Some genes with fundamental ubiquitous roles also exhibit cell type- specific functions. Since mutations that disrupt ubiquitous function are generally lethal, it has been difficult to study the specific roles of such genes. Thus, it would be useful to genetically manipulate specific functions of such genes in a directed fashion. This project investigates a highly tissue-specific function of a gene whose product is absolutely required by all eucaryotic cells. The TATA-binding protein, TBP, is required for all nuclear transcription initiation and thus is crucial for most cellular functions. In spermatids, the tbp gene is expressed at levels about 100- to 1000-fold greater than those in somatic cells. This suggests that TBP plays a specialized role in spermatogenesis. The goal of this project is to first disrupt, and then systematically rescue, the spermatid-specific function of TBP in transgenic mice. Because TBP is required for all nuclear transcription, it is important that normal somatic cell expression and function not be disrupted. To achieve this, two different approaches will be taken. In the first, the bacteriophage P1 Cre/loxP site-specific recombination system will be used to precisely excise the testis-specific promoter/ first exons without disrupting the somatic promoter/first exon. In an alternate approach, all of the alternate tbp gene promoter/first exons will be removed and replaced with the 5' region of the gene encoding the ubiquitous transcription factor, Oct1. Oct1 mRNA levels are quantitatively similar to TBP mRNA levels in somatic tissues, but are not overexpressed in testis. This later approach might prove valuable if, in the former approach, overexpression of the remaining promoter/ first exon compensates for loss of the spermatid-specific transcription units. Transgenic mouse lines that fail to overexpress TBP in spermatids will be used for complementation experiments. Vectors carrying wild type or in vitro-modified versions of the tbp gene will be expressed in the mutant germ lines to determine what domains of TBP must be intact for the exogenous protein to rescue spermatogenesis. This should provide insights into the spermatid-specific roles of TBP and provide a valuable paradigm for how specific activities of fundamental genes can be selectively disrupted.
