RESUMO
The upstream regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence known to be important in setting both basal and depressed levels of expression. We have investigated whether the CCAAT-binding HAP2/3/4 complex of the yeast Saccharomyces cerevisiae can recognise this sequence in an amdS context. Sequences from the 5' region of amdS were cloned in front of the CYC1-lacZ fusion gene bearing a minimal promoter and transformed into wild-type and hap2 strains of yeast. This study has indicated that amdS sequences are capable of promoting regulated expression of the fusion gene in response to carbon limitation. The yeast HAP2/3/4 complex can recognise the amdS CCAAT sequence and activate expression from this sequence. In addition, the results indicate that other yeast proteins can also regulate expression from the A. nidulans amdS 5'sequences under carbon-limiting conditions.
Assuntos
Amidoidrolases/genética , Aspergillus nidulans/genética , Fator de Ligação a CCAAT , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Amidoidrolases/biossíntese , Aspergillus nidulans/enzimologia , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Especificidade da Espécie , Fatores de Transcrição/genéticaRESUMO
The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally determined MU value of the urinary metabolite with those of the theoretical values established the correct stereochemistry of the steroid. This method has been compared with the use of gas chromatography-mas spectrometry in the determination of the stereochemistry of unknown metabolites.
Assuntos
Estradiol/urina , Nandrolona/metabolismo , Animais , Fenômenos Químicos , Química , Cromatografia Gasosa , Cavalos , Espectrometria de Massas , Estrutura Molecular , EstereoisomerismoRESUMO
As part of a continuing research program associated with the detection of anabolic steroid residues in horse urine, normal samples from entire male horses have now been investigated. Isomers of three C-18 neutral steroids; 4-estren-17-ol-3-one (1), estrane-3,17-diol (2) and an unsaturated estranediol having a possible structure (3), have been identified in urine samples from two male horses aged 8 and 14 years. Of these three steroids, compound (2) was not detected in the urine of a 2.5 yr old entire male nor in the majority of post-race urine samples from entire male horses average age 3.8 yrs (n = 34). Ten of these samples showed tentative indications of this compound. Although the isolation of isomers of estrane-3,17-diol from human non-pregnancy urine has been reported previously, analysis of non-pregnancy urine samples in the present study did not reveal the presence of these compounds.
