Production brewing strains of the yeast Saccharomyces cerevisiae were analysed by DNA fingerprinting, using a Southern blotting and hybridization procedure and employing the Ty1-15 transposon as a probe. The ability to differentiate readily between strains was very dependent on the restriction enzyme used to digest the DNA prior to Southern blotting and hybridization; the enzymes EcoRI, PstI and SalI were found to be particularly useful in this respect. The method was applicable to the differentiation of both ale and lager yeasts, and was sufficiently sensitive to distinguish between very closely related strains. DNA fingerprinting by this approach confirmed, for example, that a flocculent strain isolated during a production-scale fermentation with a lager yeast was genotypically different from the parent.

Malonyl-CoA decarboxylase (MLYCD) deficiency is an autosomal recessive disorder characterized by malonic aciduria, developmental delay, seizure disorder, hypoglycemia, and cardiomyopathy. Genomic sequencing of MLYCD in nine unrelated patients identified 16 of 18 pathogenic alleles, which are documented in the newly created Human MLYCD Allelic Variant Database (http://mlycd.hgu.mrc.ac.uk/). Fibroblast cell lines were available from eight of these patients and two previously reported patients with homozygous MLYCD mutations. Western blot analysis using antisera raised to a C-terminal peptide detected a 66-kDa band that was absent in six patients and substantially reduced in three patients. One patient showed an increase in protein levels with a prominent smeary 68-l83-kDa band. Immunocytochemical analysis of MLYCD-expressing patient cell lines showed apparent intracellular mislocalization. An extreme N-terminal mutation c.8G>A (p.G3D) mislocalized to the plasma membrane, suggesting that a novel targeting signal may reside in a four-amino acid conserved N-terminal motif. A 25-base deletion between the putative mitochondrial and peroxisomal initiating codons (M1 and M40) and a point mutation ablating the second of these (c.119T>C, p.M40T) both showed punctate perinuclear staining. As none of the three mislocalizing mutations are predicted to alter the catalytic function of the peptide, it seems likely that correct subcellular localization of MLYCD is critical for it to function normally.

No abstract

A cDNA clone encoding a full length putative collagen has been isolated in a screen of a mixed stage Globodera pallida expression library. Comparison of the deduced amino acid sequence of this molecule with other collagens suggests it is a cuticular collagen and a member of the col-8 subfamily of collagen genes. Northern blots show the gene is expressed specifically in gravid, adult females of the parasite as compared to second (invasive) stage juveniles and virgin females. Preliminary immunocytochemical studies indicate this collagen is present in areas other than the cuticle; these findings and the potential functional role of this collagen are discussed.

Glycerol kinase plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP dependent reaction. In humans, glycerol kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperglycerolemia and glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for glycerol kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced glycerol kinase RNA expression, reduced phosphorylation activity and elevated glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with glycerol. Due to the hygroscopic nature of glycerol, we predict glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting glycerol kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of glycerol hypersensitivity. Furthermore, we found that the glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the glycerol hypersensitive phenotype identified in our Drosophila model for glycerol kinase deficiency.