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Title: Functional analysis of the promoter regions of de novo genes_090 & 074 in Drosophila melanogaster and in vitro Topi C-terminal-DNA interaction by SELEX
Author: Dighe, Shrinivas Nivrutti
ISNI:       0000 0004 9347 5944
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2019
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de novo genes are derived from ancestral non-coding sequences, and typically show strongly testis biased expression. Sequence variation in cis-acting regulatory regions presumably generates sites that are recognised by testis-specific transcriptional regulators, such as tMAC. The causative mutations in de novo gene evolution can be identified by comparing naturally expressing and non-expressing alleles of not-yet-fixed de novo genes. Promoters of expressed and non-expressed alleles differ by the presence of SNPs and/or indels. In the first part of my thesis, I have investigated the molecular mechanisms of testisspecific expressed de novo genes_090 & 074. For gene_090, reporters showed that a 7bp deletion just upstream of the TSS is necessary and sufficient to convert a natural non-expressing allele into an expressing allele. Further constructs indicate the deletion in expressed allele does not create any binding site for regulatory proteins per se but the mutation of the two base pairs flanking the deletion site suggests that the sequence created by the deletion is critical, as well as the spacing of other flanking sequences are important for expression. Expression of 074 has evolved independently at least twice. Two expressing alleles do not share any SNPs or indels not also found in at least one nonexpressing allele, yet both are able to drive expression of a reporter gene. Intriguingly, for both 090 and 074, the reporter mRNA is translationally repressed, and only translated in late spermatids, reminiscent of many more ancient testis-specific transcripts. In the second part of my thesis, I have discovered the de novo motif of 15bp for Topi Cterminal protein. The discovered motif was characterised by EMSA and in silico analysis observed the occurrence of discovered motif in promoter regions of aly dependent genes. Altogether, these results indeed support the motif being real.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)