The molecular genetics of the raspberry locus in Drosophila melanogaster

by Hu, Song.

The raspbeny (res) locus is in region 9E1 -E4 on the X chromosome of Drosophih melanogaster. Three groups of mutations exist at the locus: spontaneous rus eye-colour mutants, defective in pteridine biosynthesis; purine auxotrophs (gualu, purl' ' 3;and recessive lethal mutants (rus-1). They constitute the "rar c~mplex'~. The rus4 mutants form a single lethal complementation group. A majority of the rad mutants fd to complementboth the eye-colour and the auxotrophic mutants, implying all three groups are firnctionally related. However, the ras mutants complementguaF and the pur1 mutants; in addition, gtral" complements pur12. Wild-type rus DNA was cloned, utilizing mutant DNA previously isolated after P- element tagging the gene (Leonard, 1986). Its DNA sequence encodes inosinate dehydrogenase (IMPDH), the rate-limiting enzyme in GMP biosynthesis. The probable function of the gene is thus in GMP biosynthesis. All mutant phenotypes in the ras complex can be rationalized as results of dtered IMPDH activity. Several cDNAs were isolated;the longest, which is close to the size (2.4kb) of the main transcriptional product of the region, was sequenced. The gene contains four introns. There is no TATA-box. Instead, there is an initiator-like element and an extremely high GC content sequence upstream. The combination of initiator and GC rich sequences is typicd of house-keeping regulation. The developmental profles of transcript and IMPDH activity were studied. The highest transcript level occurs in the 0-2 hour embryonic penod. These transcnpts are probably maternal products; extraordinariiy, their absence fiom ras' "'early embryos suggests they are dispensable. Two cis-acting regdatory sequences seem to have been identified in midies of ras mutants. One, probably a splicing enhancer, is in the second intron. Insertions in this elernent generate the classical ras eye-colour mutants, rd- ' "'. The other is located upstream of the transcriptional initiation site. A deletion in the region generates the d e specific mutant rd, suggesting involvement in dosage compensation. If house-keeping and eye-specific bctions of the WDH gene are independently regulated, tissue-specific regdatory elements seem to be extremely kted; in con- the regdatory apparatus concemed principally with houe-keeping function is probably more extensive. 1 am deeply indebted to my supe~sor Dr. D. Nash for his help in guiding my research with his helpful advice, discussion, criticism and financiai assistance during both the experimentalwork and thesis preparation. 1 also wish to acknowledge excellent advice and technical help fiom Dr. S. Tiong, Ms. E. Woloshyn and Mr. E. Chomey. 1wish to thank Dr. K. Roy particularly for his extensive advice concerning experiments involving enzymatic activity andysis; as well as his role, dong with Dr. J. Bell on my supervisory committee. Thanks also to Dr. J. Lock, Mr. W. Clark and G. Ritzel and to the many other people who helped me directly or indirectly . Finally, I am grateful to my farnily, especially my wife Jin-Xia and my son Jing, for their encouragement, support and patience throughout the long process leading to the production of this thesis. Significant segments of Section 3.2, Section 3.3.1 are derived from "The raspberv locus of DrosophiZa melanogaster includes an inosine monophosphate dehydrogenase like cobg sequence " by Nash, D., S. Hu, N. J. Leonard, S. Y. Tiong, and D. Fillips in Genome 37 (2): 333-44, 1994.