Genomic footprinting of Drosophila embryo nuclei by linker tag selection LM-PCR.
The unmatched power of Drosophila genetics revealed the complex regulatory network of gene activities that governs the development of higher eukaryotes. An understanding of gene control at the level of transcription requires insight into the protein/DNA interactions that regulate transcription in the developing embryo. Genomic footprinting allows the direct visualization of these protein/DNA interactions within intact nuclei or cells. In combination with other in vivo assays such as protein/DNA crosslinking and classical biochemistry, genomic footprinting can give valuable insight into the architecture of promoters in various states of activity. In this article we summarize our experience in analyzing Drosophila embryos by genomic footprinting and describe modifications of the ligation-mediated PCR procedure that have improved this analysis. Applications of genomic footprinting to embryos are currently limited by the fact that all target nuclei must be uniform with respect to the protein/DNA interactions at the chosen site. We discuss strategies that should allow the analysis of small numbers of cells derived from heterogeneous populations and tissues.
Quivy
J P
JP
Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Becker
P B
PB
D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004330:Drosophila / Q000196:embryology; D004625:Embryo, Nonmammalian; D016678:Genome; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D003850:Deoxyribonuclease I