Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis.

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.     

The Naturally Occurring Alleles of Mal1 in Saccharomyces Species Evolved by Various Mutagenic Processes Including Chromosomal Rearrangement

In order for a yeast strain to ferment maltose it must contain any one of the five dominant MAL loci. Each dominant MAL locus thus far analyzed contains three genes: GENE 1, encoding maltose permease, GENE 2 encoding maltase and GENE 3 encoding a positive trans-acting regulatory protein. In addition to these dominant MAL loci, several naturally occurring, partially functional alleles of MAL1 and MAL3 have been identified. Here, we present genetic and molecular analysis of the three partially functional alleles of MAL1: the MAL1p allele which can express only the MAL activator; the MAL1 g allele which can express both a maltose permease and maltase; and the mal1(0) allele which can express only maltase. Based on our results, we propose that the MAL1p, MAL1g and mal1(0) alleles evolved from the dominant MAL1 locus by a series of rearrangements and/or deletions of this yeast telomere-associated locus as well as by other mutagenic processes of gene inactivation. One surprising finding is that the MAL1g-encoded maltose permease exhibits little sequence homology to the MAL1-encoded maltose permease though they appear to be functionally homologous.     

The Constitutive, Glucose-Repression-Insensitive Mutation of the Yeast MAL4 Locus Is an Alteration of the MAL43 Gene

Mutations resulting in constitutive production of maltase have been identified at each of the five MAL loci of Saccharomyces yeasts. Here we examine a dominant constitutive, glucose-repression-insensitive allele of the MAL4 locus (MAL4-C). Our results demonstrate that MAL4-C is an alteration in the MAL43 gene, which encodes the positive regulator of the MAL structural genes, and that its product is trans-acting. The MAL43 gene from the MAL4-C strain was cloned and integrated into a series of nonfermenting strains lacking a functional regulatory gene but carrying copies of the maltose permease and maltase structural genes. Expression of the maltase structural gene was both constitutive and insensitive to glucose repression in these transformants. The MAL4-C allele also results in constitutive expression of the unlinked MAL12 gene (encoding maltase) in this strain. In addition, the cloned MAL43 gene was shown to be dominant to the wild-type MAL63 gene. We also show that most of the glucose repression insensitivity of strains carrying the MAL4-C allele results from alteration of MAL43.     

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.     

Clustering of MAL genes in Hansenula polymorpha: cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes

Hansenula polymorpha uses maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39-57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1-HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.     

Identification and characterization of the maltose permease in genetically defined Saccharomyces strain

Saccharomyces yeasts ferment several alpha-glucosides including maltose, maltotriose, turanose, alpha-methylglucoside, and melezitose. In the utilization of these sugars transport is the rate-limiting step. Several groups of investigators have described the characteristics of the maltose permease (D. E. Kroon and V. V. Koningsberger, Biochim. Biophys. Acta 204:590-609, 1970; R. Serrano, Eur. J. Biochem. 80:97-102, 1977). However, Saccharomyces contains multiple alpha-glucoside transport systems, and these studies have never been performed on a genetically defined strain shown to have only a single permease gene. In this study we isolated maltose-negative mutants in a MAL6 strain and, using a high-resolution mapping technique, we showed that one class of these mutants, the group A mutants, mapped to the MAL61 gene (a member of the MAL6 gene complex). An insertion into the N-terminal-coding region of MAL61 resulted in the constitutive production of MAL61 mRNA and rendered the maltose permease similarly constitutive. Transformation by high-copy-number plasmids containing the MAL61 gene also led to an increase in the maltose permease. A deletion-disruption of MAL61 completely abolished maltose transport activity. Taken together, these results prove that this strain has only a single maltose permease and that this permease is the product of the MAL61 gene. This permease is able to transport maltose and turanose but cannot transport maltotriose, alpha-methylglucoside, or melezitose. The construction of strains with only a single permease will allow us to identify other maltose-inducible transport systems by simple genetic tests and should lead to the identification and characterization of the multiple genes and gene products involved in alpha-glucoside transport in Saccharomyces yeasts.     

Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae.

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.     

Removal of a Mig1p Binding Site Converts a Mal63 Constitutive Mutant Derived by Interchromosomal Gene Conversion to Glucose Insensitivity

Maltose fermenting strains of Saccharomyces cerevisiae have one or more complex loci called MAL. Each locus comprises at least three genes: MALx1 encodes maltose permease, MALx2 encodes maltase, and MALx3 encodes an activator of MALx1 and MALx2 (x denotes one of five MAL loci, with x = 1, 2, 3, 4, or 6). The MAL43(c) allele is constitutive and relatively insensitive to glucose repression. To understand better this unique phenotype of MAL43(c), we have isolated several MAL63(c) constitutive mutants from a MAL6 strain. All constitutive mutants remain glucose repressible, and all have multiple amino acid substitutions in the C-terminal region, now making this region of Mal63(c)p similar to that of Mal43(c)p. These changes have been generated by gene conversion, which transfers DNA from the telomeres of chromosome II and chromosome III or XVI to chromosome VIII (MAL6). The removal of a Mig1p binding site from the MAL63(c) promoter leads to a loss of glucose repression, imitating the phenotype of MAL43(c). Conversely, addition of a Mig1p binding site to the promoter of MAL43(c) converts it to glucose sensitivity. Mig1p modulation of Mal63p and Mal43p expression therefore plays a substantial role in glucose repression of the MAL genes.     

Control of maltase synthesis in yeast

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4 and MAL6. Each MAL locus is complex consisting of at least three genes: a trans-acting activator, a maltose permease, and maltase. All the MAL loci show homology to each other both at the sequence level as determined by Southern transfer analysis and at the functional level as determined by complementation. We describe the organization of the MAL loci in yeast and the basic features of their regulation. The analysis of MAL has contributed to our understanding of the evolution of multigenic families, the global integration of carbohydrate metabolism, and gene regulation.     

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.     

Primary structure of the maltose-permease-encoding gene of Saccharomyces carlsbergensis

The MAL6 locus of Saccharomyces consists of a cluster of at least three genes: MAL6R encodes a positively acting regulatory protein; MAL6S encodes maltase; and MAL6T encodes maltose permease. A MAL6 Eco RI fragment, E1, that encompasses most of the MAL6T gene except for the first 90 bp of the ORF at its 5' end (sequenced previously), was cloned into a pGEM-Blue vector. Sequential deletions were generated and then sequenced. The MAL6T gene has a putative ORF of 1845 bp. The amino acid composition and sequence of the deduced protein shows that it is highly hydrophobic and has a size of 68.2 kDa. Computer-generated hydropathy profiles suggest that the MAL6T protein may have up to nine membrane-spanning regions. Generation of functional fusions of the MAL6T promoter region to Escherichia coli lacZ-containing vectors indicates that sequences in the intergenic region are responsible for the induction of MAL6T by maltose and for its carbon catabolite repression. We also demonstrated the suitability of E. coli lacZ as a reporter gene for promoter activity studies in yeast.     

Suppression of maltose-negative phenotype by a specific nuclear gene (PMU1) in the petite cells of the yeast Saccharomyces cerevisiae

Summary A small percentage of the primary petites isolated from strain 1403-7A-P1, constitutive for maltase synthesis, simultaneously lost the ability to utilize maltose and alpha-methylglucoside. Further studies showed that these primary petites were not stable with respect to maltose utilization. Approximately 30% of the secondary petites when isolated from the primary petites after vegetative growth were found to papillate on maltose plates. Tetrad analysis data revealed that a nuclear gene has reverted in these papillae, which is responsible for suppression of the maltose negative phenotype in primary petites. We have designated this nuclear gene as the PMU1 gene (petite maltose utilizer). The functional form of the PMU1 gene is required in addition to the MAL4 gene for both constitutive maltase synthesis and maltose utilization in cytoplasmic petite cells derived from strain 1403-7A-P1.     

Molecular Evolution of the Telomere-Associated Mal Loci of Saccharomyces

The MAL gene family of Saccharomyces consists of five multigene complexes (MAL1, MAL2, MAL3, MAL4, and MAL6) each of which encodes maltose permease (GENE 1), maltase (GENE 2) and the trans-acting MAL-activator (GENE 3). Four of these loci have been mapped and each is located at or near the telomere of a different chromosome. We compare the physical structure of the MAL loci and their flanking sequences. The MAL loci were shown to be both structurally and functionally homologous throughout an approximately 9.0-kb region. The orientation of the MAL loci was determined to be: CENTROMERE . . . GENE 3-GENE 1-GENE 2 . . . TELOMERE. Telomere-adjacent sequences were found flanking GENE 2 of the MAL1, MAL3 and MAL6 loci. No common repeated elements were found on the centromere-proximal side of all the MAL1, loci. These results suggest that, during the evolution of this polygenic family, the MAL loci translocated to different chromosomes via a mechanism that involved the rearrangement(s) of chromosome termini.