RESUMO
A xylose reductase gene was isolated from the xylose-fermenting yeast Pachysolen tannophilus as a cDNA clone by selecting clones that hybridized specifically to xylose-inducible messenger RNA. Use of the cDNA clone as a probe in Northern hybridizations identified a xylose-inducible mRNA species large enough to encode a 36 kDa xylose reductase protein known to be produced by this yeast. A corresponding genomic clone was isolated as a 3 kb EcoRI fragment that specifically hybridized to the cDNA clone. The sequence of the cDNA and the largest open reading frame of the genomic clone are identical. The predicted translation product exhibits: (1) significant sequence identity with a previously published N-terminal amino acid sequence from purified P. tannophilus xylose (aldose) reductase protein exhibiting NADH/NADPH-dependent activities (aldose reductase, EC 1.1.1.21); (2) identity with a protein composed of 317 amino acid residues with a calculated molecular mass of 36.2 kDa, equivalent to that reported for purified P. tannophilus xylose reductase; and (3) considerable sequence similarity to, and features of, a superfamily of oxidoreductases.
Assuntos
Aldeído Redutase/genética , Leveduras/enzimologia , Leveduras/genética , Aldeído Redutase/imunologia , Sequência de Aminoácidos , Anticorpos Antifúngicos/imunologia , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA Complementar/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fases de Leitura Aberta , Plasmídeos , Biossíntese de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Xilose/metabolismoRESUMO
Three plasmids, with sizes of 7.0 kbp, 6.8 kbp, and 5.0 kbp and designated pAal-1, pAal-2 and pAal-3 respectively, have been found in a tentoxin-producing isolate of Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5' ends. Plasmid pAal-1 does not hybridize to nuclear DNA, mitochondrial DNA, or double-stranded RNA from a mycovirus found in the isolate, but does hybridize weakly to a series of linear DNAs which are not visible on gels and may include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are not localized in the mitochondria. Plasmids have not been found in other tentoxin-producing isolates and there is no evidence that these plasmids have any effect on the production of tentoxin.
Assuntos
Alternaria/genética , Plasmídeos , Enzimas de Restrição do DNA/metabolismo , Mitocôndrias/metabolismoRESUMO
Double-stranded (ds) RNAs associated with viruslike particles have been found in six isolates of Alternaria alternata which produce tentoxin. Isolates had from one to three dsRNAs ranging in size from 1.0 to 5.1 kilobase pairs. In two isolates the dsRNAs were associated with 30-nm particles. No dsRNA was detected in any of six other tentoxin-producing isolates or nine isolates which did not produce tentoxin.
Assuntos
Alternaria/genética , Fungos Mitospóricos/genética , Micotoxinas/biossíntese , Peptídeos Cíclicos/biossíntese , RNA de Cadeia Dupla/análise , Vírion/genética , Alternaria/metabolismo , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , Vírion/ultraestruturaRESUMO
Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.
Assuntos
Chlamydomonas/genética , Clorofila/genética , Regulação da Expressão Gênica , Proteínas de Plantas/genética , Ciclo Celular , Regulação da Expressão Gênica/efeitos da radiação , Luz , Complexos de Proteínas Captadores de Luz , Complexo de Proteínas do Centro de Reação Fotossintética , RNA Mensageiro/genéticaAssuntos
Chlamydomonas/genética , Cloroplastos/efeitos dos fármacos , Floxuridina/farmacologia , Mutação , Antibacterianos/farmacologia , Ciclo Celular , Chlamydomonas/efeitos dos fármacos , DNA/metabolismo , Resistência a Medicamentos , Herança Extracromossômica , Genes/efeitos dos fármacos , FotossínteseRESUMO
Chloroplast components known to be coded by chloroplast DNA include chloroplast rRNAs, tRNAs, and the large subunit of ribulose-bisphosphate carboxylase. Because these components comprise less than 3% of the estimated coding capacity of the chloroplast genome, most chloroplast gene functions have yet to be identified. One approach to this problem is the isolation and characterization of mutations in the chloroplast genome affecting specific photosynthetic functions. Recently we have found that such mutations can be preferentially recovered by using arsenate selection on cells previously grown in 5-fluorodeoxyuridine. Sixteen mutants thus isolated have been localized into nine chloroplast loci, based on their ability to recombine and produce photosynthetically competent progeny. Mutants at two loci show the characteristic syndrome of photosynthetic defects that results from a deficiency in chloroplast protein synthesis. These have been found to lack chloroplast ribosome monomers. Mutants at three loci are missing chlorophyll-protein complex I in their thylakoid membranes. Mutants at three other loci are deficient in membrane polypeptides known to be associated with the chloroplast coupling factor.
