Isolation of the rudimentary locus of Drosophila melanogaster

In Drosophila melanogaster the rudimentary locus encodes the first three enzymes in de novo pyrimidine biosynthesis: CPSase, ATCase and DHOase. rudimentary is located towards the proximal end of the X chromosome at the cytogenetic locus 15A1. A variety of genetic and molecular studies have suggested that all three enzymatic activities are encoded in a single transcription unit whose mRNA is translated into a multifunctional polypeptide with a molecular weight of approximately 220,000 D. In this paper we describe the isolation of a 90 kb D. melanogaster DNA segment which contains the rudimentary gene. This was achieved by screening a D. melanogaster genomic phage library with a cDNA probe homologous to the corresponding genetic unit of hamster (pyr 1-3). One of the phage recombinants homologous to this hamster probe, BS313 was found to be derived from the 15A1 region of the X chromosome. Starting from BS313 we "walked" in both directions along the X chromosome to isolate the entire rudimentary locus plus flanking DNA segments. To positively identify the rudimentary gene we in situ hybridized the various phage recombinants obtained in this walk to an inversion chromosome that has one breakpoint at 15A1 which inactivates the gene. The phage immediately adjacent to BS313 was found to span the inversion breakpoint and we were able to determine the orientation of the phage walk on the basis of this in situ hybridization result. Finally we indentify a portion of the rudimentary mRNA coding region on the basis of homology first to the hamster cDNA probe and to total Drosophila RNA and second, to a 7.3 kb long poly A⁺ transcript which is found both in adult flies and in embryos.