Many factors may explain why certain transposable elements (TEs) spread in some species and not others. On the one hand, they include processes that affect the rate of transposition, such as differences in the regulation of expression; on the other hand, they include characteristics of a genome that affect the consequences of transposition. In particular genome size may have an effect: a genome that is large due to non-essential repetitive DNA may be permissive for TE movement, as insertion events are less likely to be deleterious. Genome size may also help explain the pattern of TE distribution between species of mosquitoes, including the important vectors of arboviruses, Aedes aegypti and Culex pipiens sensu lato. These species have genomes 3- 5 times larger than a third genus, the Anopheles mosquitoes, which includes the malaria vectors. While all mosquitoes carry a diverse range of TEs, only culicines have the super abundant retroposon, Juan which can contribute up to 3% of the genome. The genome sequences of various insect species were compared and the mosquitoes show a significant trend of increase in genome size, which can be attributed to the increase in retroposon sequences. Two variants of Juan are reported, and new information is added regarding these elements. Previous publication of these elements contained errors in their sequences. A unique triple repeat of a cysteine rich region with a CCHC motif is present in the open reading frame. This sequence is a zinc-knuckle domain, important for the replication mechanism of these elements. In comparison, a third recently active but very low copy number retroposon, termed Pip1, is also described. The results show that Pip1 is related to the Juan elements and also possess the triple CCHC motif. The PCR results also supports previous findings of polymorphism in insertion sites of this element, suggesting that Pip1 was active after the establishment of the different strains. Pip1 copies can be grouped into three distinct groups based on nucleotide differences. Pip1 could also be using an alternative start codon to initiate transcription. Full length intact copies of the three TEs in this study were been cloned into a germline transformation vector based on piggyBac and used for germline transformation in Drosophila melanogaster. Drosophila melanogaster has no Juan or Pip1 elements and an even smaller genome than anophelines mosquitoes, so insertion events from unregulated TE movement should be more detectable. We found that the elements have been successfully introduced into the Drosophila lines. The lines were inbred to obtain a homozygous population. A range of transformed lines were monitored. No effects of hybrid dysgenesis was found. Flies with black spotted eyes were identified in a Pip1 line but this phenotype was not heritable. Whole genome sequencing was carried out on the flies using next generation sequencing (NGS) technology. Retroposon sequences was detected at a high frequency. Insertion junctions were not detected but this result does not eliminate the possibility that a junction is present but the sequencing was not sensitive enough. A possible explanation is the retroposon is present as extrachromosomal plasmid DNA.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:667349 |
| Date | January 2014 |
| Creators | Yeoh, Joseph Guan Chong |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/8972 |
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