Masters Theses

Sunil Tuppale

Date of Award

12-1995

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Ranjan Ganguly

Committee Members

Mary Ann Handel, Don Dougall

Abstract

Arrestin protein (Art) is an important component of the phototransduction or visual transduction process in both vertebrates and invertebrates. It is required for normal vision and structural integrity of the retina. It has been shown from studies in Drosophila that under expression or overexpression of arrestin causes impaired vision. In Drosophila underexpression of arrestin B (ArrB), one of the two arrestins found in this species has been shown to cause light dependent retinal degeneration. Studies in rds (retinal degeneration slow ) mutant mouse showed that overexpression of Arr causes light dependent retinal degeneration. The maintenance of proper level of Arr is important for normal vision and structural integrity of the retina. The focus of this study has been to map the cis-regulatory sequences in the upstream DNA for correct level and head-specific expression of the ArrB gene. For this purpose genomic DNA fragments containing ArrB gene of Drosophila melanogaster with 1.5-kb and 0.8-kb upstream sequences were transferred to ry506 host strain via P element mediated germ line transformation. To be able to detect the transgene derived mRNA, a 0.4-kb BamHI mouse DNA fragment was introduced into the exon I of the ArrB transgene as a reporter sequence. Two transformant lines, A3 and A5, with 1.5-kb upstream DNA and one transformant line T1, with 0.8-kb upstream DNA were obtained. RNA blot analysis using mouse reporter DNA as a probe showed that the transgenes were transcriptionally active in all three transformant lines. However, the level of activity was found to be much lower than that of the resident ArrB gene. Also, transformant lines with 1.5-kb and 0.8-kb upstream DNA showed the same level of ArrB transgene expression and the expression was found in the head but not in the body of the transformed flies. Thus, the cis-regulatory elements for head-specific expression of the ArrB gene of D melanogaster are present within 0.8-kb upstream DNA. However, the cis-regulatory elements required for high level of ArrB gene expression appears to be distal to the 1.5-kb upstream DNA. Alternatively, if such sequences are present in the transgene, it must be located in the exon I which got disrupted due to the insertion of mouse DNA.

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