Active poly(ADPribose) metabolism in DNAase- and salt-resistant rat testis chromatin with high transcriptional activity/competence.
A chromatin fraction, named pP fraction, was prepared from rat testis nuclei, which had been digested with nuclease in order to separate soluble and insoluble chromatin. This fraction resembled nuclear matrix as it was highly resistant to DNAase digestion, had a high content of proteins compared to the low DNA percentage, and a noticeable transcriptional activity. Moreover, poly(ADPribosyl)ation system (i.e., poly(ADPR)polymerase, poly(ADPribose), and acceptor proteins) was still present at high levels. In order to study whether it might be identified as the protein support surrounding chromatin loops, this pP fraction was further analyzed after 3 M NaCl extraction. The 3 M NaCl extract and the highly insoluble pellet, named Nuclear Matrix Pellet, were characterized as it regards DNA, newly synthesized RNA and proteins. Furthermore, poly(ADPribose) metabolism was analyzed by measuring both poly(ADPribose) polymerase and poly(ADPribose) glycohydrolase activities, poly(ADPribose) distribution and by identifying protein acceptors. The final pellet had features of nuclear matrix containing less than 10% DNA and high percentage of proteins; 28% of newly synthesized RNA was still associated with this fraction. Long and branched polyADPribose were found in the nuclear matrix-like pellet, although ADPribose acceptors (mainly H1 and core histones) appeared to be modified mostly with short ADPribose oligomers. Longest and branched polymers were retained on the top of protein gel, likely bound to automodified poly(ADPribose) polymerase.
Mennella
Maria Rosaria Faraone
MR
Dipartimento di Chimica Biologica, Facoltá di Scienze, Universitá Federico II, Via Mezzocannone, 16-80134 Napoli, Italy. faraone@unina.it
Roma
Guglielmo
G
Farina
Benedetta
B
D000818:Animals; D015153:Blotting, Western; D005591:Chemical Fractionation / Q000379:methods; D002843:Chromatin / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000032:analysis / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006657:Histones / Q000032:analysis; D008297:Male; D015530:Nuclear Matrix / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology*; D012995:Solubility; D013737:Testis / Q000187:drug effects / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D002843:Chromatin; D006657:Histones; D011064:Poly Adenosine Diphosphate Ribose; D012965:Sodium Chloride; D012313:RNA; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D003851:Deoxyribonucleases; D006026:Glycoside Hydrolases