Genome-Wide Identification and Analysis of Genes Encoding Proteolytic Enzymes in Pineapple

Pineapple, Ananas comosus, is an economically important fruit crop. Recently its genome was completely sequenced and a total of 27,024 protein coding genes were predicted. Using a set of well evaluated bioinformatics tools we have predicted the protein subcellular locations and comparatively analyzed the protein conserved domains of the predicted proteomes in pineapple, Oryza sativa (rice), Sorghum bicolor (sorghum), and Brachypodium distachyson. Our analysis revealed that ~24–26 % of proteins were located in nucleus, 17–21 % in cytosol, 9–11 % in chloroplast, and 8–11 % proteins were secreted in these monocot plants. The secretomes in the four species were analyzed comparatively and a large number of secreted glycosyl hydrolases were identified. As pineapple proteolytic enzymes, knowns as bromelains, have been used for medical treatments, we focused on genome-wide identification and analysis of pineapple genes encoding proteases. A total of 512 pineapple genes encoding putative proteolytic enzymes were identified, with 152 secreted, 74 localized in cytosol, 67 in nucleus, 60 in chloroplast, 18 in mitochondria, and the remaining in other subcellular locations. The top large protease families in pineapple were papain family cysteine protease (62 genes), peptidase S8 family (56 genes), aspartyl protease family (38 genes), and serine carboxypeptidase (33 genes). Gene expression analysis revealed that among 512 protease genes 432 were expressed in various tissues and 72 genes were differentially expressed. The highly expressed protease genes were identified including 7 papain family cysteine proteases. The protease genes with the predicted protein subcellular locations will facilitate the efforts for examining their biological roles in pineapple growth and development and for expressing the recombinant proteases for medical use. The information of protein subcellular location of all plant species can be accessed at the PlantSecKB website (http://proteomics.ysu.edu/secretomes/plant.php).     

蚯蚓纤溶酶组分及其基因的多态性

生化制备证明,蚯蚓纤溶酶为一组含有10个以上同工酶组分的混合酶.为了研究这些组分的DNA和蛋白质序列的异同,本文通过对数据库中已报道的28条蚯蚓纤溶酶基因按相似性进行归类,将它们分为4组(基因型).同一组中的序列具有共同特征,即保守的N-末端、C-末端和相同的结构域,而且这些结构域在序列中分布的位置也相同;但它们之间在中间部分存在明显差异,这些差异说明了基因型中存在多态性.这种多态性可能是它们在体内溶栓的药理药效作用存在差异的结构基础.     

Isolation of Extremely AT-Rich Genomic DNA and Analysis of Genes Encoding Carbohydrate-Degrading Enzymes from Orpinomyces sp. Strain PC-2

An effective method for extraction of intact genomic DNA from the extremely AT-rich polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogen-like storage polysaccharides using hexadecyltrimethylammonium bromide (CTAB) and high salt washes. The DNA was digested with various restriction enzymes and was suitable for use as a PCR template, for Southern blotting, and for genomic library construction. Genomic DNA analysis of three representative genes (celE, bgl1, and xynA) encoding (hemi-) cellulolytic enzymes of the fungus revealed multiplicity of family 5 endocellulase genes (celE-like), and family 1 β-glucosidase genes (bgl1-like), but only a single copy of family 11 xylanase gene (xynA).     

毕赤酵母碳源阻遏因子编码基因PpMIG1和PpMIG2的克隆与序列分析

通过MIG基因的同源性设计简并引物,采用PCR方法从毕赤酵母(Pichia pastoris)中成功克隆了两个MIG同源基因的核心片段,同时利用Genome Walking的方法获得了基因的全长及其侧翼序列,并分别命名为PpMIG1和PpMIG2.序列分析显示,PpMIG1基因全长1 335 bp,编码444个氨基酸;PpMIG2基因全长1 365 bp,编码454个氨基酸.这个两个基因所编码的蛋白质均与酿酒酵母碳源阻遏因子ScMig1同源,在氨基末端具有两个典型的C2H2锌指结构域,该结构域能够与受葡萄糖阻遏的许多基因的启动子相结合.PpMig阻遏因子的获得为毕赤酵母碳源阻遏机制的研究奠定了基础.     

Polymorphic Genes of Xenobiotic-Metabolizing Enzymes Associated with Predisposition to Bronchial Asthma in Hereditarily Burdened and Nonburdened Children

The frequencies of theCYP1A1valine allele, homozygous deletions of GSTM1 and GSTT1, and two point mutations of the NAT2 gene, NAT2: S1(C⁴⁸¹ T) and S2 (G⁵⁹⁰ A), were compared in healthy children and children having bronchial asthma. The S1 mutation was associated with resistance, and all of the other traits, with predisposition to the disease. In families of patients with diseased progenitors and in those with healthy progenitors, the estimates of the asthma risk were similar. In both groups, parameters of the trait association with the disease depended on passive smoking. At passive smoking, a trend to an overrepresentation (high odds ratio, OR) of the GSTM1 null genotype and S2 mutation of theNAT2 gene was observed, whereas the odds ratio of the GSTT1 null genotype decreased, and those of the CYP1A1 and S1 mutation of the NAT2 gene remained unchanged. The highest OR = 36.25 (P < 0.01) was characteristic of theGSTT1 null genotype in nonsmoking hereditary burdened patients. The results obtained suggest an important role of xenobiotic-metabolizing enzymes in development of bronchial asthma.     

子宫内膜异位症中hMLH1、hMSH2基因启动子区的甲基化研究

目的：探讨错配修复基因1（hMLH1）、错配修复基因2（hMSH2）启动子区甲基化与子宫内膜异位症的关系。方法：采用甲基化特异性PCR方法检测23例子宫内膜异位症组织和20例正常子宫内膜中hMLH1、hMSH2基因启动子区的甲基化状态。结果：hMLH1基因在子宫内膜异位症中的甲基化率为39％（9/23）,在正常子宫内膜组织中的甲基化率为5％（1／20）,两者比较,差异有统计学意义（P〈0．05）;hMSH2基因在子宫内膜异位症中的甲基化率为8％（2／23）,在正常子宫内膜组织中的甲基化率为5％（1／20）,两者比较,无明显差异（P〉0．05）。结论：hMLH1基因启动子区甲基化可能与子宫内膜异位症发病有关;hMSH2基因启动子区甲基化与子宫内膜异位症之间不存在显著联系。     

Polymorphic Markers at the Genes Encoding the Endothelial NO-Synthase and Angiotensin II Type 1 Receptor and the Susceptibility to Ischemic Heart Disease

Allele and genotype frequency distribution patterns of the polymorphic regions at the genes for human endothelial NO-synthase (NOS3) (theecNOS4a/4b VNTR and the Glu298Asp substitution) and the angiotensin II type 1 receptor (AT ₁)(the A1166C substitution) were compared in 83 unrelated healthy individuals and 88 patients with ischemic heart disease (IHD). In the group of patients statistically significantly higher frequencies of the NOS3 allele4a (45.5 versus 19.3%), as well as the 4a/4a (15.9 versus 2.4%) and 4a/4b (59.1 versus 33.7%) genotypes were observed. Frequencies of the allele4b (54.5% versus 80.7%) and the 4b/4b homozygotes (25.0 versus 63.9%) were statistically significantly lower in the group of IHD patients than in healthy individuals. The IHD patients were statistically significantly different from the healthy subjects also in the distributions of the AT ₁ genotypes. In the former group, a significantly decreased frequency of the AA homozygotes (51.1 versus 65.1%) and an increased frequency of AC heterozygotes (40.9 versus 27.7%) were observed. Thus, in the Moscow population the ecNOS4a/4b VNTR of theNOS3 gene and the A1166C polymorphism of the AT ₁ gene are associated with the IHD development. Furthermore, the correlation with the IHD revealed was much stronger for the NO3 VNTR locus.     

Composition and Expression of Genes Encoding Carbohydrate-Active Enzymes in the Straw-Degrading Mushroom Volvariella volvacea

Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 285 genes encoding carbohydrate-active enzymes (CAZymes) in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation) and GH43 (hemicellulose and pectin degradation), and the lyase families PL1, PL3 and PL4 (pectin degradation) but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3′-tag digital gene expression (DGE) reveals that 239 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryotic strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryotic strains.     

Several Genes Encoding Enzymes with the Same Activity Are Necessary for Aerobic Fungal Degradation of Cellulose in Nature

The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.     

Comparative Analysis of Expression of Genes Encoding Enzymes of Catecholamine Catabolism and Renalase in Tissues of Normotensive and Hypertensive Rats

Comparative analysis of expression of genes encoding enzymes of catecholamine catabolism (monoamine oxidases A and B (MAO A and MAO B) and catechol-O-methyl transferase (COMT)) and renalase has been carried out in tissues of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Among investigated tissues the highest level of mRNA of genes encoding key enzymes of catecholamine catabolism (MAO A, MAO B, COMT) was found in the heart of WKY rats. In SHR the mRNA levels of these genes were lower (p < 0.05–0.01), however, no similar changes were observed in the tissues studied in dependence of hypertension. The relative mRNA levels of the studied genes normalized versus actin mRNA significantly varied. In heart and kidney the relative level of COMT mRNA significantly exceeded the relative levels of both MAO A mRNA and MAO B mRNA. In the brain differences in mRNAs of MAOA, MAOB, and COMT were less pronounced. However, in all examined tissue the renalase mRNA level was much (at least 10–20-fold) lower than any other mRNA studied. Taking into consideration known correlations between mRNAs and corresponding protein products reported in the literature for many genes these results suggest that in the case of any catalytic scenarios proposed or even proved for renalase this protein cannot contribute to catecholamine degradation. It is also unlikely that the products of the renalase reaction, β-NAD(P)⁺ and hydrogen peroxide, can exhibit a hypotensive effect due to low expression of the renalase encoding gene.     

The Two Genes Encoding Starch-Branching Enzymes IIa and IIb Are Differentially Expressed in Barley

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5′ portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (β/α)₈-barrel topology of the α-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.     

Alteration in the Expression of Genes Encoding Primary Metabolism Enzymes and Plastid Transporters during the Culture Growth of Chlamydomonas reinhardtii

Molecular Biology - In a mixotrophic Chlamydomonas reinhardtii culture, the expression levels of genes encoding primary metabolic enzymes and chloroplast plastid transporters were analyzed. For the...     

Genes Encoding Proteolytic Enzymes Fungalysin and Subtilisin in Dermatophytes of Human and Animal Origin: A Comparative Study

Mycopathologia - Dermatophytes are among the most successful fungal pathogens in humans, but their virulence mechanisms have not yet been fully characterized. Dermatophytic fungi secrete proteases...     

诱导子宫内膜异位症患者子宫内膜上皮细胞调亡研究

目的:研究促性腺激素释放激素激动剂(GnRHα)对子宫内膜异位症(EMs)患者在位内膜蛋白表达的影响,为EMs的治疗提供依据。方法:94例EMs患者随机分为两组,每组47例,均实施腹腔镜手术治疗,对照组未给予药物治疗,观察组给予曲普瑞林皮下注射,28日/次,注射3次,术中及治疗三个月后观察组分别用取内膜器取少量子宫内膜组织标本,采用免疫组化法法、TUNEL染色法和Western blot检测组织内Bcl-2、Caspase3、GRP78等表达情况,并观察治疗效果。结果:治疗后观察组疼痛视觉模拟评分(VAS)为(2.13±0.69)分,低于对照组的(2.62±0.71)分(P0.05);观察组1年内妊娠率及总妊娠率分别为38.30%、51.06%,均高于对照组的19.15%、29.79%(P0.05);治疗后观察组Bcl-2、Caspase3表达量分别为(1.54±0.28)、(3.20±0.65),均高于对照组的(1.38±0.15)、(1.24±0.27)(P0.05);治疗后观察组GRP78表达量为(0.46±0.17),低于对照组的(0.61±0.24)(P0.05)。结论:GnRHα可缓解EMs患者下腹痛、痛经、性交痛等症状,提高妊娠率,其机制可能与GnRHα调控上皮细胞Bcl-2、Caspase3、GRP78等的表达有关。     

Identification and Chromosomal Location of Two Human Genes Encoding Enzymes Potentially Involved in Proteolytic Maturation of Farnesylated Proteins

Two human cDNAs encoding proteins similar to yeast enzymes involved in proteolytic processing of farnesylated proteins like a-factor mating pheromone and Ras2p have been cloned from an ovary cDNA library. These proteins have been tentatively called Face-1 and Face-2 (farnesylated protein-converting enzymes 1 and 2), respectively, and are integral membrane proteins, belonging to distinct families of metalloproteinases. Northern blot analysis of poly(A)+ RNAs isolated from a wide variety of human tissues demonstrated that both genes are expressed in all examined tissues, which suggests that these enzymes play housekeeping roles in normal processes. Fluorescence in situ hybridization experiments showed that the human FACE-1 gene maps to 1p34, whereas FACE-2 is located at 11q13, a region frequently amplified in human carcinomas and lymphomas. On the basis of these results, we suggest that inhibition of Face-1 and/or Face-2 could be part of strategies directed to block the functioning of prenylated proteins activated in oncogenic processes, including Ras proteins.