Changing Patterns of Single-Stranded-DNA-Binding Proteins in Differentiating Brain Cortex and Cerebellar Neurons

Abstract

Single-stranded-DNA-binding proteins were analyzed in nuclei of differentiating rat cortex and cerebellar neurons. The developmental period investigated ranged from gestational day 19 (i.e., 3 days before term) to postnatal day 30. During this time both types of neurons undergo transition from proliferating, undifferentiated precursor cells to non-proliferating, terminally differentiated neurons. For comparison, nuclei from mature cortex glia and liver were also examined. Nuclei were isolated according to cell type, the proteins were 14C-labeled in vitro by reductive methylation and were fractionated by affinity chromatography on tandemly arranged columns of double-stranded [ds] and single-stranded [ss] DNA-cellulose. The columns were uncoupled and the proteins adsorbed to the ss DNA were eluted with salt. They were then analyzed by high resolution 2-dimensional gel electrophoresis followed by fluorography. This strategy ensured the selective detection of proteins that recognize ss DNA specifically, and eliminated interference by proteins binding to DNA by simple ionic interaction as well as by proteins with affinity for ds DNA. Many ss-DNA-binding proteins showed conspicuous developmental fluctuations. In cortex neurons these took place around the time of birth and the 1st postnatal week; in cerebellar neurons they occurred later and in a more protracted fashion. In both cortex and cerebellar neurons the protein changes followed a time course closely paralleling the arrest of cell division and the beginning of terminal differentiation. This approach may lead to the detection of putative regulatory proteins of the cell nucleus.