PDD1P ASSOCIATES WITH GERMLINE-RESTRICTED CHROMATIN AND A 2ND-NOVEL ANLAGEN-ENRICHED PROTEIN IN DEVELOPMENTALLY PROGRAMMED DNA ELIMINATION STRUCTURES

Programmed DNA rearrangements, including DNA diminution, characterize the differentiation of somatic from germline nuclei in several develop mental systems, Pdd1p (Programmed DNA degradation protein 1), a develo pment-restricted polypeptide, has been implicated in heterochromatin a ssembly and DNA degradation during ciliate macronuclear development, H ere, cross-linking and co-immunoprecipitation were used to verify that Pdd1p-associated chromatin is enriched in germline-restricted DNA, Pd d1p-associated proteins include general core histones and a second anl agen-enriched polypeptide (Pdd2p, formerly known as p13), Immunoblotti ng analyses demonstrate that, like Pdd1p, Pdd2p is developmentally reg ulated and present in conjugating cells during the time of germline DN A rearrangements and degradation, Pdd2p is post-translationally modifi ed by phosphorylation at a time in development corresponding to dephos phorylation of Pdd1p and the formation of heterochromatic DNA eliminat ion structures, Following gene cloning, the derived amino acid sequenc e of the PDD2 gene predicts a novel polypeptide containing multiple pu tative phosphorylation sites, In situ analyses, using both light and e lectron microscopy, demonstrate that Pdd1p and Pdd2p co-localize in DN A elimination structures within developing macronuclei, However, unlik e Pdd1p, which also localizes to apoptotic macronuclei, Pdd2p appears to be restricted to a higher degree to germline DNA elimination struct ures, Taken together, the data presented here demonstrate a physical l ink between Pdd1p and germline-restricted chromatin and establish Pdd2 p as the second member of a small group of developmentally restricted polypeptides implicated in programmed DNA elimination.