Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance

PCR amplification of 16S rRNA gene by universal primers followed by restriction fragment length polymorphism analysis using RsaI, CfoI and HinfI endonucleases, distinctly differentiated closely related Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus from Bacillus subtilis sensu stricto. This simple, economical, rapid and reliable protocol could be an alternative to misleading phenotype-based grouping of these closely related species.     

中华猕猴桃基因组可变剪接事件鉴定及分析

可变剪接使一个基因能产生多种m RNA成熟体,极大地增加蛋白多样性.采用中华猕猴桃基因组数据做参考数据,利用中华猕猴桃叶片和果实3个不同发育时期(未成熟、半成熟和成熟期)的转录组数据,从中华猕猴桃基因组(39040个基因)中共鉴定出11651个基因(占总基因数的29%)对应的32180个可变剪接事件.在可变剪接不同类型中,内含子保留类型的发生频率最高,占50%以上;3′可变位点类型频率约为5′端可变类型的2倍.GO富集分析结果表明,可变剪接的基因主要富集于酶调控及核苷酸结合相关功能的GO类别中,而组织特有可变剪接基因功能富集热点与组织的重要功能关联,叶片多为肌动蛋白及微管相关;未成熟果实与双组分信号系统相关;半成熟果实多与磷脂合成过程相关;成熟果实多与信号传递过程相关.另外,55.6%的维生素合成相关基因发生可变剪接事件,显著高于基因组水平的29.6%,暗示着可变剪接参与维生素合成相关基因代谢过程中的重要作用.通过对中华猕猴桃全基因组可变剪接的分析,为解析中华猕猴桃基因组及进一步开展相关分子育种工作提供依据.     

Improvement of the Coprinopsis cinerea molecular toolkit using new construct design and additional marker genes

This paper describes the optimisation of an existing basidiomycete molecular toolkit through the development of new versatile vectors. These vectors enable the straightforward and rapid construction of gene expression and silencing cassettes by allowing the easy exchange of promoters, coding regions and terminator elements. The constructs contain multiple cloning sites (MCS) allowing any gene to be inserted using a range of restriction sites, with the option of a 5′ integral intron for efficient gene expression. We describe the testing of these vectors through marker gene expression in Coprinopsis cinerea. This work also extends the range of marker genes available for use in C. cinerea with the first report of DsRed and monomeric red fluorescent protein (mRFP) expression in C. cinerea and further demonstrates the requirement for an intron in the expression cassette for some marker genes. However, analysis of transformants containing either β-glucuronidase (GUS) or luciferase (LUC) genes, with and without an intron revealed no detectable marker gene expression. The inclusion of an intron does therefore not guarantee expression and other genetic factors may be involved.     

Construction of bacterial artificial chromosome libraries from the parasitic nematode Brugia malayi and physical mapping of the genome of its Wolbachia endosymbiont

The parasitic nematode, Brugia malayi, causes lymphatic filariasis in humans, which in severe cases leads to the condition known as elephantiasis. The parasite contains an endosymbiotic alpha-proteobacterium of the genus Wolbachia that is required for normal worm development and fecundity and is also implicated in the pathology associated with infections by these filarial nematodes. Bacterial artificial chromosome libraries were constructed from B. malayi DNA and provide over 11-fold coverage of the nematode genome. Wolbachia genomic fragments were simultaneously cloned into the libraries giving over 5-fold coverage of the 1.1 Mb bacterial genome. A physical framework for the Wolbachia genome was developed by construction of a plasmid library enriched for Wolbachia DNA as a source of sequences to hybridise to high-density bacterial artificial chromosome colony filters. Bacterial artificial chromosome end sequencing provided additional Wolbachia probe sequences to facilitate assembly of a contig that spanned the entire genome. The Wolbachia sequences provided a marker approximately every 10 kb. Four rare-cutting restriction endonucleases were used to restriction map the genome to a resolution of approximately 60 kb and demonstrate concordance between the bacterial artificial chromosome clones and native Wolbachia genomic DNA. Comparison of Wolbachia sequences to public databases using BLAST algorithms under stringent conditions allowed confident prediction of 69 Wolbachia peptide functions and two rRNA genes. Comparison to closely related complete genomes revealed that while most sequences had orthologs in the genome of the Wolbachia endosymbiont from Drosophila melanogaster, there was no evidence for long-range synteny. Rather, there were a few cases of short-range conservation of gene order extending over regions of less than 10 kb. The molecular scaffold produced for the genome of the Wolbachia from B. malayi forms the basis of a genomic sequencing effort for this bacterium, circumventing the difficult challenge of purifying sufficient endosymbiont DNA from a tropical parasite for a whole genome shotgun sequencing strategy.     

Phylogenetic footprinting and genome scanning identify vertebrate BMP response elements and new target genes

The complex gene regulatory networks governed by growth factor signaling are still poorly understood. In order to accelerate the rate of progress in uncovering these networks, we explored the usefulness of interspecies sequence comparison (phylogenetic footprinting) to identify conserved growth factor response elements. The promoter regions of two direct target genes of Bone Morphogenetic Protein (BMP) signaling in Xenopus, Xvent2 and XId3, were compared with the corresponding human and/or mouse counterparts to identify conserved sequences. A comparison between the Xenopus and human Vent2 promoter sequences revealed a highly conserved 21 bp sequence that overlaps the previously reported Xvent2 BMP response element (BRE). Reporter gene assays using Xenopus animal pole ectodermal explants (animal caps) revealed that this conserved 21 bp BRE is both necessary and sufficient for BMP responsiveness. We combine the same phylogenetic footprinting approach with luciferase assays to identify a highly conserved 49 bp BMP responsive region in the Xenopus Id3 promoter. GFP reporters containing multimers of either the Xvent2 or XId3 BREs appear to recapitulate endogenous BMP signaling activity in transgenic Xenopus embryos. Comparison of the Xvent2 and the XId3 BRE revealed core sequence features that are both necessary and sufficient for BMP responsiveness: a Smad binding element (SBE) and a GC-rich element resembling an OAZ binding site. Based on these findings, we have implemented genome scanning to identify over 100 additional putative target genes containing 2 or more BRE-like sequences which are conserved between human and mouse. RT-PCR and in situ analyses revealed that this in silico approach can effectively be used to identify potential BMP target genes.     

Genome Wide Analysis for Rapid Identification of Vibrio Species

The highly conserved 16S rRNA (rrs) gene is generally used for bacterial identification. In organisms possessing multiple copies of rrs, high intra-genomic heterogeneity does not allow easy distinction among different species. In order to identify Vibrio species, a wide range of genes have been employed. There is an urgent requirement of a consensus gene, which can be used as biomarker for rapid identification. Eight sequenced genomes of Vibrio species were screened for selecting genes which were common among all the genomes. Out of 108 common genes, 24 genes of sizes varying from 0.11 to 3.94 kb were subjected to in silico digestion with 10 type II restriction endonucleases (RE). A few unique genes—dapF, fadA, hisD, ilvH, lpxC, recF, recR, rph and ruvB in combination with certain REs provided unique digestion patterns, which can be used as biomarkers. This protocol can be exploited for rapid diagnosis of Vibrio species.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0553-5) contains supplementary material, which is available to authorized users.     

Misexpression of acetylcholinesterases in the C. elegans pha-2 mutant accompanies ultrastructural defects in pharyngeal muscle cells

pha-2 is the Caenorhabditis elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here, we performed a detailed characterization of the pha-2 phenotype using cell-type-specific reporters, physical manipulation of the nuclei in pharyngeal muscle cells using "optical tweezers", electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are (i) the pha-2 (ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) in the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker than in normal cells and is severely disrupted by large tubular structures and organelles; (iii) the pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) the anc-1 mutant with mislocalized pm5 cell nuclei occasionally shows an isthmus phenotype similar to that of pha-2 worms.     

The diversity of Bt resistance genes in species of Lepidoptera

Although the mode of action of Cry1A toxins produced by Bacillus thuringiensis is fairly well understood, knowledge of the molecular mechanisms by which lepidopteran species have evolved resistance to them is still in its infancy. The most common type of resistance has been called "Mode 1" and is characterized by recessive inheritance, >500-fold resistance to and reduced binding by at least one Cry1A toxin, and negligible cross-resistance to Cry1C. In three lepidopteran species, Heliothis virescens, Pectinophora gossypiella, and Helicoverpa armigera, Mode 1 resistance is caused by mutations in a toxin-binding 12-cadherin-domain protein expressed in the larval midgut. These mutations all interrupt the primary sequence of the protein and prevent its normal localization in the membrane, presumably removing a major toxic binding target of the Cry1A toxins. In Plutella xylostella, however, Mode 1 resistance appears to be caused by a different genetic mechanism, as Cry1A resistance is unlinked to the cadherin gene. Mapping studies in H. virescens have detected an additional major Cry1A resistance gene, which on the basis of comparative linkage mapping is distinct from the one in P. xylostella. An additional resistance mechanism supported by genetic data involves a protoxin-processing protease in Plodia interpunctella, and this is likely to be different from the genes mapped in Plutella and Heliothis. Thus, resistance to Cry1A toxins in species of Lepidoptera has a complex genetic basis, with at least four distinct, major resistance genes of which three are mapped in one or more species. The connection between resistance genes and the mechanisms they encode remains a challenging task to elucidate.     

A new class of tetraspanins in fungi

Tetraspanins are animal proteins involved in membrane complexes that are involved in cell adhesion, differentiation, and motility. The PLS1 gene from rice blast fungus Magnaporthe grisea encodes a protein (Pls1p) structurally related to tetraspanins that is required for pathogenicity. In Botrytis cinerea public sequences, we identified an EST homologous to PLS1. Using degenerated oligonucleotides, we amplified sequences homologous to PLS1 in fungi Colletotrichum lindemuthianum and Neurospora crassa. Analysis of N. crassa and M. grisea genome sequences revealed the presence of a single tetraspanin gene. Thus, fungi differ from animals, which contain between 20 and 37 paralogous tetraspanin genes. Fungal proteins encoded by BcPLS1, ClPLS1, and NcPLS1 display all the structural hallmarks of tetraspanins (predicted topology with four transmembrane domains, extra- and intracellular loops; conserved cysteine-based patterns in second extracellular loop). Phylogenetic analysis suggests that these genes define a new family of orthologous genes encoding fungal-specific tetraspanins.     

Glorund interactions in the regulation of gurken and oskar mRNAs

Precise temporal and spatial regulation of gene expression during Drosophila oogenesis is essential for patterning the anterior-posterior and dorsal-ventral body axes. Establishment of the anterior-posterior axis requires posterior localization and translational control of both oskar and nanos mRNAs. Establishment of the dorsal-ventral axis depends on the precise restriction of gurken mRNA and protein to the dorsal-anterior corner of the oocyte. We have previously shown that Glorund, the Drosophila hnRNP F/H homolog, contributes to anterior-posterior axis patterning by regulating translation of nanos mRNA, through a direct interaction with its 3′ untranslated region. To investigate the pleiotropy of the glorund mutant phenotype, which includes dorsal-ventral and nuclear morphology defects, we searched for proteins that interact with Glorund. Here we show that Glorund is part of a complex containing the hnRNP protein Hrp48 and the splicing factor Half-pint and plays a role both in mRNA localization and nurse cell chromosome organization, probably by regulating alternative splicing of ovarian tumor. We propose that Glorund is a component of multiple protein complexes and functions both as a translational repressor and splicing regulator for anterior-posterior and dorsal-ventral patterning.     

Taenia asiatica and Taenia saginata: genetic divergence estimated from their mitochondrial genomes

We conducted a differential identification of Taenia asiatica and Taenia saginata, through the mapping of mitochondrial genomes and the sequencing of the cox1 and cob genes. The entire mitochondrial genomes of T. asiatica and T. saginata were amplified by long-extension PCR and cloned; each was approximately 14 kb in size. Restriction maps of T. asiatica and T. saginata mitochondrial genomes were then constructed using 13 restriction enzymes. The resulting restriction patterns enable us to estimate their genetic divergence at 4.8%. The actual sequence divergence was computed 4.5% from the cox1 gene, and 4.1% from the cob gene. These results support the designation of T. asiatica as a separate species from T. saginata.     

Allele-mining of rice blast resistance genes at AC134922 locus

The AC134922 locus is one of the most rapidly evolving nucleotide binding site-leucine-rich repeat (NBS-LRR) gene family in rice genome. Six rice blast resistance (R) genes have been cloned from this locus and other two resistance candidate genes, Pi34 and Pi47, are also mapped to this complex locus. Therefore, it seems that more functional R genes could be identified from this locus. In this study, we cloned 22 genes from 12 cultivars based on allele-mining strategy at this locus and identified 6 rice blast R genes with 4 of them recognizing more than one isolates. Our result suggests that gene stacking might be the evolutionary strategy for complex gene locus to interact with rapidly evolving pathogens, which might provide a potential way for the cloning of durable resistance genes. Moreover, the mosaic structure and ambiguous ortholog/paralog relationships of these homologous genes, caused by frequent recombination and gene conversion, indicate that multiple alleles of this complex locus may serve as a reservoir for the evolutionary novelty of these R genes.     

The Caenorhabditis elegans homolog of the putative prostate cancer susceptibility gene ELAC2, hoe-1, plays a role in germline proliferation

The potential prostate cancer susceptibility gene ELAC2 has a Caenorhabditis elegans homolog (which we call hoe-1, for homolog of ELAC2). We have explored the biological role of this gene using RNAi to reduce gene activity. We found that worms subjected to hoe-1 RNAi are slow-growing and sterile. The sterility results from a drastic reduction in germline proliferation and cell-cycle arrest of germline nuclei. We found that hoe-1 is required for hyperproliferation phenotypes seen with mutations in three different genes, suggesting hoe-1 may be generally required for germline proliferation. We also found that reduction of hoe-1 by RNAi suppresses the multivulva (Muv) phenotype resulting from activating mutations in ras and that this suppression is likely to be indirect. This is the first demonstration of a biological role for this class of proteins in a complex eukaryote and adds important information when considering the role of ELAC2 in prostate cancer.