Date of Graduation

Spring 2009

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Colette Witkowski

Keywords

C. elegans, promoter, GFP, reporter constructs, DNA sequencing

Subject Categories

Medical Molecular Biology

Abstract

With published genome sequences from C. elegans to humans readily available and with genes being identified, the genetic control of transcription is now at the forefront of molecular biology. Small nucleotide sequences known as cis-acting transcription factors contained in the upstream intergenic regions of genes are necessary for transcription promotion and pre-initiation complex formation. In the model organism C. elegans, these promoter elements are currently predicted by in silico methods and have yet to be fully confirmed. Previous computational analysis has led to the identification of a specific motif (Motif 2) element reoccurring in muscle-specific genes and present in the emb-9 promoter. DNA sequence confirmation of the upstream intergenic region cloned into the pJJ359 plasmid provided some assurance that the analyzed promoter sequence was consistent with published genomes and trustworthy for promoter::gfp reporter fusion constructs. Varying lengths of the upsteam promoter were amplified by PCR and fused to a transcriptional reporting gene gfp. Expression of gfp in C. elegans by way of the fusion constructs provided insight into the necessary promoter elements of emb-9. Microinjection of the engineered full-length promoter::gfp fusion construct into the syncytial gonad of C.elegans yielded a single transgenic progeny worm identified by expression of the dominant selective coinjection marker rol-6. Epifluorescence detection of mosaic gfp expression in the neuronal ring and nerve ganglions and not in the body wall muscle cells could be explained by injected DNA incorporation in the extrachromosomal array. However, GFP expression supported the hypothesis that cis-acting elements are present in the upstream intergenic region of the emb-9 gene.

Copyright

© Joseph P. Williams

Campus Only

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