Large scale screen for transposon insertions into cloned genes.
We describe a method of screening for transposon insertions in or near Drosophila loci that correspond to cloned DNA sequences. We mobilize a modified P element transposon that carries a bacterial plasmid origin of replication and a drug-resistance marker. The genomic sequences flanking each transposon insertion site can then be rescued as a plasmid in Escherichia coli. Libraries of such plasmids, representing pools of transposon-mutagenized individuals, are used as hybridization probes against cloned sequences to determine whether a transposon has inserted next to a particular site in the genome. The number of loci that can be screened simultaneously by this procedure is quite large. We have screened an array of cDNA clones representing almost 700 distinct loci against libraries representing 760 mutagenized flies, and we obtained hybridization signals to 7 different cDNAs. Three of these events have been analyzed in detail and represent genuine insertions near genomic sequences that correspond to the cDNAs.
Hamilton
B A
BA
Division of Biology, California Institute of Technology, Pasadena 91125.
Palazzolo
M J
MJ
Chang
J H
JH
VijayRaghavan
K
K
Mayeda
C A
CA
Whitney
M A
MA
Meyerowitz
E M
EM
D000818:Animals; D001483:Base Sequence; D002875:Chromosomes / Q000502:physiology / Q000648:ultrastructure; D003001:Cloning, Molecular / Q000379:methods*; D003433:Crosses, Genetic; D004247:DNA / Q000235:genetics; D004251:DNA Transposable Elements; D004330:Drosophila / Q000235:genetics*; D005260:Female; D015723:Gene Library; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction
D004251:DNA Transposable Elements; D015345:Oligonucleotide Probes; D004247:DNA