In this twice amended renewal proposal, the P.I. requests $507,142 in direct costs for a period of three years to investigate the organization of DNA in the mammalian sperm nucleus. Prior work by the P.I. has defined two kinds of sperm chromosomal organization: chromosomal anchoring at the sperm nuclear annulus, and DNA loop domains. The P.I. has isolated four distinct short clones of DNA specifically associated with the sperm nuclear annulus. In Specific Aim 1, he proposes to study them further and will: a) isolate and characterize longer clones from a genomic library, b) determine if the sequences are associated with more than one chromosome by FISH mapping to mitotic chromosomes, and c) use FISH techniques to determine if the sequences begin to co-localize at the same time at the nuclear annulus to provide support for the idea that the annulus is involved in the chromatin condensation that occurs as spermatids differentiate. The P.I. has previously shown that the DNA loop domain is different, and smaller, in sperm cells than in somatic cells. In Specific Aim 2, the P.I. will test the hypothesis that the sperm DNA loop domain is unique to the germ cell lineage and is passed to the embryo. He will do this by using five probes for different gene sequences to map loop domain structure in spermatogonia, spermatocytes and spermatids; in oocytes; and, finally, to determine if it is inherited, in fertilized eggs and in early and late-stage embryonic cell lines. In Specific Aim 3, the hypothesis that one of the sperm DNA loop domains can function as a replicon will be examined by using an "in vitro" DNA replication system.