The major objective of this research program is to identify and characterize DNA determinants of mammalian gene regulation using the murine Beta glucuronidase (GUS) gene complex as a model. This gene complex, designated [Gus], is defined by the GUS structural gene, Gus-s, and by three elements which specifically control the expression of Gus-s (reviewed in ref. 1). Configurations of alleles of each of these elements define three common GUS haplotypes, [Gus]alpha, [Gus]b and [Gus]h. The GUS regulatory elements include a cis-active androgen response element (Gus-r) identified by variants, which differ in the kinetics of GUS response to androgen in kidney epithelial cells. Another variant, which may or may not be an allele of Gus-r, is characterized by the failure of kidney GUS to respond to androgen and partially defines the rare haplotype, [Gus]or. Two other GUS regulatory elements are defined by mutations affecting GUS expression in [Gus]h mice. One (Gus- u) is a cis-active systemic regulator of GUS, while the other (Gus-t) regulates the postnatal accumulation of GUS in certain tissues. Gus-s has been cloned in single isolates from DNA libraries of each of the GUS haplotypes. The entire gene from [Gus]b and [Gus]h targets the DNA determinant of Gus-u to a region around position 272 of the mRNA which corresponds to sequence from within the second exon of Gus-s. Another Pol II transcriptional unit which is located on the same strand and initiated within the first intron of Gus-s has been identified. Designated Gig, this """"""""gene-within-a-gene"""""""" produces a 2.2 kb mature liver transcript sharing little, if any, sequence with the 2.7 kb GUS mRNA. The battery of cloned and characterized DNA sequences from the informative [Gus] haplotypes will be used as reagents to identify and analyze DNA determinants of those elements which underlie GUS regulatory variation and to further characterize the structure and function of the Gig gene. Androgen response elements and other regulatory regions will be targeted within [Gus] by comparing the DNAse hypersensitivity of kidney nuclei between androgen-treated and untreated mice, and among mice of each informative GUS haplotype. Concurrently, experiments will be conducted in transgenic mice to test whether those genomic clones which contain Gus-s and substantial flanking sequence are capable as transgenes of being appropriately expressed and of responding to androgen; and to what extent this responsiveness resembles the haplotypic pattern observed in nontransgenic mice. Since the effects of Guys-t and Gus-u on the rates of GUS synthesis are not mediated through effects on GUS mRNA levels, experiments are proposed to examine the possible effects of these elements on translation of GUS mRNA by comparing liver polysome profiles of GUS mRNA between the variant haplotypes. If Gus-t and Gus-u exert control over the translatability of GUS mRNA, then direct tests are proposed to determine whether the [Gus]b - [Gus]h mRNA compared between transfected and untransfected cells. Experiments are also proposed in transgenic mice to determine whether the Gus-t function is located within available genomic clones of [Gus]. To determine the location of Gig exons relative to those of Gus-s, Gig cDNAs will be identified and their sequences compared to that of Gus-s. Experiments are proposed to establish whether Gig and Gus-s are coexpressed within the same cells.
Showing the most recent 10 out of 11 publications
