Identification of novel in vitro oligonucleotide-primed labelling sites in the DNA of terminally differentiated myeloid cells.

Abstract

Cells are first embedded in agarose beads, a technique similar to that used in pulse-field-gel electrophoresis. Embedded cells are lysed in situ, subjected to mild alkali treatment and the single-stranded DNA used as template for labelling by the oligonucleotide-primer labelling method. The labelled DNA is then used to probe a battery of genes immobilized on a membrane. As a control, a similar membrane is probed with randomly labelled, completely denatured DNA. Using this new method, I attempted to identify sequences at or near oxyradical-induced breaks in human granulocyte DNA. In human granulocyte DNA, five genes (FOS, CSF2, JUN ERB-B2 and TRF) were found to be 100-1000x more intensely labelled than other genes tested. These sites of labelling were named Selective Labelling Points (SLPs). Further experiments showed that SLPs were present in PMN DNA even in the absence of oxyradical-induced breaks. (Abstract shortened by UMI.)