Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette.
As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.
Eggert
H
H
Biologie, Abteilung Cytogenetik, Humboldt Universität, 10115 Berlin, Germany. harald=eggert@rz.hu-berlin.de
Bergemann
K
K
Saumweber
H
H
D000818:Animals; D001483:Base Sequence; D003433:Crosses, Genetic; D017931:DNA Primers; D004251:DNA Transposable Elements; D004331:Drosophila melanogaster / Q000235:genetics*; D005260:Female; D016678:Genome; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping
D017931:DNA Primers; D004251:DNA Transposable Elements