Decreased zinc ion accumulation by the basidiomycete Lentinus edodes over-expressing L. edodes PriA gene

The information that the deduced expression product of Lentinus edodes priA gene consists of N-terminal hydrophobic sequence, putative zinc-binding motifs and C-terminal membrane-binding-promoting unique sequence led us to analyze its function in L. edodes. Here L. edodes monokaryotic cells over-expressing priA gene were found to exhibit a remarkably decreased accumulation of zinc ion, indicating the involvement of the priA gene in regulation of the intracellular zinc concentration.     

Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus

Two cDNAs and two genomic DNAs coding for the allelic forms of the ligninolytic phenoloxidase were isolated from the white-rot fungus Coriolus hirsutus. The cloned genes were identified in genetic libraries by hybridization screening using four deoxyoligonucleotide probes which corresponded to the partial amino acid sequence of the purified enzyme. Each cDNA encoded the full-length of the phenoloxidase, a protein consisting of 499 amino acid residues, and its putative signal peptide of 21 amino acid residues. The nucleotide sequences of the two alleles differed by 18 single base changes within the open reading frames resulting in one amino acid substitution. Ten small introns interrupted both genomic DNAs as indicated by direct comparison with the corresponding cDNAs. Putative eukaryotic regulatory sequences, "CAAT" and "TATA," were observed in the 5'-flanking region of both genomic DNAs. Each of the phenoloxidase cDNAs was successfully expressed in an active form in Saccharomyces cerevisiae using the useful yeast expression vector YEp51.     

Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNA^Asn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol?γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol?γ from yeast to human.     

Composition and biological activity of submerged mycelium of the xylotrophic basidiomycete Lentinus edodes

The composition of submerged mycelium of Lentinus edodes, grown in laboratory fermenters, has been studied. The mycelium contained 23-24% proteins, 8-9% lipids, up to 1800 mg% phenolic substances, and a significant amount of inorganic substances, including calcium and iron. The fungus produced up to 5.0% intracellular and 3.5-4.0 g/l extracellular polysaccharides. The submerged mycelium stimulated the development of humoral immune response elicited by sheep red blood cells.     

棉铃虫HaTO-like基因的克隆、序列分析和表达特征

【目的】克隆和分析了棉铃虫Helicoverpa armigera HaTO-like基因的编码框序列,检测了该基因的时空表达谱以及在棉铃虫感染核型多角体病毒HaSNPV后的转录变化,为深入研究该基因的功能提供理论依据。【方法】本研究利用RT-PCR的方法首次克隆获得HaTO-like基因的全长cDNA序列,通过几种生物信息学软件对该基因的核苷酸序列和氨基酸序列进行了分析,并利用荧光定量PCR技术检测了该基因在棉铃虫不同发育阶段、幼虫组织和成虫组织的表达情况,以及HaSNPV感染对HaTO-like基因表达的影响。【结果】棉铃虫HaTO-like基因cDNA全长为994 bp,开放阅读框为756 bp,编码251个氨基酸,其蛋白序列的N端含有23个氨基酸的信号肽。进一步的序列分析表明棉铃虫HaTO-like与其他昆虫同源蛋白的氨基酸序列一致性不是太高,大概在39%~61%之间,其中与家蚕和脐橙螟在系统进化上关系最近。荧光定量PCR结果表明该基因在棉铃虫的5龄0 h和成虫第1天的的表达量相对较高,在幼虫的头部和表皮内的表达量较其他幼虫组织较高,在成虫的头部和足的表达量也相对较高。而病毒感染则显著地诱导了该基因在棉铃虫幼虫头部和表皮内的表达。【讨论】本研究克隆了棉铃虫HaTO-like基因的全长cDNA序列,分析了该基因的序列特征和表达谱,为进一步阐释该基因的功能奠定理论基础。     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

The basidiomycete Lentinus edodes linear mitochondrial DNA plasmid contains a segment exhibiting a high autonomously replicating sequence activity in Saccharomyces cerevisiae.

A linear DNA plasmid, designated pLLE1, has been isolated from a mitochondrial fraction of a strain of Lentinus edodes. pLLE1(11.0 kbp) was sensitive to the 3'----5'-acting exonuclease III and resistant to the 5'----3'-acting lambda exonuclease. It showed no homology with mitochondrial and nuclear genomic DNAs of plasmidless strain as well as the pLLE1-harboring host strain of L. edodes. The 1434-bp fragment (sequences) capable of autonomous replication in the yeast Saccharomyces cerevisiae (ARSs) was cloned from pLLE1 DNA with YIp32 (pBR322 containing yeast LEU2 DNA), which displayed a high ARS activity. The cloned 1434-bp fragment was shown to lie near to the end of pLLE1 DNA (nucleotides about 800-2200) and contained three consecutive ARS consensus sequences (A/T)TTTAT(A/G)TTT(A/T) of S. cerevisiae and dispersive eight ARS consensus-like sequences. The subcloned 366-bp fragment containing the three ARSs retained original ARS activity of the 1434-bp fragment.     

Cloning, sequence analysis, and expression of the bacteriophage T4 cd gene

The cd gene of bacteriophage T4, which encodes the enzyme deoxycytidylate deaminase, was isolated as a 1.9-kilobase DNA fragment and completely sequenced. The deduced amino acid sequence was found to be 193 residues long compared with 188 for the corresponding enzyme from bacteriophage T2. There were nine amino acid differences between the two enzymes in addition to a 5-residue insert near the carboxyl terminus of the T4 deaminase which was not present in the T2 deaminase. The cd-containing fragment also contained all of gene 31 (Nivinskas, R., and Black, L. W. (1988) Gene (Amst.) 73, 251-257) and thus precisely locates the two genes relative to one another within the T4 phage genomic map. Attempts to place the cd gene within a high expression vector have not been successful so far due to possible toxic effects of the gene product. However, placement of the gene within pUC18 resulted in a degree of expression which is about 10-20 times that found in T4-infected Escherichia coli. The enzyme was purified to homogeneity and found to possess properties similar to T2 phage deoxycytidylate deaminase.     

Antibiotic properties of the strains of the basidiomycete Lentinus edodes (Berk.) sing]

Antibiotic properties of the extracts from the fermentation broth and mycelium of 15 strains of the edible and medicinal basidiomycete L. edodes were studied and it was shown that the extracts were active against grampositive and gramnegative bacteria, yeasts and mycelial fungi, including dermatophytes and phytopathogens. The strains differed by the set of the organisms susceptible to the action of the extracts. Strains of L. edodes combining marked antibiotic properties and high yields of water soluble polysaccharides were screened. The active compounds were detected by preparative TLC. Two of them were identified with UV- and mass spectrometry as lentinamycin B and erytadenine (lentinacin). Lentinamycin B was found to be the main component responsible for the antibiotic activity of the L. edodes strains.