Restriction enzymes have limited access to DNA sequences in Drosophila chromosomes.
Sequence specific DNA binding proteins in eukaryotic cells must efficiently locate their binding sites in chromosomes. Restriction enzymes provide a simple model system with which to investigate the factors which influence this process. We have used P element mediated transformation to introduce a DNA fragment containing a set of characterized restriction sites into the Drosophila germline. Embryonic nuclei prepared from these transgenic animals were treated with restriction enzymes to probe the accessibility of the target restriction sites. The results show that the insert is within an accessible region of the chromosome and that restriction sites within the inserted sequence can be cut. However, the rate of cutting is biphasic. At each restriction site, a fraction of the chromosomes is cut rapidly after which the remainder is refractory. Similar levels of incomplete cutting are obtained when the same P element construct is examined at a different chromosomal location, when different sequence elements are introduced into the P element vector or when the experiment is carried out on nuclei from different embryonic stages. These results are discussed in terms of how sequence specific DNA binding proteins may locate their genomic targets in vivo.
Jack
R S
RS
Institut für Genetik der Universität zu Köln, FRG.
Eggert
H
H
D000818:Animals; D002875:Chromosomes; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics*; D005822:Genetic Vectors; D007700:Kinetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013379:Substrate Specificity; D014170:Transformation, Genetic
D004268:DNA-Binding Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII