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.
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