RESUMO
Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.
Assuntos
Minas de Carvão , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Variação Genética , Metais Pesados , Mineração , Brasil , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Ribossômico/análise , DNA Ribossômico/genética , Gammaproteobacteria/isolamento & purificação , Resíduos Industriais , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Ribotipagem , Análise de Sequência de DNARESUMO
Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.
Assuntos
Proteínas de Bactérias/metabolismo , Cobre/farmacologia , Metais Pesados/farmacologia , Thiobacillus/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Cádmio/farmacologia , Eletroforese em Gel Bidimensional , Níquel/farmacologia , Consumo de Oxigênio , Fosforilação , Thiobacillus/crescimento & desenvolvimento , Thiobacillus/metabolismo , Zinco/farmacologiaRESUMO
Two-dimensional electrophoresis of Cereus peruvianus callus tissues grown in culture media containing two different 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin combinations was used to identify minor differences in polypeptide composition of these cell clones. Altered expression during growth in the two 2,4-D and kinetin combinations was apparent for 13 polypeptides when calluses in the two media were compared. The number of proteins with differential expression (presence or absence of specific spots) was higher in callus tissues cultured in the 4.0 mg/L 2,4-D and 8.0 mg/L kinetin combination than in callus tissues cultured in the 4.0 mg/L 2,4-D and 4.0 mg/L kinetin combination. The present results show that the callus tissues maintained at 4.0 mg/L 2,4-D and 8.0 mg/L kinetin can be used as a matrix for in vitro selection programs.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas de Plantas/análise , Plantas/químicaRESUMO
Transient expression and electrophoretic mobility shift assay were used to investigate the cis elements and the DNA-binding proteins involved in the regulation of expression of a 22 kDa zein-like alpha-coixin gene. A set of unidirectional deletions was generated in a 962 bp fragment of the alpha-coixin promoter that had been previously fused to the reporter gene GUS. The constructs were assayed by transient expression in immature maize endosperm. There was no significant decrease in GUS activity as deletions progressed from -1084 to -238. However, deletion from -238 to -158, which partially deleted the O2c box, resulted in a dramatic decrease in GUS activity emphasizing the importance of the O2 box in the quantitative expression of the gene. The -238 promoter fragment interacted with Coix endosperm nuclear proteins to form 5 DNA-protein complexes, C1-C5, as detected by EMSA. The same retarded complexes were observed when the -158 promoter fragment was used in the binding reactions. Reactions with nuclear extracts isolated from Coix endosperms harvested from 6 to 35 days after pollination revealed that the 5 DNA-protein complexes that interact with the alpha-coixin promoter are differentially assembled during seed development. Deletion analysis carried out on the -238/ATG promoter fragment showed that a 35 bp region from -86 to -51 is essential for the formation of the complexes observed. When nuclear extracts were incubated with an antiserum raised against the maize Opaque-2 protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented indicating that an Opaque-2 like protein participates in the formation of those complexes. Complex C2 was not affected by the addition of the O2 antibody, suggesting the existence of a novel nuclear factor, CBF1, that binds to the promoter and makes protein-protein associations with other proteins present in Coix endosperm nuclei.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Zíper de Leucina , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Proteínas Nucleares/metabolismo , Plasmídeos , Deleção de Sequência , Zea maysRESUMO
Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0-93 â 10-6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 â 10-6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 â 10-6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1.
RESUMO
The maize Opaque2 (O2) protein is a "leucine zipper" DNA binding factor that interacts with the sequence TCCACGTAGA in the promoters of the 22-kD alpha-zein genes and activates its transcription. A completely different consensus sequence (GATGAPyPuTGPu) identified in b-32, a gene that encodes an abundant albumin that is also under control of the O2 locus, can also be bound by the O2 protein. We showed that the gene encoding the 22-kD-like alpha-coixin, the alpha-prolamin of the maize-related grass Coix, can also be transactivated by the O2 protein. A binding assay in vitro and footprint analysis demonstrated that the GACATGTC sequence of the alpha-coixin promoter can be recognized and protected by the maize O2 protein. Employing transient expression experiments in immature maize endosperm and tobacco mesophyll protoplasts, we demonstrated that the O2 protein can activate expression of the beta-glucuronidase reporter gene placed under the control of the 22-kD-like alpha-coixin promoter. We also demonstrated that a 22-kD-like alpha-coixin pseudogene promoter is transactivated by the maize O2 protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Zea mays/genética , Zea mays/metabolismo , Sequência de Bases , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Zíper de Leucina , Dados de Sequência Molecular , Prolaminas , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Zeína/genéticaAssuntos
Aminoácidos/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Plantas/fisiologia , Proteínas/metabolismo , Fatores de Transcrição/fisiologia , Zea mays/metabolismo , Ácido Aspártico/metabolismo , Sequência de Bases , Lisina/metabolismo , Dados de Sequência Molecular , Sementes/metabolismo , Zeína/biossínteseRESUMO
Several genomic and cDNA clones encoding the 22 kDa-like alpha-coixin, the alpha-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like alpha-coixin genes designated alpha-3A, alpha-3B and alpha-3C were found in the 15 kb alpha-3 genomic clone. The alpha-3A and alpha-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the alpha-3B gene, suggesting that the three alpha-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of alpha-coixin clones with the published sequences of 22 kDa alpha-zein and 22 kDa-like alpha-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like alpha-prolamins and the 19 kDa alpha-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5' and 3' flanking regions of alpha-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in alpha-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in alpha-3B that occupies approximately the same positions as those identified for the 22 kDa alpha-zein and alpha-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like alpha-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.
Assuntos
Proteínas de Plantas/genética , Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Biblioteca Genômica , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Zea mays/genética , Zeína/genéticaRESUMO
Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain alpha-, beta-, and gamma-zein and alpha-, beta-, and gamma-coixin. The alpha-coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa alpha-zeins. Like the alpha-zeins, the C1 and C2 alpha-coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to gamma-coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of gamma-zein and represents 15% of the total coixin. The beta-zein fraction was composed of a major 17 kDa protein band, while the beta-coixin fraction consisted of a mixture of alpha- and gamma-coixins. Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa alpha-zein, as did C4 and C5 antisera. The antiserum against gamma-coixin showed strong cross-reaction with gamma-zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa alpha-zeins as well as the 28 and 16 kDa gamma-zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa alpha-zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone.(ABSTRACT TRUNCATED AT 250 WORDS)