A long term objective of this project is to determine mechanisms regulating transcription of testis-specific genes. An immediate objective of this project is to isolate and characterize a testis-specific DNA-binding protein (TE-binding protein) and clone the gene encoding this putative tissue-specific transcriptional factor. Recently, an element (TE) that binds TE-binding protein with high affinity was found located between the H1/AC box and H1/CCAAT box of the testis-specific histone H1t gene. The testis-specific TE-binding protein is first detectable in primary spermatocytes and a temporal correlation exists between the onset of H1t gene transcription and the expression of the TE-binding protein. To establish the biochemical nature of this testis-specific DNA-binding protein and its role in regulating transcription, in Specific Aim 1 the protein will be purified. Amino acid sequence analysis of purified polypeptides will be determined and compared to known polypeptides. To further establish the nature of the protein, the gene encoding the protein will be cloned and sequenced in Specific Aim 2.
In Specific Aim 3, polyclonal antibodies will be prepared against the protein and against synthetic polypeptides to facilitate expression library screening. Sequences of peptides from the purified DNA-binding proteins will be used to design mixed oligonucleotide probes that can be used to screen cDNA and genomic libraries. To determine the role of the TE-binding protein in regulating testis-specific transcription, its activity will be examined in transcription assays.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD029381-06
Application #
2889060
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Tasca, Richard J
Project Start
1993-04-01
Project End
2001-07-31
Budget Start
1999-08-01
Budget End
2001-07-31
Support Year
6
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Overton Brooks VA Medical Center
Department
Type
DUNS #
City
Shreveport
State
LA
Country
United States
Zip Code
71101
Wolfe, Steven A; Grimes, Sidney R (2003) Transcriptional repression of the testis-specific histone H1t gene mediated by an element upstream of the H1/AC box. Gene 308:129-38
Wilkerson, Donald C; Wolfe, Steven A; Grimes, Sidney R (2003) TE2 and TE1 sub-elements of the testis-specific histone H1t promoter are functionally different. J Cell Biochem 88:1177-87
Wolfe, Steven A; Grimes, Sidney R (2003) Specific binding of nuclear proteins to a bifunctional promoter element upstream of the H1/AC box of the testis-specific histone H1t gene. Biol Reprod 68:2267-73
Noss, Kenneth R; Wolfe, Steven A; Grimes, Sidney R (2002) Upregulation of prostate specific membrane antigen/folate hydrolase transcription by an enhancer. Gene 285:247-56
Wilkerson, Donald C; Wolfe, Steven A; Grimes, Sidney R (2002) H1t/GC-box and H1t/TE1 element are essential for promoter activity of the testis-specific histone H1t gene. Biol Reprod 67:1157-64
Noss, Kenneth R; Singal, Rakesh; Grimes, Sidney R (2002) Methylation state of the prostate specific membrane antigen (PSMA) CpG island in prostate cancer cell lines. Anticancer Res 22:1505-11
Wilkerson, Donald C; Wolfe, Steven A; Grimes, Sidney R (2002) Sp1 and Sp3 activate the testis-specific histone H1t promoter through the H1t/GC-box. J Cell Biochem 86:716-25
Grimes, S R; Wolfe, S A; Singal, R (2001) Macro for analysis of CpG and CpNpG methylation in plants. Biotechniques 30:1248
Singal, R; Grimes, S R (2001) Microsoft Word macro for analysis of cytosine methylation by the bisulfite deamination reaction. Biotechniques 30:116-20
Singal, R; vanWert, J; Bashambu, M et al. (2000) Testis-specific histone H1t gene is hypermethylated in nongerminal cells in the mouse. Biol Reprod 63:1237-44

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