One of the main reasons for the power of Drosophila in addressing problems of biological importance is that it allows forward genetic screens of chemically induced mutations to be carried out relatively easily. The problem of phototransduction was addressed by forward genetic screens of Drosophila mutants with defective electroretinogram (ERG). However, identifying and isolating the gene that carries the lesion responsible for the mutant phenotype are difficult and time consuming. Recently, a number of strategies have been proposed to improve the accuracy and resolution of mutation mapping so that the isolation of the lesion-bearing genes may be facilitated. However, these strategies generally involve fine mapping steps that are also labor intensive and time consuming, particularly for mapping mutants whose phenotypes can only be detected by electroretinogram (ERG) recording. Here, we describe a strategy for gene cloning that is based on DNA microarrays and that does not require fine mapping of mutations. It is based on the observation that almost all ERG defect-causing mutations also cause alterations in the mRNA levels. DNA microarrays are used to detect all annotated genes that are altered in mRNA levels in a target mutant compared to a wild-type control. Using information based solely on deficiency mapping, we then look for genes within the mapped interval that show the largest and most statistically significant alterations in mRNA levels. We describe applications of this strategy to five genes, two previously identified ones and three new ones. In all cases, it was possible to identify one or two candidate genes within the mapped interval reliably and rapidly. It generally took 4 months or less from the time of fly stock expansion for RNA isolation to the time of candidate gene identification. If there were more than one candidate gene in the mapped interval, the correct gene was identified by sequencing.
|Original language||English (US)|
|Title of host publication||Signal Transduction in the Retina|
|Number of pages||24|
|State||Published - Jan 1 2007|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)