Hos2p/Set3p deacetylase complex signals secretory stress through the Mpk1p cell integrity pathway.

Perturbations in secretory function activate stress response pathways critical for yeast survival. Here we report the identification of the Hos2p/Set3p deacetylase complex (SET3C) as an essential component of the secretory stress response. Strains lacking core components of the Hos2p/Set3p complex exhibit hypersensitivity to secretory stress. Although not required for the unfolded protein response (UPR) and ribosomal gene repression, the Hos2p complex is required for proper activation of the Mpk1p/Slt2p cell integrity kinase cascade. Disruption of the Hos2p complex results in abrogated Mpk1p phosphorylation, whereas constitutive activation of the Mpk1p pathway rescues the hos2Delta mutant growth defect in response to secretory stress. Furthermore, Hos2p activity is required for the Mpk1p-mediated activation of stress-responsive transcription factor Rlm1p, but not for the stress-induced degradation of the C-type cyclin Ssn8p. Our results identify the Hos2p complex as a critical component of the secretory stress response and support the existence a coordinated stress response consisting of the UPR, ribosomal gene repression, and mitogen-activated protein kinase signaling in response to defects in secretory function.

Ama1p is a meiosis-specific regulator of the anaphase promoting complex/cyclosome in yeast.

Meiosis is the developmental program by which diploid organisms produce haploid gametes capable of sexual reproduction. Here we describe the yeast gene AMA1, a new member of the Cdc20 protein family that regulates the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). AMA1 is developmentally regulated in that its transcription and splicing occur only in meiotic cells. The meiosis-specific processing of AMA1 mRNA depends on the previously described MER1 splicing factor. Several results indicate that Ama1p is required for APC/C function during meiosis. First, coimmunoprecipitation assays indicate that Ama1p associates with the APC/C in vivo. Second, Ama1p is required for the degradation of the B-type cyclin Clb1p, an APC/C substrate in both meiotic and mitotic cells. Third, ectopic overexpression of AMA1 is able to stimulate ubiquitination of Clb1p in vitro and degradation of Clb1p in vivo. Mutants lacking AMA1 revealed that it is required for the first meiotic division but not the mitotic-like meiosis II. In addition, ama1 mutants are defective for both spore wall assembly and the expression of late meiotic genes. In conclusion, this study indicates that Ama1p directs a meiotic APC/C that functions solely outside mitotic cell division. The requirement of Ama1p only for meiosis I and spore morphogenesis suggests a function for APC/C(Ama1) specifically adapted to germ cell development.

Functional analysis of the Ume3p/ Srb11p-RNA polymerase II holoenzyme interaction.

The yeast C-type cyclin Ume3p/Srb11p and its cyclin-dependent kinase (Cdk) Ume5p are required for the full repression of genes involved in the stress response or meiosis. This cyclin-Cdk kinase copurifies with the RNA polymerase II holoenzyme complex, suggesting it functions through modification of the transcriptional machinery. This report describes two domains required for Ume3p-RNA Pol II holoenzyme association. One domain contains the highly conserved cyclin box that directs cyclin-Cdk interaction and requires Ume5p for holoenzyme binding. The second domain, termed HAD for holoenzyme associating domain, is located within the amino-terminal region of the cyclin and is sufficient for holoenzyme binding independent of Ume5p or the cyclin box. In addition to its role in RNA Pol II holoenzyme association, the HAD is also required for Ume3p-dependent repression in vivo. Finally, HAD mutations do not affect the ability of the Ume3p-Ume5p kinase to phosphorylate in vitro the carboxy-terminal domain (CTD) of RNA polymerase II, a reported target of cyclin C-Cdk activity. In conclusion, this study demonstrates that the association of the Ume3p to the holoenzyme is complex, involving two independent domains, both of which are required for full Ume3p-dependent repression in vivo. Furthermore, HAD-dependent repression does not appear to involve CTD phosphorylation, suggesting a different role for this domain in directing Ume3p-Ume5p activity.

Oxidative stress-induced destruction of the yeast C-type cyclin Ume3p requires phosphatidylinositol-specific phospholipase C and the 26S proteasome.

The yeast UME3 (SRB11/SSN3) gene encodes a C-type cyclin that represses the transcription of the HSP70 family member SSA1. To relieve this repression, Ume3p is rapidly destroyed in cells exposed to elevated temperatures. This report demonstrates that Ume3p levels are also reduced in cultures subjected to ethanol shock, oxidative stress, or carbon starvation or during growth on nonfermentable carbons. Of the three elements (RXXL, PEST, and cyclin box) previously shown to be required for heat-induced Ume3p destruction, only the cyclin box regulates Ume3p degradation in response to these stressors. The one exception observed was growth on nonfermentable carbons, which requires the PEST region. These findings indicate that yeast cells contain multiple, independent pathways that mediate stress-induced Ume3p degradation. Ume3p destruction in response to oxidative stress, but not to ethanol treatment, requires DOA4 and UMP1, two factors required for 26S proteasome activity. This result for the first time implicates ubiquitin-mediated proteolysis in C-type cyclin regulation. Similarly, the presence of a membrane stabilizer (sorbitol) or the loss of phosphatidylinositol-specific phospholipase C (PLC1) protects Ume3p from oxidative-stress-induced degradation. Finally, a ume3 null allele suppresses the growth defect of plc1 mutants in response to either elevated temperature or the presence of hydrogen peroxide. These results indicate that the growth defects observed in plc1 mutants are due to the failure to downregulate Ume3p. Taken together, these findings support a model in which Plc1p mediates an oxidative-stress signal from the plasma membrane that triggers Ume3p destruction through a Doa4p-dependent mechanism.

Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA.

The yeast SIR2 gene maintains inactive chromatin domains required for transcriptional repression at the silent mating-type loci and telomeres. We previously demonstrated that SIR2 also acts to repress mitotic and meiotic recombination between the tandem ribosomal RNA gene array (rDNA). Here we address whether rDNA chromatin structure is altered by loss of SIR2 function by in vitro and in vivo assays of sensitivity to micrococcal nuclease and dam methyltransferase, respectively, and present the first chromatin study that maps sites of SIR2 action within the rDNA locus. Control studies at the MAT alpha locus also revealed a previously undetected MNase-sensitive site at the a1-alpha 2 divergent promoter which is protected in sir2 mutant cells by the derepressed a1-alpha 2 regulator. In rDNA, SIR2 is required for a more closed chromatin structure in two regions: SRR1, the major SIR-Responsive Region in the non-transcribed spacer, and SRR2, in the 18S rRNA coding region. None of the changes in rDNA detected in sir2 mutants are due to the presence of the a1-alpha 2 repressor. Reduced recombination in the rDNA correlates with a small, reproducible transcriptional silencing position effect. Deletion and overexpression studies demonstrate that SIR2, but not SIR1, SIR3 or SIR4, is required for this rDNA position effect. Significantly, rDNA transcriptional silencing and rDNA chromatin accessibility respond to SIR2 dosage, indicating that SIR2 is a limiting component required for chromatin modeling in rDNA.

Stress and developmental regulation of the yeast C-type cyclin Ume3p (Srb11p/Ssn8p).

The ume3-1 allele was identified as a mutation that allowed the aberrant expression of several meiotic genes (e.g. SPO11, SPO13) during mitotic cell division in Saccharomyces cerevisiae. Here we report that UME3 is also required for the full repression of the HSP70 family member SSA1. UME3 encodes a non-essential C-type cyclin (Ume3p) whose levels do not vary through the mitotic cell cycle. However, Ume3p is destroyed during meiosis or when cultures are subjected to heat shock. Ume3p mutants resistant to degradation resulted in a 2-fold reduction in SPO13 mRNA levels during meiosis, indicating that the down-regulation of this cyclin is important for normal meiotic gene expression. Mutational analysis identified two regions (PEST-rich and RXXL) that mediate Ume3p degradation. A third destruction signal lies within the highly conserved cyclin box, a region that mediates cyclin-cyclin-dependent kinase (Cdk) interactions. However, the Cdk activated by Ume3p (Ume5p) is not required for the rapid destruction of this cyclin. Finally, Ume3p destruction was not affected in mutants defective for ubiquitin-dependent proteolysis. These results support a model in which Ume3p, when exposed to heat shock or sporulation conditions, is targeted for destruction to allow the expression of genes necessary for the cell to respond correctly to these environmental cues.

[Dynamic skin suture for secondary skin closure and treatment of skin defects]. / Dynamische Hautnaht zum sekundären Hautverschluss und zur Behandlung von Hautdefekten.

Dynamic skin suture exerts progressive traction to the wound margins which allows a stepwise closure of a defect. It consists of interrupted sutures with two additional plastic tubes lying parallel to the wound margins on the surface of the skin. The extracuticular slopes of the sutures and the knots pass over these tubes. This decreases local pressure on the skin. The knots are performed in a way that, once they have been tied, permits further tightening without opening them. A viscoelastic property of the skin--stress-relaxation- reduces the tension on the sutures over time and allows a further tightening. As an example for the use of this technique, the results of closures of fasciotomies in compartment syndromes are presented. In 1993, 50 fasciotomies in 35 patients were treated with dynamic suture. In 42 defects (84%) a complete obliteration was possible. In the residual 8 defects reduction of size was significant. The mean time until obliteration was 11.5 days, in average sutures were tightened 4 times. The results suggest that dynamic suture is a useful technique for repair in fasciotomies. It helps avoiding tissue transplantations. The operative procedure itself is speedy and simple. Possible indications in limited excisional defects are demonstrated in a case report.

SMK1, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cerevisiae.

Mitogen-activated protein (MAP) kinases comprise a family of conserved, eukaryotic enzymes that mediate responses to a wide variety of extracellular stimuli. In yeast, different signal transduction pathways utilize distinct MAP kinase family members. We have identified a new yeast MAP kinase gene (named SMK1) that is required for the completion of sporulation. Molecular and cytologic markers indicate that meiotic development proceeds normally in homozygous smk1-delta 1 diploids through meiosis II. However, light and electron microscopy show that smk1 asci are defective in organizing spore wall assembly. Consistent with a defect in spore wall assembly, smk1-delta 1 mutant asci display enhanced sensitivities to enzymatic digestion, heat shock, and exposure to ether. SMK1 mRNA, which is not detectable in vegetative cells, is derepressed at least 200-fold just prior to prospore enclosure. We propose that the SMK1 MAP kinase participates in a developmentally regulated signal transduction pathway that coordinates cytodifferentiation events with the transcriptional program.

[Dynamic skin suture for closure of the incision defect after compartment incision]. / Dynamische Hautnaht zum Verschluss des Inzisionsdefektes nach Kompartmentspaltung.

Dynamic skin suture consists of a technique that diminishes local pressure on skin and permits progressive tightening of the sutures for stepwise incisional closure. A clinical series of 28 repairs after release of compartment syndromes shows complete closure in 22 incisions, the other 6 defects could be reduced in size. Technique, results and biological basics are discussed.

The yeast UME5 gene regulates the stability of meiotic mRNAs in response to glucose.

We reported previously that early meiotic transcripts are highly unstable. These mRNAs exhibit half-lives of approximately 3 min when expressed during vegetative growth in glucose medium and are stabilized twofold during sporulation in acetate medium. Two genes, UME2 and UME5, that regulate the stability of meiosis-specific transcripts have been identified. The wild-type UME5 gene, which has been analyzed in detail, decreases the stability of all meiotic mRNAs tested approximately twofold when expressed during vegetative growth but has no effect on the half-lives of a number of vegetative mRNAs examined. The UME5 gene is dispensable for mitotic and meiotic development. Cells in which the entire UME5 gene has been deleted are viable, although the generation time is slightly longer and sporulation is less efficient. The UME5 transcript is constitutively expressed, and its stability is not autoregulated. The UME5 gene encodes a predicted 63-kDa protein with homology to the family of CDC28 serine/threonine-specific protein kinases. The kinase activity appears to be central to the function of the UME5 protein, since alteration of a highly conserved amino acid in the kinase domain results in a phenotype identical to that of a ume5 deletion. Genetic epistasis studies suggest that the UME2 and UME5 gene products act in the same pathway to regulate meiotic transcript stability. This pathway is independent of deadenylation and translation, two factors known to be important in regulating mRNA turnover. Significantly, the UME5-mediated destabilization of meiotic mRNAs occurs in glucose- but not in acetate-containing medium. Thus, the UME5 gene appears to participate in a glucose signal transduction pathway governing message stability.

UME6 is a key regulator of nitrogen repression and meiotic development.

This report describes the identification, cloning, and molecular analysis of UME6 (CAR80/CARGRI), a key transcriptional regulator of early meiotic gene expression. Loss of UME6 function results in the accumulation of fully derepressed levels (70- to 100-fold increase above basal level) of early meiotic transcripts during vegetative growth. In contrast, mutations in five previously identified UME loci (UME1 to UME5), result in low to moderate derepression (2- to 10-fold increase) of early meiotic genes. The behavior of insertion and deletion alleles indicates that UME6 is dispensable for mitotic division but is required for meiosis and spore germination. Despite the high level of meiotic gene expression during vegetative growth, the generation times of ume6 mutant haploid and diploid cells are only slightly reduced. However, both ascus formation and spore viability are affected more severely. The UME6 gene encodes a 91-kD protein that contains a C6 zinc cluster motif similar to the DNA-binding domain of GAL4. The integrity of this domain is required for UME6 function. It has been reported recently that a mutation in CAR80 fails to complement an insertion allele of UME6. CAR80 is a gene required for nitrogen repression of the arginine catabolic enzymes. Here, through sequence analysis, we demonstrate that UME6 and CAR80 are identical. Analyses of UME6 mRNA during both nitrogen starvation and meiotic development indicate that its transcription is constitutive, suggesting that regulation of UME6 activity occurs at a post-transcriptional level.

RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes.

We show that the extent of transcriptional regulation of many, apparently unrelated, genes in Saccharomyces cerevisiae is dependent on RPD1 (and RPD3 [M. Vidal and R. F. Gaber, Mol. Cell. Biol. 11:6317-6327, 1991]). Genes regulated by stimuli as diverse as external signals (PHO5), cell differentiation processes (SPO11 and SPO13), cell type (RME1, FUS1, HO, TY2, STE6, STE3, and BAR1), and genes whose regulatory signals remain unknown (TRK2) depend on RPD1 to achieve maximal states of transcriptional regulation. RPD1 enhances both positive and negative regulation of these genes: in rpd1 delta mutants, higher levels of expression are observed under repression conditions and lower levels are observed under activation conditions. We show that several independent genetic screens, designed to identify yeast transcriptional regulators, have detected the RPD1 locus (also known as SIN3, SD11, and UME4). The inferred RPD1 protein contains four regions predicted to take on helix-loop-helix-like secondary structures and three regions (acidic, glutamine rich, and proline rich) reminiscent of the activating domains of transcriptional activators.

Identification of negative regulatory genes that govern the expression of early meiotic genes in yeast.

Mutations in Saccharomyces cerevisiae have been identified that derepress early meiotic genes functioning in separable pathways required for normal meiotic development. The phenotypes of these ume (unscheduled meiotic gene expression) mutations suggest that their wild-type alleles encode negative regulators acting downstream of both the cell-type and nutritional controls of meiosis. These newly defined loci do not affect either general transcription or transcription of meiotic genes expressed later in meiosis and spore formation.

Anti-fluorescein antibody 3-13 VH gene rearrangement in idiotypically cross-reactive hybridomas.

The deduced amino acid sequence of anti-fluorescein (F1) antibody 3-13 VH region (residues 1-95) was 78% homologous to the alpha-1----3-dextran binding myeloma protein J558 VH region and was in the Wu-Kabat Subgroup II or Dildrop Group I classifications. The 3-13 VH region was rearranged to a D segment with only 8 of 30 bp in common with DFL16.1 germ line D gene and less homologous to all other previously identified D sequences. This sequence was joined to the third codon of JH4. The sequence encoding VH residues 5-91 was subcloned into pSP65 and used as a probe in Southern analyses to monitor 3-13 VH gene rearrangements in 12 other anti-F1 hybridomas differentially expressing (or not at all) the 3-13 idiotype. Three clones which inhibited the 3-13 idiotype-anti-idiotype interaction as effectively as 3-13 (3-12, 3-17 and 3-35), all had rearranged a gene which hybridized to the cloned 3-13 fragment, however, each was contained on a different size restriction fragment. Analyses of five other idiotypically related (but not identical) hybridomas indicated that four had rearranged a cross hybridizing VH gene while no such rearrangements were detected among four idiotypically negative cell lines. A restriction site assay indicated five clones examined had all rearranged a Vk gene to the Jk1 or Jk2 gene segment. The sequence of the antibody 3-13 VH gene and its use in hybridization studies represent the first molecular analysis of a recurrently expressed repertoire specific idiotype within an unrestricted immune response.

Mutations in ARS1 increase the rate of simple loss of plasmids in Saccharomyces cerevisiae.

Autonomously replicating sequence (ARS) elements are DNA sequences that promote extrachromosomal maintenance of plasmids in yeast. Mutations generated in vitro in the ARS1 region were examined for their effect on plasmid maintenance in a yeast centromeric plasmid. Our data show that mutations in the regions surrounding the ARS1 consensus sequence cause increases in the frequency of simple loss (1:0) events without affecting the rate of nondisjunction (2:0). Removal of the consensus sequence itself causes a drastic increase in the rate of simple loss. Sequences sensitive to mutagenesis were identified in each flanking region and differ with respect to their location and importance to ARS function. These results suggest that the role ARS1 plays in plasmid maintenance deals with the replication and/or localization of the plasmid in yeast.