Mutational analysis of the MAL1 locus of Saccharomyces: identification and functional characterization of three genes

Summary Fermentation of maltose by Saccharomyces strains depends on the presence of any one of five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 or MAL6). Earlier mutational analyses of MAL2 and MAL6 containing strains have identified a single complementation group at each of these two loci. However complementation analysis between naturally occurring Mal^– Saccharomyces strains isolated from the wild demonstrated the presence of two complementation groups (designated MALp and MALg) at the MAL1, MAL3 and MAL6 loci. The available evidence suggests that the MALp gene is functionally equivalent to the complementation group identified by mutational analysis at the MAL6 locus and that this gene encodes a protein involved in the regulation of the coordinate induction of both maltase and maltose permease synthesis.In this paper we report the isolation, in a well characterized MAL1 strain, of 47 mutants unable to ferment maltose. All the mutants, with one exception, map at the MAL1 locus. These mal1 mutants, except for one, are recessive to MAL1 and fall into two major complementation groups. Evidence is presented that these two classes of mutants identify both a gene involved in the regulation of maltose fermentation (MAL1R) and a gene involved in maltose transport (MAL1T). We also report here the isolation of a temperature sensitive maltose nonfermenting mutant mapping at the MAL1 locus identifying a third gene (MAL1S) at this locus. The maltase synthesized by this mutant, when assayed in cell-free extracts, is significantly more thermolabile than the wild type enzyme. Our findings demonstrate that MAL1 is a complex locus comprising at least three genes: MAL1R, a gene involved in the coordinate regulation of the synthesis of maltase and maltose transport; MAL1T, a gene encoding a component of the maltose transport system; and MAL1S, a likely candidate for the structural gene for maltase.     

Genome-wide identification of Phytophthora sojae SNARE genes and functional characterization of the conserved SNARE PsYKT6

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.     

Isolation and characterization of maltose non utilizing (mnu) mutants mapping outside the MAL1 locus in Saccharomyces cerevisiae

The MAL1 locus of Saccharomyces cerevisiae comprises three genes necessary for maltose utilization. They include regulatory, maltose transport and maltase genes designated MAL1R, MAL1T and MAL1S respectively. Using a MAL1 strain transformed with an episomal, multicopy plasmid carrying the MAL2 locus, five recessive and one dominant mutant unable to grow on maltose, but still retaining a functional MAL1 locus were isolated. All the mutants could use glycerol, ethanol, raffinose and sucrose as a sole carbon source; expression of the maltase and maltose permease genes was severely and coordinately reduced. Only the dominant mutant failed to accumulate the MAL1R mRNA.     

Identification of deletion mutations and three new genes at the familial polyposis locus.

Small (100-260 kb), nested deletions were characterized in DNA from two unrelated patients with familial adenomatous polyposis coli (APC). Three candidate genes located within the deleted region were ascertained and a previous candidate gene, MCC, was shown to be located outside the deleted region. One of the new genes contained sequence identical to SRP19, the gene coding for the 19 kd component of the ribosomal signal recognition particle. The second, provisionally designated DP1 (deleted in polyposis 1), was found to be transcribed in the same orientation as MCC. Two other cDNAs, DP2 and DP3, were found to overlap, forming a single gene, DP2.5, that is transcribed in the same orientation as SRP19.     

MAL11 and MAL61 encode the inducible high-affinity maltose transporter of Saccharomyces cerevisiae.

We have investigated the transport of maltose in a genetically defined maltose-fermenting strain of Saccharomyces cerevisiae carrying the MAL1 locus. Two kinetically different systems were identified: a high-affinity transporter with a Km of 4 mM and a low-affinity transporter with a Km of 70 to 80 mM. The high-affinity maltose transporter is maltose inducible and is encoded by the MAL11 (and/or MAL61) gene of the MAL1 (and/or MAL6) locus. The low-affinity maltose transporter is expressed constitutively and is not related to MAL11 and/or MAL61. Both maltose transporters are subject to glucose-induced inactivation.     

Genetic characterization of three species of Onchocerca at 23 enzyme loci

The technique of allozyme electrophoresis was applied to species of Onchocerca from cattle to increase the number of enzyme loci established and therefore provide a genetic basis for a rational species-level taxonomy. Twenty-three enzyme loci were established and provided unequivocal genetic evidence for the taxonomic validity of Onchocerca gibsoni, O. gutturosa and O. lienalis. Furthermore, the diagnostic enzyme markers detected form the basis for identification of life-cycle stages, individuals and species and population structure analyses.     

High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating-type locus HMRa

Genetic and biochemical evidence implicates chromatin structure in the silencing of the two quiescent mating-type loci near the telomeres of chromosome III in yeast. With high-resolution micrococcal nuclease mapping, we show that the HMRa locus has 12 precisely positioned nucleosomes spanning the distance between the E and I silencer elements. The nucleosomes are arranged in pairs with very short linkers; the pairs are separated from one another by longer linkers of approximately 20 bp. Both the basic amino-terminal region of histone H4 and the silent information regulator protein Sir3p are necessary for the organized repressive chromatin structure of the silent locus. Compared to HMRa, only small differences in the availability of the TATA box are present for the promoter in the cassette at the active MATa locus. Features of the chromatin structure of this silent locus compared to the previously studied HMLalpha locus suggest differences in the mechanisms of silencing and may relate to donor selection during mating-type interconversion.     

Cloning and characterization of the glucokinase gene of Brucella abortus 19 and identification of three other genes.

A clone from Brucella abortus 19 complemented an Escherichia coli strain deficient in phosphorylation of glucose. Open reading frames similar to E. coli mepA, glk, and genes encoding ATP-coupled exporters were found in the sequence. A fourth affected growth on minimal media of the ptsI glk strain with various carbon sources.     

Human metallothionein genes: structure of the functional locus at 16q13

The functional human metallothionein (MT) genes are located on chromosome 16q13. We have physically mapped the functional human MT locus by isolation and restriction digest mapping of cloned DNA. The mapped region contains all sequences on chromosome 16 that hybridize to metallothionein gene probes and comprises 14 tightly linked MT genes, 6 of which have not been previously described. This analysis defines the genetic limits of metallothionein functional diversity in the human genome.     

Physical, biochemical and functional characterization of haemoglobin from three strains of Artemia

The brine shrimp, Artemia, an inhabitant of coastal and inland salterns, encounter fluctuations in the salinity which in turn influences the oxygen availability of their habitat. Hence, experiments were performed to analyze variations in haemoglobin structure and patterns of three strains of Artemia from South India and also to reflect the effect of varying oxygen levels in their habitat. Haemoglobins were purified on a DEAE-Sephadex column and haemoglobin types were analyzed by comparing their relative mobility on a non-denaturing medium. Furthermore, their molecular masses were determined by gel filtration in Sepharose column and by dodecylsulfate polyacrylamide gel electrophoresis. Results clearly reveal the presence of three distinct extracellular haemoglobins Hb I, Hb II and Hb III in Tuticorin strain while the other strains displayed only trails or the complete absence of Hb III and Hb II. Estimated molecular masses of these haemoglobins are 235,000-250,000 Da. Denaturation of the reduced and alkylated haemoglobins revealed apparently one polypeptide chain with a molecular mass of 124,000 Da. Upon denaturing gel electrophoresis of native haemoglobin Hb II, it was found that the 124,000 Da, polypeptide was cleaved specifically into two unequally-sized fragments of 50,400 and 79,800 Da. With regard to oxygen affinity, Hb III has a very high affinity for oxygen, an almost negligible Bohr effect and a good physiological adaptation to temperature changes. By combining the three haemoglobins in different proportions Artemia strains must be able to withstand diverging environmental conditions. In particular, the absence of Hb III in Puthalam and its occurrence as a faint band in Thamaraikulam could be correlated to the oxygen levels of their habitats.     

Genetic characterization of three strains of Hymenolepis diminuta at 39 enzyme loci

The technique of allozyme electrophoresis was applied to three strains of Hymenolepis diminuta to distinguish between three hypotheses [(1) multiple species, (2) genetically distinct founder stocks and (3) response to differential selection among similar stocks] proposed to account for metabolic differences among strains. There was no evidence from the 39 enzyme loci established that the three strains represented more than one species. In the absence of knowledge of the population structure of H. diminuta in the wild, electrophoretic data herein could not distinguish between the latter hypotheses. Nevertheless, all three strains were distinguishable on electrophoretic profiles and allelic similarities between strains question the view of their proposed origins.