A cis-acting genetic element, designated Gus-r, regulates the androgen-induced rates of murine glucuronidase (EC 3.2.1.31) synthesis in kidney tubule cells and is tightly linked to the glucuronidase structural gene, Gus-s. To investigate the molecular mechanism underlying this regulation, we have cloned a glucuronidase-specific cDNA sequence in plasmid pBR322. This cloned DNA has been utilized as a probe in blot hybridization analyses to determine whether the control of androgen responsiveness of kidney glucuronidase synthesis by Gus-r is exerted over the level or the translatability of glucuronidase mRNA. Three important observations emerged from these studies: (i) glucuronidase mRNA exists as a single size class of approximately 2,800 nucleotides; (ii) androgen stimulation of glucuronidase synthesis is directly related to the level of glucuronidase mRNA; and (iii) strain differences in levels of kidney glucuronidase mRNA accumulated in response to androgen are controlled by alleles of Gus-r. Thus, Gus-r regulates the androgen responsiveness of glucuronidase synthesis by controlling the amount of glucuronidase mRNA available for translation and is a cis-acting genetic element that regulates the hormonal responsiveness of a specific mRNA.

An iodometric assay for β-lactamase has been employed for identifying colonies of Escherichia coli transformed to tetracycline resistance (Tcr) by pBR322 carrying inserts at the PstI site. This assay is based upon the ability of β-lactamase produced by ampicillin-resistant (Apr) cells to convert penicillin to penicilloic acid which in turn binds iodine. Growth and selection of E. coli transformed to AprTcr or ApsTr are obtained on Luria agar plates containing soluble starch and tetracycline. When indicator solution containing penicillin and iodine is added to the colonized plates, β-lactamase-producing (Apr) colonies rapidly clear the overlying indicator solution whereas non-β-lactamase-producing (Aps) colonies exhibit no clearing effect. This reaction persists and substantial numbers of viable cells remain well beyond the end of the 15-min observation period. In post-test assessment of phenotype, all nonclearing colonies exhibited the ApsTcr phenotype while those that cleared the indicating solution exhibited the AprTcr phenotype. Application of this assay to an actual transformation experiment permitted rapid and unambiguous identification of the ApsTcr phenotype.