Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae

1983 ◽  
Vol 29 (6) ◽  
pp. 681-688 ◽  
Author(s):  
Daniel J. Nieto ◽  
Robin A. Woods
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Ultraviolet Light ◽  
Temperature Sensitivity ◽  
De Novo ◽  
Feedback Inhibition ◽  
Temperature Sensitive ◽  
Ethylmethane Sulphonate ◽  
Interallelic Complementation ◽  
Different Levels

Mutants at the ade4 locus of yeast were isolated following mutagenesis of ade+ and ade2 with ultraviolet light (UV), ethylmethane sulphonate, and the acridine half mustard ICR-170. Tests for interallelic complementation, osmotic remediality, temperature sensitivity, and mutagen-specific reversion were carried out on 19 mutants. Six mutants showed interallelic complementation and fell into four groups, defining three complons. Three mutants were osmotic remedial and the same three were temperature sensitive. Three mutants induced by ICR-170 gave purine-excreting revertants, designated Pur6 or ade4.RCF, after exposure to UV. Activity of amidophosphoribosyltransferase (PRPPAT) was assayed in the ade4 mutants and other alleles at this locus. The ade4 mutants lacked activity of the enzyme; the alleles su-pur+, su-pur, PUR6, and Pur6, showed different levels of activity. The enzyme was subject to feedback inhibition by AMP and IMP in su-pur+ and PUR6; su-pur was hypersensitive to inhibition by AMP, whereas Pur6 was slightly resistant. Purine synthesis de novo was shown to be repressible in su-pur+ and constitutive in PUR6 and Pur6 by following the accumulation of aminoimidazole ribotide in the presence and absence of cycloheximide. These observations were confirmed by direct assay of enzyme activity.

Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities

2021 ◽  
Author(s):  
Adetunji Alex Adekanmbi ◽  
Laurence Dale ◽  
Liz Shaw ◽  
Tom Sizmur
Keyword(s):  
Enzyme Activity ◽  
Soil Temperature ◽  
Temperature Sensitivity ◽  
Extracellular Enzymes ◽  
Extracellular Enzyme ◽  
Daily Maximum ◽  
Temperature Sensitive ◽  
Intracellular Enzyme ◽  
Temperature Changes ◽  
Som Decomposition

<p>Predicting the pattern of soil organic matter (SOM) decomposition as a feedback to climate change, via release of CO<sub>2</sub>, is extremely complex and has received much attention. However, investigations often do not differentiate between the extracellular and intracellular processes involved and work is needed to identify their relative temperature sensitivities. Samples were collected from a grassland soil at Sonning, UK with average daily maximum and minimum soil temperature of 15 °C and 5 °C. We measured potential activities of β-glucosidase (BG) and chitinase (NAG) (extracellular enzymes) and glucose-induced CO<sub>2 </sub>respiration (intracellular enzymes) at a range of assay temperatures (5 °C, 15 °C, 26 °C, 37<sup>  </sup>°C, and 45 °C). The temperature coefficient Q<sub>10</sub> (the increase in enzyme activity that occurs after a 10 °C increase in soil temperature) was calculated to assess the temperature sensitivity of intracellular and extracellular enzymes activities. Between 5 °C and 15 °C intracellular and extracellular enzyme activities had equal temperature sensitivity, but between 15 °C and 26°C intracellular enzyme activity was more temperature sensitive than extracellular enzyme activity and between 26 °C and 37 °C extracellular enzyme activity was more temperature sensitive than intracellular enzyme activity. This result implies that extracellular depolymerisation of higher molecular weight organic compounds is more sensitive to temperature changes at higher temperatures (e.g. changes to daily maximum summer temperature) but the intracellular respiration of the generated monomers is more sensitive to temperature changes at moderate temperatures (e.g. changes to daily mean summer temperature). We therefore conclude that the extracellular and intracellular steps of SOM mineralisation are not equally sensitive to changes in soil temperature. The finding is important because we have observed greater increases in average daily minimum temperatures than average daily mean or maximum temperatures due to increased cloud cover and sulphate aerosol emission. Accounting for this asymmetrical global warming may reduce the importance of extracellular depolymerisation and increase the importance of intracellular catalytic activities as the rate limiting step of SOM decomposition.</p>

scholarly journals TEN1 Is Essential for CDC13-Mediated Telomere Capping

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 793-810 ◽  
Author(s):  
Ling Xu ◽  
Ruben C. Petreaca ◽  
Hovik J. Gasparyan ◽  
Stephanie Vu ◽  
Constance I. Nugent
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
High Temperatures ◽  
De Novo ◽  
Temperature Sensitive ◽  
Single Stranded Dna ◽  
Growth Defects ◽  
Telomere Capping ◽  
Telomere Replication ◽  
Essential Function

Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLα complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.

scholarly journals Hypomodified tRNA in evolutionarily distant yeasts can trigger rapid tRNA decay to activate the general amino acid control response, but with different consequences

2020 ◽  
Author(s):  
Thareendra De Zoysa ◽  
Eric M. Phizicky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Amino Acid ◽  
Temperature Sensitivity ◽  
Temperature Sensitive ◽  
Temperature Resistance ◽  
General Amino Acid Control ◽  
Growth Defects ◽  
Acid Control ◽  
Body Modifications

AbstractAll tRNAs are extensively modified, and modification deficiency often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of any of several tRNA body modifications results in rapid tRNA decay (RTD) of certain mature tRNAs by the 5’-3’ exonucleases Rat1 and Xrn1. As tRNA quality control decay mechanisms are not extensively studied in other eukaryotes, we studied trm8Δ mutants in the evolutionarily distant fission yeast Schizosaccharomyces pombe, which lack 7-methylguanosine at G46 of tRNAs. We report here that S. pombe trm8Δ mutants are temperature sensitive primarily due to decay of tRNATyr(GUA) and that spontaneous mutations in the RAT1 ortholog dhp1+ restored temperature resistance and prevented tRNA decay, demonstrating conservation of the RTD pathway. We also report for the first time evidence linking the RTD and the general amino acid control (GAAC) pathways, which we show in both S. pombe and S. cerevisiae. In S. pombe trm8Δ mutants, spontaneous GAAC mutations restored temperature resistance and tRNA levels, and the temperature sensitivity of trm8Δ mutants was precisely linked to GAAC activation due to tRNATyr(GUA) decay. Similarly, in the well-studied S. cerevisiae trm8Δ trm4Δ RTD mutant, temperature sensitivity was closely linked to GAAC activation due to tRNAVal(AAC) decay; however, in S. cerevisiae, GAAC mutations increased tRNA decay and enhanced temperature sensitivity. Thus, these results demonstrate a conserved GAAC activation coincident with RTD in S. pombe and S. cerevisiae, but an opposite impact of the GAAC response in the two organisms. We speculate that the RTD pathway and its regulation of the GAAC pathway is widely conserved in eukaryotes, extending to other mutants affecting tRNA body modifications.Author SummarytRNA modifications are highly conserved and their lack frequently results in growth defects in the yeast Saccharomyces cerevisiae and neuorological disorders in humans. S. cerevsiaie has two tRNA quality control decay pathways that sense tRNAs lacking modifications in the main tRNA body. One of these, the rapid tRNA decay (RTD) pathway, targets mature tRNAs for 5’-3’ exonucleolytic decay by Rat1 and Xrn1. It is unknown if RTD is conserved in eukaryotes, and if it might explain phenotypes associated with body modification defects. Here we focus on trm8Δ mutants, lacking m7G46, in the evolutionarily distant yeast Schizosaccharomyces pombe. Loss of m7G causes temperature sensitivity and RTD in S. cerevisiae, microcephalic primordial dwarfism in humans, and defective stem cell renewal in mice. We show that S. pombe trm8Δ mutants are temperature sensitive due to tY(GUA) decay by Rat1, implying conservation of RTD among divergent eukaryotes. We also show that the onset of RTD triggers activation of the general amino acid control (GAAC) pathway in both S. pombe and S. cerevisiae, resulting in exacerbated decay in S. pombe and reduced decay in S. cerevisiae. We speculate that RTD and its regulation of the GAAC pathway will be widely conserved in eukaryotes including humans.

Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations.

Genetics ◽  
1993 ◽  
Vol 135 (1) ◽  
pp. 35-44 ◽  
Author(s):  
M A Hoyt ◽  
L He ◽  
L Totis ◽  
W S Saunders
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitivity ◽  
Motor Domain ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Loss Of Function ◽  
Spindle Poles ◽  
Essential Function ◽  
Extragenic Suppressors ◽  
Genotype A

Abstract The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

scholarly journals PMI40, an intron-containing gene required for early steps in yeast mannosylation.

1992 ◽  
Vol 12 (7) ◽  
pp. 2924-2930 ◽  
Author(s):  
D J Smith ◽  
A Proudfoot ◽  
L Friedli ◽  
L S Klig ◽  
G Paravicini ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Enzyme Activity ◽  
Sole Carbon Source ◽  
Amino Acid Sequences ◽  
Transfer Reactions ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Phosphomannose Isomerase ◽  
Protein Levels

We have previously described a temperature-sensitive pmi40-1 mutant of Saccharomyces cerevisiae which is defective in glycosylation and secretion because of a thermolabile phosphomannose isomerase (PMI) activity. Inactivation of PMI at the restrictive temperature of 37 degrees C prevents synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death. Here, we report the isolation of the PMI40 gene by complementation of the corresponding mutation. The PMI40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S. cerevisiae in that it is short and the branch-forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC. The 48.2-kDa protein predicted to be encoded by PMI40 contains amino acid sequences corresponding to those of internal peptides derived from purified S. cerevisiae PMI. Deletion of the PMI40 coding sequence results in a strain requiring D-mannose for growth. The PMI40 gene is located on chromosome V, and its transcription is increased 12-fold when cells are grown on D-mannose as sole carbon source instead of D-glucose. PMI enzyme activity, however, is not increased in D-mannose-grown cells, and PMI protein levels remain constant, suggesting that the PMI40 gene is subject to additional levels of regulation.

scholarly journals A general screen for mutant of Saccharomyces cerevisiae deficient in tRNA biosynthesis.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 55-68 ◽  
Author(s):  
W H van Zyl ◽  
N Wills ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Broad Spectrum ◽  
De Novo ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
New Genes ◽  
Trna Species ◽  
Suppression Assay ◽  
Trna Locus

Abstract We have devised a general screen for isolating conditional lethal mutants defective in synthesis of mature tRNA in Saccharomyces cerevisiae. Using this screen, we have identified several new genes in yeast that are required for production of mature tRNA. These genes most likely encode essential functions, since the mutations we isolated are recessive and cause temperature-sensitive growth. One of the mutants, tpd3, is defective in de novo transcription of 4S RNA at the nonpermissive temperature. A second mutant, tpd1, is specifically defective in excision of intervening sequence from a variety of tRNA species. Finally, two other mutants are defective in production of tRNA from a suppressor tRNA locus, as measured by an in vitro suppression assay. The specific lesion in these strains, though, is not known. These data confirm that the screen does, in fact, yield a broad spectrum of mutants defective in tRNA maturation.

scholarly journals Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid

2020 ◽  
Author(s):  
Pingping Zhou ◽  
Chunlei Yue ◽  
Bin Shen ◽  
Yi Du ◽  
Nannan Xu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Caffeic Acid ◽  
Acid Production ◽  
De Novo ◽  
Feedback Inhibition ◽  
Biological Activities ◽  
Acid Product ◽  
Enhanced Production ◽  
Microbial Biosynthesis

Abstract Background As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently, Saccharomyces cerevisiae has been reported sporadically for biosynthesis of caffeic acid via free plasmid‑mediated pathway assembly. However, the production was far from satisfactory and even relied on the addition of precursor. Results In this study, we first established a controllable caffeic acid pathway by employing a modified GAL regulatory system in S. cerevisiae and realized de novo biosynthesis of 313.8 mg/L caffeic acid from glucose. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback inhibition, deleting genes involved in competing pathways and overexpressing rate-limiting enzymes led to about 2.5-fold improvement in the caffeic acid production, reaching up to 769.3 mg/L in shake-flask cultures. To our knowledge, this is the highest ever reported titer of caffeic acid de novo synthesized by engineered yeast. Conclusions Caffeic acid production in S. cerevisiae strain was successfully improved by adopting a glucose-regulated GAL system and comprehensive metabolic engineering strategies. This work showed the prospect for microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of its derived metabolites.

FINE STRUCTURE ANALYSIS OF THE ade3 LOCUS IN SACCHAROMYCES CEREVISIAE

Genetics ◽  
1972 ◽  
Vol 70 (2) ◽  
pp. 233-250
Author(s):  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fine Structure ◽  
Diagnostic Criteria ◽  
Structure Analysis ◽  
Temperature Sensitivity ◽  
Temperature Sensitive ◽  
Unexpected Result ◽  
Missense Mutations ◽  
Nonsense Mutations ◽  
Spontaneous Mutants

ABSTRACT Twenty-six spontaneous mutants at the ade3 locus of Saccharomyces cerevisiae have been mapped and characterized with respect to revertibility, osmotic remediability and temperature sensitivity. Twelve of the twenty-six are temperature sensitive, 25 of 26 are osmotic remedial and 21 of 26 revert. Two of the mutants map as deletions. At least five of the 26 are nonsense mutations but are also, unexpectedly, osmotic remedial. Three nonsense mutations are also temperature sensitive, again an unexpected result. The two multisite mutations are both temperature sensitive and osmotic remedial. For mutants at this locus osmotic remediability and temperature sensitivity cannot be considered diagnostic criteria for missense mutations.

PMI40, an intron-containing gene required for early steps in yeast mannosylation

1992 ◽  
Vol 12 (7) ◽  
pp. 2924-2930
Author(s):  
D J Smith ◽  
A Proudfoot ◽  
L Friedli ◽  
L S Klig ◽  
G Paravicini ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Enzyme Activity ◽  
Sole Carbon Source ◽  
Amino Acid Sequences ◽  
Transfer Reactions ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Phosphomannose Isomerase ◽  
Protein Levels

We have previously described a temperature-sensitive pmi40-1 mutant of Saccharomyces cerevisiae which is defective in glycosylation and secretion because of a thermolabile phosphomannose isomerase (PMI) activity. Inactivation of PMI at the restrictive temperature of 37 degrees C prevents synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death. Here, we report the isolation of the PMI40 gene by complementation of the corresponding mutation. The PMI40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S. cerevisiae in that it is short and the branch-forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC. The 48.2-kDa protein predicted to be encoded by PMI40 contains amino acid sequences corresponding to those of internal peptides derived from purified S. cerevisiae PMI. Deletion of the PMI40 coding sequence results in a strain requiring D-mannose for growth. The PMI40 gene is located on chromosome V, and its transcription is increased 12-fold when cells are grown on D-mannose as sole carbon source instead of D-glucose. PMI enzyme activity, however, is not increased in D-mannose-grown cells, and PMI protein levels remain constant, suggesting that the PMI40 gene is subject to additional levels of regulation.

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