| First Author | Ganschow R | Year | 1986 |
| Journal | Mouse News Lett | Volume | 76 |
| Pages | 53 | Mgi Jnum | J:32408 |
| Mgi Id | MGI:79903 | Citation | Ganschow R, et al. (1986) The molecular genetics of murine beta-glucuronidase. Mouse News Lett 76:53 |
| abstractText | Full text of MNL contribution: THE MOLECULAR GENETICS OF MURINE Beta-GLUCURONIDASE Ganschow R., Abbott R., D 'Amore M., Gallagher P. Korfhagen T., McNeish J., Potter S., Wang B. Division of Basic Science Research, The Children's Hospital Research Foundation, Cincinnati, OH 45229, USA Our research program attempts to identify and characterize DNA determinants of genetic and hormonal regulation of mammalian gene expression through biochemical, genetic and molecular analyses of the murine Beta-glucuronidase (GUS) gene complex. In addition to the GUS structural gene (Gus-s), three regulatory functions have been identified within this complex which are specific to GUS (for review see Paigen, K., Ann. Rev Genet. 13: 417, 1979). Two represent cis-active elements, one controlling the androgen-responsiveness of kidney GUS mRNA and the other regulating GUS levels in all tissues at all times. The third specifies the developmental timing of GUS expression and probably represents the only example of a trans-acting cellular genetic element which is tightly linked to the gene over which it exerts control. We have isolated cDNA and genomic clones representing regions within this gene complex and have subjected such cloned DNAs to sequence analysis in order to characterize the structure of Gus-s. Of approximately 2,900 nucleotides in GUS mRNA, all but 450 at the 5' end can be accounted for in the cloned cDNAs. An open reading frame (ORF) of 1,718 nucleotides extends from the 5' end of the composite cDNAs to the beginning of a 516 nucleotide untranslated region at the 3' end of the composite. Within this ORF is a sequence of 130 amino acids which is 40 percent homologous to an amino acid sequence of similar length in Beta-galactosidase of E. coli. Gus-s is a single gene with at least eleven exons and no closely related pseudogenes. While the 5' end of Gus-s is undefined at this time, the 3' untranslated region of the GUS mRNA and all but the first two amino acids of GUS are encoded within 14 kb of DNA. Moreover, a short genomic sequence located 32 kb upstream of the Gus-s poly-A addition signal hybridizes to a single, 2 kb mRNA clearly distinguished from that of GUS and probably represents a portion of an exon from the gene immediately upstream of Gus-s. Sequence analysis of cloned genomic DNA within Gus- s reveals appropriately-spaced and -oriented consensus promoter sequences. These sequences do not represent the Gus-s promoter since exons of Gus-s are found a considerable distance upstream and downstream of this site. Three lines of evidence suggest, however, that these sequences represent an active promoter. First, restriction fragments of genomic clones containing these sequences act as RNA polymerase II promoters in HeLa cell extracts. Second, when such fragments are inserted in the proper orientation immediately upstream of the bacterial chloramphenicol acetyl transferase (CAT) gene in the shuttle vector, pSVOCAT, promotion of CAT activity is observed in transfected L cells. Third, preliminary studies of mice transgenic for a construct containing the putative promoter region immediately upstream of the CAT gene suggest that expression of this element in adult male mice may be testis-specific (these studies also show that liver, and probably other mouse tissues, contain a potent inhibitor of bacterial CAT as well as low levels of CAT activity, all of which are destroyed by heating at 65 degrees for 1 hr). |
