Differentiated Gene Expression in Cells within Yeast Colonies

Yeast cells growing on solid media organize themselves into multicellular structures, colonies, exhibiting patterns specific for particular yeast strains. With the aim of identifying genes involved in regulations of the colony formation, we applied a new approach enabling the extensive screening of Saccharomyces cerevisiae genes, the expression of which is changed during colony development. We used the library of S. cerevisiae DNA fragments inserted in front of the lacZ gene lacking its own promoter. Colonies of transformants with a blue/white patterned morphotype, implying that the expression of the lacZ gene from the inserted yeast promoter is switched on and off during the colony formation, were isolated. We identified several genes with variable expression during colony morphogenesis, including CCR4, PAM1, MEP3, ADE5,7 and CAT2. S. cerevisiae strain deleted in the CCR4 gene forms colonies with less organized morphology when compared with the isogenic parental strain. The synchronization of the expression patterns of some of the isolated genes in neighboring colonies was observed.     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.     

Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae

The Saccharomyces cerevisiae ADE16 and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway. Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine. In this work, we show that an ade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy of ade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17 gene products (Ade16p and Ade17p). Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies. Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining. Epitope-tagged constructs were used to study expression of the two isozymes. The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine. Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation.     

Identification of mutations in the asporogenic yeast Candida tropicalis using intrageneric fusion of protoplasts

The possibility of genetic identification of mutations in asporogenic yeast by the technique of intrageneric fusion of yeast protoplasts of Candida tropicals and Saccharomyces cerevisiae has been demonstrated for Candida tropicals strains G5-9 (Ade- Leu-) and G32-4 (Leu-). The mutations to auxotrophy ade- in the strain G5-9 and leu- in G32-4 of Candida tropicals are allelic to ade2 and leu1 mutations in the genes of Saccharomyces cerevisiae yeast. The allelic character of adenine auxotrophy mutation in Candida tropicals and ade2 mutation in Saccharomyces cerevisiae is confirmed by the absence of AIR-carboxylase activity in cellular extract from the strain G5-9.     

A general method for generation and analysis of defined mutations in enzymes involved in a tetrahydrofolate-interconversion pathway

In eukaryotes, 10-formyltetrahydrofolate (THF) synthetase, 5,10-methenyl-THF cyclohydrolase and 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. These reactions are generally catalyzed by three separate monofunctional enzymes in prokaryotic cells. In this report a general method for the generation, detection and analysis of specific mutations affecting the catalytic activity of any of the reactions catalyzed by C1-THF synthase or its monofunctional counterparts is described. The method relies on plasmid-borne expression of genes in strains of the yeast Saccharomyces cerevisiae that are missing one or more of the activities of C1-THF synthase. Specific segments of the gene are subjected in vitro to random mutagenesis, the mutant genes expressed in yeast and screened by phenotype for inactivating mutations. Plasmids encoding mutant enzymes are recovered for sequence analysis. One-step purification of C1-THF synthase from the yeast expression system is demonstrated. The feasibility and versatility of the method is shown with the yeast ADE3 gene encoding the cytoplasmic C1-THF synthase and the gene encoding the monofunctional 10-formyl-THF synthetase from Clostridium acidiurici.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the ADE1 gene.

The ADE1 gene of Saccharomyces cerevisiae was isolated by complementation in S. cerevisiae from a yeast genomic DNA library carried on plasmid YEp13. Electron microscopy of R-loop-containing DNA indicated the location of the ADE1 gene on the plasmid insert. Gene disruption and gene replacement were used to demonstrate that the ade1-complementing sequence was the actual ADE1 gene that maps on chromosome I. ade1 strains which normally form red colonies form white ones when transformed with the cloned ADE1 gene. This property should be very useful, since it enables detection of plasmids carrying this gene under nonselective conditions.     

Genetic control of purine biosynthesis in Pichia methanolica yeast. The ADE5 (PUR4) gene, controlling 5'-phosphoribosylformyl glycineamide synthetase

By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system "from red to white," it was shown that the PUR4 gene encoding 5'-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeast Saccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe (the ade3 gene), and Pichia methanolica (the ADE5 gene). This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group. Study of characteristics of spontaneous mutations in early BPN genes of P. methanolica demonstrated that the vast majority of unstable ade5sU alleles (mutations with a high reversion frequency ranging from 0.2 x 10(-6) to 2 x 10(-6)) appeared solely among mutants for the ADE5 gene. Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene. The DNA sequence that can compensate for the P. methanolica ade5 mutation and probably is the structural P-ADE5 gene, was cloned from a genomic library of P. methanolica by the ade6 mutation complementation in the recipient S. cerevisiae strain.     

Molecular nature of the direct mutations induced by 4-nitro-quinoline-N-oxide and 3-methyl-4-nitroquinoline-N-oxide in the ADE2 gene of Saccharomyces cerevisiae

The lethal and mutagenic effects and the nature of forward mutations in ADE2 gene induced by highly carcinogenic agent 4-nitroquinoline-N-oxide (4NQO) and its noncarcinogenic analogue 3-methyl-4-nitroquinoline-N-oxide (3M4NQO) have been examined in Saccharomyces cerevisiae. It is shown that 3M4NQO is more toxic than 4NQO. Both are very efficient mutagens: the mutagenic efficiency for ADE1 and ADE2 genes was 7.9 X 10(-5) for 4NQO and 10.5 X 10(-5) for 3M4NQO. The base pair substitutions are the main type of induced mutations in ADE2 gene (95 and 89% for 4NQO and 3M4NQO, respectively); among these 40% transversions for 4NQO and 63% for 3M4NQO, GC----AT transitions-32 and 31% for 4NQO and 3M4NQO, respectively, AT----GC transitions-23 and 22% for 4NQO and 3M4NQO, respectively. The results obtained indicate that 4NQO and 3M4NQO induce the same spectrum of mutations in ADE2 gene and that both mutagens are nonspecific in yeast cells.     

Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.     

The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control.

The ADE1 gene of the yeast Saccharomyces cerevisiae has been cloned by complementation of the ade1 mutation. The nucleotide sequence has been determined for the 918-bp coding region, 240-bp 5'-noncoding region and 292-bp 3'-noncoding region. The sequenced region includes a single large open reading frame coding for a protein of 306 amino acid (aa) residues. The promoter of the ADE1 gene contains a copy of the 5'-TGACTC hexanucleotide, a feature characteristic of promoters under general aa control. Subsequent search of other published purine biosynthesis gene sequences revealed that all of them also contain general aa control signals in their promoter regions. An expression plasmid containing the ADE1 coding region under control of the PHO5 promoter produced N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase in yeast cells at a level of 40% of total cellular protein. One-step purification resulted in an almost homogeneous preparation of SAICAR synthetase.     

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.     

New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris

We describe the isolation and characterization of three new biosynthetic genes-ARG4, ADE1, and URA3-from the methylotrophic yeast Pichia pastoris. The predicted products of the genes share significant sequence similarity to their Saccharomyces cerevisiae counterparts, namely argininosuccinate lyase, PR-aminoimidazolesuccinocarboxamide synthase, and orotidine-5'-phosphate decarboxylase, respectively. Along with the previously described HIS4 gene, each gene was incorporated as the yeast selectable marker into a set of shuttle vectors designed to express foreign genes in P. pastoris. In addition, we have constructed a series of host strains containing all possible combinations of ade1, arg4, his4, and ura3 auxotrophies to be used with these new vectors.     

Genome-wide expression profile of sake brewing yeast under shaking and static conditions

To identify the genes responsible for characteristics, that are different as between sake brewing yeasts and laboratory yeast strains, we used a DNA microarray to compare the genome-wide gene expression profiles of a sake yeast, Saccharomyces cerevisiae K-9 (kyokai 9), and a laboratory yeast, S. cerevisiae X2180-1A, under shaking and static conditions.The genes overexpressed in K-9 more than in X2180-1A were related to C-metabolism, including the HXT, ATP, and COX genes, ergosterol biosynthesis, ERG genes, and thiamine metabolism, THI genes. These genes may contribute to higher growth rates and fermentation ability and the ethanol tolerance of sake yeast.The genes underexpressed in K-9 more than in X2180-1A were CUP1-1 and CUP1-2, PHO genes, which may explain the low copper tolerance and low acid phosphatase activity of sake yeast. These underexpressed genes agree with the features and the alteration of the genome structure of sake yeast.     

CUP1为筛选标记的酿酒酵母整合型多基因表达载体的构建

为了赋予工业酿酒酵母对淀粉和纤维素的降解活性,提高酿酒酵母对粗木薯粉进行酒精发酵时的酒精产率;另一方面,为了解决工业酿酒酵母不适于使用营养缺陷型筛选标记对转化子进行筛选的问题,以及避免引入抗药性标记基因带来的安全性问题,构建了以抗铜蛋白基因CUP1为筛选标记的酿酒酵母整合型多基因表达载体.以载体pYES2-PMF-rDNA为基础,以新的筛选标记基因CUP1替换原有的尿嘧啶Ura-基因,得到载体pYES2M.再顺序插入葡聚糖内切酶基因eg3、葡萄糖淀糖酶基因gal和β-葡萄糖苷酶基因bgl1,构建得到以CUP1为筛选标记的酵母整合型三价表达载体pYES2M-eg3-ga1 -bgl1,其中每个基因都具有独立而完整的表达盒,包括启动子、信号肽和终止子,从而实现多基因单表达载体一次转化.     

Inulase-secreting strain of Saccharomyces cerevisiae produces fructose

The gene encoding inulase of the yeast Kluyveromyces marxianus (INU1Km) was cloned and expressed in the inulin-negative yeast Saccharomyces cerevisiae. Cells of S. cerevisiae transformed with the INU1Km gene have acquired extracellular inulase activity and were able to grow in the medium with inulin as a sole carbon source. The S. cerevisiae strain was constructed that is capable of heterologous expression of secreted K. marxianus inulase and is defective in fructose uptake due to null-mutations of the hexokinase structural genes HXK1 and HXK2. When grown in inulin-containing media, this strain is capable of accumulating at least 10% glucose-free fructose in the culture liquid.     

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.