Glycerol 3-phosphate Dehydrogenase Gene Research Articles

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene.

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.

Fermentation properties of a wine yeast over-expressing the Saccharomyces cerevisiae glycerol 3-phosphate dehydrogenase gene (GPD2)

Wine yeasts efficiently convert sugar into ethanol. The possibility of diverting some of the sugar into compounds other than ethanol by using molecular genetic methods was tested. Over-expression of the yeast glycerol 3-phosphate dehydrogenase gene (GPD2) in a laboratory strain of Saccharomyces cerevisiae led to an approximate two-fold increase in the extracellular glycerol concentration. In the medium fermented with the modified strain, acetic acid concentration also increased approximately two-fold when respiration was blocked. A strain deleted for the GPD2 gene had the opposite phenotype, producing lower amounts of glycerol and acetic acid, with the latter compound only reduced during non-respiratory growth. A commercial wine yeast over-expressing GPD2 produced 16.5 g/L glycerol in a wine fermentation, compared to 7.9 g/L obtained with the parent strain. As seen for the laboratory strain, acetic acid concentrations were also increased when using the genetically modified wine yeast. A panel of wine judges confirmed the increase in volatile acidity of these wines. The altered glycerol biosynthetic pathway sequestered carbon from glycolysis and reduced the production of ethanol by 6 g/L.

Retinoic acid and dexamethasone interact to regulate S14 gene transcription in 3T3-F442A adipocytes

We have examined the effects of retinoic acid (RA) on S14 gene expression in 3T3-F442A preadipocytes and adipocytes. RA induced mRNA s14 14-fold in adipocytes, but had no effect on S14 gene expression in preadipocytes. Northern analysis of retinoic acid receptor (RAR) isoforms revealed the presence of RARα, but not RARβ, in both preadipocytes and adipocytes. These results suggest that adipocytespecific factors expressed during differentiation may be required for RA control of S14 gene expression in cultured adipocytes. When compared to dexamethasone (DEX), RA was a weak inducer of S14 gene expression. However, when added together, RA and DEX acted synergistically to induce S14 gene expression. Since changes in S14 gene transcription paralleled changes in mRNA s14 levels, the principal target for RA and DEX action on S14 gene expression was at the level of gene transcription. In the presence of DEX (0.1 nM), RA effects on S14 gene expression were dose-dependent (ED 50 = 5 nM) and rapid (within 4 h). In contrast to S14, RA inhibited glycerol 3-phosphate dehydrogenase (GPD) gene transcription and mRNA GDP abundance by 75%. The effects of RA on S14 and GPD gene transcription in cultured adipocytes suggest that RA action may extend beyond growth and differentiation to include effects on lipid metabolism.

Glycerol 3-phosphate dehydrogenase gene expression in cultured 3T3-L1 adipocytes: regulation by insulin, dexamethasone and dibutyryl cAMP at the level of mRNA abundancem transcription and mRNA stability

In fully differentiated 3T3-L1 adipocytes, glycerol 3-phosphate dehydrogenase (G3PDH:Sn-glycerol 3-phosphate:NAD + oxidoreductase, EC 1.1.1.8) is subject to regulation by hormones and dibutyryl cAMP. An increase by insulin (4-fold) and decrease by dexamethasone (by 50%) and dibutyryl cAMP (by 70%) was observed for G3PDH mRNA abundance as analyzed by Northern blot hybridization. In addition, incubation of adipocytes with dibutyryl cAMP resulted in 3-fold increase in G3PDH gene transcription as measured by nuclear transcript elongation assay. The effects of these modulators on the control of G3PDH mRNA stability were also investigated. The G3PDH mRNA has a half-life of about 125 min. Dibutyryl cAMP caused an increase in G3PDH mRNA degradation by > 2-fold ( t 1 2 = 55 min ) whereas insulin had an opposite effect ( t 1 2 = 240 min ) and dexamethasone was without any effect on G3PDH mRNA stability. Taken together, our results directly demonstrate that in fully differentiated 3T3-L1 adipocytes the regulation of G3PDH gene expression by dibutyryl cAMP and insulin is exerted by alterations in transcription as well as mRNA stability.

Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans.

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.

Sequence Conservation and Structural Organization of the Glycerol-3-Phosphate Dehydrogenase Promoter in Mice and Humans

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.