Hamilton, Bruce A. (1993) Assessing molecular function in the Drosophila nervous system: a reverse genetic approach. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:12072012-092856340
I describe the application of new methods for reverse genetic analyses in Drosophila melanogaster to genes expressed in the nervous system. The methods can be devided into two classes: tools for molecular analysis and tools for genetic analysis.
The first set of methods is designed to facilitate rapid, large-scale molecular analysis of cDNA clones. This set of tools begins with a family of bacteriophage λ cDNA cloning vectors and E. coli host cell strains that allow automatic plasmid subcloning by in vivo site-specific recombination. A high density filter hybridization and diagnostic PCR assay technique applied to size-selected libraries then substantially simplifies the isolation of full-length cDNA clones in these vectors. Next, a transposon γδ-facilitated DNA sequencing procedure minimizes the labor required to isolate a nested set of initiation sites for chain termination sequencing of each strand of a cloned DNA segment. I also describe the characterization of 250 cDNA clones isolated from adult heads on the basis of gross expression patterns in the embryonic ventral nerve cord and larval fat body.
The second set of procedures facilitates the isolation of mutations in the chromosomal genes that correspond to isolated cDNA clones. I describe three such experiments. A plasmid rescue and hybridization strategy allowed the isolation of PlacW elements inserted adjacent to 4 cloned genes in an array of nearly 700. Modifications of this procedure to take advantage of P-element local transposition allowed the isolation and characterization of an apparent null mutation in the receptor-linked protein tyrosine phosphatase gene DPTP99A. In a pilot study, I demonstrate the feasibility of isolating chemically induced mutations that remove targeted restriction enzyme cleavage sites with a PCR-based assay. In addition, I describe the serendipitous isolation of a PlacW-induced mutation, encumbered, that affects the morphogenesis of imaginal wing discs as well as adult longevity, activity, and fertility.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||19 February 1993|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Dan Anguka|
|Deposited On:||10 Dec 2012 16:23|
|Last Modified:||26 Dec 2012 04:46|
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