Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action

The relationship between co-fitness and co-inhibition of genes in chemicogenomic yeast screens provides insights into gene function and drug target prediction

Differential gene expression in abdomens of the malaria vector mosquito, Anopheles gambiae, after sugar feeding, blood feeding and Plasmodium berghei infection

BACKGROUND: Large scale sequencing of cDNA libraries can provide profiles of genes expressed in an organism under defined biological and environmental circumstances. We have analyzed sequences of 4541 Expressed Sequence Tags (ESTs) from 3 different cDNA libraries created from abdomens from Plasmodium infection-susceptible adult female Anopheles gambiae. These libraries were made from sugar fed (S), rat blood fed (RB), and P. berghei-infected (IRB) mosquitoes at 30 hours after the blood meal, when most parasites would be transforming ookinetes or very early oocysts. RESULTS: The S, RB and IRB libraries contained 1727, 1145 and 1669 high quality ESTs, respectively, averaging 455 nucleotides (nt) in length. They assembled into 1975 consensus sequences – 567 contigs and 1408 singletons. Functional annotation was performed to annotate probable molecular functions of the gene products and the biological processes in which they function. Genes represented at high frequency in one or more of the libraries were subjected to digital Northern analysis and results on expression of 5 verified by qRT-PCR. CONCLUSION: 13% of the 1965 ESTs showing identity to the A. gambiae genome sequence represent novel genes. These, together with untranslated regions (UTR) present on many of the ESTs, will inform further genome annotation. We have identified 23 genes encoding products likely to be involved in regulating the cellular oxidative environment and 25 insect immunity genes. We also identified 25 genes as being up or down regulated following blood feeding and/or feeding with P. berghei infected blood relative to their expression levels in sugar fed females

Update of the Anopheles gambiae PEST genome assembly

BACKGROUND: The genome of Anopheles gambiae, the major vector of malaria, was sequenced and assembled in 2002. This initial genome assembly and analysis made available to the scientific community was complicated by the presence of assembly issues, such as scaffolds with no chromosomal location, no sequence data for the Y chromosome, haplotype polymorphisms resulting in two different genome assemblies in limited regions and contaminating bacterial DNA. RESULTS: Polytene chromosome in situ hybridization with cDNA clones was used to place 15 unmapped scaffolds (sizes totaling 5.34 Mbp) in the pericentromeric regions of the chromosomes and oriented a further 9 scaffolds. Additional analysis by in situ hybridization of bacterial artificial chromosome (BAC) clones placed 1.32 Mbp (5 scaffolds) in the physical gaps between scaffolds on euchromatic parts of the chromosomes. The Y chromosome sequence information (0.18 Mbp) remains highly incomplete and fragmented among 55 short scaffolds. Analysis of BAC end sequences showed that 22 inter-scaffold gaps were spanned by BAC clones. Unmapped scaffolds were also aligned to the chromosome assemblies in silico, identifying regions totaling 8.18 Mbp (144 scaffolds) that are probably represented in the genome project by two alternative assemblies. An additional 3.53 Mbp of alternative assembly was identified within mapped scaffolds. Scaffolds comprising 1.97 Mbp (679 small scaffolds) were identified as probably derived from contaminating bacterial DNA. In total, about 33% of previously unmapped sequences were placed on the chromosomes. CONCLUSION: This study has used new approaches to improve the physical map and assembly of the A. gambiae genome

Comparative Analysis of the Global Transcriptome of Anopheles funestus from Mali, West Africa

Background: Anopheles funestus is a principal vector of malaria across much of tropical Africa and is considered one of the most efficient of its kind, yet studies of this species have lagged behind those of its broadly sympatric congener, An. gambiae. In aid of future genomic sequencing of An. funestus, we explored the whole body transcriptome, derived from mixed stage progeny of wild-caught females from Mali, West Africa. Principal Findings: Here we report the functional annotation and comparative genomics of 2,005 expressed sequence tags (ESTs) from An. funestus, which were assembled with a previous EST set from adult female salivary glands from the same mosquito. The assembled ESTs provided for a nonredundant catalog of 1,035 transcripts excluding mitochondrial sequences. Conclusions/Significance: Comparison of the An. funestus and An. gambiae transcriptomes using computational and macroarray approaches revealed a high degree of sequence identity despite an estimated 20–80 MY divergence time between lineages. A phylogenetically broader comparative genomic analysis indicated that the most rapidly evolving proteins – those involved in immunity, hematophagy, formation of extracellular structures, and hypothetical conserved proteins – are those that probably play important roles in how mosquitoes adapt to their nutritional and externa

Mechanisms of Haploinsufficiency Revealed by Genome-Wide Profiling in Yeast

Haploinsufficiency is defined as a dominant phenotype in diploid organisms that are heterozygous for a loss-of-function allele. Despite its relevance to human disease, neither the extent of haploinsufficiency nor its precise molecular mechanisms are well understood. We used the complete set of Saccharomyces cerevisiae heterozygous deletion strains to survey the genome for haploinsufficiency via fitness profiling in rich (YPD) and minimal media to identify all genes that confer a haploinsufficient growth defect. This assay revealed that ∼3% of all ∼5900 genes tested are haploinsufficient for growth in YPD. This class of genes is functionally enriched for metabolic processes carried out by molecular complexes such as the ribosome. Much of the haploinsufficiency in YPD is alleviated by slowing the growth rate of each strain in minimal media, suggesting that certain gene products are rate limiting for growth only in YPD. Overall, our results suggest that the primary mechanism of haploinsufficiency in yeast is due to insufficient protein production. We discuss the relevance of our findings in yeast to human haploinsufficiency disorders

Gene expression patterns associated with blood-feeding in the malaria mosquito Anopheles gambiae

Abstract Background Blood feeding, or hematophagy, is a behavior exhibited by female mosquitoes required both for reproduction and for transmission of pathogens. We determined the expression patterns of 3,068 ESTs, representing ~2,000 unique gene transcripts using cDNA microarrays in adult female Anopheles gambiae at selected times during the first two days following blood ingestion, at 5 and 30 min during a 40 minute blood meal and at 0, 1, 3, 5, 12, 16, 24 and 48 hours after completion of the blood meal and compared their expression to transcript levels in mosquitoes with access only to a sugar solution. Results In blood-fed mosquitoes, 413 unique transcripts, approximately 25% of the total, were expressed at least two-fold above or below their levels in the sugar-fed mosquitoes, at one or more time points. These differentially expressed gene products were clustered using k-means clustering into Early Genes, Middle Genes, and Late Genes, containing 144, 130, and 139 unique transcripts, respectively. Several genes from each group were analyzed by quantitative real-time PCR in order to validate the microarray results. Conclusion The expression patterns and annotation of the genes in these three groups (Early, Middle, and Late genes) are discussed in the context of female mosquitoes' physiological responses to blood feeding, including blood digestion, peritrophic matrix formation, egg development, and immunity