Endonuclease for apurinic sites in yeast comparison of the enzyme activity in the wild type and in rad mutants of Saccharomyces cerevisiae to MMS

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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Influences of Histone Stoichiometry on the Target Site Preference of Retrotransposons Ty1 and Ty2 in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.3.761 ◽

1996 ◽

Vol 142 (3) ◽

pp. 761-776 ◽

Cited By ~ 2

Author(s):

Lori A Rinckel ◽

David J Garfinkel

Keyword(s):

Saccharomyces Cerevisiae ◽

Promoter Region ◽

Target Site ◽

Site Preference ◽

Wild Type ◽

Histone Proteins ◽

Protein Levels ◽

In The Wild ◽

Type Strains ◽

Orientation Bias

Abstract In Saccharomyces cerevisiae, the target site specificity of the retrotransposon Ty1 appears to involve the Ty integration complex recognizing chromatin structures. To determine whether changes in chromatin structure affect Ty1 and Ty2 target site preference, we analyzed Ty transposition at the CAN1 locus in mutants containing altered levels of histone proteins. A Δhta1-htb1 mutant with decreased levels of H2A and H2B histone proteins showed a pattern of Ty1 and Ty2 insertions at CAN1 that was significantly different from that of both the wild-type and a Δhta2-htb2 mutant, which does not have altered histone protein levels. Altered levels of H2A and H2B proteins disrupted a dramatic orientation bias in the CAN1 promoter region. In the wild-type strains, few Ty1 and Ty2 insertions in the promoter region were oriented opposite to the direction of CAN1 transcription. In the Δhta1-htb1 background, however, numerous Ty1 and Ty2 insertions were in the opposite orientation clustered within the TATA region. This altered insertion pattern does not appear to be due to a bias caused by selecting canavanine resistant isolates in the different HTA1-HTB1 backgrounds. Our results suggest that reduced levels of histone proteins alter Ty target site preference and disrupt an asymmetric Ty insertion pattern.

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Suppressors of Saccharomyces cerevisiae his3 promoter mutations lacking the upstream element

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.1901-1909.1985 ◽

1985 ◽

Vol 5 (8) ◽

pp. 1901-1909

Author(s):

M A Oettinger ◽

K Struhl

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Tata Box ◽

Micrococcal Nuclease ◽

Promoter Element ◽

Wild Type ◽

Upstream Promoter ◽

In The Wild ◽

Promoter Mutations ◽

His3 Gene

Transcription of the Saccharomyces cerevisiae his3 gene requires an upstream promoter element and a TATA element. A strain containing his3-delta 13, an allele which deletes the upstream promoter element but contains the TATA box and intact structural gene, fails to express the gene and consequently is unable to grow in medium lacking histidine. In this paper we characterize His+ revertants of his3-delta 13 which are due to unlinked suppressor mutations. Recessive suppressors in three different ope genes allow his3-delta 13 to be expressed at wild-type levels. In all cases, the suppression is due to increased his3 transcription. However, unlike the wild-type his3 gene, whose transcripts are initiated about equally from two different sites (+1 and +12), transcription due to the ope mutations is initiated only from the +12 site, ope-mediated transcription is regulated in a novel manner; it is observed in minimal medium, but not in rich broth. Although ope mutations restore wild-type levels of transcription, his3 chromatin structure, as assayed by micrococcal nuclease sensitivity of the TATA box, resembles that found in the his3-delta 13 parent rather than in the wild-type strain. This provides further evidence that TATA box sensitivity is not correlated with transcriptional activation. ope mutations are pleiotropic in that cells have a crunchy colony morphology and lyse at 37 degrees C in conditions of normal osmolarity. ope mutations are allele specific because they fail to suppress five other his3 promoter mutations. We discuss implications concerning upstream promoter elements and propose some models for ope suppression.

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Conversion of Waste Agriculture Biomass to Bioethanol by Recombinant Saccharomyces cerevisiae

Journal of Scientific Research ◽

10.3329/jsr.v2i2.2882 ◽

2010 ◽

Vol 2 (2) ◽

pp. 351-361

Author(s):

A. A. Saleh ◽

S. Hamdan ◽

N. Annaluru ◽

S. Watanabe ◽

M. R. Rahman ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzyme Activity ◽

Protein Engineering ◽

Agricultural Waste ◽

Xylitol Dehydrogenase ◽

Ethanol Conversion ◽

Waste Biomass ◽

Wild Type ◽

Coenzyme Specificity ◽

Recombinant Yeasts

Agricultural waste biomass has already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR) and the protein engineered xylitol dehydrogenase (XDH) (with NADP) of Pichia stipitis. These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomyces cerevisiae as judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARS strain, in which NADP+-dependent XDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild-type XDH. Keywords: Agricultural waste biomass; Protein engineering; Xylitol dehydrogenase; Xylose-fermentation; Eethanol production. © 2010 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. DOI: 10.3329/jsr.v2i2.2882 J. Sci. Res. 2 (2), 351-361 (2010)

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Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/122.3.535 ◽

1989 ◽

Vol 122 (3) ◽

pp. 535-542 ◽

Cited By ~ 1

Author(s):

B A Kunz ◽

M G Peters ◽

S E Kohalmi ◽

J D Armstrong ◽

M Glattke ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Base Pair ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Mutator Phenotype ◽

In The Wild ◽

Single Base Pair ◽

Almost All

Abstract Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

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Suppressors of Saccharomyces cerevisiae his3 promoter mutations lacking the upstream element.

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.1901 ◽

1985 ◽

Vol 5 (8) ◽

pp. 1901-1909 ◽

Cited By ~ 7

Author(s):

M A Oettinger ◽

K Struhl

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Tata Box ◽

Micrococcal Nuclease ◽

Promoter Element ◽

Wild Type ◽

Upstream Promoter ◽

In The Wild ◽

Promoter Mutations ◽

His3 Gene

Transcription of the Saccharomyces cerevisiae his3 gene requires an upstream promoter element and a TATA element. A strain containing his3-delta 13, an allele which deletes the upstream promoter element but contains the TATA box and intact structural gene, fails to express the gene and consequently is unable to grow in medium lacking histidine. In this paper we characterize His+ revertants of his3-delta 13 which are due to unlinked suppressor mutations. Recessive suppressors in three different ope genes allow his3-delta 13 to be expressed at wild-type levels. In all cases, the suppression is due to increased his3 transcription. However, unlike the wild-type his3 gene, whose transcripts are initiated about equally from two different sites (+1 and +12), transcription due to the ope mutations is initiated only from the +12 site, ope-mediated transcription is regulated in a novel manner; it is observed in minimal medium, but not in rich broth. Although ope mutations restore wild-type levels of transcription, his3 chromatin structure, as assayed by micrococcal nuclease sensitivity of the TATA box, resembles that found in the his3-delta 13 parent rather than in the wild-type strain. This provides further evidence that TATA box sensitivity is not correlated with transcriptional activation. ope mutations are pleiotropic in that cells have a crunchy colony morphology and lyse at 37 degrees C in conditions of normal osmolarity. ope mutations are allele specific because they fail to suppress five other his3 promoter mutations. We discuss implications concerning upstream promoter elements and propose some models for ope suppression.

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SPO71 Encodes a Developmental Stage-Specific Partner for Vps13 in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00239-13 ◽

2013 ◽

Vol 12 (11) ◽

pp. 1530-1537 ◽

Cited By ~ 20

Author(s):

Jae-Sook Park ◽

Yuuya Okumura ◽

Hiroyuki Tachikawa ◽

Aaron M. Neiman

Keyword(s):

Saccharomyces Cerevisiae ◽

De Novo ◽

Ectopic Expression ◽

Membrane Vesicles ◽

Specific Gene ◽

Wild Type ◽

Protein Mutation ◽

Prospore Membrane ◽

In The Wild ◽

Membrane Morphogenesis

ABSTRACT The creation of haploid gametes in yeast, termed spores, requires the de novo formation of membranes within the cytoplasm. These membranes, called prospore membranes, enclose the daughter nuclei generated by meiosis. Proper growth and closure of prospore membranes require the highly conserved Vps13 protein. Mutation of SPO71 , a meiosis-specific gene first identified as defective in spore formation, was found to display defects in membrane morphogenesis very similar to those seen in vps13 Δ cells. Specifically, prospore membranes are smaller than in the wild type, they fail to close, and membrane vesicles are present within the prospore membrane lumen. As in vps13 Δ cells, the levels of phophatidylinositol-4-phosphate are reduced in the prospore membranes of spo71 Δ cells. SPO71 is required for the translocation of Vps13 from the endosome to the prospore membrane, and ectopic expression of SPO71 in vegetative cells results in mislocalization of Vps13. Finally, the two proteins can be coprecipitated from sporulating cells. We propose that Spo71 is a sporulation-specific partner for Vps13 and that they act in concert to regulate prospore membrane morphogenesis.

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The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

Biochemical Journal ◽

10.1042/bj3120273 ◽

1995 ◽

Vol 312 (1) ◽

pp. 273-280 ◽

Cited By ~ 40

Author(s):

M Haraguchi ◽

S Yamashiro ◽

K Furukawa ◽

K Takamiya ◽

H Shiku ◽

...

Keyword(s):

Enzyme Activity ◽

Intracellular Localization ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Kinetics Analysis ◽

Glycosylation Sites ◽

In The Wild ◽

Polymerase Chain ◽

The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

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Biological role of the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.1.7.584 ◽

1981 ◽

Vol 1 (7) ◽

pp. 584-593 ◽

Cited By ~ 73

Author(s):

P Niederberger ◽

G Miozzari ◽

R Hütter

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Biosynthesis ◽

Wild Type ◽

General Control ◽

Acid Biosynthesis ◽

Mutant Strains ◽

In The Wild ◽

Amino Acid Imbalance

The biological role of the "general control of amino acid biosynthesis" has been investigated by analyzing growth and enzyme levels in wild-type, bradytrophic, and nonderepressing mutant strains of Saccharomyces cerevisiae. Amino acid limitation was achieved by using either bradytrophic mutations or external amino acid imbalance. In the wild-type strain noncoordinate derepression of enzymes subject to the general control has been found. Derepressing factors were in the order of 2 to 4 in bradytrophic mutant strains grown under limiting conditions and only in the order of 1.5 to 2 under the influence of external amino acid imbalance. Nonderepressing mutations led to slower growth rates under conditions of amino acid limitation, and no derepression of enzymes under the general control was observed. The amino acid pools were found to be very similar in the wild type and in nonderepressing mutant strains under all conditions tested. Our results indicate that the general control affects all branched amino acid biosynthetic pathways, namely, those of the aromatic amino acids and the aspartate family, the pathways for the basic amino acids lysine, histidine, and arginine, and also the pathways of serine and valine biosyntheses.

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Improving 3-methylphenol (m-cresol) production in yeast via in vivo glycosylation or methylation

FEMS Yeast Research ◽

10.1093/femsyr/foaa063 ◽

2020 ◽

Vol 20 (8) ◽

Author(s):

Julia Hitschler ◽

Eckhard Boles

Keyword(s):

Saccharomyces Cerevisiae ◽

De Novo ◽

Wild Type ◽

Biotechnological Production ◽

Yeast Saccharomyces Cerevisiae ◽

In Situ Extraction ◽

Methylsalicylic Acid Synthase ◽

In The Wild

ABSTRACT Heterologous expression of 6-methylsalicylic acid synthase (MSAS) together with 6-MSA decarboxylase enables de novo production of the platform chemical and antiseptic additive 3-methylphenol (3-MP) in the yeast Saccharomyces cerevisiae. However, toxicity of 3-MP prevents higher production levels. In this study, we evaluated in vivo detoxification strategies to overcome limitations of 3-MP production. An orcinol-O-methyltransferase from Chinese rose hybrids (OOMT2) was expressed in the 3-MP producing yeast strain to convert 3-MP to 3-methylanisole (3-MA). Together with in situ extraction by dodecane of the highly volatile 3-MA this resulted in up to 211 mg/L 3-MA (1.7 mM) accumulation. Expression of a UDP-glycosyltransferase (UGT72B27) from Vitis vinifera led to the synthesis of up to 533 mg/L 3-MP as glucoside (4.9 mM). Conversion of 3-MP to 3-MA and 3-MP glucoside was not complete. Finally, deletion of phosphoglucose isomerase PGI1 together with methylation or glycosylation and feeding a fructose/glucose mixture to redirect carbon fluxes resulted in strongly increased product titers, with up to 897 mg/L 3-MA/3-MP (9 mM) and 873 mg/L 3-MP/3-MP as glucoside (8.1 mM) compared to less than 313 mg/L (2.9 mM) product titers in the wild type controls. The results show that methylation or glycosylation are promising tools to overcome limitations in further enhancing the biotechnological production of 3-MP.

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