The respiratory response to heat shock in Neurospora crassa

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.     

Purification and properties of a low molecular weight DNA polymerase from Neurospora crassa

A third DNA polymerase ‘C’ with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to β- and γ-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40 000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K⁺ at 2–20 mmol/l, for Mn²⁺ at 0.8 mmol/l, for Mg²⁺ at 4.0 mmol/l, the pH optimum is 8.0. Its K_m is 1.5 μmol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease.     

Characterization of a 45-kDa polypeptide as the precursor of subunit 1 of cytochrome c oxidase in Neurospora crassa

In a previous paper (Van 't Sant, P., Mak, J.F.C. and Kroon, A.M. (1981) Eur. J. Biochem. 121, 21–26) we showed the existence of three elongated precursor proteins (45, 36 and 25 kDa) of mitochondrial translation products in Neurospora crassa. We presented some indications that the largest precursor could be related to subunit 1 of cytochrome c oxidase. Here we present conclusive evidence that the 45-kDa polypeptide is indeed this precursor by demonstrating that an immunodetectable 45-kDa polypeptide displays the same behaviour as the labeled 45-kDa precursor; both accumulate after long incubation with cycloheximide or by decreasing the temperature and both are not tightly membrane bound. Moreover the antibody against subunit 1 of cytochrome c oxidase also recognizes, in immunoadsorption experiments, besides subunit 1, the 45-kDa polypeptide accumulated by cycloheximide incubation. Furthermore, we developed a small scale purification of antibodies against subunit 1 of cytochrome c oxidase. By means of these purified antibodies it is demonstrated that the 45-kDa polypeptide and subunit 1 have corresponding antigenic determinants. Under the various conditions tested, all three precursors are less firmly membrane-bound than the mature subunits. Finally, it is observed that in short incubations in vivo, chloramphenicol inhibits the processing of the mitochondrially synthesized precursors, under conditions where mitochondrial translation is only partially inhibited.     

Gene pho-2 codes for the multiple active forms of Pi-repressible alkaline phosphatase in the mould Neurospora crassa

The mycelial Pi-repressible alkaline phosphatase of the wild-type strain 74A of Neurospora crassa was separated into at least ten isoforms by isoelectric focusing. The components visualized by activity with sodium -naphthyl phosphate as the substrate were predominantly acidic proteins with isoelectric points ranging from pH 4.5 to 7.6. The number of these isoforms was a function of growth pH. Strain pho-2A did not produce active Pi-repressible alkaline phosphatase (the pho-2 gene codes for its amino acid sequence), which gives an indication that these isoforms are encoded by the same structural gene.     

Effect of growth temperature on lipid fatty acids of four fungi (Aspergillus niger,Neurospora crassa,Penicillium chrysogenum,andTrichoderma reesei)

The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10° C with 5° C intervals in four exponentially growing fungi: Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Trichoderma reesei. Fatty acid unsaturation increased in A. niger, P. chrysogenum, and T. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. In A. niger and T. reesei, this was due to the increase in linolenic acid content. In P. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to 20° C. In T. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid content decreased. In A. niger and P. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, in N. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities found may indicate common regulatory mechanisms causing the responses. Received: 1 March 1995 / Accepted: 8 May 1995     

温度、盐度对强壮箭虫耗氧率和窒息点的影响

研究了不同温度、盐度下强壮箭虫的耗氧率和窒息点.结果表明:温度和盐度均对强壮箭虫的耗氧率和比耗氧率有显著影响.试验温度在5℃-25℃,强壮箭虫个体耗氧率(IO)和比耗氧率(SO)开始随温度的升高而升高,之后呈明显下降趋势.其回归方程分别为y=0.0058x3 -0.2956x2+4.415x-8.7816(R2=0.99,P＜0.05)和y=0.0011x3 -0.0546x2+0.8161x-1.6232(R2=0.99,P＜0.05),数值分别为6.30～11.71μg·ind-1·h-1和1.22 ～2.16μg· mg-1·h-1,窒息点为4.18～6.87 mg·L-1.盐度10 ～40条件下,IO和SO随盐度的升高逐渐下降,回归方程分别为y=-0.0068x2 -0.1412x+21.702(R2=0.89,P＜0.05)和y=-0.0013x2-0.0261x+4.0114( R2 =0.89,P＜0.05),数值分别为4.98～17.73μg ·ind-1·h-1和0.92～3.56 μg·mg-1 ·h-1,窒息点为4.02～6.24 mg·L-1.对强壮箭虫与其他水生动物的耗氧率和窒息点进行比较得出,强壮箭虫是一种狭氧性浮游动物.     

粗糙脉孢菌基因组分泌蛋白的初步分析

文章报道利用信号肽预测软件SignalP v3.0和PSORT,跨膜螺旋结构预测软件TMHMMv2.0和THUMBUP,GPI-锚定位点预测软件big-PI Predictor和亚细胞器中蛋白定位分布预测软件TargetP v1.01对粗糙脉孢菌全基因组数据库中已公布的10 082个氨基酸序列进行预测分析。结果表明在粗糙脉孢菌中有437个蛋白为分泌蛋白,编码这些蛋白最小的可读框（open reading frame,ORF）为252 bp,最大为6 604 bp,平均1 433 bp,分泌蛋白信号肽长度介于15~59个氨基酸之间。在437个分泌蛋白中,205个具有功能描述,主要包括各种酶类、细胞能量生成、运转以及自身修复、防卫等多种功能。这些蛋白所参与的生化过程可能发生在膜外的周质空间或是菌体外的场所,为该物种营养的摄取,以及对环境做出响应服务。      

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyze the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analyzed by light-, confocal- and electron microscopy. Changes in PA distribution were analyzed in E. festucae using a PA biosensor and the impact of these changes on the endocytic recycling and superoxide production investigated. We found that E. festucae PldB, and the N. crassa ortholog, PLA-7, are required for polarized growth and cell fusion and contribute to ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network for hyphal morphogenesis and growth of filamentous fungi.     

Cloning and Expression of an Endo-1,6-β-D-glucanase Gene (neg1) from Neurospora crassa

A gene (neg1) encoding an endo-1,6-β-D-glucanase from Neurospora crassa was cloned. The putative neg1 was 1443-bp long and encoded a mature endo-1,6-β-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-β-D-glucanase activity. No genes similar in sequence were found in yeasts and fungi.