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PCR primers for the amplification of four insect mitochondrial gene fragments.

Insect mitochondrial genome (mtDNA) analysis is a powerful tool for the study of population genetics and phylogenetics. In the past few years primer sequences for the PCR amplification of various insect mtDNA genes have been published. The objectives of this study were (1) present new primer sequences for six insect mitochondrial genes and (2) test primers designed in our laboratory and some previously published primers on a wide range of insects to determine if amplification of the target fragment could be obtained. The primers for the amplification of the two ribosomal RNA gene (16S and 12S rRNA) fragments are universal for insects and related groups; the primers for NADH5 and NADH4 dehydrogenase gene fragments and cytochrome c oxidase I gene fragment are applicable broadly.

Animals↗

Model systems to evaluate the use of transgenic haematophagous insects to deliver protective vaccines.

Insect vector control has proved an effective method for reducing the transmission of disease-causing organisms to human populations in many tropical countries. We are interested in employing direct genetic manipulation of insect vector genomes to use them in beneficial ways so as to have a profound and long-lasting effect on disease transmission. Our research focuses on assessing whether haematophagous insects may be used as a means to deliver protective proteins, such as an antimalarial vaccine, when they take a blood meal. The progress which has been made towards assessing this concept using a number of model systems is described.

Aedes↗

A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity.

We have previously cloned a cellulase [beta-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Ag-EGase I) belonging to glycoside hydrolase family (GHF) 45 from the mulberry longicorn beetle, Apriona germari. We report here the gene structure, expression and enzyme activity of an additional celluase (Ag-EGase II) from A. germari and also described the gene structure of Ag-EGase I. The Ag-EGase II gene spans 1033 bp and consisted of two introns and three exons coding for 239 amino acid residues. The 2713-bp-long genomic DNA of Ag-EGase I also consisted of two introns and three exons. The Ag-EGase II showed 61% protein sequence identity to Ag-EGase I and 51% to another beetle, Phaedon cochleariae, cellulase, belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase II. The Ag-EGase II has 14 conserved cysteine residues and three putative N-glycosylation sites. Northern blot analysis confirmed midgut-specific expression of Ag-EGase II, suggesting that the midgut is the prime site for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase II was expressed as a 36-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase II was approximately 812 U/mg of recombinant Ag-EGase II. The enzymatic properties of the purified recombinant Ag-EGase II showed the highest activity at 50 degrees C and pH 6.0, and were stable at 60 degrees C at least for 10 min.

Amino Acid Sequence↗

The gene search system: its application to functional genomics in Drosophila melanogaster.

In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been established, allowing identification of genes that may not be easily detectable by loss-of-function screening approaches. The conditional features of misexpression systems are especially useful for studying late-stage biological processes, such as those involving adult behavior or lifespan. The gene search system, incorporating a bidirectional misexpression vector, was used to screen for genes critical for longevity determination. We have identified several genes whose misexpression in adulthood extends the fly's lifespan. Phenotypic characterization of fly lines carrying a mis-expression vector, in conjunction with obtaining information about the genomic insertion sites, creates valuable resources for the systematic functional genomics in Drosophila.

Animals↗

Construction of a BAC library and generation of BAC end sequence-tagged connectors for genome sequencing of the African malaria mosquito Anopheles gambiae.

A Bacterial Artificial Chromosome (BAC) genomic DNA library of Anopheles gambiae, the major human malaria vector in sub-Saharan Africa, was constructed and characterized. This library (ND-TAM) is composed of 30,720 BAC clones in eighty 384-well plates. The estimated average insert size of the library is 133 kb, with an overall genome coverage of approximately 14-fold. The ends of approximately two-thirds of the clones in the library were sequenced, yielding 32,340 pair-mate ends. A statistical analysis (G-test) of the results of PCR screening of the library indicated a random distribution of BACs in the genome, although one gap encompassing the white locus on the X-chromosome was identified. Furthermore, combined with another previously constructed BAC library (ND-1), ~2,000 BACs have been physically mapped by polytene chromosomal in situ hybridization. These BAC end pair mates and physically mapped BACs have been useful for both the assembly of a fully sequenced A. gambiae genome and for linking the assembled sequence to the three polytene chromosomes. This ND-TAM library is now publicly available at both http://www.malaria.mr4.org/mr4pages/index.html/ and http://hbz.tamu.edu/, providing a valuable resource to the mosquito research community.

Animals↗

Codon usage and genome evolution.

The rates and patterns of evolution at silent sites in codons reveal much about the basic features of molecular evolution. Recent increases in the amount of sequence data available for various species and more precise knowledge of the chromosomal locations of those sequences, coming in particular from genome projects, reveal that some features of molecular evolution vary around the genome.

Animals↗

The mitochondrial genome of the entomophagous endoparasite Xenos vesparum (Insecta: Strepsiptera).

In this study, the nearly complete sequence (14,519 bp) of the mitochondrial DNA (mtDNA) of the entomophagous endoparasite Xenos vesparum (Insecta: Strepsiptera) is described. All protein coding genes (PCGs) are in the arrangement known to be ancestral for insects, but three tRNA genes (trnA, trnS(gcu), and trnL(uag)) have transposed to derived positions and there are three tandem copies of trnH, each of which is potentially functional. All of these rearrangements except for that of trnL(uag) is within the short span between nad3 and nad4 and there are numerous blocks of unassignable sequence in this region, perhaps as remnants of larger scale predisposing rearrangements. X. vesparum mtDNA nucleotide composition is strongly biased toward A and T, as is typical for insect mtDNAs. There is also a significant strand skew in the distribution of these nucleotides, with the J-strand being richer in A than T and in C than G, and the N-strand showing an opposite skew for complementary pairs of nucleotides. The hypothetical secondary structure of the LSU rRNA has also been reconstructed, obtaining a structural model similar to that of other insects.

Animals↗

Highly conserved piggyBac elements in noctuid species of Lepidoptera.

The piggyBac transposable element was originally discovered in a Trichoplusia ni cell line and nearly identical elements were subsequently discovered in the tephritid fly, Bactrocera dorsalis. This suggested the existence of piggyBac in additional insects and this study shows highly conserved, though not identical, piggyBac sequences in the noctuid species Heliocoverpa armigera, H. zea, and Spodoptera frugiperda, as well as new piggyBac sequences from the T. ni organismal genome. Genomic piggyBac elements could not be unambiguously identified in several other moth species indicating a discontinuous presence of piggyBac in the Lepidoptera. Most sequences have greater than 95% nucleotide identity to the original IFP2 piggyBac, except for a more diverged sequence in S. frugiperda, having approximately 78% identity. Variants of 1.3 and 0.8kb sequences found in both H. armigera and H. zea most likely became established by interbreeding, supporting the notion that the species are conspecific. None of the independent piggyBac sequences isolated from T. ni larval genomes are identical to IFP2, though all have an uninterrupted reading frame with the potential for encoding a functional transposase. The piggyBac sequences from T. ni and the Helicoverpa species, as well as those previously reported from B. dorsalis, all share three common nucleotide substitutions resulting in a single amino acid substitution in the transposase. This suggests that the original IFP2 piggyBac is a related variant of a predecessor element that became widespread. The existence of conserved piggyBac elements, some of which may have been transmitted horizontally between lepidopteran species, raises important considerations for the stability and practical use of piggyBac transformation vectors.

Animals↗

[Cloning and expression of a novel ubiquitin fusion gene Uba256].

Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene is the only Ub-gp37 fusion gene in the genome of insect viruses. With the specific primers designed for Uba256 gene that was reported recently, the coding regions of Uba256, N-terminal ubiquitin and C-terminal GP37 that lacking the signal sequence, were amplified from SpltMNPV genomic DNA by PCR. The Uba256 coding region was expressed using the expression vector pBV220, and a band of 38 kD was detected with Western blot analysis, indicating that ubiquitin-GP37 fusion protein did not undergo post-transcriptional processing in E. coli. The ubiquitin and GP37 coding region were highly expressed, respectively, using pQE30 expression vector. Antibody to the purified ubiquitin reacted not only to the recombinant ubiquitin but also to bovine ubiquitin, and an antibody to the purified GP37 also reacted to the recombinant GP37. The results indicated that the purified ubiquitin and GP37 retained their antigenicity. Western blot analysis results of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed in this insect cell line to yield free ubiquitin and GP37 protein.

Animals↗

Evolutionary changes in cis and trans gene regulation.

Differences in gene expression are central to evolution. Such differences can arise from cis-regulatory changes that affect transcription initiation, transcription rate and/or transcript stability in an allele-specific manner, or from trans-regulatory changes that modify the activity or expression of factors that interact with cis-regulatory sequences. Both cis- and trans-regulatory changes contribute to divergent gene expression, but their respective contributions remain largely unknown. Here we examine the distribution of cis- and trans-regulatory changes underlying expression differences between closely related Drosophila species, D. melanogaster and D. simulans, and show functional cis-regulatory differences by comparing the relative abundance of species-specific transcripts in F1 hybrids. Differences in trans-regulatory activity were inferred by comparing the ratio of allelic expression in hybrids with the ratio of gene expression between species. Of 29 genes with interspecific expression differences, 28 had differences in cis-regulation, and these changes were sufficient to explain expression divergence for about half of the genes. Trans-regulatory differences affected 55% (16 of 29) of genes, and were always accompanied by cis-regulatory changes. These data indicate that interspecific expression differences are not caused by select trans-regulatory changes with widespread effects, but rather by many cis-acting changes spread throughout the genome.

Alleles↗

Evolutionary conservation and molecular characteristics of repetitive sequences of Drosophila koepferae.

Thirteen middle repetitive DNA clones obtained from the genome of Drosophila koepferae have been tested for their evolutionary conservation in the other seven species of the buzzatii and martensis clusters (repleta group). All but two of these clones exhibit qualitatively similar patterns of hybridization in the eight species. The average interspecific hybridization signal is 85 per cent of that found intraspecifically, ranging from 73 to 93 per cent. Partial sequencing of six of these clones has shown sequences related to the retrotransposon Gypsy, first characterized in D. melanogaster, as well as to the Anopheles gambiae LINE elements T1Ag and Q. A fragment of a hitherto unknown, short inverted repeat transposable element has also been found. The evolutionary conservation of repetitive D. koepferae sequences seems to be related to the high proportion of simple DNA and inactive mobile elements in the genome of this species.

Amino Acid Sequence↗

The complete nucleotide sequence and gene organization of the mitochondrial genome of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae).

The complete nucleotide sequences of the mitochondrial genome (mitogenome) of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae), were determined. The 15,521-bp-long G. orientalis mitogenome contains typical gene complement, base composition, and codon usage found in metazoan mitogenomes. The G. orientalis mitogenome contains the third lowest A+T content (70.5%) among the complete insects mt genome sequences. The initiation codon for the G. orientalis COI gene appears to be ATG, instead of the tetranucleotides, which have been postulated to act as initiation codon for Locusta migratoria and some lepidopteran COI genes. The initiation codon for ND2 appears to be GTG, which is rare, but has been designated as an initiator of Tricholepidion gertschi ND2. All anticodons of G. orientalis tRNAs were identical to Drosophila yakuba and L. migratoria. The tRNA(Ser)(AGN) could not form a stable stem loop structure in the DHU arm as shown in many other insect tRNA(Ser)(AGN). Phylogenetic analysis of nucleotide sequence information from all mt genes supported a monophyletic Diptera, a monophyletic Lepidoptera, a monophyletic Coleoptera, a monophyletic Mecopterida (Diptera+Lepidoptera), and a monophyletic Endopterygota (Diptera+Lepidoptera+Coleoptera), suggesting that the complete insect mitogenome sequence has a resolving power to the diversification events within Endopterygota. However, the relationships of ancient insect orders were unstable, indicating the limited use of mitogenome information at deeper phylogenetic depth.

Animals↗

Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo.

Metazoan genomes contain vast tracts of cis-regulatory DNA that have been identified typically through tedious functional assays. As a result, it has not been possible to uncover a cis-regulatory code that links primary DNA sequences to gene expression patterns. In an initial effort to determine whether coordinately regulated genes share a common "grammar," we have examined the distribution of Dorsal recognition sequences in the Drosophila genome. Dorsal is one of the best-characterized sequence-specific transcription factors in Drosophila. The homeobox gene zerknullt (zen) is repressed directly by Dorsal, and this repression is mediated by a 600-bp silencer, the ventral repression element (VRE), which contains four optimal Dorsal binding sites. The arrangement and sequence of the Dorsal recognition sequences in the VRE were used to develop a computational algorithm to search the Drosophila genome for clusters of optimal Dorsal binding sites. There are 15 regions in the genome that contain three or more optimal sites within a span of 400 bp or less. Three of these regions are associated with known Dorsal target genes: sog, zen, and Brinker. The Dorsal binding cluster in sog is shown to mediate lateral stripes of gene expression in response to low levels of the Dorsal gradient. Two of the remaining 12 clusters are shown to be associated with genes that exhibit asymmetric patterns of expression across the dorsoventral axis. These results suggest that bioinformatics can be used to identify novel target genes and associated regulatory DNAs in a gene network.

Animals↗

Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element.

The gap gene hunchback (hb) is required for the formation and segmentation of two regions of the Drosophila embryo, a broad anterior domain and a narrow posterior domain. Accumulation of hb transcript in the posterior of the embryo occurs in two phases, an initial cap covering the terminal 15% of the embryo followed by a stripe at the anterior edge of this region. By in situ hybridization with transcript-specific probes, we show that the cap is composed only of mRNA from the distal transcription initiation site (P1), while the later posterior stripe is composed of mRNA from both the distal and proximal (P2) transcription initiation sites. Using a series of genomic rescue constructs and promoter-lacZ fusion genes, we define a 1.4 kb fragment of the hb upstream region that is both necessary and sufficient for posterior expression. Sequences within this fragment mediate regulation by the terminal gap genes tailless (tll) and a huckebein, which direct the formation of the posterior hb stripe. We show that the tll protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by tll. We propose a model in which the anterior border of the posterior hb stripe is determined by tll concentration in a manner analogous to the activation of anterior hb expression by bicoid.

Animals↗

Large-scale taxonomic profiling of eukaryotic model organisms: a comparison of orthologous proteins encoded by the human, fly, nematode, and yeast genomes.

Comparisons of DNA and protein sequences between humans and model organisms, including the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, are a significant source of information about the function of human genes and proteins in both normal and disease states. Important questions regarding cross-species sequence comparison remain unanswered, including (1) the fraction of the metabolic, signaling, and regulatory pathways that is shared by humans and the various model organisms; and (2) the validity of functional inferences based on sequence homology. We addressed these questions by analyzing the available fractions of human, fly, nematode, and yeast genomes for orthologous protein-coding genes, applying strict criteria to distinguish between candidate orthologous and paralogous proteins. Forty-two quartets of proteins could be identified as candidate orthologs. Twenty-four Drosophila protein sequences were more similar to their human orthologs than the corresponding nematode proteins. Analysis of sequence substitutions and evolutionary distances in this data set revealed that most C. elegans genes are evolving more rapidly than Drosophila genes, suggesting that unequal evolutionary rates may contribute to the differences in similarity to human protein sequences. The available fraction of Drosophila proteins appears to lack representatives of many protein families and domains, reflecting the relative paucity of genomic data from this species.

Animals↗