Structural modification of spindle pole bodies during meiosis II is essential for the normal formation of ascospores in Schizosaccharomyces pombe: Ultrastructural analysis of spo mutants

In order to characterize the morphological steps defined by sporulation (spo) genes during the formation of ascospores in the fission yeast Schizosaccharomyces pombe, we performed an electron microscopic study of the ultrastructure of the spindle pole body (SPB) and of the development of the forespore membrane during the second meiotic division (meiosis II) in sporulation-deficient (spo) mutants (spo4, spo5, spo14 and spo18). No difference was found in terms of the function and the structure of the SPB during the first meiotic division (meiosis I) between the four mutants and wild-type cells. However, during meiosis II, the spo4 and spo18 mutants underwent nuclear division but in neither case were the SPBs modified nor were forespore membranes formed. The SPBs of the spo18 mutant diminished in size after meiosis II and eventually disappeared after 18 h in sporulation medium. By contrast, the SPBs of the spo4 mutant remained unchanged even after an 18-h incubation. The outer plaques of SPBs of spo5 and spo14 mutants were sufficiently modified to allow them to initiate development of the forespore membrane, but the membrane had an abnormally expanded lumen and did not enclose the nuclei during meiosis II. The spo5 mutant produced anucleate spore-like bodies while the spo14 mutant formed unorganized structures with irregular peripheries which, presumably, contained spore-wall precursors, instead of anucleate spore-like bodies. We conclude that the modification of the SPB is essential for the formation of ascospores and at least two genes (spo5 and spo14) participate in the development of the forespore membrane. The defective phenotypes define discrete steps in the development of ascospores, which proceeds via steps defined by the mutant spo4, spo18, spo14 and spo5 genes respectively. Our observations provide further substantial evidence that the SPB plays a pivotal role in the normal development of ascospores in yeasts.     

Sterility (ste) genes of Schizosaccharomyces pombe

In previous experiments of Girgsdies (1982), eight sterile (ste) mutants of Schizosaccharomyces pombe did not sporulate when fused with h⁺ or h^? protoplasts. We succeeded in achieving sporulation with these mutants. Two hitherto unknown ste genes, ste7 and ste8, were found.     

Nuclear pore complex antigens delineate nuclear envelope dynamics in vegetative and conjugating Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the nucleus undergoes dramatic shape changes during mitosis and mating. We have studied nuclear envelope dynamics during the processes of mitosis and conjugation using nuclear pore complexes as a marker for the nuclear envelope in wild-type cells and several cell-division-cycle (cdc) mutants. Three monoclonal antibodies are described that recognize nuclear pore complex-related antigens in S. cerevisiae. One of these antibodies, RL1, has been extensively characterized by Gerace and colleagues and recognizes nuclear pore complexes in mammalian and amphibian cells. By indirect immunofluorescence of yeast cells, all three antibodies yield a discontinuous nuclear rim stain. All three react with multiple nuclear-enriched proteins in immunoblots, including the nucleoporin protein encoded by the NSP1 gene. When the antibodies were used in immunofluorescence experiments on mating cells, the nuclear pore complex staining pattern proved to be a sensitive indicator of nuclear fusion. Nuclei with closely apposed spindle pole bodies and unfused nuclear envelopes could be readily distinguished. Marked shape changes were observed in nuclei during fusion and segregation of the diploid nucleus into the zygotic bud. In cdc14 and cdc15 mutants that arrest late in mitosis, the elongated nuclear envelope extension that stretches between daughter nuclei during telophase was preserved. In cytokinesis-defective mutants (cdc3, cdc10, cdc11 and cdc12), the elongated nuclear envelope was usually resolved into two daughter nuclei in the absence of cytokinesis. These results indicate that nuclear envelope division is mechanistically distinguishable from chromosome segregation, nucleolar segregation and cytokinesis.     

Boric acid destabilizes the hyphal cytoskeleton and inhibits invasive growth of Candida albicans

Exposure of Candida albicans to sub‐lethal concentrations of boric acid (BA) restricts the dimorphic fungus to its yeast morphology and prevents the formation of invasive hyphae on solid substrates. Exposure to BA causes a rapid and reversible disappearance of polarisome and Spitzenkörper in growing hyphae. In BA‐treated hyphae of C. albicans, actin quickly reorganizes from cytoplasmic cables to cortical patches and cell wall growth switches from an apical to an isotropic pattern. As a result of the cytoskeletal changes, the hyphal tips broaden and directional growth of hyphae ceases in the presence of BA. An analysis of homozygous deletion strains showed that mutants with constitutive or enhanced hyphal growth (tup1, nrg1, ssn6, rbf1) are BA‐sensitive, demonstrating that cellular morphology is a major determinant of BA tolerance. The screening of deletion mutants also showed that deficiencies of the main activator of hyphal gene expression, Efg1, and the Rim101‐signalling cascade, leading to Efg1 activation, cause BA resistance. Taken together, the data presented show that the selective inhibitory effect on BA on C. albicans hyphae is rooted in a disruption of apical cytoskeletal elements of growing hyphae. Copyright © 2015 John Wiley & Sons, Ltd.     

β‐1,6‐glucan synthesis‐associated genes are required for proper spore wall formation in Saccharomyces cerevisiae

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β ‐1,6‐glucan synthesis, including kre1? , kre6? , kre9? and big1? , were sporulated to analyse the effect of β ‐1,6‐glucan defects on the spore wall. Except for kre6? , these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild‐type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1? spores. The majority of the big1?chs3? mutants failed to form visible spores at a higher temperature. Given that the big1? mutation caused a failure to attach a GPI‐anchored reporter, Cwp2‐GFP, to the spore wall, β ‐1,6‐glucan is involved in tethering of GPI‐anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β ‐1,6‐glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd.     

Essential role of Ubr11, but not Ubr1, as an N‐end rule ubiquitin ligase in Schizosaccharomyces pombe

The N‐end rule pathway degrades proteins bearing a destabilization‐inducing amino acid at the N‐terminus. In this proteolytic system, Ubr ubiquitin ligases recognize and ubiquitylate substrates intended for degradation. Schizosaccharomyces pombe has two similar Ubr proteins, Ubr1 and Ubr11. Both proteins have unique roles in various cellular processes, although the ubr1? strain shows more severe defects. However, their involvement in the N‐end rule pathway is unclear, and even the N‐end rule pathway‐dependent proteolytic activity has not been demonstrated in Sz. pombe. Here, we show that: (a) Sz. pombe has the N‐end rule pathway in which only Ubr11, but not Ubr1, is responsible; and (b) the C‐terminal fragment of the meiotic cohesin Rec8 (denoted as Rec8c) generated by separase‐mediated cleavage is an endogenous substrate of the N‐end rule pathway. Forced overexpression of stable Rec8c was deleterious in mitosis and caused a loss of the mini‐chromosome. In unperturbed mitosis without overexpression, the rate of mini‐chromosome loss was five‐fold higher in the ubr11? strain. Since Rec8 is normally produced in meiosis, we examined whether meiosis and sporulation were affected in the ubr11? strain. In unperturbed meiosis, chromosome segregation occurred almost normally and viable spores were produced in the ubr11? cells, irrespective of the presence of undegraded endogenous Rec8c peptides. Copyright © 2012 John Wiley & Sons, Ltd.     

Bicarbonate-mediated social communication stimulates meiosis and sporulation of Saccharomyces cerevisiae

Meiosis and sporulation in the yeast Saccharomyces cerevisiae requires social communication, mediated by an extracellular factor which is secreted from cells during sporulation and accumulates in a cell density-dependent manner. We show here genetic and biochemical analyses supporting our conclusion that the extracellular factor is bicarbonate acting as an alkali to elevate extracellular pH. Sporulation defects of mdh1 (mitochondrial malate dehydrogenase) mutants and of wild-type cells at low density were rescued extracellularly by addition of bicarbonate or other alkaline solutions to raise medium pH. Addition of bicarbonate (or alkalization of medium) raised steady-state levels of mRNA in respiration-deficient mdh1 mutants and inhibited proliferation of wild-type cells at low density. These results indicate that the two conditions (respiration competency and high cell density), required for meiosis and sporulation, are essential for extracellular accumulation of bicarbonate and resulting alkalization of medium. © 1998 John Wiley & Sons, Ltd.     

Phytosterol content in seven oat cultivars grown at three locations in Sweden

The total and individual sterol content in 21 oat samples (seven cultivars grown at three different locations in Sweden) were analysed by gas chromatography after acid hydrolysis. The total sterol content in these oat cultivars varied between 350–491 µg g⁻¹ of dry weight of kernel. The most abundant phytosterol was β‐sitosterol (237–321 µg g⁻¹) followed by campesterol (32–46 µg g⁻¹), Δ⁵‐avenasterol (15–47 µg g⁻¹) and stigmasterol (11–21 µg g⁻¹). There was a statistically significant difference in total sterol content between cultivars (p < 0.05) but no effect was found for cultivation location. Furthermore when contents of Δ⁵‐avenasterol in hexane‐extracted oat oil and acid‐hydrolysed oat samples were compared, it was noticed that the content of Δ⁵‐avenasterol was lowered due to acid hydrolysis. © 1999 Society of Chemical Industry     

Characterization of the Saccharomyces cerevisiae cdc42-1ts Allele and New Temperature-Conditional-Lethal cdc42 Alleles

Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1^ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1^ts allele and identified multiple mutations in the coding region and 5′ promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42^W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1^ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23°C. These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe, the cdc42^W97R mutant displayed a cold-sensitive, loss-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42^T58A and cdc42^S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S. pombe; these mutants did not display a conditional phenotype when expressed in S. cerevisiae. These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts. © 1997 John Wiley & Sons, Ltd.     

In vitro protein digestibility and mineral availability of green beans (Phaseolus vulgarisL) as influenced by variety and pod size

Three varieties of green beans (Cleo, Strike and Sentry) were harvested and sorted into four fractions according to pod size (diameter <7 mm; 7–8·5 mm; 8·6–10 mm and >10 mm). Ash content and dietary fibre increased significantly as pod size increased mainly in Cleo and Strike beans. Strike showed the highest fibre content (378·0 g kg⁻¹) but the lowest carbohydrate (364·6 g kg⁻¹) and ash (68·4 g kg⁻¹) values. Mean values for Fe and Mg content were higher in Cleo beans (70·9 and 27·1 mg kg⁻¹, respectively), Zn, Cu and Mg were higher in Strike beans (48·7 mg kg⁻¹, 22·4 mg kg⁻¹ and 3·15 g kg⁻¹, respectively) while Na and Ca values were maximum in Sentry (459·1 mg kg⁻¹ and 7·11 g kg⁻¹, respectively). Trypsin inhibitor was negatively related to in vitro protein digestibility but no relationship was found between this last parameter and phytic acid content. This antinutrient, together with dietary fibre, and a negative influence on in vitro mineral dialysability of green beans. © 1998 SCI     

Control of speargrass (Imperata cylindrica (L) Raeuschel) with glyphosate and fluazifop‐butyl for soybean (Glycine max (L) Merr) production in savanna zone of Nigeria

A field trial was conducted in 1996 and 1997 to control speargrass (Imperata cylindrica (L) Raeuschel) for soybean production. The treatments comprised four levels of glyphosate, 1.08, 1.44, 1.80 and 2.16 kg ha⁻¹, applied pre‐tillage and followed by one hoe‐weeding (HW) at 6 weeks after planting (WAP); fluazifop‐butyl, 0.125, 0.25 and 0.375 kg ha⁻¹, at 3WAP; 1HW at 3WAP; 2HW at 3 and 6WAP; and an unweeded control. Glyphosate and fluazifop‐butyl controlled 57–85% and 51–83% respectively of I cylindrica compared with 64–67% by traditional hoe‐weeding. The highest grain yield (1.88 t ha⁻¹) was obtained from plots treated with glyphosate (1.44 kg ha⁻¹) + 1HW. The highest profit, however, was obtained with fluazifop‐butyl. It was unprofitable to apply glyphosate at rates higher than 1.44 kg ha⁻¹ to control I cylindrica at 30 cm foliage. © 2000 Society of Chemical Industry     

Purification and some properties of a novel raw starch‐digesting amylase from Aspergillus carbonarius

A medium was developed to obtain the maximum yield of raw starch‐digesting amylase from Aspergillus carbonarius (Bainier) Thom IMI 366159 in submerged culture with raw starch as the sole carbon source. The amylase was purified to apparent homogeneity by sucrose concentration and ion exchange chromatography on S‐ and Q‐Sepharose (fast flow) columns. SDS‐PAGE revealed two migrating protein bands corresponding to relative molecular masses of 31.6 and 32 KDa. The enzyme was optimally active at pH 6.0–7.0 and 40 °C, was uninfluenced across a relatively broad pH range of 3.0–9.0 and retained over 85% activity between 30 and 80 °C after 20 min incubation. The enzyme was strongly activated by Co²⁺ and only slightly by Fe²⁺, while Ca²⁺, Hg²⁺, EDTA and N‐bromosuccinamide elicited significant repression of the enzyme activity. The enzyme hydrolysed amylopectin (K_m 0.194 mg ml ⁻¹), glycogen (K_m 0.215 mg ml ⁻¹), pullulan (K_m 0.238 mg ml ⁻¹), amylose (K_m 0.256 mg ml ⁻¹) and raw potato starch (K_m 0.260 mg ml ⁻¹), forming predominantly maltose and relatively smaller amounts of glucose. © 2000 Society of Chemical Industry     

Nutrient contents,rumen protein degradability and antinutritional factors in some colour- and white-flowering cultivars of Vicia faba beans

Six colour-flowering (Scirocco, Alfred, Carola, Condor, Tina and Herz Freya) and six white-flowering (Caspar, Albatros, Gloria, Tyrol, Vasco and Cresta) cultivars of Vicia faba were studied. The crude protein contents of colour- and white-flowering cultivars were 267±13·6 and 283±18·8 g kg⁻¹, respectively, which did not differ significantly at P<0·05. The levels of lipids, crude fibre, starch and ash varied from 14 to 22 g kg⁻¹, 88 to 143 g kg⁻¹, 407 to 485 g kg⁻¹ and 32 to 42 g kg⁻¹, respectively. The calculated organic matter digestibility (OMD) and metabolisable energy (ME) of the white-flowering cultivars were significantly higher (P<0·001) than those of the colour-flowering cultivars (OMD: 889·1±26·6 g kg⁻¹ vs 797·5±17·1 g kg⁻¹; ME: 13·97±0·49 vs 12·30±0·34 MJ kg⁻¹). In all cultivars, sulphur amino acids were lower than adequate concentration when compared with recommended amino acid pattern of FAO/WHO/UNO reference protein for a 2–5-year-old child. The in vitro rumen nitrogen degradability of colour-flowering cultivars was significantly lower (P<0·01) compared to that of white-flowering cultivars (71·4±9·3% vs 88·0±11·1%). Amongst colour-flowering varieties, the contents of total phenols (TP), tannins (T) and condensed tannins (CT) were highest in Alfred (28·3, 21·0 and 35·4 g kg⁻¹, respectively). The contents of TP and T were similar (about 15 and 10 g kg⁻¹, respectively) in Carola, Tina and Herz Freya, and the CT were in the order: Condor>Herz Freya>Carola. The CT were not detected in white-flowering varieties, T were virtually absent and TP were extremely low (4·0–4·9 g kg⁻¹). The activities of other antinutritional factors (white- and colour-flowering cultivars, respectively: trypsin inhibitor activity 3·05±0·34 and 1·85±0·09 mg trypsin inhibited g⁻¹; lectin 27·2±9·4 and 27·1±5·1 mg ml⁻¹ assay medium producing haemagglutination; phytate 15·0±2·7 and 16·6±2·3 g kg⁻¹) were very low. A strong negative correlation (r=-0·92, P<0·001) between tannins and in vitro rumen protein degradability was observed which suggested that tannins have adverse effect on protein degradability. Similarly negative correlations between tannin levels and metabolisable energy (r=-0·89; P<0·001) and organic matter digestibility (r=-0·89; P<0·001) were observed. The correlation coefficient between trypsin inhibitor activity and tannins was negative and highly significant (r=-0·88, P<0·001), whereas between tannins and saponins it was significantly positive (r=0·96, P<0·001). ©1997 SCI     

Production of fungal biomass from cormel process waste‐water of cocoyam (Xanthosoma sagittifolium (L) Schott) using Aspergillus oryzae obtained from cormel flour

Aspergillus oryzae obtained from spoilt cormel flour was subjected to mutation treatments using X‐rays, solar radiation and bleach. Exposure of A oryzae spores to X‐radiation of 50 kV at 20 mA s and other treatments induced both mutation and lethality in the organism. Following selection and screening of viable colonies on a medium containing cormel process waste‐water as the only carbon source, two strains, A oryzae No 15 and A oryzae No 8, which significantly (P ≤ 0.05) produced more biomass at a higher growth rate than the wild parent, were chosen for single‐cell protein production. Nutrient content of the single‐cell protein produced by the mutants was comparable to that of the wild type. Addition of 3.0 g each of (NH₄)₂SO₄, NH₄NO₃, NH₄Cl, and urea in 1 l of Xanthosoma process waste‐water increased the growth rate of mutants, with the highest increase observed with urea. Medium amended with urea also produced fungal biomass with the highest protein level of 7.97 g l^?1 for A oryzae No 8 strain compared with a protein yield of 3.97 g l^?1 obtained in the control. Total biomass produced after 54 h was 22.47 g l^?1 for A oryzae No 15 strain when urea was added, whereas only 15.20 g l^?1 was produced when no nitrogen source was added. The optimal temperature for single‐cell protein production was found to be 35 °C and the optimal pH was 3.50. A speed of 100 rpm gave the largest quantity of fungal biomass for the mutants tested. Copyright © 2003 Society of Chemical Industry     

Proximate Composition and Seed Lipid Components of Sorghum Cultivars from Argentina

Seeds of 11 sorghum cultivars ( Sorghum bicolor ) from Argentina were analysed for proximate composition, fatty acids and sterols. Oil, protein, carbohydrate and ash contents varied between 41 and 66 g kg⁻¹, 111 and 156 g kg⁻¹, 670 and 730 g kg⁻¹ and 13·8 and 20·6 g kg⁻¹ of dry matter, respectively. Fatty acid profiles revealed that the major acids were palmitic (15·1–24·8%), oleic (29·9–41·8%) and linoleic (35·9–51·3%). Unsaponifiable matter was examined for sterols. Sitosterol was the prominent component in all cultivars (43·8–57·9%), followed by campesterol (18·7–29·1%) and stigmasterol (12·4–20·5%).     

GSG1, a yeast gene required for sporulation

We have identified a gene, GSG1 (g eneral s porulation g ene 1), required for sporulation in Saccharomyces cerevisiae. Diploids homozygous for a disruption of GSG1 fail to sporulate. The gene has an open reading frame of 2094 bp, encoding a polypeptide with an expected size of 77 kDa. GSG1 is expressed mitotically in both a and α haploids, and both mitotically and meiotically in diploids. The message level of GSG1 increases approximately two-fold after 4–6 h of sporulation. gsg1 mutants enter pre-meiotic DNA synthesis later than wild-type diploids. Mutant diploids are not rescued by spo13. These results suggest that GSG1 has a role late in meiosis following DNA replication. The sequence reported here has the GenBank Accession Number U26674.     

Rag−mutations involved in glucose metabolism in yeast: Isolation and genetic characterization

Some natural isolates and many laboratory strains of the yeast Kluyveromyces lactis cannot grow on glucose when respiration is inhibited by antimycin A. The ability or inability to grow on glucose in the absence of mitochondrial respiration has been called Rag⁺ or Rag^? phenotype (resistance to antimycin on glucose, respectively). Rag^? strains, unable to grow on glucose in the presence of the respiratory drug, behave as if they were defective in fermentation. The Rag phenotype was first found to be determined by variant alleles of either of the two nuclear genes, RAG1 and RAG2, which code for a low-affinity glucose transport protein and for phosphoglucose isomerase, respectively. These findings suggested that the Rag^? phenotype can be used to obtain mutations of genes involved in glucose metabolism in K. lactis. We thus looked for other Rag^? mutants. Seventy-four mutants were isolated and genetically characterized. All of the mutations were nuclear recessive alleles, defining 11 new complementation groups, which we designate rag3 through rag13.     

Does u.v. light affect the total phenolic compound,anthocyanin, antioxidant capacity,and sensory profiles in wines?

In the study, by applying ultraviolet (u.v.) lights (254 nm) for 45 min to red wines from fermented Cabernet Franc, Cabernet Sauvignon, Merlot, and Petit Verdot (Vitis vinifera L.) grapes, we examined the total phenolic compound, total anthocyanin, and antioxidant capacity by four different methods, trans-resveratrol, (+)-catechin, gallic acid content, and the change in tasting profiles of the wines after application. According to the results of the research, the u.v.-C application has caused an increase in all parameters examined. If we talk about averages without distinguishing between varieties, after the application the total phenolic compound content of 3206 increased to 3356 mg gallic acid equivalent (GAE) per L and after u.v.-C application, the total anthocyanin content increased from 411 to 780 mg L⁻¹, the ABTS [2,2′-azinobis(3-ethyl-benzothiazoline-6-sulphonic acid)] value increased from 30 to 33 μmol trolox mL⁻¹, the DPPH ((2,2-diphenyl-1-picrylhydrazyl) level increased from 13 to 16 μmol trolox mL⁻¹, the FRAP (ferric reducing antioxidant power) level increased from 15 to 16 μmol trolox mL⁻¹, and the CUPRAC (cupric reducing antioxidant capacity) level increased from 40 to 45 μmol trolox mL⁻¹. The u.v. application affected trans-resveratrol the most among the phenolic compounds and an average increase of 12.99% was obtained. The rate of increase after u.v. application was 3.33% in (+)-catechin and 5.57% in gallic acid. The highest correlation between antioxidant activity measurement methods was measured to be 0.998 between FRAP and DPPH. In addition, u.v.-C application has had a positive effect on taste-based sensory profiles. In recent years, the search for a diet with a high antioxidant content has become even more important.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

Functional interrelationships between carbohydrate and lipid storage,and mitochondrial activity during sporulation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae under conditions of nutrient stress, meiosis precedes the formation of spores. Although the molecular mechanisms that regulate meiosis, such as meiotic recombination and nuclear divisions, have been extensively studied, the metabolic factors that determine the efficiency of sporulation are less understood. Here, we have directly assessed the relationship between metabolic stores and sporulation in S. cerevisiae by genetically disrupting the synthetic pathways for the carbohydrate stores, glycogen (gsy1/2Δ cells), trehalose (tps1Δ cells), or both (gsy1/2Δ and tps1Δ cells). We show that storage carbohydrate-deficient strains are highly inefficient in sporulation. Although glycogen and trehalose stores can partially compensate for each other, they have differential effects on sporulation rate and spore number. Interestingly, deletion of the G₁ cyclin, CLN3, which resulted in an increase in cell size, mitochondria and lipid stores, partially rescued meiosis progression and spore ascus formation but not spore number in storage carbohydrate-deficient strains. Sporulation efficiency in the carbohydrate-deficient strain exhibited a greater dependency on mitochondrial activity and lipid stores than wild-type yeast. Taken together, our results provide new insights into the complex crosstalk between metabolic factors that support gametogenesis.