Expression of human inositol monophosphatase suppresses galactose toxicity in Saccharomyces cerevisiae: possible implications in galactosemia. (1/269)

A suppressor of galactose toxicity in a gal7 yeast strain (lacking galactose 1-phosphate uridyl transferase) has been isolated from a HeLa cell cDNA library. Analysis of the plasmid clone indicated that the insert has an ORF identical to that of hIMPase (human myo-inositol monophosphatase). The ability of hIMPase to suppress galactose toxicity is sensitive to the presence of Li(+) in the medium. A gal7 yeast strain harboring a plasmid containing cloned hIMPase grows on galactose as a sole carbon source. hIMPase mediated galactose metabolism is dependent on the functionality of GAL1 as well as GAL10 encoded galactokinase and epimerase respectively. These results predicted that the UDP-glucose/galactose pyrophosphorylase mediated pathway may be responsible for the relief of galactose toxicity. Experiments conducted to test this prediction revealed that expression of UGP1 encoded UDP-glucose pyrophosphorylase can indeed overcome the relief of galactose toxicity. Moreover, expression of UGP1 allows a gal7 strain to grow on galactose as a sole carbon source. Unlike the hIMPase mediated relief of galactose toxicity, UGP1 mediated relief of galactose toxicity is lithium insensitive. Based on our results and on the basis of available information on galactose toxicity, we suggest an alternative explanation for the molecular mechanism of galactose toxicity.  (+info)

A founder mutation in the GK1 gene is responsible for galactokinase deficiency in Roma (Gypsies). (2/269)

Galactokinase deficiency is an inborn error in the first step of galactose metabolism. Its major clinical manifestation is the development of cataracts in the first weeks of life. It has also been suggested that carriers of the deficiency are predisposed to presenile cataracts developing at age 20-50 years. Newborn screening data suggest that the gene frequency is very low worldwide but is higher among the Roma in Europe. Since the cloning of the galactokinase gene (GK1) in 1995, only two disease-causing mutations, both confined to single families, have been identified. Here we present the results of a study of six affected Romani families from Bulgaria, where index patients with galactokinase deficiency have been detected by the mass screening. Genetic linkage mapping placed the disease locus on 17q, and haplotype analysis revealed a small conserved region of homozygosity. Using radiation hybrid mapping, we have shown that GK1 is located in this region. The founder Romani mutation identified in this study is a single nucleotide substitution in GK1 resulting in the replacement of the conserved proline residue at amino acid position 28 with threonine (P28T). The P28T carrier rate in this endogamous population is approximately 5%, suggesting that the mutation may be an important cause of early childhood blindness in countries with a sizeable Roma minority.  (+info)

Overexpression of Erg11p by the regulatable GAL1 promoter confers fluconazole resistance in Saccharomyces cerevisiae. (3/269)

The contribution of the dosage of target enzyme P-450 14alpha-demethylase (14alphaDM) to fluconazole resistance in both Candida albicans and Saccharomyces cerevisiae remains unclear. Here, we show that overexpression of Saccharomyces P-450 14alphaDM in S. cerevisiae, under the control of the regulatable promoter GAL1, results in azole resistance.  (+info)

The Spt components of SAGA facilitate TBP binding to a promoter at a post-activator-binding step in vivo. (4/269)

The SAGA complex of Saccharomyces cerevisiae is required for the transcription of many RNA polymerase II-dependent genes. Previous studies have demonstrated that SAGA possesses histone acetyltransferase activity, catalyzed by the SAGA component Gcn5. However, the transcription of many genes, although SAGA dependent, is Gcn5 independent, suggesting the existence of distinct SAGA activities. We have studied the in vivo role of two other SAGA components, Spt3 and Spt20, at the well-characterized GAL1 promoter. Our results demonstrate that both Spt3 and Spt20 are required for the binding of TATA-binding protein but not of the activator Gal4 and that this role is Gcn5 independent. These results suggest a coactivator role for Spt3 and Spt20 in the recruitment of TBP.  (+info)

Influence of the lactose plasmid on the metabolism of galactose by Streptococcus lactis. (5/269)

Streptococcus lactis strain DR1251 was capable of growth on lactose and galactose with generation times, at 30 degrees C, of 42 and 52 min, respectively. Phosphoenolpyruvate-dependent phosphotransferase activity for lactose and galactose was induced during growth on either substrate. This activity had an apparent K(m) of 5 x 10(-5) M for lactose and 2 x 10(-2) M for galactose. beta-d-Phosphogalactoside galactohydrolase activity was synthesized constitutively by these cells. Strain DR1251 lost the ability to grow on lactose at a high frequency when incubated at 37 degrees C with glucose as the growth substrate. Loss of ability to metabolize lactose was accompanied by the loss of a 32-megadalton plasmid, pDR(1), and Lac(-) isolates did not revert to a Lac(+) phenotype. Lac(-) strains were able to grow on galactose but with a longer generation time. Galactose-grown Lac(-) strains were deficient in beta-d-phosphogalactoside galactohydrolase activity and phosphoenolpyruvate phosphotransferase activity for both lactose and galactose. There was also a shift from a predominantly homolactic to a heterolactic fermentation and a fivefold increase in galactokinase activity, relative to the Lac(+) parent strain grown on galactose. These results suggest that S. lactis strain DR1251 metabolizes galactose primarily via the tagatose-6-phosphate pathway, using a lactose phosphoenolpyruvate phosphotransferase activity to transport this substrate into the cell. Lac(-) derivatives of strain DR1251, deficient in the lactose phosphoenolpyruvate phosphotransferase activity, appeared to utilize galactose via the Leloir pathway.  (+info)

A case-control study of galactose consumption and metabolism in relation to ovarian cancer. (6/269)

Consumption or metabolism of dairy sugar and ovarian cancer have been linked based on evidence that galactose may be toxic to ovarian germ cells and that ovarian cancer is induced in animals by depletion of oocytes. We assessed consumption of dairy products and obtained blood for biochemical and molecular genetic assessment of galactose metabolism in 563 women with newly diagnosed epithelial ovarian cancer and 523 control women selected either by random digit dialing or through lists of residents in eastern Massachusetts and New Hampshire. We observed no significant differences between cases and controls in usual consumption of various types of dairy products or total daily lactose (the principal source of galactose in the diet); nor did we find that RBC activity of either galactose-1-phosphate uridyl transferase (GALT) or galactokinase differed. The mean (and SE) activity of uridine diphospho-galactose 4'-epimerase (in micromoles per hour per gram of hemoglobin) was, however, significantly lower (P < 0.005) in cases compared with controls, 20.32 (0.31) versus 21.64 (0.36). Ovarian cancer cases were also more likely to carry the N314D polymorphism of the GALT gene, generally predisposing to lower GALT activity. The difference was most evident for endometrioid and clear cell types of ovarian cancer, in which 3.9% of cases were found to be homozygous for N314D compared with 0.4% of controls, yielding an odds ratio and 95% confidence interval of 14.17 (2.62-76.60). We conclude that, whereas adult consumption of lactose carries no clear risk for the disease, certain genetic or biochemical features of galactose metabolism may influence disease risk for particular types of ovarian cancer.  (+info)

The insertion of two amino acids into a transcriptional inducer converts it into a galactokinase. (7/269)

The transcriptional induction of the GAL genes of Saccharomyces cerevisiae occurs when galactose and ATP interact with Gal3p. This protein-small molecule complex associates with Gal80p to relieve its inhibitory effect on the transcriptional activator Gal4p. Gal3p shares a high degree of sequence homology to galactokinase, Gal1p, but does not itself possess galactokinase activity. By constructing chimeric proteins in which regions of the GAL1 gene are inserted into the GAL3 coding sequence, we have been able to impart galactokinase activity upon Gal3p as judged in vivo and in vitro. Remarkably, the insertion of just two amino acids from Gal1p into the corresponding region of Gal3p confers galactokinase activity onto the resultant protein. The chimeric protein, termed Gal3p+SA, retains its ability to efficiently induce the GAL genes. Kinetic analysis of Gal3p+SA reveals that the K(m) for galactose is similar to that of Gal1p, but the K(m) for ATP is increased. The chimeric enzyme was found to have a decreased turnover number in comparison to Gal1p. These results are discussed in terms of both the mechanism of galactokinase function and that of transcriptional induction.  (+info)

Growth-phase regulation of the Escherichia coli thioredoxin gene. (8/269)

The two promoters of Escherichia coli trxA gene were separately cloned into pKO100 as well as pJEL170. Galactokinase expression in cells containing the pKO100 derivatives was found to be negatively correlated with growth rate and was 6- to 20-fold higher in stationary cultures than in exponential cultures. The expression of trxA-galK was induced by amino acid starvation in a RelA(+) strain but not in an isogenic Rel(-) strain indicating that the control involves guanosine 3',5'-bispyrophosphate (ppGpp). RpoS, which appears to be essential for expression of most stationary phase expressed genes, is not required for trxA expression. Increased expression of relA, which increases ppGpp concentration, increases trxA expression.  (+info)