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PubMed2004

Unusual microbial xylanases from insect guts.

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Amino Acid Sequence↗

PubMed2006

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome.

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.

Animals↗

PubMed2003

Genomes at the interface between bacteria and organelles.

The topic of the transition of the genome of a free-living bacterial organism to that of an organelle is addressed by considering three cases. Two of these are relatively clear-cut as involving respectively organisms (cyanobacteria) and organelles (plastids). Cyanobacteria are usually free-living but some are involved in symbioses with a range of eukaryotes in which the cyanobacterial partner contributes photosynthesis, nitrogen fixation, or both of these. In several of these symbioses the cyanobacterium is vertically transmitted, and in a few instances, sufficient unsuccessful attempts have been made to culture the cyanobiont independently for the association to be considered obligate for the cyanobacterium. Plastids clearly had a cyanobacterial ancestor but cannot grow independently of the host eukaryote. Plastid genomes have at most 15% of the number of genes encoded by the cyanobacterium with the smallest number of genes; more genes than are retained in the plastid genome have been transferred to the eukaryote nuclear genome, while the rest of the cyanobacterial genes have been lost. Even the most cyanobacteria-like plastids, for example the "cyanelles" of glaucocystophyte algae, are functionally and genetically very similar to other plastids and give little help in indicating intermediates in the evolution of plastids. The third case considered is the vertically transmitted intracellular bacterial symbionts of insects where the symbiosis is usually obligate for both partners. The number of genes encoded by the genomes of these obligate symbionts is intermediate between that of organelles and that of free-living bacteria, and the genomes of the insect symbionts also show rapid rates of sequence evolution and AT (adenine, thymine) bias. Genetically and functionally, these insect symbionts show considerable similarity to organelles.

Animals↗

PubMed2001

Determination of Wolbachia genome size by pulsed-field gel electrophoresis.

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.

Chromosomes, Bacterial↗

PubMed1995

Genomic organization and expression of a trypsin gene from the spruce budworm, Choristoneura fumiferana.

A 7 kb (kilobase) genomic fragment containing a trypsin gene from the spruce budworm Choristoneura fumiferana was isolated and sequenced. The coding sequence is interrupted by two introns; these occur at the same positions as the first two introns of mammalian trypsin genes. The three exons encode 256 amino acids. The deduced protein sequence displays all of the structural features that characterize trypsin enzymes in other eukaryotic organisms. Genomic Southern hybridization showed that there is only one copy of the trypsin gene in the Choristoneura genome. This gene is expressed in the insect midgut, where the pH is extremely high. The complete lack of Lysine residues may be an adaptation to the high pH conditions. Extensive sequencing of the flanking regions did not reveal the presence of any linked trypsin-encoding genes. Instead, several short repetitive sequences and a sequence homologous to a Drosophila reverse transcriptase gene was identified in this genomic region.

Amino Acid Sequence↗

PubMed2003

Taste, movement, and death: varying effects of new prospero mutants during Drosophila development.

The PGal4 transposon inserted upstream of the pan-neural gene prospero (pros) causes several neural and behavioral defects in the Voila(1) strain. The precise excision of the transposon simultaneously rescued all these defects whereas its unprecise excision created new pros(V) alleles, including the null allele pros(V17). Here, we describe the relationship between the genetic structure of pros locus, larval locomotion, and larval gustatory response. These two behaviors showed varying degrees of variation depending upon the pros allele. We also found a good relation between behavioral alteration, the level of Pros protein in the embryo, and the degree of disorganization in the larval neuromuscular junction. These data suggest that the complete development of the nervous system requires a full complement of Pros, and that a gradual decrease in the levels of this protein can proportionally alter the development and the function of the nervous system.

Alleles↗

PubMed1999

Structural and functional characterization of the Drosophila glycogen phosphorylase gene.

We identified a P element insertional mutant of the Drosophila glycogen phosphorylase (DGPH) gene. Glycogen phosphorylase protein concentration and enzyme activity are decreased while glycogen content is increased in flies homozygous for the mutant allele. The DGPH gene has been cloned and sequenced; its open reading frame codes for a protein of 844 amino acids with a predicted molecular mass of 97 kDa. Comparison of the conceptual amino acid sequence of the Drosophila glycogen phosphorylase with glycogen phosphorylase sequences from other organisms shows a high degree of homology to mammalian enzymes. All the residues of the allosteric effector binding sites, the active site, and the site of phosphorylation are exactly conserved, but some of the residues of the glycogen storage site are not.

Amino Acid Sequence↗

PubMed1996

Isolation of a gene (DLG3) encoding a second member of the discs-large family on chromosome 17q12-q21.

The discs-large family is a collection of proteins that have a common structural organization and are thought to be involved in signal transduction and mediating protein-protein interactions at the cytoplasmic surface of the cell membrane. The defining member of this group of proteins is the gene product of the Drosophila lethal (1) discs large (dlg) 1 locus, which was originally identified by the analysis of recessive lethal mutants. Germline mutations in dlg result in loss of apical-basolateral polarity, disruption of normal cell-cell adhesion, and neoplastic overgrowth of the imaginal disc epithelium. We have isolated and characterized a novel human gene, DLG3, that encodes a new member of the discs-large family of proteins. The putative DLG3 gene product has a molecular weight of 66 kDa and contains a discs-large homologous region, a src oncogene homology motif 3, and a domain with homology to guanylate kinase. The DLG3 gene is located on chromosome 17, in the same segment, 17q12-q21, as the related gene, DLG2. The products of the DLG2 and DLG3 genes show 36% identity and 58% similarity to each other, and both show nearly 60% sequence similarity to p55, an erythroid phosphoprotein that is a component of the red cell membrane. We suggest that p55, DLG2, and DLG3 are closely related members of a gene family, whose protein products have a common structural organization and probably a similar function.

Amino Acid Sequence↗

PubMed1997

Genomic structure, evolution, and expression of human FLII, a gelsolin and leucine-rich-repeat family member: overlap with LLGL.

The Drosophila melanogaster flightless-I gene is involved in cellularization processes in early embryogenesis and in the structural organization of indirect flight muscle. The encoded protein contains a gelsolin-like actin binding domain and an N-terminal leucine-rich repeat protein-protein interaction domain. The homologous human FLII gene encodes a 1269-residue protein with 58% amino acid sequence identity and is deleted in Smith-Magenis syndrome. We have cloned the FLII gene and determined its nucleotide sequence (14.1 kb). FLII has 29 introns, compared with 13 in Caenorhabditis elegans and 3 in D. melanogaster. The positions of several introns are conserved in FLII-related genes and in the domains and subdomains of the gelsolin-like regions giving indications of gelsolin gene family evolution. In keeping with its function in indirect flight muscle in Drosophila, the human FLII gene was most highly expressed in muscle. The FLII gene lies adjacent to LLGL, the human homologue of the D. melanogaster tumor suppressor gene lethal(2) giant larvae. The 3' end of the FLII transcript overlaps the 3' end of the LLGL transcript, and the corresponding mouse genes Fliih and Llglh also overlap. The overlap region contains poly(A) signals for both genes and is strongly conserved between human and mouse.

Animals↗

PubMed2002

Novel paralogy relations among human chromosomes support a link between the phylogeny of doublesex-related genes and the evolution of sex determination.

Recent advances in the evolutionary genetics of sex determination indicate that DMRT1 may be a vertebrate equivalent of the Drosophila melanogaster master sex regulator gene, doublesex. The role of DMRT1 seems to be confined to some aspects of male sex differentiation, whereas in Drosophila, doublesex has wider developmental effects in both sexes. This suggests other homologs of doublesex may exist in the vertebrate genome and encode sex-specific functions not displayed by DMRT1. We identified and characterized five novel human DM genes, distinct from previously described family members. Human DM genes map to three well-defined regions of chromosomes 1, 9, and 19 (one gene on chromosome 19 having an additional homolog on chromosome X). We collated data indicating these chromosomal regions harbor multiple syntenic genes sharing highly specific paralogy relations, suggesting that they arose early during vertebrate evolution. The 9p21-p24.3 bands represent the ancestral copy and harbor closely linked DM genes that may reflect the overall diversity of the fruit fly DM gene family. The human genome contains a small number of potential doublesex homologs that may be involved in human sexual development. Identifying highly conserved chromosomal regions, such as distal 9p, is an important tool to trace complex ancient evolutionary processes inaccessible by other approaches.

Amino Acid Sequence↗

PubMedDate not supplied

Enhanced functional annotation of protein sequences via the use of structural descriptors.

In order to circumvent limitations of sequence based methods in the process of making functional predictions for proteins, we have developed a methodology that uses a sequence-to-structure-to-function paradigm. First, an approximate three-dimensional structure is predicted. Then, a three-dimensional descriptor of the functional site, termed a Fuzzy Functional Form, or FFF, is used to screen the structure for the presence of the functional site of interest (Fetrow et al., 1998; Fetrow and Skolnick, 1998). Previously, a disulfide oxidoreductase FFF was developed and applied to predicted structures obtained from a small structural database. Here, using a substantially larger structural database, we expand the analysis of the disulfide oxidoreductase FFF to the B. subtilis genome. To ascertain the performance of the FFF, its results are compared to those obtained using both the sequence alignment method BLAST and three local sequence motif databases: PRINTS, Prosite, and Blocks. The FFF method is then compared in detail to Blocks and it is shown that the FFF is more flexible and sensitive in finding a specific function in a set of unknown proteins. In addition, the estimated false positive rate of function prediction is significantly lower using the FFF structural motif, rather than the standard sequence motif methods. We also present a second FFF and describe a specific example of the results of its whole-genome application to D. melanogaster using a newer threading algorithm. Our results from all of these studies indicate that the addition of three-dimensional structural information adds significant value in the prediction of biochemical function of genomic sequences.

Algorithms↗

PubMed1995

Gene 2 of the sigma rhabdovirus genome encodes the P protein, and gene 3 encodes a protein related to the reverse transcriptase of retroelements.

The nucleotide sequence of the genes 2 and 3 of the Drosophila rhabdovirus sigma was determined from cDNAs to viral genome and poly(A)+ mRNAs. Gene 2 comprises 1032 nucleotides and contains a long ORF encoding a molecular weight 35,208 polypeptide present in infected cells and in virions which migrates in SDS-PAGE as a doublet of M(r) about 60 kDa. The distribution of acidic charges as well as the electrophoretic properties of the protein are characteristic of the rhabdovirus P proteins. Gene 3 comprises 923 nucleotides and contains a long ORF capable of coding a polypeptide of 298 amino acids of MW 33,790. The putative protein (PP3) is similar in size to a minor component of the virions. Computer analysis shows that the sequence of PP3 contains three motifs related to the conserved motifs of reverse transcriptases.

Amino Acid Sequence↗

PubMed1998

Emergence of epidemic O'nyong-nyong fever in Uganda after a 35-year absence: genetic characterization of the virus.

O'nyong-nyong (ONN) virus is an alphavirus (family Togaviridae, genus Alphavirus) classified in the Semliki Forest virus (SFV) antigenic complex. ONN was initially isolated in northern Uganda in 1959 during the early stages of an explosive arbovirus epidemic in which > 2 million cases were reported. No additional epidemics or human isolations of ONN were reported until 1996, when it was isolated from an epidemic in southern Uganda. We report the complete nucleotide and deduced amino acid sequence of one of these 1996-1997 ONN isolates (SG650) and that of the related alphavirus Igbo Ora virus. The data indicate that the recent ONN virus isolate is closely related to the previously published ONN strain isolated in 1959. In addition, phylogenetic analysis of the sequence data reveals that Igbo Ora virus, previously thought to be a separate virus closely related to ONN and Chikungunya (CHIK), clearly is a strain of ONN. The sequence data also reveal that unlike the published ONN (1959) sequence, all ONN strains from the 1996-1997 epidemic possess a stop codon at the nsp3-nsp4 junction.

Adolescent↗

PubMed2004

Complete nucleotide sequence of Iranian tomato yellow leaf curl virus isolate: further evidence for natural recombination amongst begomoviruses. Brief Report.

Complete nucleotide sequence of the Iranian strain of tomato yellow leaf curl virus (TYLCV-IR) was determined and compared with some begomoviruses. The complete sequence of TYLCV-IR clustered together with TYLCV and TYLCV-MId from Israel. A similar relationship holds when the deduced amino acid sequences of V1, V2, C2 and C3 and nucleotide sequences of IR, and RIR were compared. In contrast, phylogenetic analyses of amino acid sequences of C4, C1, and nucleotide sequences of LIR revealed that TYLCV-IR clustered with TLCIRV and two Indian species: ToLCBV- [Ban4], and ToLCKV. The phylogenetic analyses, Recombination Detection Program analyses, and sequence alignment survey provided evidence of the occurrence of recombination between an Israeli TYLCV-MId, as major parent, and TLCIRV, as minor parent. In this recombination event, a region (from nt 2149 to 2766) of TYLCV-MId genome were replaced with corresponding genome sequences of TLCIRV (RDP P-value = 5.976 x 10(-72)), which include LIR, C4, and N-terminal of C1. Infectivity of the cloned TYLCV-IR genome was demonstrated by successful agroinoculation of tomato ( Lycopersicon esculentum) and other plant species. The disease was transmitted by the natural vector Bemisia tabaci from agroinoculated plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-IR consists of only one circular single-stranded DNA molecule.

Animals↗

PubMed1995

Molecular cloning and chromosomal localization of a pseudogene related to the human acyl-CoA binding protein/diazepam binding inhibitor.

The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5' ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-related sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1.

Amino Acid Sequence↗

PubMed1994

Cloning, characterization, and genetics of the juvenile hormone esterase gene from Heliothis virescens.

The gene for juvenile hormone esterase (JHE) was cloned from Heliothis virescens (Lepidoptera: Noctuidae). A genomic library was constructed from embryonic DNA and screened with a homologous N-terminal probe from the JHE cDNA. Five genomic clones were isolated and analyzed by dot blot hybridization using regions of the JHE cDNA as probes. Clone C hybridized to both 5' and 3' probes from the JHE cDNA, suggesting that clone C contains both ends of JHE gene. This was verified by sequencing the ends of the JHE gene from clone C using primers from both the 5' and 3' ends of the JHE cDNA. Additional sequencing and restriction mapping were used to characterize the gene. The gene is c. 8 kb long and contains four introns with consensus intron-exon junctions. One of the introns is relatively large (4 kb) and is situated near the extreme 5' end of the gene. Genetic analysis of RFLP variation in interspecific and intraspecific crosses shows that the JHE locus is single-copy with no closely related paralogs and is autosomally encoded in Heliothis. Therefore the developmental pattern of expression of this gene and the previously documented sequence variation in cDNA clones is not explainable by reference to a JHE gene family with distinct structural loci for the different forms.

Animals↗

PubMed2005

Melanogenesis and associated cytotoxic reactions: applications to insect innate immunity.

Insects transmit the causative agents for such debilitating diseases as malaria, lymphatic filariases, sleeping sickness, Chagas' disease, leishmaniasis, river blindness, Dengue, and yellow fever. The persistence of these diseases provides testimony to the genetic capacity of parasites to evolve strategies that ensure their successful development in two genetically diverse host species: insects and mammals. Current efforts to address the problems posed by insect-borne diseases benefit from a growing understanding of insect and mammalian immunity. Of considerable interest are recent genomic investigations that show several similarities in the innate immune effector responses and associated regulatory mechanisms manifested by insects and mammals. One notable exception, however, is the nearly universal presence of a brown-black pigment accompanying cellular innate immunity in insects. This response, which is unique to arthropods and certain other invertebrates, has focused attention on the elements involved in pigment synthesis as causing or contributing to the death of the parasite, and has even prompted speculation that the enzyme cascade mediating melanogenesis constitutes an ill-defined recognition mechanism. Experimental evidence defining the role of melanin and its precursors in insect innate immunity is severely lacking. A great deal of what is known about melanogenesis comes from studies of the process occurring in mammalian systems, where the pigment is synthesized by such diverse cells as those comprising portions of the skin, hair, inner ear, brain, and retinal epithelium. Fortunately, many of the components in the metabolic pathways leading to the formation of melanin have been found to be common to both insects and mammals. This review examines some of the factors that influence enzyme-mediated melanogenic responses, and how these responses likely contribute to blood cell-mediated, target-specific cytotoxicity in immune challenged insects.

Animals↗

PubMed2006

Detection and characterisation of three novel species of reovirus (Reoviridae), isolated from geographically separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) on Orkney.

Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles in order to investigate the prevalence of virus pathogens. Reoviruses were isolated in 11 of the 13 winter moth populations sampled, with 3 novel species being detected. Two species of Cypoviridae (CPV) were isolated, Operophtera brumata CPV18 and O. brumata CPV19, with one host population suffering 46% infection prevalence of OpbuCPV19. A third virus, O. brumata Reovirus (OpbuRV), was isolated from both winter moth and a hymenopteran parasitoid wasp, Phobocampe tempestiva, which is abundant in these populations. This was identified as a non-occluded reovirus, which was clearly able to infect and persist in both the lepidopteran and the hymenopteran host. The genomes of the three viruses were characterised using gel electrophoresis and the virus structure was investigated using transmission electron microscopy. The relationship of these viruses with a baculovirus that also infects winter moth, OpbuNPV, was investigated, as well as the association of OpbuRV with P. tempestiva. The detection of such viruses is discussed with reference to studies of similar viruses in other lepidopteran and hymenopteran host systems.

Animals↗