UDP-Beta4-galactosyltransferase (Beta4-(GT) is a trans-Golgi membrane- bound enzyme that performs both a housekeeping function (glycan biosynthesis) and a cell-specific function (lactose biosynthesis). Beta- GT has also been localized to the plasma membrane of many mammalian cells, including murine sperm. This cell surface localization on sperm has resulted in a model in which it is proposed that Beta4-(GT serves a functional role in sperm/egg binding. In somatic tissues, transcription of the single copy Beta4-GT gene, from two different start sites separated by about 200 bp, results in two mRNAs of 4.1 kb and 3.9 kb, distinguished by the length and Gc content of their respective 5'- untranslated regions. In all somatic tissues, the 4.l kb start site is either exclusively, or predominately used. The only exception to this pattern is the lactating mammary gland where there is a switch to the predominant use of the 3.9 kb start site, coincident with an increase in both steady state Beta4-GT mRNA levels and enzyme levels required for lactose biosynthesis. We have proposed a model of transcriptional and translational regulation for the Beta4-GT gene in somatic cells in which the distal region upstream of the 4.1 kb start site functions as a housekeeping promoter (glycan biosynthesis) and the proximal region adjacent to the 3.9 kb start site functions as a mammary cell-specific promoter (lactose synthesis). In murine male germ cells there is an additional level of transcriptional regulation. As male germ cells enter meiosis, there is a switch from the use of the 4.1 kb transcriptional start site to the exclusive use of a germ cell specific start site. This germ cell specific start site is located about 600 bp upstream of the 4.1 kb start site. We have three complementary goals in this application: (1) To experimentally validate our model of transcriptional regulation of the Beta4-GT gene in somatic tissues. Both the cis-elements and the trans- acting factors that regulate transcription from each start site will be identified using complementary in vitro analyses, including mobility shift and DNaseI footprinting assays. Potential somatic regulatory regions identified by in vitro analysis will be confirmed in vivo, using transgenic mice. A similar strategy will be used to define the male germ cell promoter elements. (2) To demonstrate that the 3.9 kb Beta4-GT transcript, due to its short 5'-untranslated region (30 nt) is more efficiently translated relative to the 4.1 kb mRNA. (3) To directly analyze the role of Beta4-GT in male germ cell development and function by developing transgenic mice in which the male germ cell specific promoter is selectively deleted by homologous recombination, without disturbing somatic cell expression.