Identification and physical characterization of yeast glucoamylase structural genes

Summary Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta⁺ strains, each carrying a single genetically defined STA locus, were crossed with a Sta^– strain and the segregation behavior of the functional locus (i.e. Sta⁺) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5 and 3 homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta^–), but has retained extensive sequence homology to the 5 end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.     

The MAT A locus of the dimorphic yeast Yarrowia lipolytica consists of two divergently oriented genes

The MAT A locus of Yarrowia lipolytica, which was on the basis of its ability to induce sporulation in a diploid B/B strain, represses the mating capacity of this strain. The gene functions required for induction of sporulation and repression of conjugation could be separated by subcloning. Sequence analysis revealed two ORFs in the MAT A locus. One of them (MAT A1) codes for a protein of 119 amino acids which is required to induce sporulation. The other (MAT A2) codes for a protein of 291 amino acids that is able to repress conjugation. Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes. The product of MAT A1 shows no homology to any known protein and seems to represent a new class of mating-type genes. MAT A2 contains a HMG box with homology to other mating-type genes. Both MAT A1 and MAT A2 are mating-type specific. In cells of both mating types, the regions flanking the MAT A locus contain sequences with homology to either S. cerevisiae SLA2 and ORF YBB9, respectively. From hybridization and subcloning data we estimate that the MAT A region is approximately 2 kb long and is present only once in the genome. Received: 25 January 1999 / Accepted: 16 April 1999     

Control of yeast cell type by the mating type locus: Positive regulation of the α-specific STE3 gene by the MATα 1 product

The mating type locus (MAT) determines the three yeast cell types, a, α, and a/α. It has been proposed that alleles of this locus, MATa and MATα, encode regulators that control expression of unlinked genes necessary for mating and sporulation. Specifically, the α1 product of MATα is proposed to be a positive regulator of α-specific genes. To test this view, we have assayed RNA production from the α-specific STE3 gene in the three cell types and in mutants defective in MATα. The STE3 gene was cloned by screening a yeast genomic clone bank for plasmids that complement the mating defect of ste3 mutants. Using the cloned STE3 gene as a probe, we find that a cells produce STE3 RNA, whereas a and a/a cells do not. Furthermore, matα 1 mutants do not produce STE3 RNA, whereas matα 2 mutants do. These results show that the STE3 gene, required for mating only by α cells, is expressed only in α cells. They show also that production of RNA from the STE3 gene requires the α1 product of MATα. Thus α1 positively regulates at least one α-specific gene by increasing the level of that gene's RNA product.     

Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica

Summary The mating type gene MA TA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.     

Mating Signals Control Expression of both Starch Fermentation Genes and a Novel Flocculation Gene FLO 8 in the Yeast Saccharomyces

In the yeast Saccharomyces diastaticus, expression of both glucoamylase-producing (STA) genes and a novel flocculation gene FLO 8 was greatly diminished by the mating-type locus MATa/MAT α.     

Localization of glucoamylase genes of Saccharomyces cerevisiae by pulsed field gel electrophoresis

The chromosomal locations of four glucoamylase-specifying genes in the yeastSaccharomyces cerevisiae have been determined. Chromosomes were separated by pulsed field gel electrophoresis and blots were probed with radiolabelledSTA2 and marker DNA from specific yeast chromosomes. The three genes encoding extracellular glucoamylases,STA1 (DEX2), STA2 (DEX1) andSTA3 (DEX3) are located on chromosomes IV, II and XIV, respectively.SGA, specifying the sporulation-specific glucoamylase, was positioned on chromosome IX.     

The MAT A locus of the dimorphic yeast Yarrowia lipolytica consists of two divergently oriented genes

The MAT A locus of Yarrowia lipolytica, which was on the basis of its ability to induce sporulation in a diploid B/B strain, represses the mating capacity of this strain. The gene functions required for induction of sporulation and repression of conjugation could be separated by subcloning. Sequence analysis revealed two ORFs in the MAT A locus. One of them (MAT A1) codes for a protein of 119 amino acids which is required to induce sporulation. The other (MAT A2) codes for a protein of 291 amino acids that is able to repress conjugation. Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes. The product of MAT A1 shows no homology to any known protein and seems to represent a new class of mating-type genes. MAT A2 contains a HMG box with homology to other mating-type genes. Both MAT A1 and MAT A2 are mating-type specific. In cells of both mating types, the regions flanking the MAT A locus contain sequences with homology to either S. cerevisiae SLA2 and ORF YBB9, respectively. From hybridization and subcloning data we estimate that the MAT A region is approximately 2?kb long and is present only once in the genome.     

The trans action of HMRa in mating type interconversion

Summary HML and HMR are the sites of cryptic mating type genes in the yeast Saccharomyces cerevisiae. In the presence of the HO gene, the information from HML or HMR (an a or cassette) is transferred to the mating type locus (MAT). HML, HMR, and MAT are located on chromosome III, yet are widely separeted. Similarly, in other yeasts, at least some of the genes involved in mating type interconversion are linked to the mating type locus. We demonstrate here that a cassette donor (HMR) and the cassette target (MAT) need not be physically linked for successful mating type interconversion. In particular, we show that HMR a on one chromosome can donate an a cassette to the mating type locus on a homologous chromosome III.     

The sporulation capable (sca) mutation of Saccharomyces cerevisiae is an allele of the SIR2 gene

Summary We have used the special properties of the spo13-1 mutation in order to study the regulation of yeast meiosis by the mating type loci. We have found that both the rme1-1 mutation and the sca mutation allow haploid meiosis in spo13-1 strains. Therefore, haploid meiosis is regulated in the same manner as diploid meiosis. Unlike rme1-1, the sca mutation allows meiosis through derepression of the silent mating type cassettes; sca strains can sporulate only because they express both MAT a and MAT information. We have found further that sca is an allele of SIR2, one of the genes involved in repression of the silent cassettes. Therefore, the RME1 gene is the only known candidate for a master negative regulator through which the MAT locus controls meiosis.     

A long region upstream of the IME1 gene regulates meiosis in yeast

Summary Meiosis and sporulation in yeast are subject to two types of regulation. The first depends on environmental conditions. The second depends on a genetic pathway which involves the control of the positive regulatory gene IME1 by RME1, which is in turn controlled by the MAT locus. The presence of IME1 on a multicopy plasmid enables cells to undergo meiosis regardless of their genotype at MAT or RME1. We show here that a multicopy plasmid carrying IME1 also enables meiosis, regardless of the environment. Therefore, both kinds of regulation appear to act through IME1. Furthermore, the behavior of multicopy plasmids carrying various segments from the IME1 region suggests that the region upstream of IME1 contains both positive and negative regulatory sites. Control of IME1 by the environment and by the MAT pathway both act through negative regulatory sites.     

Allelism within the DEX and STA gene families in Saccharomyces diastaticus

Summary Saccharomyces diastaticus produces an extracellular glucoamylase and is therefore capable of hydrolyzing and fermenting starch. Tamaki (1978) studied starch utilization in S. diastaticus and found three polymeric genes controlling this function: STA1, STA2 and STA3. Independently, Erratt and Stewart (1978) studied dextrin utilization by the yeast S. diastaticus and designated the gene, which they identified, DEX1. Erratt and Stewart (1981a, b) later described two other genes which controlled glucoamylase production in S. diastaticus: DEX2 and a third which was allelic to STA3. At that time STA1 and STA2 were not available to test for allelism in the DEX gene family. In this study strains containing the remaining 4 genes have been examined to determine if further allelism exists between the two gene families. It was ascertained that DEX1 is allelic to STA2 and DEX2 is allelic to STA1. Therefore, no new gene controlling starch utilization has been identified and these two nomenclatures can now be consolidated into one. Based on the fact that the glucoamylase from S. diastaticus can hydrolyze both dextrin and starch, dextrin being the term used to described partially hydrolyzed starch, and the more wide use of the nomenclature STA, we propose to retain STA as the designation for genes coding for glucoamylase production in S. diastaticus.     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

Expression of glucoamylase gene usingSUC2 promoter inSaccharomyces cerevisiae

Summary The cloning of glucoamylase geneSTA using theSUC2 promoter intoSaccharomyces cerevisiae was performed. The signal sequence ofSTA gene was used for the secretion of glucoamylase protein. The plasmid constructed in this way was named YEpSUCSTA and its expression was identified. The expression of YEpSUCSTA was repressed in the presence of glucose in growth medium, but derepressed when glucose became depleted. YEpSUCSTA showed the similar efficiency of glucoamylase secretion as YEpSTA-F which has the entireSTA gene. Glucoamylase activity in starch-glucose medium was largely increased because cell mass and plasmid stability were high in biosynthesis phase compared to extracellular glucoamylase activities in media which starch or glucose was the only carbon source.     

Cloning and expression of a glucoamylase gene from Lactobacillus amylovorus ATCC 33621 in Escherichia coli

Summary The glucoamylase gene from Lactobacillus amylovorus was cloned and expressed in Escherichia coli. A genomic DNA library from Lactobacillus amylovorus was prepared by partially digesting genomic DNA with EcoRI and ligating random fragments to the EcoRI digested cloning vector, pZErO-1.1. Three E. coli transformants expressing glucoamylase were identified using a probe prepared from the STA2 glucoamylase gene from Saccharomyces cerevisiae var. diastaticus. The physical maps of the recombinant plasmids were constructed. These plasmids contained inserts of about 5.2 Kb, 5.9 Kb and 6.4 Kb respectively. Temperature and pH optima of 45°C and 6.0, respectively, were obtained for both recombinant and purified wild type glucoamylases. Also, the enzymes were found to be thermolabile at temperatures above 50°C.     

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.     

Overexpression of the glucoamylase-encoding STA1 gene of Saccharomyces cerevisiae var. diastaticus in laboratory and industrial strains of Saccharomyces

Production of glucoamylase encoded by the Saccharomyces cerevisiae (var. diastaticus) STA1 gene has been assayed in laboratory S. cerevisiae strains of different ploidy and in different industrial Saccharomyces strains, in which STA1 was expressed under control of an inducible promoter. Highest enzyme activity was achieved with a tetraploid strain constructed by crossing preselected parental strains. Maximal glucoamylase production correlated with heterogeneity in enzyme mass, likely due to incomplete glycosylation, suggesting that the secretion-glycosylation process is the limiting step in the production of the STA-encoded glucoamylase by Saccharomyces. Industrial strains showed quite different capacity to produce glucoamylase. High production was achieved with a S. pastorianus brewer’s strain. Overall, our results allowed the selection of strains capable of yielding a high level of glucoamylase and suggest specific approaches for further enhancing this capability.     

Single mating type-specific genes and their 3′ UTRs control mating and fertility in Cochliobolus heterostrophus

To determine the number of proteins required for mating type (MAT) locus-regulated control of mating in Cochliobolus heterostrophus, MAT fragments of various sizes were expressed in MAT deletion strains. As little as 1.5 kb of MAT sequence, encoding a single unique protein in each mating type (MAT-1 and MAT-2), conferred mating ability, although an additional 160 bp of 3^′ UTR was needed for production of ascospores. No other mating type-specific genes involved in mating identity or fertility were found. Thus, although homologs of the C. heterostrophus MAT-1 and MAT-2 genes exist in the filamentous ascomycetes Neurospora crassa and Podospora anserina, C. heterostrophus does not appear to have mating type-specific homologs of two additional genes required by both N. crassa and P.␣anserina for successful sexual reproduction. Three genes were identified in the common DNA flanking the MAT locus: a gene encoding a GTPase-activating protein and an ORF of unknown function lie 5^′ while a β-glucosidase encoding gene lies found 3^′. None of these genes appears to be involving in the mating process. Received: 21 November 1997 / Accepted: 28 April 1998     

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis

We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and $\text{a}\text{α}$. First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of $\text{a}\text{α}$ cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1^?matα2^? mutants should have the mating ability of an a cell and that matα1^?matα2^?/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MATa cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

A putative pheromone signaling pathway is dispensable for self-fertility in the homothallic ascomycete Gibberella zeae

Gibberella zeae, a homothallic ascomycetous fungus, does not seek a partner for mating. Here, we focused on the role(s) of putative pheromone and receptor genes during sexual development in G. zeae. Orthologs of two pheromone precursor genes (GzPPG1 and GzPPG2), and their cognate receptor genes (GzPRE2 and GzPRE1) were transcribed during sexual development. The expression of these genes was controlled by the mating-type (MAT) locus and a MAP kinase gene, but not in a MAT-specific manner. Targeted gene deletion and subsequent outcrosses generated G. zeae strains lacking these putative pheromone/receptor genes in various combinations (from single to quadruple deletions). All G. zeae deletion strains were similar to the self-fertile progenitor in both male- and female fertility and other traits. Sometimes, the deletions including ΔGzPPG1;ΔGzPRE2 caused increased numbers of immature perithecia. Taken together, it is clear that these putative pheromones/receptors play a non-essential role in the sexual development of G. zeae.