Decreased cellular permeability to H2O2 protects Saccharomyces cerevisiae cells in stationary phase against oxidative stress.

The higher resistance of stationary-phase Saccharomyces cerevisiae to H2O2 when compared with exponential phase is well characterized, but the molecular mechanisms underlying it remain mostly unknown. By applying the steady-state H2O2-delivery model, we show that (a) cellular permeability to H2O2 is five times lower in stationary--than in exponential phase; (b) cell survival to H2O2 correlates with H2O2 cellular gradients for a variety of cells; and, (c) cells in stationary phase are predicted to be more susceptible to intracellular H2O2 than in exponential phase. In conclusion, limiting H2O2 diffusion into cells is a key protective mechanism against extracellular H2O2.

Microtubule cytoskeleton perturbation induced by taxol and colchicine affects chaperonin containing TCP-1 (CCT) subunit gene expression in Tetrahymena cells.

We report the existence of a CCT epsilon subunit gene that encodes subunit epsilon of the chaperonin CCT (chaperonin containing TCP-1) in Tetrahymena pyriformis. This work focuses on the study of the effects of the microtubule polymerizing agent taxol and the depolymerizing agent colchicine on microtubule dynamics and their role in the regulation of tubulin and CCT subunit genes. Under taxol treatment some TpCCT and tubulin genes are distinctly expressed until 30 min of treatment. Cytoplasmic TpCCT mRNA levels slightly decrease while tubulin transcripts are increasing. In colchicine treated cells TpCCT and tubulin transcripts decrease in the initial 30 min of treatment and then start to increase. However, both antimitotic agents induce TpCCT and tubulin gene transcription. This induction does not correlate with increased steady-state levels of TpCCT proteins and seems to be necessary to replete cytoplasmic TpCCT mRNAs. Moreover, we found that TpCCT epsilon and TpCCT alpha but not TpCCT eta are present in the insoluble fraction after a postmitochondrial fractionation that contains components of the ciliate cortex structure, basal bodies and cilia. This suggests that some TpCCT subunits may be associated with these structures. The association of TpCCT epsilon subunit is stimulated either by taxol or colchicine treatment. These observations support the idea that CCT subunits could have additional roles in vivo.

Structure of Tetrahymena CCT theta gene and its expression under colchicine treatment.

We report here the cloning and the characterization of the Tetrahymena pyriformis chaperonin-containing-TCP1 theta gene (TpCCT theta), an orthologue of the mouse chaperonin gene CCT theta. TpCCT theta gene is interrupted by eight introns, ranging in size between 91 and 419 nucleotides, and encodes a protein consisting of 540 amino acid residues (59.1 kDa), with a putative pI of 5.73. The amino acid sequence of TpCCT theta reveals 39.4-46.0% identity with the sequences of Candida albicans and mouse CCT theta subunits and 28.0-32.6% identity with the other TpCCT subunits known so far. We have studied the expression of this gene in exponentially growing Tetrahymena cells and in cells treated with colchicine for different times. The steady-state levels of CCT theta mRNA rapidly decrease in the first 30 min of colchicine treatment. Interestingly, treatment for subsequent 60 min gives expression levels higher than those found in exponentially growing cells.

The third member of the Tetrahymena CCT subunit gene family, TpCCT alpha, encodes a component of the hetero-oligomeric chaperonin complex.

The sequence of a third member of the Tetrahymena pyriformis chaperonin CCT ('chaperonin containing TCP1') subunit gene family is presented. This gene, designated TpCCT alpha, is the orthologue of the mouse chaperonin gene TCP1/CCT alpha. To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCT alpha, TpCCT gamma and TpCCT eta subunits. We have also used polyclonal antibodies produced against recombinant yeast CCT alpha and CCT beta subunits. Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCT beta, suggesting that this gene is also present in the ciliate. The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins. TpCCT alpha gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCT gamma and TpCCT eta, and also to tubulin genes.

The Tetrahymena chaperonin subunit CCT eta gene is coexpressed with CCT gamma gene during cilia biogenesis and cell sexual reproduction.

We report here the cloning and the characterization of the T. pyriformis CCT eta gene (TpCCT eta) and also a partial sequence of the corresponding T. thermophila gene (TtCCT eta). The TpCCt eta gene encodes a protein sharing a 60.3% identity with the mouse CCT eta. We have studied the expression of these genes in Tetrahymena exponentially growing cells, cells regenerating their cilia for different periods and during different stages of the cell sexual reproduction. These genes have similar patterns of expression to those of the previously identified TpCCt gamma gene. Indeed, the Tetrahymena CCT eta and CCT gamma genes are up-regulated at 60-120 min of cilia recovery, and in conjugation when vegetative growth was resumed and cell division took place. Our results seem to indicate that both CCT subunits play an important role in the biogenesis of the newly synthesized cilia of Tetrahymena and during its cell division.

Sequencing of a 17.6 kb segment on the right arm of yeast chromosome VII reveals 12 ORFs, including CCT, ADE3 and TR-I genes, homologues of the yeast PMT and EF1G genes, of the human and bacterial electron-transferring flavoproteins (beta-chain) and of the Escherichia coli phosphoserine phosphohydrolase, and five new ORFs.

A 17.6 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains twelve open reading frames (ORFs) longer than 100 amino acids. Three genes had already been cloned and sequenced: CCT, ADE3 and TR-I. Two ORFs are similar to other yeast genes: G7722 with the YAL023 (PMT2) and PMT1 genes, encoding two integral membrane proteins, and G7727 with the first half of the genes encoding elongation factors 1gamma, TEF3 and TEF4. Two other ORFs, G7742 and G7744, are most probably yeast orthologues of the human and Paracoccus denitrificans electron-transferring flavoproteins (beta chain) and of the Escherichia coli phosphoserine phosphohydrolase. The five remaining identified ORFs do not show detectable homology with other protein sequences deposited in data banks. The sequence has been deposited in the EMBL data library under Accession Number Z49133.

Different patterns of expression of beta-tubulin genes in Tetrahymena pyriformis during reciliation.

The ciliate Tetrahymena pyriformis contains one alpha-tubulin (alpha TT) and two beta-tubulin (beta TT1 and beta TT2) genes. The specific expression of these genes was investigated by Northern blot hybridization using oligonucleotide probes complementary to beta TT1 and beta TT2 genes and the coding region of the alpha-tubulin gene. The three genes are expressed producing 1.8-kb mRNAs but the level of beta TT1 mRNA is much higher than that of beta TT2 mRNA. During cilia regeneration, we found that the expression patterns of the alpha TT and beta TT1 genes are similar whereas that of the beta TT2 gene is different. The alpha TT and beta TT1 transcripts reached higher values between 60-120 min after the onset of reciliation than in exponentially growing cells, while beta TT2 transcripts were maintained at low levels during the whole period. The differences in the amounts of steady-state populations of the both beta-tubulin mRNAs do not correspond to the copy number per haploid genome. These differences could result from the fact that the promoter region of beta TT2 may contain highly structured sequences which would affect the binding of the respective trans-acting factor(s). The apparent transcription rate revealed a significant increase at 15 min of reciliation which could be responsible for the high levels of alpha TT and beta TT1 transcripts in the cytoplasm between 60-120 min of reciliation. This coordinated response to cilia regeneration of the alpha TT and beta TT1 tubulin genes is also a relevant aspect of our findings. Several conserved motifs found in their promoter regions led us to think that some of them may function as cis-elements in the specific binding of nuclear protein factor(s).

Sequence of one alpha- and two beta-tubulin genes of Tetrahymena pyriformis. Structural and functional relationships with other eukaryotic tubulin genes.

Macronuclear DNA of the ciliate Tetrahymena pyriformis contains only one size class of fragments coding for alpha-tubulin, alpha TT. We have isolated alpha TT from a partial plasmid library, using Chlamydomonas reinhardtii alpha-tubulin gene as a probe. This gene as well as the two beta-tubulin genes, beta TT1 and beta TT2, have been sequenced. None of these genes contains introns and all use TGA as the stop codon. In the coding region of the two beta-tubulin genes, there are several TAA and TAG stop codons that probably code for glutamine. The codon usage is very biased. Regions flanking the tubulin coding sequences are A + T-rich (75%) and quite different among themselves. In these regions there are several putative transcription-regulatory sequences. Nuclear transcripts begin and terminate at multiple sites. The beta-tubulin proteins differ only in two amino acid residues. Primary structure of Tetrahymena tubulins as well as their hydropathy indexes show a high degree of homology with tubulins from other organisms. Two-dimensional electrophoretic analysis of the ciliary tubulins shows the presence of eight alpha-tubulins and four beta-tubulins. The alpha-tubulins migrate faster than the beta-tubulins, in contrast with what happens with brain tubulins. We suggest that there are several alpha- and beta-tubulin isoforms and the migratory inversion observed may be due to post-translational modifications.