Dominant effects of tubulin overexpression in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (3) ◽  
pp. 1049-1059
Author(s):  
D Burke ◽  
P Gasdaska ◽  
L Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Loss ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Inducible Promoters ◽  
Selective Degradation ◽  
Novel Structure ◽  
Genes Encoding ◽  
Transient Overexpression ◽  
Mutant Phenotypes

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.

scholarly journals Dominant effects of tubulin overexpression in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (3) ◽  
pp. 1049-1059 ◽  
Author(s):  
D Burke ◽  
P Gasdaska ◽  
L Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Loss ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Inducible Promoters ◽  
Selective Degradation ◽  
Novel Structure ◽  
Genes Encoding ◽  
Transient Overexpression ◽  
Mutant Phenotypes

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.

Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin

1990 ◽  
Vol 10 (10) ◽  
pp. 5295-5304
Author(s):  
B Weinstein ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Data Support ◽  
Cell Biol

Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division. However, other consequences of overexpression of these genes differ in several ways. As shown previously (D. Burke, P. Gasdaska, and L. Hartwell, Mol. Cell. Biol. 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability. Eventually, the excess beta-tubulin forms abnormal structures. We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation. Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed. The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation

1992 ◽  
Vol 12 (2) ◽  
pp. 791-799
Author(s):  
N G Theodorakis ◽  
D W Cleveland
Keyword(s):  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Selective Degradation ◽  
Cellular Factors ◽  
Nascent Peptide ◽  
Tubulin Mrna ◽  
The Stability ◽  
Necessary And Sufficient ◽  
Specific Degradation

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.

scholarly journals Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number.

1990 ◽  
Vol 10 (10) ◽  
pp. 5286-5294 ◽  
Author(s):  
W Katz ◽  
B Weinstein ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cellular Response ◽  
Gene Copy Number ◽  
Microtubule Assembly ◽  
Gene Copy ◽  
Microtubule Organization ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Tubulin Protein

Microtubule organization in the cytoplasm is in part a function of the number and length of the assembled polymers. The intracellular concentration of tubulin could specify those parameters. Saccharomyces cerevisiae strains constructed with moderately decreased or increased copy numbers of tubulin genes provide an opportunity to study the cellular response to a steady-state change in tubulin concentration. We found no evidence of a mechanism for adjusting tubulin concentrations upward from a deficit, nor did we find a need for such a mechanism: cells with no more than 50% of the wild-type tubulin level were normal with respect to a series of microtubule-dependent properties. Strains with increased copies of both alpha- and beta-tubulin genes, or of alpha-tubulin genes alone, apparently did down regulate their tubulin levels. As a result, they contained greater than normal concentrations of tubulin but much less than predicted from the increase in gene number. Some of this down regulation occurred at the level of protein. These strains were also phenotypically normal. Cells could contain excess alpha-tubulin protein without detectable consequences, but perturbations resulting in excess beta-tubulin genes may have affected microtubule-dependent functions. All of the observed regulation of levels of tubulin can be explained as a response to toxicity associated with excess tubulin proteins, especially if beta-tubulin is much more toxic than alpha-tubulin.

Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number

1990 ◽  
Vol 10 (10) ◽  
pp. 5286-5294
Author(s):  
W Katz ◽  
B Weinstein ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cellular Response ◽  
Gene Copy Number ◽  
Microtubule Assembly ◽  
Gene Copy ◽  
Microtubule Organization ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Tubulin Protein

Microtubule organization in the cytoplasm is in part a function of the number and length of the assembled polymers. The intracellular concentration of tubulin could specify those parameters. Saccharomyces cerevisiae strains constructed with moderately decreased or increased copy numbers of tubulin genes provide an opportunity to study the cellular response to a steady-state change in tubulin concentration. We found no evidence of a mechanism for adjusting tubulin concentrations upward from a deficit, nor did we find a need for such a mechanism: cells with no more than 50% of the wild-type tubulin level were normal with respect to a series of microtubule-dependent properties. Strains with increased copies of both alpha- and beta-tubulin genes, or of alpha-tubulin genes alone, apparently did down regulate their tubulin levels. As a result, they contained greater than normal concentrations of tubulin but much less than predicted from the increase in gene number. Some of this down regulation occurred at the level of protein. These strains were also phenotypically normal. Cells could contain excess alpha-tubulin protein without detectable consequences, but perturbations resulting in excess beta-tubulin genes may have affected microtubule-dependent functions. All of the observed regulation of levels of tubulin can be explained as a response to toxicity associated with excess tubulin proteins, especially if beta-tubulin is much more toxic than alpha-tubulin.

scholarly journals Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin.

1990 ◽  
Vol 10 (10) ◽  
pp. 5295-5304 ◽  
Author(s):  
B Weinstein ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Data Support ◽  
Cell Biol

Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division. However, other consequences of overexpression of these genes differ in several ways. As shown previously (D. Burke, P. Gasdaska, and L. Hartwell, Mol. Cell. Biol. 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability. Eventually, the excess beta-tubulin forms abnormal structures. We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation. Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed. The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.

scholarly journals Genetic Analysis of the TOR Pathway in Aspergillus nidulans

2005 ◽  
Vol 4 (9) ◽  
pp. 1595-1598 ◽  
Author(s):  
Gregory J. Fitzgibbon ◽  
Igor Y. Morozov ◽  
Meriel G. Jones ◽  
Mark X. Caddick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Aspergillus Nidulans ◽  
Minor Role ◽  
Tor Signaling ◽  
Tor Pathway ◽  
Genes Encoding ◽  
Tor Kinase ◽  
Mutant Phenotypes ◽  
A Minor

ABSTRACT We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.

scholarly journals Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

1992 ◽  
Vol 12 (2) ◽  
pp. 791-799 ◽  
Author(s):  
N G Theodorakis ◽  
D W Cleveland
Keyword(s):  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Selective Degradation ◽  
Cellular Factors ◽  
Nascent Peptide ◽  
Tubulin Mrna ◽  
The Stability ◽  
Necessary And Sufficient ◽  
Specific Degradation

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.

scholarly journals The Schizosaccharomyces pombe hst4 + Gene Is a SIR2 Homologue with Silencing and Centromeric Functions

1999 ◽  
Vol 10 (10) ◽  
pp. 3171-3186 ◽  
Author(s):  
Lisa L. Freeman-Cook ◽  
Joyce M. Sherman ◽  
Carrie B. Brachmann ◽  
Robin C. Allshire ◽  
Jef D. Boeke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Regulation ◽  
Molecular Level ◽  
Reporter Genes ◽  
Chromosome Loss ◽  
Wild Type ◽  
Centromere Function ◽  
Mutant Strains ◽  
Mutant Phenotypes ◽  
Dna Mutant

Although silencing is a significant form of transcriptional regulation, the functional and mechanistic limits of its conservation have not yet been established. We have identified theSchizosaccharomyces pombe hst4 + gene as a member of the SIR2/HST silencing gene family that is defined in organisms ranging from bacteria to humans.hst4Δ mutants grow more slowly than wild-type cells and have abnormal morphology and fragmented DNA. Mutant strains show decreased silencing of reporter genes at both telomeres and centromeres. hst4 + appears to be important for centromere function as well because mutants have elevated chromosome-loss rates and are sensitive to a microtubule-destabilizing drug. Consistent with a role in chromatin structure, Hst4p localizes to the nucleus and appears concentrated in the nucleolus.hst4Δ mutant phenotypes, including growth and silencing phenotypes, are similar to those of the Saccharomyces cerevisiae HSTs, and at a molecular level,hst4 + is most similar toHST4. Furthermore, hst4 + is a functional homologue of S. cerevisiae HST3 andHST4 in that overexpression ofhst4 + rescues the temperature-sensitivity and telomeric silencing defects of an hst3Δ hst4Δdouble mutant. These results together demonstrate that aSIR-like silencing mechanism is conserved in the distantly related yeasts and is likely to be found in other organisms from prokaryotes to mammals.

Structure and arrangement of the beta-tubulin genes of Leishmania tropica

1984 ◽  
Vol 4 (7) ◽  
pp. 1372-1383
Author(s):  
P L Huang ◽  
B E Roberts ◽  
D M Pratt ◽  
J R David ◽  
J S Miller
Keyword(s):  
Protozoan Parasite ◽  
Leishmania Tropica ◽  
Beta Tubulin ◽  
S1 Nuclease ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Repeat Units ◽  
Genes Encoding ◽  
Tubulin Mrna ◽  
Parasite Leishmania

We studied the organization and arrangement of the genes encoding beta-tubulin in the protozoan parasite Leishmania tropica and examined the structure and orientation of the beta-tubulin mRNA relative to the gene. There were found to be eight to nine beta-tubulin genes arranged in an array of direct tandem repeat units with a length of 3.8 kilobase pairs, and they were extremely homologous, if not identical, in sequence. These repeat units did not contain the alpha-tubulin genes. The transcribed sequences within the beta-tubulin genes were localized, and the orientation of the major alpha-tubulin mRNA was mapped on the gene by S1 nuclease analysis.

Sign in / Sign up

Export Citation Format

Share Document