Cloning and Activity Determination of Enhancer-like Sequence from Proboscis Monkey (Nasalis larvatus)

Lee, Ying (2017) Cloning and Activity Determination of Enhancer-like Sequence from Proboscis Monkey (Nasalis larvatus). [Final Year Project Report] (Unpublished)

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Abstract

Enhancer is a distal regulatory element which plays a significant role in activating gene transcription. The hallmark of enhancer is critical, such that its spatiotemporal pattern in gene regulation has encouraged various research interests on this molecule. Enhancer identification has remained the greatest challenge as different enhancer possesses distinct function in different cell types. Nasalis larvatus which was indigenous in island of Borneo is a candidate species for us to explore its regulatory gene element due to some similarity that share within its genome and that of human. The main aim of this research is to isolate and clone potential enhancer sequence from Nasalis larvatus and subsequently analyse the transcription factor binding sites (TFBS) present in liver. Initially, putative enhancer was determined in-silico via the software iEnhancer-2L and strong enhancer of more than 500 bp was selected with at least 90% similarity as compared to other primates. Primers were designed for PCR amplification based on conserved domains from multiple alignments of four primate species. The amplicon was then cloned into pGL 3.0 basic vector modified with SV40 promoter insertion. The size of enhancer after sequencing was 795 bp which is deviated from expected size of 823 bp. Alignment analysis reveals that the enhancer was not conserve that it contains a number of nucleotide variations. The study of TFBS was then conducted by using MATCH"" programme and several liver specific TFBSs such as AP-l, cIEBP~, CHOP clEBPu, HNF-l, HNF-313 and NF-l. Finding of synonymous and non-synonymous nucleotide variation within TFBSs have given some novel insights on their relationship with gene expression output. Further enhancer activity determination is necessary to confirm the above status but due to time limitation it is not achievable.

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