Protease inhibitors clitocypin and macrocypin are differentially expressed within basidiomycete fruiting bodies

Clitocypin and macrocypin are cysteine protease inhibitors of the mycocypin family which is unique to basidiomycetes. We have established that Clitocybe nebularis and Macrolepiota procera each contain genes for both macrocypin and clitocypin. Both are expressed in M. procera but only clitocypin in C. nebularis. Further analysis of mycocypin expression at the mRNA and protein levels in mature fruiting bodies of M. procera revealed that clitocypin is expressed evenly throughout the fruiting body, while the level of expression of macrocypins varies, and, at the protein level, is much higher in the veil fragments and the ring. The expression patterns of various mycocypins were determined in Coprinopsis cinerea, using promoters linked to a reporter gene. The expression profile of the clitocypin promoter was similar to that of the constitutive promoter gpdII from Agaricus bisporus, while that of the macrocypin 4 promoter was limited to the outer edges of the fruiting body throughout development. In addition, the activity of the macrocypin 3 promoter was different, indicating different regulation of expression for different macrocypin genes. The complex, tissue specific expression patterns for mycocypin genes suggest different biological roles for the products, either in regulation of endogenous proteases or in defense against pathogens or predators.     

Characterisation of the <Emphasis Type="Italic">Aspergillus niger dapB</Emphasis> gene,which encodes a novel fungal type IV dipeptidyl aminopeptidase

We have cloned the Aspergillus niger dapB gene. Analysis of its nucleotide sequence and the corresponding protein sequence indicates that the gene encodes a type IV dipeptidyl aminopeptidase (DPP IV). Based upon its deduced sequence we predict the presence of a transmembrane domain in the protein. Furthermore, dapB-overexpressing transformants display an increase in intracellular DPP IV activity. This is the first reported characterisation of a dipeptidyl aminopeptidase with a transmembrane domain from a filamentous fungus. Using the dapB sequence as a query, we were able to identify 14 DPP IV-encoding genes, and 12 additional DPPIV proteases in public genomic databases. Phylogenetic analysis reveals that in yeasts there are two clades of genes that encode DPP IV proteases with a transmembrane domain. In this study we demonstrate that, as in yeasts, two classes of DPP IV-encoding genes exist in filamentous fungi. However, only one of these codes for DPP IV proteases with a transmembrane domain. The second type present in filamentous fungi encodes extracellular DPP IV proteases. The dapB gene belongs to the first cluster. We propose that DapB plays a role in the proteolytic maturation of enzymes produced by A. niger.Electronic Supplementary Material Supplementary material is available for this article at     

Homology Modeling of Hemagglutinin/Protease [HA/P (vibriolysin)] from <Emphasis Type="Italic">Vibrio Cholerae</Emphasis>: Sequence Comparision,Residue Interactions and Molecular Mechanism

Vibrio cholerae produces a zinc-containing and calcium-stabilized soluble hemagglutinin/protease, which has been earlier shown to have the ability to cleave several physiologically important substrates including mucin, fibronectin and lactoferin. This study presents homology modeling of hemagglutinin/protease (vibriolysin) from Vibrio cholerae in the presence of inhibitor HPI [N-(1-carboxy-3-phenylpropyl)-phenylalanyl-alpha-aspargine]. The 3D structure was predicted based on its sequence homology with Pseudomonas aeruginosa elastase (PAE). Comparison of the 3D structures of PAE and HA/P reveals a remarkable similarity having a conserved α + β domain. The inhibitor shows similar binding features as seen in other metalloproteases of M4 peptidase family. The study also highlights the key catalytic residues as well as the residues at the S₁ and binding sub-sites. The similarities between the two proteins provide support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. The fact that both enzymes are secreted as zinc-containing proteases, led us to further hypothesize that they may play similar role in pathogenesis.     

Identification of papain-like cysteine proteases from the bovine piroplasm Babesia bigemina and evolutionary relationship of piroplasms C1 family of cysteine proteases

Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases.     

Genetic diversity of populations of <Emphasis Type="Italic">Merodon aureus</Emphasis> and <Emphasis Type="Italic">M. cinereus</Emphasis> species complexes (Diptera,Syrphidae): integrative taxonomy and implications for conservation priorities on the Balkan Peninsula

The genetic structure of 10 populations of the Merodon aureus group from the Balkan Peninsula was examined through allozyme electrophoresis and mitochondrial DNA sequencing of the cytochrome c oxidase subunit I (COI). Six diagnosable cryptic taxa were identified within the morphologically defined species M. aureus Fabricius, 1805 and M. cinereus (Fabricius, 1794), with clear separation of the populations (((M. aureus A + M. aureus B) + cinereus complex) + M. aureus C). The parsimony analysis of COI sequence data of the aureus–cinereus complex using Merodon avidus A species as an outgroup resulted in two main clades, (M. aureus A + M. aureus B) and ((M. aureus C + M. cinereus B + M. cinereus C) + M. cinereus A), which differed on average by 5.7%. The observed spatial distribution of the taxonomic diversity of the group suggested that these taxa originated from a common ancestral population in the Mediterranean. Identification of genetic uniqueness and genetic endemism emphasizes the importance of molecular markers and estimation of genetic diversity in recognition of conservation units. The primary goals of the conservation measures that we propose are the protection of phylogenetic lineages within the highly diverse M. aureus group taxa and conservation of the genetic variation through management of important areas.     

Molecular characterization,relatedness and antifungal susceptibility of the basidiomycetous hormographiella species and Coprinus cinereus from clinical and environmental sources

Hormographiella-like strains, isolated from different natural substrates and producing sclerotia and occasionally basidiomata of Coprinus cinereus, were compared morphologically and using molecular techniques with clinical strains of Hormographiella aspergillata and H. verticillata. Analysis of restriction fragment length polymorphisms of ribosomal and mitochondrial-like DNA confirmed interspecific differences between H. aspergillata and H. verticillata, supporting the morphological data, and helped demonstrate that H. aspergillata is the anamorph of C. cinereus. The latter was confirmed also by crossing tests. The analysis of the mtDNA restriction profiles revealed intraspecific variability in C. cinereus, which allowed differentiation of clinical and environmental strains. Due to the implication of C. cinereus and Hormographiella in human opportunistic infections, the antifungal susceptibility test is included. Results show that all strains were susceptible to miconazole, itraconazole and ketoconazole but not to flucytosine and fluconazol. Susceptibility against amphotericin B was variable; while H. verticillata was susceptible, four out of seven C. cinereus strains tested were resistant.     

Ultrastructure of chitin in hyphae ofCandida albicans and other dimorphic and mycelial fungi

Summary Chitin microfibrils exposed by chemical extraction of hyphal walls ofCandida albicans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidiodes brasiliensis, Coprinus cinereus andMucor mucedo were of variable morphology but gave identical infrared spectra and behaved as pure chitin in chromatographic analyses. The microfibrils of the four dimorphic fungi studied were shorter than those in the mouldsC. cinereus andM. mucedo but were similar to those reported for the yeastSaccharomyces cerevisiae. InC. albicans the microfibrils in the septal plates of hyphae were predominantly tangentially orientated and were longer than those in the lateral walls. Microfibrils produced by chitin synthasein vitro were very much longer than any observed from hyphal preparations.     

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from <Emphasis Type="Italic">Asclepias fruticosa</Emphasis> latex

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.     

Cloning and characterization of the histidine kinase gene<Emphasis Type="Italic"> Dic1</Emphasis> from<Emphasis Type="Italic"> Cochliobolus heterostrophus</Emphasis> that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel     

基于nad5的西施舌漳州群体遗传分化水平分析——以蛤蜊属2个物种差异水平为参照

扩增了西施舌日照、连云港、北海、漳州4个野生群体、四角蛤蜊和中国蛤蜊各1个群体共73个样本的NAD5基因片段,测序获得了480bp核苷酸序列,分析核苷酸的多态性,旨在评估福建漳州西施舌与日照、连云港、北海西施舌之间的分化水平。结果:从73个序列中共检测到44种单倍型(Hap),其中西施舌4个群体有29种Haps,四角蛤蜊和中国蛤蜊分别有10种和5种Haps,漳州群体与北海、日照、连云港群体单倍型有明显差异;将西施舌分为北海、日照、连云港组(GP1)和漳州组(GP2)2个组,分析核苷酸差异,GP1与GP2间的T、A、G含量差异极显著(P0.01)。GP1与GP2间的遗传距离与组内(GP1、GP2)遗传距离之比为25.1—41.8,四角蛤蜊与中国蛤蜊之间的遗传距离与种内个体间遗传距离之比为24.4—36.7,GP1、GP2间的差异达到了四角蛤蜊和中国蛤蜊种间差异水平,而日照、北海群体间的遗传距离只有0.009,北海与日照群体地理位置虽远,但遗传差异则很小;AMOVA分析显示漳州西施舌发生了极显著遗传分化(FST=0.966—0.978,P0.01)。     

The canine kallikrein-related peptidase 14: Structural characterization, alternative splicing and differential expression in mammary cancer

Human kallikrein-related peptidases (KLKs) represent a family of 15 serine proteases with diverse roles in many physiological and pathological processes, including carcinogenesis. In the dog, only two KLK genes are known; dKLK1 and canine arginine esterase. Recently, 12 other genes have been predicted using computational methods, but none of them has ever been experimentally validated in canine tissues. In this study we investigated the expression of Canis familiaris KLK14, (CANFA)KLK14, in normal and cancerous mammary tissues. First, it was demonstrated that the in-silico determined canine KLK14 mRNA (GenBank accession no: XM_541464) has been wrongfully predicted on its 5′-end (nucleotides 1–88). The (CANFA)KLK14 mRNA sequence presented here, has high homology to its human counterpart and exhibits all defining-KLK features. In addition to the classical form of the gene, five splice variants were also identified. The splicing events involved 5′-truncation or complete elimination of exon 4 and/or retention of intron I. All encoded protein products of the splice variants were predicted to be truncated and catalytically inactive. The classical form and variant 3 were almost ubiquitously expressed in both normal and neoplastic tissues. Variant 1 was predominantly detected in normal tissues. The classical form and variants 1 and 2 exhibited lower expression levels in tumor compared to normal tissues. Moreover, an Ile155Asn polymorphism was identified. This is the first report on the structural characterization, alternative splicing and tissue expression of canine KLK14 mRNA. These findings may form the basis for the establishment of comparative studies investigating KLK functions in health and disease using the dog as a model.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

cDNA-derived identification of novel thionin precursors in Viscum album that contain highly divergent thionin domains but conserved signal and acidic polypeptide domains

The existence of new thionin variants in Viscum album has been deduced from cDNA sequences. Unlike the viscotoxins and related thionins previously found in different members of the Viscaceae, these novel thionins contain eight rather than six cysteine residues. In this respect they resemble thionins described previously from various cereals and from Pyrularia pubera, which also contain eight cysteine residues at identical positions. All of the new thionins of V. album are encoded as higher-molecular-weight precursors consisting of a signal peptide, a thionin domain and an acidic polypeptide domain. While the deduced amino acid sequences of the thionin domains of different precursor molecules are highly divergent, the two other domains are conserved among all of the variants and are distinct from the corresponding domains of thionin precursors of other plant species.     

Development Models for the Filth Fly ParasitoidsSpalangia gemina, S. cameroni,andMuscidifurax raptor(Hymenoptera: Pteromalidae) under Constant and Variable Temperatures

Development rates were determined for three pteromalid parasitoids of houseflies under constant and varying temperatures from 15 to 35°C.Muscidifurax raptorGirault and Sanders was the fastest developing species, with females completing development in 13.8 days at 32.5°C and 66.5 days at 15°C.Spalangia geminaBoucek females completed development in 20.8 days at 30.0°C and 161 days at 15.0°C, whereasS. cameroniPerkins females completed development in 20.6 days at 30.0°C and 155.5 days at 15.0°C. Male development times were 90.3% of those for femaleS. geminaand 92.7 and 88.6% of those for femaleS. cameroniandM. raptor,respectively. Parasitoid survival was very low at 35°C for all species and noSpalangiasurvived constant exposure to 15.0°C. Exposure to these lethal temperatures for shorter periods indicated that the parasitoids can tolerate them well under conditions more typical of the field. Development rates were modeled using biophysical and degree-day models and the models were tested for their ability to predict development under fluctuating conditions (24–36°C). Neither model was superior for all three species because of interspecific differences in the parasitoids' responses to high temperatures. Agreement between predicted and observed development times for all three species was achieved by small empirical adjustments of a key parameter in the biophysical model.     

Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana

A family of calcium-responsive protein kinases is abundant in plant cell extracts but has not been identified in animals and fungi. These enzymes have a unique structure consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. In this report, we present the amino acid sequences for eight new Arabidopsis cDNA clones encoding isoforms of this enzyme. Three isoforms were expressed as fusion proteins in Escherichia coli and exhibited calcium-stimulated protein kinase activity. We propose CPK as the gene designation for this family of enzymes and describe a phylogenetic analysis for all known isoforms.     

Cloning and characterization of the gene encoding phosphoketolase in <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> isolated from kimchi

The gene encoding phosphoketolase, which is 2749 bp long and contains 814 amino acid polypeptides with a total molecular mass of 91.9 kDa, was cloned from Leuconostoc mesenteroides C7 (LMC7) and expressed in Escherichia coli. It exhibited a homology of >58% with phosphoketolases from other lactic acid bacteria. The phosphoketolase of LMC7 belongs to the xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp) family, which is an enzyme with dual specificity for X5P and F6P. The members of this family contain typical thiamin pyrophosphate (TPP) binding sites as reported for other TPP-dependent enzymes, and several highly conserved regions as signature patterns for phosphoketolases. The plasmid pGPK containing the Xfp gene (xfp) exhibits phosphoketolase activity in E. coli. The specific activities of the enzyme from E. coli BL21 and E. coli EC101 harboring xfp were 0.28 and 0.14 units/mg, respectively. They both exhibited a 1.5-fold increase in the production of acetic acid from acetyl phosphate compared with their corresponding original strain.An erratum to this article can be found at     

<Emphasis Type="Italic">ASYMMETRIC LEAVES2-LIKE1</Emphasis>gene,a member of the AS2/LOB family,controls proximal–distal patterning in <Emphasis Type="Italic">Arabidopsis</Emphasis> petals

The formation and the development of the floral organs require an intercalate expression of organ-specific genes. At the same time, meristem-specific genes are repressed to complete the differentiation of the organs in the floral whorls. In an Arabidopsis activation tagging population, a mutant affected in inflorescence architecture was identified. This gain-of-function mutant, designateddownwards siliques1 (dsl1-D), has shorter internodes and the lateral organs such as flowers are bending downwards, similar to the loss-of-function brevipedicellus (bp) mutant. The affected gene in dsl1-D appeared to be ASYMMETRIC LEAVES2-LIKE1 (ASL1)/LATERAL ORGAN BOUNDARIESdomain gene 36 (LBD36), which is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. Analysis of the loss-of-function mutant asl1/lbd36 did not show morphological aberration. Double mutant analysis of asl1/lbd36 together with as2, the ASL1/LBD36 closest homologue, demonstrates that these two members of the AS2/LOB family act partially redundant to control cell fate determination in Arabidopsis petals. Moreover, molecular analysis revealed that overexpression of ASL1/LBD36 leads to repression of the homeobox gene BP, which supports the model that an antagonistic relationship between ASL/LBD and homeobox members is required for the differentiation of lateral organs.