Abstract
Two formaldehyde-induced mutations at the DrosophilaAdh locus (Adh fn45 andAdh fn46) were analyzed by determining RNA structures at different developmental stages, polymerase chain reaction (PCR) amplification of the affected genomic regions, and direct sequencing of the resulting double-stranded DNA fragments.Adh fn46 adults and larvae accumulate abundant ADH-like distal (adult) and proximal (larval) transcripts that are shorter than transcripts in wild-type flies by a lesion located in the second ADH protein-coding exon. Direct sequencing of the amplified DNA region showed thatAdh fn46 contains a 69-bp in-frame deletion that removes 23 amino acids near one border of the second exon. Consistent with these findings, we observed a shorter ADHfn46 protein present at only 3% of wild-type levels. In contrast,Adh fn45 adults and larvae accumulate much smaller amounts of ADH-like distal and proximal transcripts. Both RNAs have an identical aberration in RNA splicing of the 65-base intron sequence. Direct sequencing of the amplified mutated DNA region showed thatAdh fn45 contains a 21-bp deletion that removed and rearranged DNA at the 5′ splice junction of the 65-bp intron. No ADH cross-reacting material is detected inAdh fn45 flies. Direct-repeat sequences (3–11 bp) are present flanking and within the mutated DNA regions. The patterns of DNA deletion and deletion accompanied by sequence addition at the mutant sites suggest a slipped mispairing mechanism during DNA replication or repair that involves local DNA homology.
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This work was supported by Grant GM34850 from the National Institutes of Health to C.B. and Grant PCM-8110819 from the National Science Foundation to A.R.P. Long Le was an undergraduate de Kiewiet Summer Research Fellow in the Program in Biology and Medicine at the University of Rochester in the summer of 1988. This is contribution No. 129 from the Center of Marine Biotechnology, University of Maryland.
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Le, L., Ayer, S., Place, A.R. et al. Analysis of formaldehyde-inducedAdh mutations inDrosophila by RNA structure mapping and direct sequencing of PCR-amplified genomic DNA. Biochem Genet 28, 367–387 (1990). https://doi.org/10.1007/BF02401426
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DOI: https://doi.org/10.1007/BF02401426