RESUMO
In the present report we show that unknown DNA fragments are easily amplified in a single PCR reaction from an oligo-cassette library with a single genome-specific primer in combination with a cassette-specific primer. The novelty of the system, in comparison to the vectorette PCR method, lies in the use of unphosphorylated in contrast with phosphorylated oligo-cassettes in the ligation to the chromosomal DNA fragments. After denaturation of the DNA library, all chromosomal fragments carry a single-stranded linker attached to the 5'-end only. Therefore, the presence of the vectorette mismatched region is not required when unphosphorylated cassettes are used. As an example we report the amplification of the era gene from Lactococcus lactis.
Assuntos
Passeio de Cromossomo/métodos , Primers do DNA , DNA Bacteriano/genética , Proteínas de Escherichia coli , Reação em Cadeia da Polimerase/métodos , Proteínas de Ligação a RNA , Proteínas de Bactérias/genética , Cromossomos Bacterianos , Proteínas de Ligação ao GTP/genética , Biblioteca Gênica , Genoma , Lactococcus lactis/genética , Dados de Sequência Molecular , FosforilaçãoRESUMO
A full-length cDNA clone encoding barley dihydroflavonol-4-reductase was isolated from a kernel-specific cDNA library by screening with the cDNA of the structural gene (A1) for this enzyme from maize. Subsequently, the gene corresponding to the barley dihydroflavonol-4-reductase cDNA was cloned and sequenced. The gene contains three introns at the same positions as in the Zea mays gene, corresponding to the positions of the first three of the five introns present in the genes of Petunia hybrida and Antirrhinum majus. In vitro transcription and translation of the Hordeum vulgare cDNA clone yielded a protein which converts dihydroquercetin into 2,3-trans-3,4-cis-leucocyanidin with NADPH as cofactor. The protein has a deduced amino acid sequence of 354 residues and a molecular weight of 38,400 daltons. Dihydroflavonol reductases of barley, maize, petunia and snapdragon are highly polymorphic in the NH2- and C-terminal parts of the polypeptide chain while a central region of 324 residues contains 51% identical amino acids. This identity increases to 81% when only the barley and maize enzymes are compared. Recessive mutants in the Ant18 gene tested so far lack dihydroflavonol-4-reductase activity and accumulate small amounts of dihydroquercetin but have retained activity for at least two other enzymes in the flavonoid pathway. In testa-pericarp tissue of mutants ant18-159, ant18-162 and ant18-164, wild-type levels of steady state mRNA for dihydroflavonol reductase have been measured, while mRNA for this enzyme is not transcribed in mutant ant18-161. These data are consistent with the proposal that the Ant18 locus carries the structural gene for dihydroflavonol-4-reductase of barley.
Assuntos
Oxirredutases do Álcool/genética , Flavonoides/biossíntese , Hordeum/enzimologia , Mutação , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Southern Blotting , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA/genética , Genes de Plantas , Genes Reguladores , Dados de Sequência Molecular , Poli A/genética , RNA/genética , RNA Mensageiro , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
A novel method is presented for the synthesis and purification of (+)-2,3-trans-3,4-cis-[4-3H]leucocyanidin. Soluble enzyme extracts from developing barley grains and leaves of the forage legume Onobrychis viciifolia (sainfoin) catalyzed the NAD(P)H-dependent reduction of (+)-2,3-trans-3,4-cis-[4-3H]leucocyanidin to (+)-[4-3H]catechin. NADPH was the preferred substrate. With extracts of barley the rate of reaction with 1 mM NADH was 20% of the rate found with NADPH. With extracts from both tissues there was a broad pH optimum around pH 6.6.
Assuntos
Oxirredutases do Álcool/química , Catequina/química , Flavonoides/síntese química , Fabaceae/química , Fabaceae/enzimologia , Flavonoides/biossíntese , Flavonoides/química , Hordeum/química , Hordeum/enzimologia , Concentração de Íons de Hidrogênio , NADP/química , Oxirredução , Plantas Medicinais , Especificidade por Substrato , TrítioRESUMO
delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-alpha-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 microM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.