[Use of the modified method of chromatin immunoprecipitation for the isolation of actively transcribed loci].

A modified version of the chromosomal immunoprecipitation (ChIP) assay was implemented for discrete isolation and characterization of actively transcribed genes. Specifically, it was demonstrated with the gene II/9-1 of Sciara coprophila as a model locus that significant enhancement in the isolation of actively transcribed versus repressed and inactive genes can be achieved through the ChIP methodology. A combination of solid-phase magnetic bead technology with chromosomal immunoprecipitation using antibodies that recognize the large subunit (c) of RNA polymerase II resulted in efficient isolation of the promoter region of gene II/9-1 exclusively during the amplification stage of larval development, when the gene is actively transcribed.

[Identification of two new p53 target genes through implementation of the modified chromatin immunoprecipitation method and inverse PCR].

Direct target loci for the transcription factor p53 were identified through the employment of a combination of a modified version of chromosomal immunoprecipitation and inverse PCR. Irradiation of Hela cells to drive DNA damage response was followed by sequential chromosomal immunoprecipitation utilizing antibodies which recognize the large subunit of RNA polymerase II and p53. Inverse PCR with degenerate oligonucleotides specific for the p53 binding site was subsequently performed on immunoprecipitated DNA and fragments containing putative p53 target genes were subcloned and sequenced.

[Segments initiating replication of the dihydrofolate reductase domain and the DNA fragment DARC146 interact with the same nuclear proteins].

Comparison analysis was made of the putative replication origin DARC146 and the origin determined in the DNFR domain of CHO cells. We failed to observe extensive homology between these two sequences. However, several short (8-10 bp) areas of homology were identified.

[DNA fragment DARC146 from a complex form of DNA polymerase alpha contains several nuclear protein binding segments].

Earlier, a number of DNA fragments were identified in the complex form of DNA polymerase alpha. One of them, DARC146, can support autonomous replication in mammalian cells. We have subcloned 146 bp from DARC146 (here called DARC146).