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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD028504-03
Application #
2201149
Study Section
Reproductive Endocrinology Study Section (REN)
Project Start
1992-04-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1996-03-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Wayne State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Kramer, J A; Krawetz, S A (1996) Analysis of transgenes by polymerase chain reaction: establishing and maintaining the PRM1-->PRM2-->TNP2 line. Anal Biochem 235:110-1
Kramer, J A; Krawetz, S A (1996) Nuclear matrix interactions within the sperm genome. J Biol Chem 271:11619-22
Choudhary, S K; Wykes, S M; Kramer, J A et al. (1995) A haploid expressed gene cluster exists as a single chromatin domain in human sperm. J Biol Chem 270:8755-62
Nelson, J E; Krawetz, S A (1995) Mapping the clonally unstable recombinogenic PRM1-->PRM2-->TNP2 region of human 16p13.2. DNA Seq 5:163-8
Kramer, J A; Krawetz, S A (1995) Matrix-associated regions in haploid expressed domains. Mamm Genome 6:677-9
Wykes, S M; Nelson, J E; Visscher, D W et al. (1995) Coordinate expression of the PRM1, PRM2, and TNP2 multigene locus in human testis. DNA Cell Biol 14:155-61
Nelson, J E; Krawetz, S A (1994) Easy-read DNA sequencing gels. Biotechniques 17:416, 418
Nelson, J E; Krawetz, S A (1994) Characterization of a human locus in transition. J Biol Chem 269:31067-73
De Jonckheere, J; Nelson, J E; Ginsburg, K A et al. (1994) GA repeat polymorphism at the PRM2 male fertility locus. Hum Mol Genet 3:1915
Nelson, J E; Krawetz, S A (1993) Linkage of human spermatid-specific basic nuclear protein genes. Definition and evolution of the P1-->P2-->TP2 locus. J Biol Chem 268:2932-6

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