A screen for mutations affecting mRNA processing in Drosophila

This thesis reports: (i) the design and construction of a white gene reporter system in D. melanogaster intended to detect mutations with small effects on mRNA processing, particularly on cleavage and polyadenylation; (ii) the characterization of transcripts from the white reporter gene; and (iii) the isolation and characterization of mutations affecting the expression of the reporter gene.;The reporter gene, designated white-sensor ( ws) consists of a white gene with two copies of a 301 bp modified white cleavage and polyadenylation signal segment inserted in tandem in the untranslated leader of the white transcription unit. The reporter fly, showing a pale yellow eye color, consists of a third-chromosome (ws) insertion in a white deletion background.;Dominant and recessive X-linked and dominant autosomal EMS-induced mutations affecting ws eye color were selected. Eye color darkening mutations closely linked to (ws) (cis-dark) resulted from precise excision of one of the two inserted 301 bp segments. A further mutation with a near wild-type eye color (cis-dark-dark) arose spontaneously from one of the cis-dark lines. The DNA sequence of its solo inserted 301 bp segment is identical to that of wild-type white and differs from the corresponding segment of the cis-dark and (w s) lines.;RNA studies and DNA sequencing indicated the presence of putative 0.7 kb and 0.4 kb introns in the ws and cis-dark genes, respectively, with a donor site within the inserted 301 bp segment and an acceptor site downstream of the translation start site. Splicing of either intron deletes the translation start site and part of the coding sequence, causing hypomorphic expression. There is no such splicing in the cis-dark-dark gene, transcripts of which include a 0.3 kb minor species truncated at the inserted cleavage and polyadenylation signal segment and a 2.8 kb major species corresponding to functional message.;A second-chromosome eye color darkening mutation decreased the splicing efficiency of the 0.7 kb intron in the (ws) reporter gene. Therefore, the current (ws) system is able to detect trans-acting mutations that affect splicing and, if suitably modified, would be of use in isolating weak mutations that affect cleavage and polyadenylation.