Ligation and synthesis of chromatin deoxyribonucleic acid in vitro in neuronal, glial and liver nuclei isolated from adult guinea pig.
Neuron-rich and glial nuclear preparations and liver nuclei were isolated from adult guinea pigs. These nuclei were incubated to carry out DNA-ligation and -synthesis reactions. Before and after incubation, the sizes of single-standed DNA and DNA-synthesis patterns in single strands were analysed by using alkaline sucrose-density-gradient centrifugation. Isolation of nuclei by cell-fractionation technique shortened chromatin DNA and decreased markedly the number-average molecular weight of DNA strands. Chromatin DNA in neuronal and glial nuclei was ligated at the nicks during incubation in a reaction mixture containing ATP, Mg(2+), dithiothreitol and four deoxyribonucleotides. The number-average molecular weights were estimated to increase 1.1-and 2.1-fold in neuronal and glial nuclei respectively. DNA strands in liver nuclei were shortened during incubation, but elongated under conditions that inhibit deoxyribonuclease. Since the endogenous deoxyribounuclease activity was conspicuously higher in liver nuclei than in neuronal and glial nuclei, the shortening and elongation were thought to depend on the balance between DNA ligase and deoxyribonuclease reactions. DNA synthesis occurred at the gaps in chromatin DNA and about 50% of the total synthesized DNA was found in the shorter strands having 6 to 297 bases in all species of nuclei. Based on these results, it was concluded that in nuclei isolated from non-dividing cells (neurons) and slowly dividing cells (glial and liver cells) DNA-ligation and -synthesis reactions proceeded in parallel at the breaks in single-stranded DNA, which was produced mainly by endogenous deoxyribonuclease during isolation and incubation processes.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D004260:DNA Repair; D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases / Q000378:metabolism; D005260:Female; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism*; D009562:Nissl Bodies / Q000378:metabolism*
D002843:Chromatin; D004277:DNA, Single-Stranded; D004247:DNA; D003851:Deoxyribonucleases