比较分析草菇与其他真菌直系同源基因的关系

担子菌类的食用菌种类多、价值高、产量大,然而其产业的升级发展需要对食用菌生长发育相关生物学问题进行深入解析。目前多种食用菌完成了全基因组测序,然而作为非模式种其与模式丝状真菌间的直系同源基因目前尚缺乏全基因组水平的系统研究,在一定程度上限制了其分子生物学研究的深入。本研究以草菇为参照物种,将其与几种食用菌和模式丝状真菌进行两两直系同源基因分析,并对多物种间不同类型的直系同源基因进行功能富集。结果显示：一对一直系同源基因较多富集于基因复制、转录、翻译、修饰、加工等保守的基本功能类别;非一对一直系同源基因多属于基因家族,且包含了65%的转录因子,功能上富集在碳水化合物、脂质、氨基酸、次生代谢物及外源物质的代谢通路。无直系同源基因则较多富集在与基因重组、修复、信号转导相关的功能类别、特导性转录因子以及未知的预测基因。结果为食用菌分子生物学的深入研究提供有价值的参考。     

RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune

Disruption of genes by homologous recombination occurs at a low frequency in the basidiomycete Schizophyllum commune. For instance, the SC3 and SC15 genes were inactivated at frequencies of 1 and 5%, respectively. As an alternative to disruption, we used gene silencing through the introduction of a hairpin construct. The SC15 gene, which encodes an abundantly secreted structural protein, was silenced at a frequency of 80% in monokaryons of S. commune after introduction of a hairpin construct of the gene. Silencing also occurred in dikaryons in which one of the partners was not a silenced strain. The silencing mechanism resembles RNAi in other filamentous fungi and is a powerful tool for the functional analysis of genes expressed in monokaryons or dikaryons.     

Differentiated Gene Expression in Cells within Yeast Colonies

Yeast cells growing on solid media organize themselves into multicellular structures, colonies, exhibiting patterns specific for particular yeast strains. With the aim of identifying genes involved in regulations of the colony formation, we applied a new approach enabling the extensive screening of Saccharomyces cerevisiae genes, the expression of which is changed during colony development. We used the library of S. cerevisiae DNA fragments inserted in front of the lacZ gene lacking its own promoter. Colonies of transformants with a blue/white patterned morphotype, implying that the expression of the lacZ gene from the inserted yeast promoter is switched on and off during the colony formation, were isolated. We identified several genes with variable expression during colony morphogenesis, including CCR4, PAM1, MEP3, ADE5,7 and CAT2. S. cerevisiae strain deleted in the CCR4 gene forms colonies with less organized morphology when compared with the isogenic parental strain. The synchronization of the expression patterns of some of the isolated genes in neighboring colonies was observed.     

Effects of indole and caffeine on cAMP in the ind1 and cfn1 mutant strains of Schizophyllum commune during sexual development

The ind1 and cfn1 mutations of Schizophyllum commune express resistance to high concentrations of indole and caffeine respectively, and also affect sexual development. To clarify molecular events caused by the mutations, it was investigated how cAMP levels in S. commune strains respond to externally supplied indole and caffeine. Both compounds increased the cAMP levels in wild-type strains under several culture conditions. During sexual development of the ind1 mutant, the cAMP level in an early stage (hyphal aggregation) was highly increased by addition of indole, and the phenomenon disappeared in a later stage (fruit body formation). For the cfn1 mutants, the incremental increase in cAMP levels by addition of caffeine was smaller than that of wild-type strains.     

Study of the inactivation of antibiotics by bacterial colonies

A procedure of bacteria application to disks from the colonies was used for determining antibiotic inactivation in the disks by the bacteria colonies after the disk direct contact with the colonies. Changes in the antibiotic activity in the disks were registered after incubation at 37 degrees C for 2 hours. It was shown that ampicillin resistant strains of E. coli K12 carrying R plasmids and strains of S. typhimurium and S. aureus inactivated the antibiotics in the disks and their population were homogenous in this respect. It is advisable to use the procedure in assaying drug resistance of bacterial populations.     

In situ hybridisation in filamentous fungi using peptide nucleic acid probes

Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.     

Scooter, a new active transposon in Schizophyllum commune, has disrupted two genes regulating signal transduction

Two copies of scooter, a DNA-mediated transposon in the basidiomycetous fungus Schizophyllum commune, were characterized. Scooter is the first transposon isolated from S. commune. Scooter creates 8-bp target site duplications, comparable to members of the hAT superfamily, and has 32-bp terminal inverted repeats. Both copies of scooter are nonautonomous elements capable of movement. Southern blot hybridizations show that scooter-related sequences are present in all S. commune strains tested. Scooter-1 was identified initially as an insertion in the Bbeta2 pheromone receptor gene, bbr2, leading to a partial defect in mating. Scooter-2 spontaneously disrupted a gene to produce the frequently occurring morphological mutant phenotype known as thin. The scooter-2 insert permitted cloning of the disrupted gene, thn1, which encodes a putative regulator of G protein signaling (RGS) protein. Spontaneous insertion of scooter into genes with identifiable mutant phenotypes constitutes the first evidence of active transposition of a DNA-mediated transposon in a basidiomycete.     

Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae

Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.     

Severe impairment of growth and differentiation in a Neurospora crassa mutant lacking all heterotrimeric G alpha proteins

Heterotrimeric G alpha proteins play a critical role in regulating growth and differentiation in filamentous fungi. No systematic analysis of functional relationships between subunits has been investigated. This study explores the relative contributions of Neurospora crassa G alpha subunits, gna-1, gna-2, and gna-3, in directing development by analyzing strains deleted for various combinations of these genes. Although viable, mutants lacking all G alpha subunits or gna-1 and gna-3 are severely restricted in apical growth, forming small colonies. These strains form little aerial hyphae during asexual development on solid medium and exhibit inappropriate sporulation in submerged cultures. Similar to all strains carrying the Delta gna-1 mutation, these mutants are female sterile. Defects attributed to gna-2 are observed only in conjunction with the loss of gna-1 or gna-3, suggesting a minor role for this G alpha in N. crassa biology. Results from analysis of adenylyl cyclase and epistatic studies with the cAMP-dependent protein kinase regulatory subunit (mcb) indicate separate functions for GNA-1 and GNA-3 in cAMP metabolism and additional cAMP-independent roles for GNA-1. These studies indicate that although G alpha subunits are not essential for viability in filamentous fungi, their loss results in an organism that cannot effectively forage for nutrients or undergo asexual or sexual reproduction.