scholarly journals Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene.

1988 ◽  
Vol 8 (12) ◽  
pp. 5555-5560 ◽  
Author(s):  
H Iida
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Adenylate Cyclase ◽  
Uv Light ◽  
Yeast Cells ◽  
Wild Type ◽  
Coding Region ◽  
Resistant Mutants ◽  
Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene

1988 ◽  
Vol 8 (12) ◽  
pp. 5555-5560
Author(s):  
H Iida
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Adenylate Cyclase ◽  
Uv Light ◽  
Yeast Cells ◽  
Wild Type ◽  
Coding Region ◽  
Resistant Mutants ◽  
Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

Expression of a mammalian Na+/H+ antiporter in Saccharomyces cerevisiae

1999 ◽  
Vol 77 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Mónica Montero-Lomelí ◽  
Anna L Okorokova Façanha.
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Subcellular Fractionation ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Phosphatidylcholine Liposomes ◽  
Yeast Transformants ◽  
Wild Type Yeast ◽  
Membrane Region

The basolateral Na+/H+ antiporter (NHE) from LLC-PK1 cells was expressed in Saccharomyces cerevisiae. Two different strategies were tested for expression. In the first, we used a yeast strain that contains a temperature-sensitive mutation in the SEC-6 gene, whose product is required for the fusion of secretory vesicles with the plasma membrane. This strain was transformed with a vector containing the coding region of the NHE1 isoform under control of a heat shock (HS) promoter (pYNHE1-HS). In the second strategy, we replaced the heat shock promoter from pYNHE1-HS with a galactose (GAL) promoter (pYNHEI-GAL) and transformed wild-type yeast. In both cases, Northern blots demonstrated a transcript that hybridized against a probe containing the membrane region of the exchanger. When an antibody against the last 40 amino acids of the carboxy-terminus of NHE1 was used for immuno-blots, a protein with a Mr of 73 000 was seen in total membranes from both yeast transformants. Subcellular fractionation revealed that NHE1 was expressed in the endoplasmic reticulum. In the case of the pYNHEI-GAL transformant, the 100 000 × g membrane pellet was reconstituted in phosphatidylcholine liposomes, and ethylisopropyl-amiloride-sensitive Na+/H+ exchange was observed. These results have paved the way for expression of the Na+/H+ exchanger in a genetically well-known microorganism.Key words: Na+/H+ exchanger, NHE1, expression, yeast.

scholarly journals The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A.

1995 ◽  
Vol 15 (11) ◽  
pp. 6232-6245 ◽  
Author(s):  
J C Varela ◽  
U M Praekelt ◽  
P A Meacock ◽  
R J Planta ◽  
W H Mager
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Protein Kinase ◽  
Stationary Phase ◽  
Protein Kinase A ◽  
Yeast Cells ◽  
Wild Type ◽  
High Osmolarity ◽  
High Osmolarity Glycerol ◽  
Kinase A

The HSP12 gene encodes one of the two major small heat shock proteins of Saccharomyces cerevisiae. Hsp12 accumulates massively in yeast cells exposed to heat shock, osmostress, oxidative stress, and high concentrations of alcohol as well as in early-stationary-phase cells. We have cloned an extended 5'-flanking region of the HSP12 gene in order to identify cis-acting elements involved in regulation of this highly expressed stress gene. A detailed analysis of the HSP12 promoter region revealed that five repeats of the stress-responsive CCCCT motif (stress-responsive element [STRE]) are essential to confer wild-type induced levels on a reporter gene upon osmostress, heat shock, and entry into stationary phase. Disruption of the HOG1 and PBS2 genes leads to a dramatic decrease of the HSP12 inducibility in osmostressed cells, whereas overproduction of Hog1 produces a fivefold increase in wild-type induced levels upon a shift to a high salt concentration. On the other hand, mutations resulting in high protein kinase A (PKA) activity reduce or abolish the accumulation of the HSP12 mRNA in stressed cells. Conversely, mutants containing defective PKA catalytic subunits exhibit high basal levels of HSP12 mRNA. Taken together, these results suggest that HSP12 is a target of the high-osmolarity glycerol (HOG) response pathway under negative control of the Ras-PKA pathway. Furthermore, they confirm earlier observations that STRE-like sequences are responsive to a broad range of stresses and that the HOG and Ras-PKA pathways have antagonistic effects upon CCCCT-driven transcription.

scholarly journals Suppression of an Hsp70 Mutant Phenotype in Saccharomyces cerevisiae through Loss of Function of the Chromatin Component Sin1p/Spt2p

1998 ◽  
Vol 180 (24) ◽  
pp. 6484-6492 ◽  
Author(s):  
Bonnie K. Baxter ◽  
Elizabeth A. Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Molecular Chaperones ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Wild Type ◽  
Extragenic Suppressor ◽  
Slow Growing ◽  
Type Strains ◽  
Existing Data

ABSTRACT The Ssa subfamily of Hsp70 molecular chaperones in the budding yeast Saccharomyces cerevisiae has four members, encoded bySSA1, SSA2, SSA3, andSSA4. Deletion of the two constitutively expressed genes,SSA1 and SSA2, results in cells which are slow growing and temperature sensitive. In this study, we demonstrate that an extragenic suppressor of the temperature sensitivity of ssa1 ssa2 strains, EXA1-1, is a loss-of-function mutation in SIN1/SPT2, which encodes a nonhistone component of chromatin. Loss of function of Sin1p leads to overexpression ofSSA3 in the ssa1 ssa2 mutant background, at a level which is sufficient to mediate suppression. In a strain which is wild type for SSA genes, we detected no effect of Sin1p on Ssa3p expression except under conditions of heat shock. Existing data indicate that expression of SSA3 in thessa1 ssa2 mutant background as well as in heat-shocked wild-type strains is mediated by the heat shock transcription factor HSF. Our findings suggest that it is HSF-mediated induction of SSA3 which is modulated by Sin1p. TheEXA1-1 suppressor mutation thus improves the growth ofssa1 ssa2 strains by selectively increasing HSF-mediated expression of SSA3.

scholarly journals LEA (late embryonic abundant)-like protein Hsp 12 (heat-shock protein 12) is present in the cell wall and enhances the barotolerance of the yeast Saccharomyces cerevisiae

2004 ◽  
Vol 377 (3) ◽  
pp. 769-774 ◽  
Author(s):  
Precious MOTSHWENE ◽  
Robert KARREMAN ◽  
Gail KGARI ◽  
Wolf BRANDT ◽  
George LINDSEY
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Barometric Pressure ◽  
Yeast Cells ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rapid Changes ◽  
Wild Type Yeast

Yeast cells Saccharomyces cerevisiae, late embryogenic abundant-like stress response protein Hsp 12 (heat-shock protein 12) were found by immunocytochemistry to be located both in the cytoplasm and in the cell wall, from where they could be extracted with dilute NaOH solutions. Yeast cells with the Hsp 12 gene disrupted were unable to grow in the presence of either 12 mM caffeine or 0.43 mM Congo Red, molecules known to affect cell-wall integrity. The volume of yeast cells were less affected by rapid changes in the osmolality of the growth medium when compared with the wild-type yeast cells, suggesting a role for Hsp 12 in the flexibility of the cell wall. This was also suggested by subjecting the yeast cells to rapid changes in barometric pressure where it was found that wild-type yeast cells were more resistant to cellular breakage.

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

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 761-776 ◽  
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.

Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (4) ◽  
pp. 591-598
Author(s):  
J Cappello ◽  
C Zuker ◽  
H F Lodish
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Nucleotide Sequence ◽  
Dna Sequence ◽  
Base Pair ◽  
Shock Treatment ◽  
Yeast Cells ◽  
Genomic Segment ◽  
Heat Shock Treatment ◽  
Shock Response

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.

Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation

1987 ◽  
Vol 7 (1) ◽  
pp. 244-250
Author(s):  
D Y Shin ◽  
K Matsumoto ◽  
H Iida ◽  
I Uno ◽  
T Ishikawa
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Cyclic Amp ◽  
Heat Shock Proteins ◽  
Temperature Shift ◽  
G1 Phase ◽  
Shock Response ◽  
Dependent Protein ◽  
Shock Proteins

When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle. The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift. The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift. In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment. The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle.

Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae

1994 ◽  
Vol 14 (11) ◽  
pp. 7322-7330 ◽  
Author(s):  
N Iizuka ◽  
L Najita ◽  
A Franzusoff ◽  
P Sarnow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Translation System ◽  
Translational Efficiency ◽  
Coding Region ◽  
Initiation Mechanism ◽  
Noncoding Regions ◽  
Cell Extracts ◽  
Independent Translation

Translation extracts were prepared from various strains of Saccharomyces cerevisiae. The translation of mRNA molecules in these extracts were cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences. These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells. Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules. Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus B1 conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation. Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts. Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system. This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs.

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

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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