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Microarray studies make it possible to obtain gene expression data on a whole-genome scale. Arbeitman et al. present microarray data generated at multiple time points throughout Drosophila melanogaster development, and identify new genes engaged in a broad spectrum of processes, including the patterning of the early embryo and senescence in adults.
Research on model organisms has substantially advanced our understanding of aging. However, these studies collectively lack any examination of the element of sociality, an important feature of human biology. Social insects present a number of unique possibilities for investigating social influences on aging and potentially detecting new mechanisms for extremely prolonged, healthy life spans that have evolved naturally. Social evolution has led to life spans in reproductive females that are much longer (up to over 100-fold) than those of males or of nonreproductive worker castes. These differences are particularly dramatic because they are due to environmental influences, as all individuals develop from the same genomes. Social insect colonies consist of semi-autonomous individuals, and the relationship between the colony and the individual creates many interesting predictions in the light of the common theories of aging. Furthermore, the variety of lifestyles of social insects creates the potential for crucial comparative analyses across distinct social systems.
Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.
Although the organization and functions of the constitutive secretory pathway have been intensively studied for decades, a recent genome-wide RNAi screen in Drosophila cells has identified about 100 genes encoding novel so-called TANGO proteins (for transport and Golgi organization) that may be direct regulators of various aspects of protein exocytosis or secretion.
Defective interfering baculoviruses (DIs) lack considerable portions of the genome, interfere with the replication of helper virus, and cause the so-called "passage-effect" during serial passaging in insect cells and in bioreactor configurations. We investigated their origin by (nested) PCR and demonstrated that DIs lacking approximately 43% (d43) of their DNA are present in low-passage Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)-E2 virus stocks and in polyhedra, but not in the authentic AcMNPV isolate obtained prior to passage in cell culture. To investigate whether DIs are rapidly generated de novo in Sf21 insect cells, a genetically homogeneous AcMNPV bacmid was serially passaged, resulting in the generation of d43 DIs within two passages. AT-rich sequences of up to 66 nucleotides of partly unknown origin were found at the deletion junctions in the d43 DI genomes. These data suggest that the rapid generation of DIs is an intrinsic property of baculovirus infection in insect cell culture and involves several recombination steps.
Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.
Two invasive hemipteran adelgids are associated with widespread damage to several North American conifer species. Adelges tsugae, hemlock woolly adelgid, was introduced from Japan and reproduces parthenogenetically in North America, where it has rapidly decimated Tsuga canadensis and Tsuga caroliniana (the eastern and Carolina hemlocks, respectively). Adelges abietis, eastern spruce gall adelgid, introduced from Europe, forms distinctive pineapple-shaped galls on several native spruce species. While not considered a major forest pest, it weakens trees and increases susceptibility to additional stressors. Broad-spectrum insecticides that are often used to control adelgid populations can have off-target impacts on beneficial insects. Whole genome sequencing was performed on both species to aid in development of targeted solutions that may minimize ecological impact. Adelges abietis was sequenced using Illumina Linked-Read technology from 30 pooled individuals, with Hi-C scaffolding performed using data from a single individual collected from the same host plant. Adelges tsugae used Oxford Nanopore long-read sequencing from pooled nymphs. The assembled A. tsugae and A. abietis genomes, pooled from several parthenogenetic females, are 220.75 Mbp and 253.16 Mbp, respectively. Each consists of eight autosomal chromosomes, as well as two sex chromosomes (X1/X2), supporting the XX-XO sex determination system. The genomes are over 96% complete based on BUSCO assessment. Genome annotation identified 11,424 and 12,060 protein-coding genes in A. tsugae and A. abietis, respectively. Comparative analysis of proteins across 29 hemipteran species and 14 arthropod outgroups identified 31,666 putative gene families. Gene family evolution analysis with CAFE revealed lineage-specific expansions in immune-related aminopeptidases (ERAP1) and juvenile hormone binding proteins (JHBP), contractions in juvenile hormone acid methyltransferases (JHAMT), and conservation of nicotinic acetylcholine receptors (nAChR). These genes were explored as candidate families towards a long-term objective of developing adelgid-selective insecticides. Structural comparisons of proteins across seven focal species (Adelges tsugae, Adelges abietis, Adelges cooleyi, Rhopalosiphum maidis, Apis mellifera, Danaus plexippus, and Drosophila melanogaster) revealed high conservation of nAChR and ERAP1, while JHAMT exhibited species-specific structural divergence. The potential of JHAMT as a lineage-specific target for pest control was explored through virtual drug and pesticide screening.
A novel cellulase [beta-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA belonging to glycoside hydrolase family (GHF) 45 was cloned from the mulberry longicorn beetle, Apriona germari. The cDNA encoding EGase of A. germari (Ag-EGase) is 711 bp long with an open reading frame of 237 amino acid residues. The Ag-EGase was closely related to another beetle, Phaedon cochleariae, cellulase and one symbiotic protist cellulase in the hindgut of the termite Reticulitermes speratus, those belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase. Southern blot analysis of genomic DNA suggested the presence of Ag-EGase gene as a single copy and Northern blot analysis confirmed midgut-specific expression at transcriptional level. Similarly, the Ag-EGase enzyme assay exhibited high activity only in midgut tissue, suggesting that the midgut is the prime site where large quantities of EGase are synthesized for degrading the absorbed cellulose from the diet. The cDNA encoding Ag-EGase was expressed as a 29-kDa polypeptide in baculovirus-infected insect Sf9 cells and the culture supernatants of the recombinant baculovirus-infected cells showed EGase enzyme activity of 15.25 U/ml of medium containing 0.5 x 10(6) cells at 5 days post-infection (p.i.). The enzyme activity of the purified recombinant Ag-EGase expressed in baculovirus-infected insect cells was approximately 992 U per mg of recombinant Ag-EGase. The purified recombinant Ag-EGase showed the highest enzymatic activity at 50 degrees C and pH 6.0, and was stable at 55 degrees C at least for 10 min.
A clone of a DNA-mediated mobile element (transposon) corresponding to a mariner-like element (MLE) was obtained by carrying out the polymerase chain reaction with genomic DNA of Bombyx mori using a Hyalophora cecropia MLE sequence as a primer. This clone had a size of about 4.2 kb and, after sequencing, was found to contain an RNA-mediated, shorter retrotransposon named L1Bm, which was in turn integrated with a much longer retrotransposon named BMC1. Thus, the mobile elements made a novel tripartite structure. The BMC1 and L1Bm moieties of the composite structure each contained a 63-bp conserved sequence which was subsequently found to be highly conserved in all BMC1 and L1Bm elements registered so far. We propose that the 63-bp stretch may be a recognition site for a retrotransposition mechanism conducted by a reverse transcriptase and an endonuclease complex. On the basis of this inference, we propose a model that predicts how different types of BMC1 and L1Bm elements are dispersed in the genome. In addition, a phylogenetic tree made from the current and extant BMC1 and L1Bm sequences indicated that these elements can be classified into Subfamilies I and II.
A crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.
BACKGROUND: Factors affecting the organization and spacing of functionally unrelated genes in metazoan genomes are not well understood. Because of the vast size of a typical metazoan genome compared to known regulatory and protein-coding regions, functional DNA is generally considered to have a negligible impact on gene spacing and genome organization. In particular, it has been impossible to estimate the global impact, if any, of regulatory elements on genome architecture. RESULTS: To investigate this, we examined the relationship between regulatory complexity and gene spacing in Caenorhabditis elegans and Drosophila melanogaster. We found that gene density directly reflects local regulatory complexity, such that the amount of noncoding DNA between a gene and its nearest neighbors correlates positively with that gene's regulatory complexity. Genes with complex functions are flanked by significantly more noncoding DNA than genes with simple or housekeeping functions. Genes of low regulatory complexity are associated with approximately the same amount of noncoding DNA in D. melanogaster and C. elegans, while loci of high regulatory complexity are significantly larger in the more complex animal. Complex genes in C. elegans have larger 5' than 3' noncoding intervals, whereas those in D. melanogaster have roughly equivalent 5' and 3' noncoding intervals. CONCLUSIONS: Intergenic distance, and hence genome architecture, is highly nonrandom. Rather, it is shaped by regulatory information contained in noncoding DNA. Our findings suggest that in compact genomes, the species-specific loss of nonfunctional DNA reveals a landscape of regulatory information by leaving a profile of functional DNA in its wake.
BACKGROUND: Microarray and other high-throughput technologies are producing large sets of interesting genes that are difficult to analyze directly. Bioinformatics tools are needed to interpret the functional information in the gene sets. RESULTS: We have created a web-based tool for data analysis and data visualization for sets of genes called GOTree Machine (GOTM). This tool was originally intended to analyze sets of co-regulated genes identified from microarray analysis but is adaptable for use with other gene sets from other high-throughput analyses. GOTree Machine generates a GOTree, a tree-like structure to navigate the Gene Ontology Directed Acyclic Graph for input gene sets. This system provides user friendly data navigation and visualization. Statistical analysis helps users to identify the most important Gene Ontology categories for the input gene sets and suggests biological areas that warrant further study. GOTree Machine is available online at http://genereg.ornl.gov/gotm/. CONCLUSION: GOTree Machine has a broad application in functional genomic, proteomic and other high-throughput methods that generate large sets of interesting genes; its primary purpose is to help users sort for interesting patterns in gene sets.
Two distinct mariner-like elements, Atmar-1 and Atmar-2, were isolated from the genome of the mosquito Ochlerotatus atropalpus. Full-sized Atmar-1 elements, obtained by screening a genomic library, have a 1293-bp consensus sequence with 27-bp inverted terminal repeats and a 1047-bp open reading frame (ORF) encoding the transposase. The Atmar-2 elements were amplified by polymerase chain reaction from genomic DNA and contain the central part of the transposase ORF. Individual clones of both mariner elements contain deletions, frameshifts, and stop codons. The Atmar-1 elements are present in 370-1200 copies, while the Atmar-2 elements are present in approximately 100-300 copies per haploid genome. One of the Atmar-1 elements, Atmar-1.33, could be mobilized, suggesting the presence of functional Atmar-1 elements elsewhere in the genome. Phylogenetic analysis demonstrated that Atmar-1 elements belong to the irritans subfamily and Atmar-2 elements to the cecropia subfamily of mariner elements.
The GeneWise method for combining gene prediction and homology searches was applied to the 2.9-Mb region from Drosophila melanogaster. The results from the Genome Annotation Assessment Project (GASP) showed that GeneWise provided reasonably accurate gene predictions. Further investigation indicates that many of the incorrect gene predictions from GeneWise were due to transposons with valid protein-coding genes and the remaining cases are pseudogenes or possible annotation oversights.
The gene encoding sialokinin I, the principal vasodilatory peptide of Aedes aegypti, has been isolated and characterized. Degenerate oligonucleotide primers based on peptide amino acid sequence were used to amplify a gene fragment from messenger RNA (mRNA) isolated from female salivary glands. The amplification product was used to probe a salivary gland complementary DNA (cDNA) library, and a number of corresponding cDNAs were isolated and their primary sequence determined. Analysis of the conceptual translation product of a 406-bp cDNA indicates that sialokinin I is expressed as a preprosialokinin and is subsequently post-translationally processed to the active peptide. Northern analysis revealed a 490-bp transcription product expressed exclusively in female salivary glands, and hybridization in situ of probes to RNA in whole tissues localized gene expression to the medial lobe of female salivary glands. Screening of an Ae. aegypti genomic library with the cDNA resulted in the isolation of a clone containing the gene, designated Sialokinin I (Sia I). Comparison of the cDNA with the genomic clone reveals two introns of 62 bp and 833 bp. Primer extension analysis showed that several transcription initiation sites are present. Southern analysis of genomic DNA shows that Sia I is most probably a single-copy gene. Similarities of the Sia I gene product with other genes are confined to the region encoding the active decapeptide.
The concept of 'organismal complexity' has had a chequered career in genetics, with no rigorous operational definition available for the term. The recent finding that Drosophila melanogaster has more than four thousand fewer genes than the nematode forces a re-examination of whether gene number, in itself, can be taken as any real guide to complexity.
In the late stage of infection, virions of the Melolontha melolontha entomopoxvirus (MmEPV) are occluded into cytoplasmic paracrysalline proteinaceous occlusion bodies designated spherules (A. Amargier, C. Vago, G. Meynadier, 1964, Mikroskopie 19, 309-315). We have cloned and sequenced a 4-kpb DNA fragment of the MmEPV genome encompassing the spherule major protein gene named spherulin. The spherulin gene contains an open reading frame able to code for a 942-amino-acid (aa) polypeptide (MW 109 kDa), consistent with a size above 100 kDa determined by SDS-PAGE for purified spherulin. The MmEPV spherulin showed more than 40% as homology with the Amsacta moorei EPV (AmEPV) spheroidin and shared homologies with the partially sequenced Choristoneura biennis EPV (CbEPV) spheroidin, indicating that this biologically important polypeptide is well conserved among EPVs infecting phylogenetically as distant groups of insects as lepidoptera and coleoptera. Western blot analyses confirmed the relationships between the three polypeptides. In contrast, no homology was detected between the MmEPV spherulin and EPV fusolins or vertebrate poxvirus A-type inclusion proteins. The 45 bases upstream from the ATG initiation codon of spherulin shared 60% homology with the vaccinia virus late promoters including the highly conserved TAAATG consensus sequence. Furthermore, the 5' extremity of the spherulin mRNA consisted of a poly(A) tract of about 20 nucleotides just upstream from the AUG translational initiation codon. These are characteristic features of vertebrate poxvirus late mRNAs suggesting similar modalities of gene expression for vertebrate and insect poxvirus genomes.
Little is known about the origin and evolution of supernumerary (B) chromosomes. This study utilizes molecular markers to examine the evolutionary history and microstructural organization of the supernumerary paternal-sex-ratio (PSR) chromosome of the parasitic wasp Nasonia vitripennis. Copies of the retrotransposon NATE were previously isolated from PSR and the genomes of N. vitripennis and related wasp species. A phylogenetic analysis of sequences representing 29 elements from PSR and seven wasp species, coupled with a hybridization analysis of elements in genomic DNA provides evidence that PSR was recently transferred into N. vitripennis from a species in the genus Trichomalopsis. A linear region of the PSR chromosome was compared by Southern blot analysis with genomic DNA from N. vitripennis, Nasonia longicornis, Trichomalopsis americanus, and Trichomalopsis dubius. A region organized similarly to the region on PSR was not evident in any of the species, thus a progenitor region was not identified. However, the hybridizations revealed that this region of PSR is primarily composed of repetitive sequences that appear dispersed in these wasp genomes, and might represent additional mobile elements. At least three different dispersed repeats are present in the 18 kb region of PSR. The abundance of tandem and dispersed repetitive sequences in this relatively small region provides additional evidence for the degenerate structure of the PSR chromosome.