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Development of a physical and genetic map of the virulent Wolbachia strain wMelPop.

ABSTRACT: We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.

SUBMITTER: Sun LV

PROVIDER: S-EPMC296261 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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Publications

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop.

Sun Ling V LV Riegler Markus M O'Neill Scott L SL

Journal of bacteriology 20031201 24

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comp ...[more]

PMID: 14645266

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Project description:BackgroundWolbachia are bacterial endosymbionts of many arthropod species in which they manipulate reproductive functions. The distribution of these bacteria in the Drosophila ovarian cells at different stages of oogenesis has been amply described. The pathways along which Wolbachia influences Drosophila oogenesis have been, so far, little studied. It is known that Wolbachia are abundant in the somatic stem cell niche of the Drosophila germarium. A checkpoint, where programmed cell death, or apoptosis, can occur, is located in region 2a/2b of the germarium, which comprises niche cells. Here we address the question whether or not the presence of Wolbachia in germarium cells can affect the frequency of cyst apoptosis in the checkpoint.ResultsOur current fluorescent microscopic observations showed that the wMel and wMelPop strains had different effects on female germline cells of D. melanogaster. The Wolbachia strain wMel did not affect the frequency of apoptosis in cells of the germarium. The presence of the Wolbachia strain wMelPop in the D. melanogasterw1118 ovaries increased the number of germaria where cells underwent apoptosis in the checkpoint. Based on the appearance in the electron microscope, there was no difference in morphological features of apoptotic cystocytes between Wolbachia-infected and uninfected flies. Bacteria with normal ultrastructure and large numbers of degenerating bacteria were found in the dying cyst cells.ConclusionsOur current study demonstrated that the Wolbachia strain wMelPop affects the egg chamber formation in the D. melanogaster ovaries. This led to an increase in the number of germaria containing apoptotic cells. It is suggested that Wolbachia can adversely interfere either with the cystocyte differentiation into the oocyte or with the division of somatic stem cells giving rise to follicle cells and, as a consequence, to improper ratio of germline cells to follicle cells and, ultimately, to apoptosis of cysts. There was no similar adverse effect in D. melanogaster Canton S infected with the Wolbachia strain wMel. This was taken to mean that the observed increase in frequency of apoptosis was not the general effect of Wolbachia on germline cells of D. melanogaster, it was rather induced by the virulent Wolbachia strain wMelPop.

Project description:BackgroundIntegrated genetic and physical maps are extremely valuable for genomic studies and as important references for assembling whole genome shotgun sequences. Screening of a BAC library using molecular markers is an indispensable procedure for integration of both physical and genetic maps of a genome. Molecular markers provide anchor points for integration of genetic and physical maps and also validate BAC contigs assembled based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy and an in silico approach to anchor molecular markers onto the soybean physical map.ResultsA total of 1,470 markers (580 SSRs and 890 STSs) were anchored by PCR on a subset of a Williams 82 BstY I BAC library pooled into 208 pools in six dimensions. This resulted in 7,463 clones (approximately 1x genome equivalent) associated with 1470 markers, of which the majority of clones (6,157, 82.5%) were anchored by one marker and 1106 (17.5%) individual clones contained two or more markers. This contributed to 1184 contigs having anchor points through this 6-D pool screening effort. In parallel, the 21,700 soybean Unigene set from NCBI was used to perform in silico mapping on 80,700 Williams 82 BAC end sequences (BES). This in silico analysis yielded 9,835 positive results anchored by 4152 unigenes that contributed to 1305 contigs and 1624 singletons. Among the 1305 contigs, 305 have not been previously anchored by PCR. Therefore, 1489 (78.8%) of 1893 contigs are anchored with molecular markers. These results are being integrated with BAC fingerprints to assemble the BAC contigs. Ultimately, these efforts will lead to an integrated physical and genetic map resource.ConclusionWe demonstrated that the six-dimensional soybean BAC pools can be efficiently used to anchor markers to soybean BACs despite the complexity of the soybean genome. In addition to anchoring markers, the 6-D pooling method was also effective for targeting BAC clones for investigating gene families and duplicated regions in the genome, as well as for extending physical map contigs.

Project description:BackgroundCytosine methylation is one of several reversible epigenetic modifications of DNA that allow a greater flexibility in the relationship between genotype and phenotype. Methylation in the simplest models dampens gene expression by modifying regions of DNA critical for transcription factor binding. The capacity to methylate DNA is variable in the insects due to diverse histories of gene loss and duplication of DNA methylases. Mosquitoes like Drosophila melanogaster possess only a single methylase, DNMT2.DescriptionHere we characterise the methylome of the mosquito Aedes aegypti and examine its relationship to transcription and test the effects of infection with a virulent strain of the endosymbiont Wolbachia on the stability of methylation patterns.ConclusionWe see that methylation in the A. aegypti genome is associated with reduced transcription and is most common in the promoters of genes relating to regulation of transcription and metabolism. Similar gene classes are also methylated in aphids and honeybees, suggesting either conservation or convergence of methylation patterns. In addition to this evidence of evolutionary stability, we also show that infection with the virulent wMelPop Wolbachia strain induces additional methylation and demethylation events in the genome. While most of these changes seem random with respect to gene function and have no detected effect on transcription, there does appear to be enrichment of genes associated with membrane function. Given that Wolbachia lives within a membrane-bound vacuole of host origin and retains a large number of genes for transporting host amino acids, inorganic ions and ATP despite a severely reduced genome, these changes might represent an evolved strategy for manipulating the host environments for its own gain. Testing for a direct link between these methylation changes and expression, however, will require study across a broader range of developmental stages and tissues with methods that detect splice variants.

Dataset Information

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop.

Publications

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop.

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