A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation

In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG‐decoding tRNA^Gln_CUG. A mutant allele, sup70‐65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNA^Gln_CUG anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70‐65 tRNA^Gln_CUG is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG‐rich ORFs in the tRNA^Gln_CUG‐depleted sup70‐65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70‐65 pseudohyphal phenotype was partly complemented by overexpressing CAA‐decoding tRNA^Gln_UUG, an inefficient wobble‐decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5′ end of an ORF can reduce eukaryote translational expression, and that the mutant tRNA_CUG^Gln constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency.     

Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A.

The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.     

Trehalose biosynthetic pathway regulates filamentation response in Saccharomyces cerevisiae

Molecular Biology Reports - Diploid cells of Saccharomyces cerevisiae undergo either pseudohyphal differentiation or sporulation in response to depletion of carbon and nitrogen sources. Distinct...     

Pseudohyphal growth is induced in Saccharomyces cerevisiae by a combination of stress and cAMP signalling

In Saccharomyces cerevisiae pseudohyphae formation may be triggered by nitrogen deprivation and is stimulated by cAMP. It was observed that even in a medium with an adequate nitrogen supply, cAMP can induce pseudohyphal growth when S. cerevisiae uses ethanol as carbon source. This led us to investigate the effects of the carbon source and of a variety of stresses on yeast morphology. Pseudohyphae formation and invasive growth were observed in a rich medium (YP) with poor carbon sources such as lactate or ethanol. External cAMP was required for the morphogenetic transition in one genetic background, but was dispensable in strain 1278b which has been shown to have an overactive Ras2/cAMP pathway. Pseudohyphal growth and invasiveness also took place in YPD plates when the yeast was subjected to different stresses: a mild heat-stress (37 °C), an osmotic stress (1 m NACl), or addition of compounds which affect the lipid bilayer organization of the cell membrane (aliphatic alcohols at 2%) or alter the glucan structure of the cell wall (Congo red). We conclude that pseudohyphal growth is a physiological response not only to starvation but also to a stressful environment; it appears to require the coordinate action of a MAP kinase cascade and a cAMP-dependent pathway.     

The Saccharomyces cerevisiae mutation elm4-1 facilitates pseudohyphal differentiation and interacts with a deficiency in phosphoribosylpyrophosphate synthase activity to cause constitutive pseudohyphal growth.

Saccharomyces cerevisiae mutant E124 was selected in a visual screen based on elongated cell shape. Genetic analysis showed that E124 contains two separate mutations, pps1-1 and elm4-1, each causing a distinct phenotype inherited as a single-gene trait. In rich medium, pps1-1 by itself causes increased doubling time but does not affect cell shape, whereas elm4-1 results in a moderate cell elongation phenotype but does not affect growth rate. Reconstructed elm4-1 pps1-1 double mutants display a synthetic phenotype in rich medium including extreme cell elongation and delayed cell separation, both characteristics of pseudohyphal differentiation. The elm4-1 mutation was shown to act as a dominant factor that potentiates pseudohyphal differentiation in response to general nitrogen starvation in a genetic background in which pseudohyphal growth normally does not occur. Thus, elm4-1 allows recognition of, or response to, a pseudohyphal differentiation signal that results from nitrogen limitation. PPS1 was isolated and shown to be a previously undescribed gene coding for a protein similar in amino acid sequence to phosphoribosylpyrophosphate synthase, a rate-limiting enzyme in the biosynthesis of nucleotides, histidine, and tryptophan. Thus, the pps1-1 mutation may generate a nitrogen limitation signal, which when coupled with elm4-1 results in pseudohyphal growth even in rich medium.     

The yeast ammonium transport protein Mep2 and its positive regulator, the Npr1 kinase, play an important role in normal and pseudohyphal growth on various nitrogen media through retrieval of excreted ammonium

Three ammonium transport systems of the Mep/Amt/Rh superfamily contribute to ammonium uptake for use as a nitrogen source in Saccharomyces cerevisiae. A specific sensor role has further been proposed for Mep2 in the stimulation of pseudohyphal development during ammonium limitation. Optimal ammonium transport by the Mep proteins requires the Npr1 kinase, a potential target of the target-of-rapamycin signalling pathway. We show here that the growth impairment of cells lacking Npr1 on many nitrogen sources is shared by cells deprived of the three Mep proteins and is a consequence of deficient ammonium retrieval. Expression of a newly isolated Npr1-independent and hyperactive Mep2 in cells lacking Npr1 and/or the Mep proteins restores growth on low ammonium but also on other nitrogen sources. This hyperactive Mep2 variant efficiently counteracts ammonium excretion. Hence, ammonium uptake activity plays an important role in compensating for leakage of catabolic ammonium. Our data also reveal that the requirement of Npr1 for ammonium-induced pseudohyphal growth is an indirect consequence of its necessity for Mep2-mediated ammonium transport. Finally, we show that Mep2 participates, through ammonium leakage compensation, in pseudohyphal growth induced by amino acid starvation. This argues further in favour of tight coupling of Mep2 transport and sensor functions.     

A physiological study of Saksenaea vasiformis Saksena

Summary The physiological conditions governing growth and sporulation ofSaksenaea vasiformis Saksena, a fungus with outstanding morphological features quite peculiar for Mucorales, were determined. Earlier studies made byTiwari (1955) on a strain of the same species had shown that this fungus is incapable of sporulating on any synthetic medium normally employed for growing fungi.The fungus had been found to have a high tolerance for very low and high pH values. It showed maximum growth at two pH values, one near neutral point, at pH 6, and another at high alkaline pH value, i.e., pH 11. Reason for this behaviour of this fungus has already been discussed. The most suitable temperature for the growth of the fungus was found to be between 30–35° C.Nearly all the carbon, nitrogen and sulphur sources which generally favour growth of fungi were found to support vegetative growth of this fungus as well. However, sporulation in this fungus had peculiar nutritional needs. Only some of the carbon sources, viz., arabinose, rhamnose, sorbose, galactose, lactose and citric acid which supported poor growth, were found to support good or excellent sporulation. But it may be stated that not all carbon sources supporting poor growth could induce sporulation of the organism. Also none of the nitrogen or sulphur sources could induce the fungus to sporulate in presence of glucose as carbon source.     

Sporulation of Streptomyces antibioticus ETHZ 7451 in submerged culture.

Streptomyces antibioticus ETHZ 7451 formed spores in cultures grown in a liquid medium from either a spore or a mycelium inoculum. The spores formed were similar to those formed on surface-grown cultures, except for reduced heat resistance. Both types of spores were sensitive to lysozyme, which is unusual for Streptomyces spores. Glucose and other carbon sources, which promoted different growth rates, did not affect sporulation efficiency. Nitrogen sources, such as casamino acids, that allowed high growth rates suppressed the sporulation. A remarkable repression was also observed in media with some nitrogen sources that promoted noticeably lower growth rates. In permissive media, with nitrogen sources that permitted relatively high growth rates, sporulation was conditioned to the consumption of ammonium in the medium, but not to that of other nitrogen sources, such as asparagine. Phosphate did not show a repressive effect on sporulation in the assayed conditions.     

The Yeast Trimeric Guanine Nucleotide-Binding Protein α Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients

Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development. Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation. Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro. Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen. In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function. Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium. These observations are consistent with the following model. In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels. Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium. The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted. This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision.     

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.     

草坪草离蠕孢叶枯病菌生物学特性的研究

对草坪离蠕孢叶枯病病原菌进行分离鉴定,并对其生物学特性进行了研究。结果表明:该病害由禾草离蠕孢(Bipolaris sorokiniana)引起。该病原菌的菌丝生长及产孢的最适温度为30℃,孢子萌发最适温度为25℃,菌丝的致死温度为65℃,而孢子的致死温度则为55℃;该菌对酸碱度的适应能力较强,中性偏酸性的条件对菌丝的生长有利,而pH值为8.0时最易产孢;各碳源对菌丝的生长均有促进作用,但不同碳源对产孢量的影响很大,单糖和双糖利于产孢,多糖对产孢的影响不大。氮源对菌丝生长和产孢量非常重要,无机氮效果较好,硝态氮好于氨态氮,有机氮效果最差。花粉、叶面物质和草坪草汁液可促进孢子萌发。     

Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog.

Pseudohyphal differentiation, a filamentous growth form of the budding yeast Saccharomyces cerevisiae, is induced by nitrogen starvation. The mechanisms by which nitrogen limitation regulates this process are currently unknown. We have found that GPA2, one of the two heterotrimeric G protein alpha subunit homologs in yeast, regulates pseudohyphal differentiation. Deltagpa2/Deltagpa2 mutant strains have a defect in pseudohyphal growth. In contrast, a constitutively active allele of GPA2 stimulates filamentation, even on nitrogen-rich media. Moreover, a dominant negative GPA2 allele inhibits filamentation of wild-type strains. Several findings, including epistasis analysis and reporter gene studies, indicate that GPA2 does not regulate the MAP kinase cascade known to regulate filamentous growth. Previous studies have implicated GPA2 in the control of intracellular cAMP levels; we find that expression of the dominant RAS2(Gly19Val) mutant or exogenous cAMP suppresses the Deltagpa2 pseudohyphal defect. cAMP also stimulates filamentation in strains lacking the cAMP phosphodiesterase PDE2, even in the absence of nitrogen starvation. Our findings suggest that GPA2 is an element of the nitrogen sensing machinery that regulates pseudohyphal differentiation by modulating cAMP levels.