The correct functioning of the mammalian P1 and P2 protamine gene pair during the maturation of the spermatozoon is critical for fertilization because it ensures the precise packaging of the male genetic material. Transcriptional activity of this gene pair is modulated in a rather abrupt manner. It is activated from a quiescent state then repressed quickly as part of the termination of cellular transcription. This provides an excellent model system to study how a gene complex is readied for expression, briefly expressed then precipitously repressed. The elucidation of this mechanism will be aided by defining the domain for this haploid expressed gene pair and determining its conformational organization during differentiation. This fundamental problem has not been rigorously addressed for any reproductive system. To achieve this goal, the protamine domain will be defined by DNase-I mapping of nuclei isolated from male germ cells separated into various stages of differentiation. This will establish the boundaries of the domain and delineate when it assumes a transcriptionally competent state, i.e., primed for expression. Accordingly, the activation/repression model proposed in this study will be directly tested. The model predicts that within the pachytene spermatocytes, the protamine domain will be compacted, then upon mitotic division and differentiation into the round spermatid forming a more open conformation primed for expression. Primary sequence characterization of the protamine domain will provide the first detailed analysis of this region, filling the gaps that remain in many of the published fragmentary protamine sequences. Supplemental Northern and PCR analysis will be employed to determine whether other genes are contained within this domain and whether they are co-expressed during differentiation. Upon defining and characterizing the domain, candidate, Locus Control Regions Will be mapped by assessing DNase-I hypersensitivity. To define these cis sequence elements, the regions will be fine-mapped using Ligation Mediated PCR. Completion of this project will provide the basis from Which to isolate and purify the associated trans-activating conformational factor(s) that prepare a haploid domain for expression.
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