The response of eukaryotic cells to heat shock and other forms of stress occurs at both transcriptional and post-transcriptional levels. We used in situ hybridization to determine whether stress affected the subcellular distribution of poly(A]^ RNA in Saccharomyces cerevisiae. Following induction of stress by either heat shock (42°C) or addition of a high concentration of ethanol (10%), the nucleocytoplasmic export of most poly(A)^ RNA was blocked. In situ hybridization indicated that heat-inducible SSA4 and SSAl mRNAs were exported from nuclei under these same conditions. On the other hand, both GALl and URA3 transcripts expressed from the SSA4 promoter accumulated in nuclei following heat shock. Sequences within either the 5' 1600 or the 3' 500 nucleotides of SSA4 mRNA were sufficient to direct GALl mRNA to the cytoplasm during stress. The export of SSA4 mRNA following stress required functional nuclear pore complexes, as SSA4 mRNA accumulated in nuclei following heat shock of cells containing temperature-sensitive nucleoporins. However, the selective export of SSA4 mRNA was maintained in heat-shocked cells carrying temperature-sensitive alleles of RNAl, PRP20, or an inducible dominant-negative allele of GSPl, the S. cerevisiae homolog of RAN/TC4. The results reported here suggest that there is selective export of mRNA in yeast.

In budding yeast, many mutants defective in meiotic recombination and chromosome synapsis undergo checkpoint-mediated arrest at the pachytene stage of meiotic prophase. We recovered the NDT80 gene in a screen for genes whose overexpression bypasses the pachytene checkpoint. Ndt80 is a meiosis-specific transcription factor that promotes expression of genes required for exit from pachytene and entry into meiosis I. Herein, we show that the Ndt80 protein accumulates and is extensively phosphorylated during meiosis in wild type but not in cells arrested at the pachytene checkpoint. Our results indicate that inhibition of Ndt80 activity is one mechanism used to achieve pachytene arrest.

The yeast RNAI gene encodes a cytosolic protein that affects pre-tRNA splicing, pre-rRNA processing, the production of mRNA, and the export of RNA from the nucleus to the cytosol. In an attempt to understand how the RNA1 protein affects such a diverse set of processes, we sought second-site suppressors of a mutation, rnal-1, of the RNA] locus. Mutations in a single complementation group were obtained. These lesions proved to be in the same gene, SRNI, identified previously in a search for second-site suppressors of mutations that affect the removal of intervening sequences from pre-mRNAs. The SRNI gene was mapped, cloned, and sequenced. DNA sequence analysis and the phenotype of disruption mutations showed that, surprisingly, SRNI is identical to HEX2IREGI, a gene that negatively regulates glucose-repressible genes. Interestingly, SRNI is not a negative regulator ofRNAI at the transcriptional, translational, or protein stability level. However, SRNI does regulate the level of two newly discovered antigens, p43 and p70, one of which is not glucose repressible. These studies for the first time link RNA processing and carbon catabolite repression.Primary transcription products are often trimmed and modified to yield mature species via a series of processing steps. These processing steps may include 5' and 3' nucleotide alterations and splicing of intervening sequences, as well as addition and modification of nucleotides. In Saccharomyces cerevisiae, genes encoding some of the products necessary for RNA processing have been identified. The primary effect of mutations in these genes is usually at a specific step in the production of a single class of RNA. For example, mutations of the LOS1, SEN1, SEN2, TPD1, PTA1, and STPJ genes result in defects in the removal of intervening sequences from pre-tRNAs (for reviews, see references 5 and 11), whereas mutations in TRMI, MOD5, TRM2, and ML41 affect single nucleotide modifications of tRNAs (for a review, see reference 11). Mutations of the PRP genes affect the removal of intervening sequences from pre-mRNAs, causing a depletion of ribosomal proteins which in turn affects the accumulation of mature rRNAs (for a review, see reference 42).In contrast to the mutations in the genes described above, the mnal-1 mutation of the RNAJ gene (10) pleiotropically affects the processing of all three major classes of RNAs at the nonpermissive temperature. Processing of tRNA in rnal-1 cells is defective at the step of removal of the intervening sequence (12, 18), and rRNA is affected at the step of processing the 35S primary pre-rRNA (12). The defect in the production of mRNA in mal-1 cells is not clearly defined, but one report described elongated 3' ends (39) and another report described alterations in poly(A) lengths (33). Cells with the mal-1 allele are apparently not defective in the removal of intervening sequences from pre-mRNA (34). The transport of RNA from the nucleus to the cytosol is also affected by the mal-I mutation (15, 37).Paradoxically, it has been found that the RNA1 pr...

Sequence analysis of a mouse testicular a-tubulin partial cDNA, pRDaTTl, reveals an isotype that differs from both the somatic and the predominant testicular a tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-GlyGlu-Glu, found in the carboxyl termini of many a tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis a-tubulin cDNA, we have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular a tubulin that is not detectable in brain a tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple a-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.Microtubules, formed from heterodimers of a and ,3 tubulin, constitute the primary structural components of mitotic and meiotic spindles, eucaryotic cilia and flagella, and elongated neuronal processes (3). The differential expression of specific tubulins during development in higher eucaryotes (3), the identification of a testis-specific i tubulin in Drosophila melanogaster (6), testis-specific a tubulins in mouse (4, 16) and chicken (13) testes, and a male-specific Drosophila a tubulin (14) suggest that tissue-or cell-type-specific microtubules could exist in organs such as the testis.Microtubules play an important role in the dramatic morphological changes in cell structure and shape that occur during the differentiation events of spermatogenesis. Of the microtubular structures peculiar to spermatogenesis, the meiotic spindle and the manchette are highly notable. The manchette forms during the haploid phase of spermatogenesis, when round spermatids first begin to lose their spherical shape, and disappears as the male germ cell elongates during late spermatid differentiation (2). The manchette structure consists of a parallel arrangement of up to 1,100 microtubules attached to a circumnuclear, dense protein plate. Although the function of the manchette is not known, it effectively partitions the cytoplasm of the spermatid, may play a role in shaping the nucleus of the spermatozoon, and is likely involved with transport of cytoplasmic components from the anterior to the posterior portion of the developing male gamete.Identification of a divergent testicular a tubulin. A 1,650-nucleotide cDNA insert that contains coding and 3'-untranslated sequences of the rat brain a tubulin, pILaT1 (10), was used to isolate a novel a-tubulin cDNA from a mouse testis cDNA library (4). Approximately 20,000 colonies were screened by colony hybridization. 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.1% sodium dodecyl sulfate), and 100 ,ug of soni...