were examined using a mutant cell line which lacks lanosterol 14 alpha-methyl demethylase activity. Data are presented which suggest that 3 beta-hydroxy-lanost-8-en-32-al inhibits HMGR gene expression by reducing the translational efficiency of the HMGR mRNA. We have recently reported that 15 alpha-fluoro-3 beta-hydroxy-lanost-7-en-32-aldehyde, a compound which is structurally similar to 3 beta-hydroxy-lanost-8-en-32-aldehyde, suppresses HMGR activity in cultured Chinese hamster ovary cells by a posttranscriptional process, inhibiting translation without affecting either transcription or enzyme degradation (Trzaskos et al., 1993, J. Biol. Chem. 268, 22591-22599). In contrast to the results obtained with the 15 alpha-fluorolanostenol, the lanostenol 32-aldehyde increased the rate of degradation of HMGR in a manner similar to that reported for oxycholesterols. These data suggest that 15 alpha-fluoro-3 beta-hydroxy-lanost-7-en-32-aldehyde and 3 beta-hydroxy-lanost-8-en-32-aldehyde, although structurally similar posttranscriptional regulators of HMGR suppress enzyme activity, at least in part, by different mechanisms.
Assuntos
Hidroximetilglutaril-CoA Redutases/biossíntese , Lanosterol/análogos & derivados , Biossíntese de Proteínas/efeitos dos fármacos , Processamento Pós-Transcricional do RNA , Animais , Células CHO , Colesterol/biossíntese , Cricetinae , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Hidroximetilglutaril-CoA Redutases/genética , Lanosterol/metabolismo , Lanosterol/farmacologia , Oxirredutases/metabolismo , RNA Mensageiro/análise , Esterol 14-DesmetilaseRESUMO
The Star/asteroid (S/ast) region of Drosophila melanogaster has been cloned by P element transposon tagging using the snw chromosome as a source of defective P elements. In each mutation examined, the element integrated into the region was a 0.5-kb element from a region proximal to sn and not one of the head-to-head elements from the sn locus. Previously described spontaneous and X-ray induced mutations of S and ast were located on the molecular map by Southern analysis and restriction endonuclease mapping of genomic clones. S mutations are either large deletions of the cloned region or DNA breaks located near the P element insertions that cause ast mutations. Both S and ast mutations reduce the steady-state amounts of a 3.4-kb RNA. The molecular data, together with the phenotypic interactions observed for S and ast alleles, are consistent with the interpretation that S and ast mutations are lesions within the same gene or within genes that are functionally related.
Assuntos
Drosophila melanogaster/genética , Olho/crescimento & desenvolvimento , Animais , Clonagem Molecular , DNA , Elementos de DNA Transponíveis , Feminino , Masculino , Mutação , Mapeamento por Restrição , Sitios de Sequências RotuladasRESUMO
While studying the organization of the mouse glycerol-phosphate dehydrogenase gene (Gdc-1 on chromosome 15), we identified a novel transcriptional unit located only 3.4 kilobases (kb) upstream of the 5' end of the Gdc-1 gene. This gene has been provisionally named D15Kz1. The unusual proximity of these two genes led us to investigate the pattern of expression and sequence characteristics of the new gene for comparison with those of Gdc-1. D15Kz1 was found to have transcripts of 3.2 and 3.4 kb in length. The 3.4-kb transcript was expressed at low levels in all tissues examined, whereas the 3.2-kb transcript was detected only in the cerebral cortex and the brown fat. D15Kz1 and Gdc-1 are not coordinately regulated, as evidenced by the characteristics of their expression in several tissues and in differentiating 3T3-F442A adipocyte cultures. A cDNA sequence of 3,105 bases isolated from an embryonal carcinoma lambda gt10 cDNA library had a large open reading frame of 461 amino acids at one end followed by 1.6 kb of sequence with multiple stop codons. Algorithms used to search the protein and nucleic acid data bases detected no significant sequence similarity to any other protein or gene. Southern blot analysis of genomic DNA using the D15Kz1 cDNA as a probe indicated that D15Kz1 is a single-copy gene in the mouse genome and that it is conserved in humans, rats, and chickens. This conservation of gene sequences suggests that D15Kz1 encodes a protein with an important cellular function.
Assuntos
Glicerolfosfato Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Feminino , Amplificação de Genes , Regulação da Expressão Gênica , Ligação Genética , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Twelve dominant enhancers of position effect variegation, representing four loci on the second and third chromosomes of Drosophila melanogaster, have been induced by P-element mutagenesis. Instead of simple transposon insertions, seven of these mutations are cytologically visible duplications and three are deficiencies. The duplications define two distinct regions, each coinciding with a locus that also behaves as a dominant haplo-dependent suppressor of variegation. Conversely, two of the deficiencies overlap with a region that contains a haplo-dependent enhancer of variegation while duplications of this same region act to suppress variegation. The third deficiency defines another haplo-dependent enhancer. These data indicate that loci capable of modifying variegation do so in an antipodal fashion through changes in the wild-type gene copy number and may be divided into two reciprocally acting classes. Class I modifiers enhance variegation when duplicated or suppress variegation when deficient. Class II modifiers enhance when deficient but suppress when duplicated. From our data, and those of others, we propose that in Drosophila there are about 20 to 30 dominant loci that modify variegation. Most appear to be of the class I type whereas only two class II modifiers have been identified so far. From these observations we put forth a model, based on the law of mass action, for understanding how such suppressor-enhancer loci function. We propose that each class I modifier codes for a structural protein component of heterochromatin and their effects on variegation are a consequence of their dosage dependent influence on the extent of the assembly of heterochromatin at the chromosomal site of the position effect. It is further proposed that class II modifiers may inhibit the class I products directly, bind to hypothetical termination sites that define heterochromatin boundaries or promote euchromatin formation. Consistent with our mass action model we find that combining two enhancers together produce additive and not epistatic effects. Also, since different enhancers have different relative strengths on different variegating mutants, we suggest that heterochromatic domains are constructed by a combinatorial association of proteins. The mass action model proposed here is of general significance for any assembly driven reaction and has implications for understanding a wide variety of biological phenomena.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Mapeamento Cromossômico , Drosophila melanogaster/genética , Modelos Genéticos , Animais , Cruzamentos Genéticos , Drosophila melanogaster/anatomia & histologia , Elementos Facilitadores Genéticos , Feminino , Masculino , Mutação , Hibridização de Ácido Nucleico , Supressão GenéticaRESUMO
We have isolated from a BALB/c genomic library a 17.5-kilobase (kb) DNA fragment containing the entire mouse sn-glycerol-3-phosphate dehydrogenase (glycerol-P dehydrogenase) gene, and a substantial portion of the flanking regions. The DNA sequence of 10.8 kb of this fragment was determined. Using this information, together with the DNA sequence of several partial glycerol-P dehydrogenase cDNA clones and the rabbit glycerol-P dehydrogenase amino acid sequence, we determined the structural organization of the mouse glycerol-P dehydrogenase gene. The gene is 7.3 kb long and contains 8 exons. Transcription starts 19 base pairs 5' of the ATG initiation codon. Sequence analysis and S1 nuclease mapping indicated that the 8th exon contains coding sequences for the last 31 amino acids of glycerol-P-dehydrogenase, followed by 1.7 kb of 3' untranslated mRNA sequence. The mouse glycerol-P dehydrogenase amino acid sequence determined from the DNA sequence is 90% homologous with the rabbit enzyme. An examination of the exon/intron organization of the mouse glycerol-P dehydrogenase gene shows that intron 3 precisely separates the NAD-binding and the catalytic domains and intron 2 separates the adenine- and nicotinamide-binding regions.
Assuntos
Genes , Glicerolfosfato Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/isolamento & purificação , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , Coelhos , Especificidade da EspécieRESUMO
The chromosomal region surrounding the structural gene for alpha-glycerophosphate dehydrogenase (alpha Gpdh, 2-20.5) of Drosophila melanogaster has been studied in detail. Forty-three EMS-induced recessive lethal mutations and five previously identified visible mutations have been localized within the 25A-27D region of chromosome 2 by deficiency mapping and in some cases by a recombination analysis. The 43 lethal mutations specify 17 lethal loci. alpha Gpdh has been localized to a single polytene chromosome band, 25F5, and there apparently are no lethals that map to the alpha Gpdh locus.
Assuntos
Glicerolfosfato Desidrogenase/genética , Animais , Mapeamento Cromossômico , Drosophila melanogaster/genética , Genes , Genes Letais , Genes Recessivos , MutaçãoRESUMO
Thirty mutants of alpha-glycerophosphate dehydrogenase (alpha GPDH, EC 1.1.1.8) from Drosophila melanogaster were produced with the chemical mutagen ethyl methanesulfonate (EMS). These mutants and nine others previously obtained have been characterized with respect to level of enzymatic activity, viability, flight ability, and presence of cross-reacting material (CRM). The presence of alpha GPDH mRNA in several of the mutans has been tested by in vitro translation. There are strong correlations between the level of enzyme activity, viability and flight ability. Thirteen of the mutants are CRM- by solution immunoprecipitation experiments, but of these, only three are CRM- by a more sensitive 125I-protein A-based radioimmune gel assay. The viability of the three CRM- mutants suggests that the absence of alpha GPDH protein is not a lethal condition. The immunoprecipitated protein of the low activity mutant, alpha GpdhnGL3, has a smaller apparent molecular weight on polyacrylamide-SDS gels than does the protein from wild type. Criteria for the identification of nonsense mutations in Drosophila are discussed.