Efficient recovery of centric heterochromatin P-element insertions in Drosophila melanogaster.
Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow(+) (y(+))-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from approximately 3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for approximately 63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.
Yan
Christopher M
CM
Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Dobie
Kenneth W
KW
Le
Hiep D
HD
Konev
Alexander Y
AY
Karpen
Gary H
GH
D000818:Animals; D004247:DNA / Q000302:isolation & purification; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D005260:Female; D006570:Heterochromatin; D017404:In Situ Hybridization, Fluorescence; D019476:Insect Proteins / Q000235:genetics; D008297:Male; D017422:Sequence Analysis, DNA
D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D006570:Heterochromatin; D019476:Insect Proteins; C050680:y protein, Drosophila; D004247:DNA