Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. Received: 24 May 1999 / Accepted: 19 October 1999     

Participation of <Emphasis Type="Italic">SRM5/CDC28</Emphasis>, <Emphasis Type="Italic">SRM8/NET1</Emphasis>, and <Emphasis Type="Italic">SRM12/HFI1</Emphasis> genes in checkpoint control in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G₂/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

Identification of 21 novel <Emphasis Type="Italic">S-RNase</Emphasis> alleles and determination of <Emphasis Type="Italic">S</Emphasis>-genotypes in 66 loquat (<Emphasis Type="Italic">Eriobotrya</Emphasis>) accessions

As observed in other self-incompatible species in the Pyrinae subtribe, loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus, which encodes a polymorphic stylar ribonuclease (S-RNase). This allows the female reproductive organ (style) to recognize and reject the pollen from individuals with the same S-alleles, but allows the pollen from individuals with different S-alleles to effect fertilization. The S-genotype is therefore an important consideration in breeding strategies and orchard management. In an attempt to optimize the selection of parental lines in loquat production, the S-RNase alleles of 35 loquat cultivars and their 26 progeny, as well as five wild loquat species, were identified and characterized in this study. The best pollinizer cultivar combinations were also explored. A total of 28 S-alleles were detected, 21 of which constituted novel S-RNase alleles. The S-haplotypes S₂ and S₆ were the most frequent, followed by S ₂₉ , S ₃₁ , S ₅ , S ₂₄ , S ₂₈ , S ₃₃ , S ₃₄ , S ₃₂ , and S ₁₅ , while the rare alleles S ₁ , S ₉ , S ₁₄ , S ₁₆ , S ₁₇ , S ₁₈ , S ₁₉ , S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , S ₂₇ , and S ₃₅ were only observed in one of the accessions tested. Moreover, the S-genotypes of five wild loquat species (E. prinoides, E. bengalensis, E. prinoides var. dadunensis, E. deflexa, and E. japonica) are reported here for the first time. The results will not only facilitate the selection of suitable pollinators for optimal orchard management, but could also encourage the crossbreeding of wild loquat species to enhance the genetic diversity of loquat cultivars.     

Chromosome number variability in central european members of the<Emphasis Type="Italic">Festuca ovina</Emphasis> and<Emphasis Type="Italic">F. pallens</Emphasis> groups (sect.<Emphasis Type="Italic">Festuca</Emphasis>)

Chromosome numbers for 98 plants ofF. pallens, 19 ofF. psammophila, F. belensis andF. vaginata, and 44 ofF. ovina (originating from Austria, the Czech Republic, Germany, Slovakia and Latvia) are given. In addition to theF. ovina andF. pallens groups, chromosome counts for the following taxa are also reported:F. alpestris (2n=14) reported for the first time in this work,F. amethystina subsp.amethystina (2n=28),F. brevipila (2n=42),F. cinerea (2n=28),F. rupicola subsp.rupicola (2n=42) andF. versicolor subsp.versicolor (2n=14).InF. pallens, two ploidy levels (2n=2x=14+0-1B, 2n=4x=28+0-1B) as well as two natural triploid plants (2n=21+0-1B), were found. In addition to the fourF. pallens types that have been distinguished in Austria, one new tetraploid type (F. pallens “scabrifolia”) from the Czech Republic and Germany is reported and its taxonomy is discussed. The distributions of the Oberösterreich-Niederösterreich and Pannonisches-HügellandF. pallens types outside of Austria are documented.Only the diploid chromosome number (2n=14) was found inF. psammophila andF. vaginata. Chromosome numbers forF. psammophila subsp.muellerstollii andF. belensis (both 2n=14) were determined here for the first time. Two ploidy levels, 2n=14+0-5B corresponding toF. ovina subsp.ovina and 2n=28 corresponding toF. ovina subsp.guestphalica andF. cf.duernsteinensis were confirmed inF. ovina. Differences in chromosome structure (simple and multiple secondary constrictions) betweenF. pallens as opposed toF. psammophila andF. vaginata are discussed. A complete survey of published chromosome counts for Central European species from theF. ovina andF. pallens groups is included.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> analysis reveal a novel gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> trehalose metabolism

Background

The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p.

Results

In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain.

Conclusions

These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

     

A putative <Emphasis Type="Italic">NEM1</Emphasis> homologue regulates lipid droplet biogenesis <Emphasis Type="Italic">via PAH1</Emphasis> in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Nuclear envelope morphology protein 1 (NEM1) along with a phosphatidate phosphatase (PAH1) regulates lipid homeostasis and membrane biogenesis in yeast and mammals. We investigated four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C and TtNEM1D) in the Tetrahymena thermophila genome. Disruption of TtNEM1B, TtNEM1C or TtNEM1D did not compromise normal cell growth. In contrast, we were unable to generate knockout strain of TtNEM1A under the same conditions, indicating that TtNEM1A is essential for Tetrahymena growth. Interestingly, loss of TtNEM1B but not TtNEM1C or TtNEM1D caused a reduction in lipid droplet number. Similar to yeast and mammals, TtNem1B of Tetrahymena exerts its function via Pah1, since we found that PAH1 overexpression rescued loss of Nem1 function. However, unlike NEM1 in other organisms, TtNEM1B does not regulate ER/nuclear morphology. Similarly, neither TtNEM1C nor TtNEM1D is required to maintain normal ER morphology. While Tetrahymena PAH1 was shown to functionally replace yeast PAH1 earlier, we observed that Tetrahymena NEM1 homologues did not functionally replace yeast NEM1. Overall, our results suggest the presence of a conserved cascade for regulation of lipid homeostasis and membrane biogenesis in Tetrahymena. Our results also suggest a Nem1-independent function of Pah1 in the regulation of ER morphology in Tetrahymena.     

Clavicipitaceous entomopathogens: new species in <Emphasis Type="Italic">Metarhizium</Emphasis> and a new genus <Emphasis Type="Italic">Nigelia</Emphasis>

In several surveys in the tropical forests in Thailand, specimens that looked morphologically similar to Metarhizium martiale and Cordyceps variegata, as well as other Metarhizium species were collected and cultured in vitro. A combined phylogeny of several genes including the small (18S) and large (28S) subunits of the ribosomal DNA, elongation factor 1-α (TEF), RNA polymerase II subunits 1 and 2 (RPB1, RPB2) genes has shown these to be new taxa in the Clavicipitaceae. Nigelia is described as a new genus closely related to Metarhizium, to the scale insect pathogens Aschersonia (Hypocrella), Samuelsia and Moelleriella, and to plant pathogens in Claviceps and Balansia, and other relatives. Nigelia comprises M. martiale and a new species Nigelia aurantiaca, which has been found infecting lepidopteran larvae and which produces pseudoimmersed, obliquely arranged, obpyriform perithecia with curved or bent ostioles and with whole (non-separating) cylindric ascospores. Metarhizium chaiyaphumense, M. kalasinense, M. prachinense, M. samlanense, and M. takense are described as new species of Metarhizium. Metarhizium martiale is transferred to Nigelia, and Paecilomyces reniformis is transferred to Metarhizium.     

Influence of <Emphasis Type="Italic">UPF</Emphasis> genes on severity of <Emphasis Type="Italic">SUP45</Emphasis> mutations

Eukaryotic cells possess a special mechanism for the degradation of mRNAs containing premature termination codons (PTCs), referred to as NMD (nonsense-mediated mRNA decay). The strength of this pathway depends on the recognition of the PTCs by translational machinery and the interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins in Sachromyces cerevisiae yeast. Previously, we have shown that the decrease of eRF1 protein amounts in sup45 nonsense mutants leads to the impairment of NMD. Here we show that the deletion of UPF1 or UPF2 genes leads to an increase in the viability of sup45 mutants, while the effect of UPF3 gene deletion is allele-specific. Two-hybrid data have shown that amino acid residues 1–555 of Upf1 protein interact with eRF1. Any UPF gene deletion leads to allosupression of the adel1-14 mutation without a change in eRF1 content. The Upf1 depletion does not influence the synthetic lethality of sup45 mutations and the [PSI ⁺] prion. It is possible that the absence of Upf1 (or its activator Upf2) leads to a more effective formation of the translation termination complex and consequently to the increased viability of the cells containing mutant termination factors.     

The <Emphasis Type="Italic">yajC</Emphasis> gene from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and its role in ethanol tolerance

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-d-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

<Emphasis Type="Italic">Cantharellus</Emphasis> sect. <Emphasis Type="Italic">Amethystini</Emphasis> in Asia

In this contribution on the genus Cantharellus in Asia, C. subvaginatus is described from the Republic of Korea as a close relative to the Chinese C. vaginatus, which is here reported for the first time from India. Both species are here placed in Cantharellus subg. Cantharellus sect. Amethystini, together with the Indian C. pseudoformosus (syn.: C. umbonatus) and the Malayan C. subamethysteus. As such, Asia has suddenly become the continent with the highest diversity for Amethystini. Species delimitation in sect. Amethystini is molecularly supported by a combined phylogenetic analysis of rDNA sequences obtained for LSU and ITS and additionally suggests the existence of a still undescribed species in North America. Character variability is discussed for all known members of Amethystini, including atypical specimens of the North American C. lewisii that are morphologically more reminiscent of the South Korean C. subvaginatus.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Evaluation of the Diversity of Probiotic <Emphasis Type="Italic">Bacillus</Emphasis>, <Emphasis Type="Italic">Clostridium</Emphasis>, and <Emphasis Type="Italic">Bifidobacterium</Emphasis> Using the Illumina-Based Sequencing Method

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.     

The Role of <Emphasis Type="Italic">sho1</Emphasis> in Polarized Growth of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Aspergillus fumigatus is an opportunistic pathogen that may cause severe invasive disease in immunocompromised patients. The filamentous fungi undergo polarized growth, searching for nutrients in the environment and causing invasive growth in tissue. Sho1 is a sensor of the high osmolarity glycerol pathway, and the sho1 mutant showed a decrease in growth rate. We found that sho1 is involved in the polarized growth of A. fumigatus. The sho1 mutation resulted in extended isotropic growth of germinating conidia followed by multiple germ tubes and wide hyphae with short intercalary cells by calcofluor white staining. The mechanism by which sho1 gene affected polarized growth is investigated. A reduced number of apical vesicles with greater dispersion were observed by transmission electron microscopy in the Spitzenkörper body of the sho1 mutant. Actin patches were distributed randomly at low density at early stages of mutant strain fungal development and reaggregated to the hyphal tip of later stages when long filamentous fungi formed. Actin patches located at the tip of polarized wild-type cells. RNA levels of polarized growth-related genes Rho GTPases were detected by real-time PCR. The sho1 gene did not affect the RNA expression when strains were cultured at 37°C for 6 h. At 17 h, the RNA expression of rho1, rho3 and CDC42 in the sho1 mutant were 0.18-, 0.18- and 0.33-fold of that in the wild type. The sho1 gene affected the polarized growth through affecting the expression of Rho GTPases, the distribution of actin cytoskeleton, vesicle quantity and distribution.