Saccharomyces cerevisiae null mutants in glucose phosphorylation: metabolism and invertase expression.

Abstract A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism.

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Spontaneous Frameshift Mutations in Saccharomyces cerevisiae: Accumulation During DNA Replication and Removal by Proofreading and Mismatch Repair Activities

Genetics ◽

10.1093/genetics/159.1.65 ◽

2001 ◽

Vol 159 (1) ◽

pp. 65-75 ◽

Cited By ~ 1

Author(s):

Christopher N Greene ◽

Sue Jinks-Robertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Mismatch Repair ◽

Wild Type Strain ◽

Dna Polymerases ◽

Wild Type ◽

Triple Mutant ◽

Repair Capacity ◽

Frameshift Mutations ◽

Dna Polymerization ◽

In The Wild

Abstract The accumulation of frameshift mutations during DNA synthesis is determined by the rate at which frameshift intermediates are generated during DNA polymerization and the efficiency with which frameshift intermediates are removed by DNA polymerase-associated exonucleolytic proofreading activity and/or the postreplicative mismatch repair machinery. To examine the relative contributions of these factors to replication fidelity in Saccharomyces cerevisiae, we determined the reversion rates and spectra of the lys2ΔBgl +1 frameshift allele. Wild-type and homozygous mutant diploid strains with all possible combinations of defects in the exonuclease activities of DNA polymerases δ and ε (conferred by the pol3-01 and pol2-4 alleles, respectively) and in mismatch repair (deletion of MSH2) were analyzed. Although there was no direct correlation between homopolymer run length and frameshift accumulation in the wild-type strain, such a correlation was evident in the triple mutant strain lacking all repair capacity. Furthermore, examination of strains defective in one or two repair activities revealed distinct biases in the removal of the corresponding frameshift intermediates by exonucleolytic proofreading and/or mismatch repair. Finally, these analyses suggest that the mismatch repair machinery may be important for generating some classes of frameshift mutations in yeast.

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Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.2.383 ◽

1996 ◽

Vol 142 (2) ◽

pp. 383-391 ◽

Cited By ~ 2

Author(s):

Yasumasa Tsukamoto ◽

Jun-ichi Kato ◽

Hideo Ikeda

Keyword(s):

Saccharomyces Cerevisiae ◽

Quantitative Analysis ◽

Structural Analysis ◽

Recombination Rate ◽

Type Strain ◽

Negative Selection ◽

Wild Type Strain ◽

Illegitimate Recombination ◽

Wild Type ◽

In The Wild

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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Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2.

Genetics ◽

10.1093/genetics/135.2.321 ◽

1993 ◽

Vol 135 (2) ◽

pp. 321-326 ◽

Cited By ~ 2

Author(s):

H Mitsuzawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Slow Growth ◽

Loss Of Function ◽

Wild Type ◽

Triple Mutant ◽

Apparent Absence ◽

Camp Pathway ◽

Naturally Occurring ◽

Elevated Activity ◽

Growth Phenotype

Abstract The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases.

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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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Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽

10.1182/blood-2012-07-441824 ◽

2012 ◽

Vol 120 (16) ◽

pp. 3336-3344 ◽

Cited By ~ 36

Author(s):

Anu Laitala ◽

Ellinoora Aro ◽

Gail Walkinshaw ◽

Joni M. Mäki ◽

Maarit Rossi ◽

...

Keyword(s):

Cultured Cells ◽

Mrna Level ◽

Hypoxia Inducible Factor ◽

Mrna Levels ◽

Wild Type ◽

Α Subunit ◽

Hepcidin Expression ◽

In The Wild ◽

Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

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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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Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4467-4472.1989 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4467-4472

Author(s):

M Altmann ◽

N Sonenberg ◽

H Trachsel

Keyword(s):

Saccharomyces Cerevisiae ◽

Point Mutations ◽

Translation Initiation Factor ◽

Apparent Temperature ◽

Initiation Factor ◽

Temperature Sensitive ◽

Wild Type ◽

Free System ◽

Gal1 Promoter

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

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Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4084-4092.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4084-4092

Author(s):

P C McCabe ◽

H Haubruck ◽

P Polakis ◽

F McCormick ◽

M A Innis

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Bound States ◽

Yeast Cells ◽

Temperature Sensitive ◽

Wild Type ◽

Amino Terminal ◽

Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

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Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1805 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1805-1812 ◽

Cited By ~ 58

Author(s):

J Zhu ◽

R H Schiestl

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Aberrations ◽

Topoisomerase I ◽

Wild Type Strain ◽

Hot Spot ◽

Illegitimate Recombination ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae

Chromosome aberrations may cause cancer and many heritable diseases. Topoisomerase I has been suspected of causing chromosome aberrations by mediating illegitimate recombination. The effects of deletion and of overexpression of the topoisomerase I gene on illegitimate recombination in the yeast Saccharomyces cerevisiae have been studied. Yeast transformations were carried out with DNA fragments that did not have any homology to the genomic DNA. The frequency of illegitimate integration was 6- to 12-fold increased in a strain overexpressing topoisomerase I compared with that in isogenic control strains. Hot spot sequences [(G/C)(A/T)T] for illegitimate integration target sites accounted for the majority of the additional events after overexpression of topoisomerase I. These hot spot sequences correspond to sequences previously identified in vitro as topoisomerase I preferred cleavage sequences in other organisms. Furthermore, such hot spot sequences were found in 44% of the integration events present in the TOP1 wild-type strain and at a significantly lower frequency in the top1delta strain. Our results provide in vivo evidence that a general eukaryotic topoisomerase I enzyme nicks DNA and ligates nonhomologous ends, leading to illegitimate recombination.

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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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