家蚕核型多角体病毒水平转移基因分析

为了探讨杆状病毒基因组的遗传进化模式,文章利用家蚕核型多角体病毒(BmNPV)和其宿主家蚕全基因组数据,进行了全基因组的同源性搜索和系统进化分析,结果显示,BmNPV的几丁质酶(Chi)基因、凋亡抑制蛋白3(IAP3)基因和尿苷二磷酸葡萄糖转移酶(UGT)基因为水平转移基因。这3个基因都来源于其宿主昆虫。通过核苷酸组成、密码子偏好性、选择压力等基因特征分析,发现BmNPV水平转移基因与其基因组序列存在明显差异,进一步验证水平转移基因的外源性。对3个水平转移基因的功能分析发现它们有利于杆状病毒在宿主昆虫中的侵染与繁殖,并提高杆状病毒在昆虫中的生存能力。     

Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.     

Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.     

昆虫杆状病毒几丁质酶及其应用研究进展

杆状病毒几丁质酶基因（chitinase,,ChiA）是晚期表达的非必需基因,高度保守。表达产物几丁质酶分为3个区：N-端信号肽区,中部酶活性区和C-末端酶内质网结合区。该酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的几丁质,促进虫体液化;作为组织蛋白酶原（pro-V-Cath）的分子伴侣,参与其加工和运输过程; 影响多角体的形成,并与细胞的裂解有关;还与病毒侵染机制相关联。杆状病毒ChiA与细菌ChiA源于共同的祖先,而昆虫ChiA则可能直接来自杆状病毒。在害虫生物防治中,杆状病毒ChiA可直接作为杀虫剂,或作为苏云金杆菌和杆状病毒等微生物杀虫剂的增效剂使用;杆状病毒ChiA可转入植物,获得具有持续杀虫及抗病活性的转基因植物;将杆状病毒ChiA的内质网定位序列删除、突变,或在病毒基因组中插入外源ChiA,重组病毒的杀虫活性增强。通过基因工程手段,删除病毒基因组ChiA或V-Cath,可改善杆状病毒表达系统对分泌蛋白和膜结合蛋白的表达。     

The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a functional exochitinase that plays a role in the chitin degradation during the molting process

The silkworm, Bombyx mori, has been recently demonstrated to contain a bacterial-type chitinase gene (BmChi-h) in addition to a well-characterized endochitinase gene (BmChitinase). The deduced amino acid sequence of BmChi-h showed extensive structural similarities with chitinases from bacteria such as Serratia marcescens chiA and baculoviruses (v-CHIA). Bacterial-type chitinase genes have not been found from any eukaryotes and viruses except for lepidopteran insects and lepidopteran baculoviruses. Thus, it was suggested that BmChi-h may be derived from a bacterial or baculovirus chitinase gene via horizontal gene transfer. In this report, we investigated the biological function of BmChi-h. Our enzymological study indicated that a chitinase encoded by BmChi-h has exo-type substrate preference, which is the same as S. marcescens chiA and v-CHIA, and different from BmChitinase, which has endo-type substrate preference. An immunohistochemical study revealed that BmChi-h localizes in the chitin-containing tissues during the molting stages, indicating that it plays a role in chitin degradation during molting. These results suggest that BmChi-h (exochitinase) and BmChitinase (endochitinase) may catalyze a native chitin by a concerted mechanism. Cloning and comparison of BmChi-h orthologues revealed that bacterial-type chitinase genes are highly conserved among lepidopteran insects, suggesting that the utilization of a bacterial-type chitinase during the molting process may be a general feature of lepidopteran insects.     

Wolbachia screening in spiders and assessment of horizontal transmission between predator and prey

Recent studies have revealed that the prevalence of Wolbachia in arthropods is attributable not only to its vertical transmission, but also to its horizontal transfer. In order to assess the horizontal transmission of Wolbachia between predator and prey, arthropods belonging to 11 spider families and six insect families were collected in the same field of rice. The distribution of Wolbachia in these arthropods was detected by diagnostic PCR amplification of the wsp (Wolbachia outer surface protein gene) and 16S rDNA genes. Nurscia albofasciata Strand (Araneae: Titanoecidae), Propylea japonica Thunberg (Coleoptera: Coccinellidae), Paederus fuscipes Curtis (Coleoptera: Staphylinidae), and Nilaparvata lugens Stal (Homoptera: Delphacidae) were infected with Wolbachia. This is the first report of infection of N. albofasciata and P. fuscipes by Wolbachia. No direct evidence indicated the existence of horizontal transmission of Wolbachia between predator and prey.     

与宿主昆虫液化相关的杆状病毒基因及其蛋白

昆虫被杆状病毒感染后会发生液化现象,这有利于病毒向周围环境扩散。目前在杆状病毒苜蓿银纹夜蛾核型多角体病毒NPV和GV中,发现与昆虫宿主液化相关的基因有组织蛋白酶基因V-cath基因和几丁质酶基因。V-cath基因表达产物在苜蓿银纹夜蛾多角体病毒(AcMNPV)中能特异性降解昆虫细胞内的肌动蛋白。几丁质酶不仅参与了虫体体表面几丁质的降解,同时还参与V-CATH蛋白前体的加工过程,起分子伴侣的作用。对家蚕核型多角体病毒(BmNPV)的研究表明其FP25K基因表达产物通过影响组织蛋白酶的释放与分泌而参与虫体液化。简要综述了此3种基因及其表达产物的结构、功能与特性,并讨论了它们在生产上的应用前景。     

Evolution of DNA Polymerase Families: Evidences for Multiple Gene Exchange Between Cellular and Viral Proteins

A phylogenetic analysis of the five major families of DNA polymerase is presented. Viral and plasmid sequences are included in this compilation along with cellular enzymes. The classification by Ito and Braithwaite (Ito and Braithwaite 1991) of the A, B, C, D, and X families has been extended to accommodate the ``Y family' of DNA polymerases that are related to the eukaryotic RAD30 and the bacterial UmuC gene products. After analysis, our data suggest that no DNA polymerase family was universally conserved among the three biological domains and no simple evolutionary scenario could explain that observation. Furthermore, viruses and plasmids carry a remarkably diverse set of DNA polymerase genes, suggesting that lateral gene transfer is frequent and includes non-orthologous gene displacements between cells and viruses. The relationships between viral and host genes appear very complex. We propose that the gamma DNA polymerase of the mitochondrion replication apparatus is of phage origin and that this gene replaced the one in the bacterial ancestor. Often there was no obvious relation between the viral and the host DNA polymerase, but an interesting exception concerned the family B enzymes: in which ancient gene exchange can be detected between the viruses and their hosts. Additional evidence for horizontal gene transfers between cells and viruses comes from an analysis of the small damage-inducible DNA polymerases. Taken together, these findings suggest a complex evolutionary history of the DNA replication apparatus that involved significant exchanges between viruses, plasmids, and their hosts.     

Baculoviruses and apoptosis: the good, the bad, and the ugly

Since 1991, when a baculovirus was first shown to inhibit apoptosis of its host insect cells, considerable contributions to our knowledge of apoptosis have arisen from the study of these viruses and the anti-apoptotic genes they encode. Baculovirus anti-apoptotic genes include p35, which encodes the most broadly acting caspase inhibitor protein known, and iap (inhibitor of apoptosis) genes, which were the first members of an evolutionarily conserved gene family involved in regulation of apoptosis and cytokinesis in organisms ranging from yeast to humans. Baculoviruses also provide an ideal system to study the effects of an apoptotic response on viral pathogenesis in an animal host. In this review, I discuss a number of interesting recent developments in the areas of apoptotic regulation by baculoviruses and the effects of apoptosis on baculovirus replication and pathogenesis.     

Recurrent horizontal transfer of bacterial toxin genes to eukaryotes

In this work, we report likely recurrent horizontal (lateral) gene transfer events of genes encoding pore-forming toxins of the aerolysin family between species belonging to different kingdoms of life. Clustering based on pairwise similarity and phylogenetic analysis revealed several distinct aerolysin sequence groups, each containing proteins from multiple kingdoms of life. These results strongly support at least six independent transfer events between distantly related phyla in the evolutionary history of one protein family and discount selective retention of ancestral genes as a plausible explanation for this patchy phylogenetic distribution. We discuss the possible roles of these proteins and show evidence for a convergent new function in two extant species. We hypothesize that certain gene families are more likely to be maintained following horizontal gene transfer from commensal or pathogenic organism to its host if they 1) can function alone; and 2) are immediately beneficial for the ecology of the organism, as in the case of pore-forming toxins which can be utilized in multicellular organisms for defense and predation.     

A Survey of the bacteriophage WO in the endosymbiotic bacteria Wolbachia

Bacteriophages are common viruses infecting prokaryotes. In addition to their deadly effect, phages are also involved in several evolutionary processes of bacteria, such as coding functional proteins potentially beneficial to them, or favoring horizontal gene transfer through transduction. The particular lifestyle of obligatory intracellular bacteria usually protects them from phage infection. However, Wolbachia, an intracellular alpha-proteobacterium, infecting diverse arthropod and nematode species and best known for the reproductive alterations it induces, harbors a phage named WO, which has recently been proven to be lytic. Here, phage infection was checked in 31 Wolbachia strains, which induce 5 different effects in their hosts and infect 25 insect species and 3 nematodes. Only the Wolbachia infecting nematodes and Trichogramma were found devoid of phage infection. All the 25 detected phages were characterized by the DNA sequence of a minor capsid protein gene. Based on all data currently available, phylogenetic analyses show a lack of congruency between Wolbachia or insect and phage WO phylogenies, indicating numerous horizontal transfers of phage among the different Wolbachia strains. The absence of relation between phage phylogeny and the effects induced by Wolbachia suggests that WO is not directly involved in these effects. Implications on phage WO evolution are discussed.     

Horizontal transfer, not duplication, drives the expansion of protein families in prokaryotes

Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus), average-sized genomes (Bacillus, Enterobacteriaceae), and large genomes (Pseudomonas, Bradyrhizobiaceae) to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes--xenologs--persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes--paralogs--are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein-protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life's biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families.     

Temporal and spatial expression of mRNAs encoding pathogenesis-related proteins during ethylene-promoted leaflet abscission in Sambucus nigra*

Differential screening of a cDNA library generated from RNA extracted from ethylene-treated leaflet abscission zones of Sambucus nigra resulted in the isolation of 20 abscission-related clones. These clones could be grouped into seven families. Sequencing of members of these families revealed that the majority encoded pathogenesis-related (PR) proteins, and these could be identified by sequence homology as a polyphenol oxidase (PPO), a PR-1 type protein, a Chial type chitinase, a PR-4 type protein similar to the potato win peptides, a PR-6 type proteinase inhibitor, a Chia4 type chitinase and a metallothionein-like protein (Coupe, Taylor & Roberts 1995, Planta 197, 442–447). Northern analysis revealed that these mRNAs were not expressed in freshly excised material but accumulated primarily in the abscission zone tissue after 18 h of exposure to ethylene at a time when abscission of the leaflet explants had reached 70%. Expression of the PPO and the Chia4-type chitinase was ethylene-dependent while that of the PR-4 type was up-regulated in the abscission zone tissue in the absence of the gas. The characterization of these mRNAs and their encoded proteins is presented and their possible roles during abscission are discussed.     

昆虫几丁质酶基因家族功能研究进展

昆虫几丁质酶的研究历史很长,研究内容也非常广泛;但仅局限于传统的方法研究某个昆虫单个基因的功能及应用。近年来,随着生物信息学和RNAi技术在生命科学研究工作中的广泛应用,在昆虫几丁质酶的研究方面也取得了较大进展。本文主要以生物信息学在几丁质酶家族基因鉴定和分类研究过程中的应用,以及利用RNAi技术分析几丁质酶不同家族成员在赤拟谷盗和褐飞虱两种昆虫生长发育中的功能比较分析,对全面认识昆虫几丁质酶基因家族功能和作用机理,并利用几丁质酶基因防治害虫奠定良好的基础。     

Detection of atypical genes in virus families using a one-class SVM

Background

The diversity of viruses, the absence of universally common genes in them, and their ability to act as carriers of genetic material make assessment of evolutionary paths of viral genes very difficult. One important factor contributing to this complexity is horizontal gene transfer.

Results

We explore the possibility for the systematic identification of atypical genes within virus families, including viruses whose genome is not encoded by a double-stranded DNA. Our method is based on gene statistical features that differ in genes that were subject of recent horizontal gene transfer from those of the genome in which they are observed. We employ a one-class SVM approach to detect atypical genes within a virus family basing of their statistical signatures and without explicit knowledge of the source species. The simplicity of the statistical features used makes the method applicable to various viruses irrespective of their genome size or type.

Conclusions

On simulated data, the method can robustly identify alien genes irrespective of the coding nucleic acid found in a virus. It also compares well to results obtained in related studies for double-stranded DNA viruses. Its value in practice is confirmed by the identification of isolated examples of horizontal gene transfer events that have already been described in the literature. A Python package implementing the method and the results for the analyzed virus families are available at http://svm-agp.bioinf.mpi-inf.mpg.de.     

Evolutionary genomics of mycovirus-related dsRNA viruses reveals cross-family horizontal gene transfer and evolution of diverse viral lineages

ABSTRACT: BACKGROUND: Double-stranded (ds) RNA fungal viruses are typically isometric single-shelled particles that are classified into three families, Totiviridae, Partitiviridae and Chrysoviridae, the members of which possess monopartite, bipartite and quadripartite genomes, respectively. Recent findings revealed that mycovirus-related dsRNA viruses are more diverse than previously recognized. Although an increasing number of viral complete genomic sequences have become available, the evolution of these diverse dsRNA viruses remains to be clarified. This is particularly so since there is little evidence for horizontal gene transfer (HGT) among dsRNA viruses. RESULTS: In this study, we report the molecular properties of two novel dsRNA mycoviruses that were isolated from a field strain of Sclerotinia sclerotiorum, Sunf-M: one is a large monopartite virus representing a distinct evolutionary lineage of dsRNA viruses; the other is a new member of the family Partitiviridae. Comprehensive phylogenetic analysis and genome comparison revealed that there are at least ten monopartite, three bipartite, one tripartite and three quadripartite lineages in the known dsRNA mycoviruses and that the multipartite lineages have possibly evolved from different monopartite dsRNA viruses. Moreover, we found that homologs of the S7 Domain, characteristic of members of the genus phytoreovirus in family Reoviridae are widely distributed in diverse dsRNA viral lineages, including chrysoviruses, endornaviruses and some unclassified dsRNA mycoviruses. We further provided evidence that multiple HGT events may have occurred among these dsRNA viruses from different families. CONCLUSIONS: Our study provides an insight into the phylogeny and evolution of mycovirus-related dsRNA viruses and reveals that the occurrence of HGT between different virus species and the development of multipartite genomes during evolution are important macroevolutionary mechanisms in dsRNA viruses.     

Chitinase genes in lake sediments of Ardley Island, Antarctica

A sediment core spanning approximately 1,600 years was collected from a lake on Ardley Island, Antarctica. The sediment core had been greatly influenced by penguin guano. Using molecular methods, the chitinolytic bacterial community along the sediment core was studied over its entire length. Primers targeting conserved sequences of the catalytic domains of family 18 subgroup A chitinases detected group A chitinases from a wide taxonomic range of bacteria. Using quantitative competitive PCR (QC-PCR), chitinase gene copies in each 1-cm section of the whole sediment column were quantified. QC-PCR determination of the chitinase gene copies indicated significant correlation with phosphorus and total organic carbon concentration, suggesting a historical connection between chitinase gene copies and the amount of penguin guano input into the lake sediment. Most of the chitinase genes cloned from the historic sediment core were novel. Analysis of the chitinase gene diversity in selected sediment layers and in the fresh penguin deposits indicated frequent shifts in the chitinolytic bacterial community over time. Sequence analysis of the 16S rRNA genes of chitinolytic bacteria isolated from the lake sediment revealed that the isolates belonged to Janthinobacterium species, Stenotrophomonas species of gamma-Proteobacteria, Cytophaga species of the Cytophaga-Flexibacter-Bacteroides group, and Streptomyces and Norcardiopsis species of Actinobacteria. Chitinase gene fragments were cloned and sequenced from these cultivated chitinolytic bacteria. The phylogeny of the chitinase genes obtained from the isolates did not correspond well to that of the isolates, suggesting acquisition via horizontal gene transfer.     

Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome

MOTIVATION: Multiple chitinases as well as lectins closely related to them have been characterized previously from many insect species and the corresponding genes/cDNAs have been cloned. However, the identification of the entire assortment of genes for chitinase family proteins and their differences in biochemical properties have not been carried out in any individual insect species. The completion of the entire DNA sequence of Drosophila melanogaster (fruit fly) genome and identification of open reading frames presents an opportunity to study the structures and functions of chitinase-like proteins, and also to identify new members of this family in DROSOPHILA: We are, therefore, interested in studying the functional genomics of chitinase-like gene families in insects. METHODS: We searched the Drosophila protein sequences database using fully characterized insect chitinase sequences and BLASTP software, identified all the putative chitinase-like proteins encoded in Drosophila genome, and predicted their structures using domain analysis tools. A phylogenetic analysis of the chitinase-like proteins from Drosophila and several other insect species was carried out. The structures of these chitinases were modeled using homology modeling software. RESULTS: Our analysis revealed the presence of 18 chitinase-like proteins in the Drosophila protein database. Among these are seven novel chitinase-like proteins that contain four signature amino acid sequences of chitinases belonging to family 18 glycosylhydrolases, including both acidic and hydrophobic amino acid residues critical for enzyme activity. All the proteins contain at least one catalytic domain with one having four catalytic domains. Phylogenetic analysis of chitinase-like proteins from Drosophila and other insects revealed an evolutionary relationship among all these proteins, which indicated gene duplication and domain shuffling to generate the observed diversity in the encoded proteins. Homology modeling showed that all the Drosophila chitinase-like proteins contain one or more catalytic domains with a (alpha/beta)8 barrel-like structure. Our results suggest that insects utilize multiple family 18 chitinolytic enzymes and also non-enzymatic chitinase-like proteins for degrading/remodeling/binding to chitin in different insect anatomical extracellular structures, such as the cuticle, peritrophic membrane, trachea and mouth parts during insect development, and possibly for other roles including chitin synthesis. AVAILABILITY: Perl program and supplementary material are available at http://www.ksu.edu/bioinformatics/supplementary.htm