Androgen Regulation of Murine β-Glucuronidase Expression: Identification and Characterization of a Nonresponse Variant

One of the major features of beta-glucuronidase (GUS) expression in inbred strains of the house mouse, Mus musculus, is the responsiveness of this enzyme to androgen stimulation in tubule cells of the kidney. Both GUS-specific and nonspecific mutations have been described which define genes that serve to control this response. During examination of the expression of GUS in the interbreeding subspecies, Mus hortulanus, a new GUS haplotype was uncovered that is characterized, in part, by a lack of GUS response to androgen stimulation in an apparently responsive kidney. Blot hybridization analyses of kidney RNA with a radiolabeled murine GUS cDNA shows this lack of response to be reflected in GUS mRNA levels. The difference in heat stability of GUS activity between M. hortulanus and a responsive inbred strain, ICR/Ha, was utilized to assess the contribution of each parent to kidney levels of GUS in androgen-treated and -untreated F1 progeny of these strains. The results, together with preliminary genetic studies, suggest that the element controlling this responsiveness (or the lack thereof) is cis-active and tightly linked to the GUS structural gene on chromosome 5. It is not known whether this element is identical to another GUS-specific, cis-active element, Gus-r, which also controls the androgen response of GUS in mouse kidney.     

Androgen induction of beta-glucuronidase translational yield in submaxillary gland of B6.N mice

Androgens induce the synthesis of murine beta-glucuronidase (GUS) 10-fold in the submaxillary gland of B6.PAC-Gusn mice without a concomitant increase in GUS mRNA levels. Since the rate of GUS synthesis per mRNA molecule, or translational yield, is a function of both the efficiency with which the message is translated and the fraction of newly made polypeptides that are incorporated into GUS tetramers, we conclude that androgen induces at least one of these components in the submaxillary gland of B6.N mice. Genetic variation in the submaxillary gland induction response was tested for using six congenic mice strains, each carrying a different haplotype of the Gus gene complex on a C57BL/6J genetic background. The results indicate that the DNA sequences determining androgen responsiveness of the Gus gene in the submaxillary gland are linked to the Gus gene complex and that the DNA sequences determining the submaxillary gland response are distinct from those determining the androgen induction of GUS mRNA in kidney.     

Genetic variation in androgen regulation of ornithine decarboxylase gene expression in inbred strains of mice

Previous studies have indicated that androgen regulation of certain gene products in murine kidney is genetically controlled. In the present work, the expression of renal ornithine decarboxylase (ODC) gene(s) was used as a biological marker to study androgen responsiveness of eight inbred strains of mice (A/J, C57BR/cdJ, 129/J, C57L/J, BALB/cJ, SM/J, RF/J, and C57BL/6J). Kidneys of untreated females from these strains did not have significantly different basal ODC activities or ODC mRNA concentrations. However, renal enzyme concentrations in intact male mice exhibited marked strain-dependent variation; three strains (RF/J, SM/J, and C57BR/cdJ) had 5- to 20-fold higher activities than the other five strains. Renal ODC mRNA content showed similar genetic variability in the male mice; animals with highest enzyme activity had higher mRNA levels than those with low activity. These results could not be explained by differences in either serum testosterone levels or renal nuclear androgen receptor content, suggesting that the animals were differentially sensitive to endogenous androgens. To evaluate further the androgen regulation of ODC gene expression, female mice were treated with testosterone-releasing implants for 5-7 days. The two strains (A/J and C57BL/6J) that had low enzyme activity in response to endogenous testosterone in male mice also showed blunted responses to exogenous androgen administration, as measured by the induction of ODC and its mRNA. The relative distribution of the two mRNA species coding for ODC (2.2 and 2.7 kb in size) exhibited strain-dependent variation that did not, however, correlate with the androgen responsiveness. Studies of the mRNA levels in reciprocal F1 hybrids of C57BR/cdJ and C57BL/6J mice suggested that androgen sensitivity of ODC gene expression, at least in these crosses, was inherited in an autosomal dominant manner.     

mRNA synthesis rates in vivo for androgen-inducible sequences in mouse kidney.

A method was developed for measuring in vivo rates of mRNA synthesis in mice by pulse-labeling with the RNA precursor [3H]orotate and then using hybridization to recover specific mRNAs. The efficiency of recovery is determined with synthetic RNAs as internal hybridization standards. The method is particularly applicable to the kidney since this organ shows a strong preferential uptake of the label. Rates of synthesis, expressed as a fraction of total RNA synthesis, were measured for the androgen-inducible mRNAs coding for beta-glucuronidase (GUS), ornithine decarboxylase (ODC), the protein coded by the RP-2 gene, and the so-called kidney androgen-regulated protein (KAP). Control mRNAs coded for beta-actin, phosphoenolpyruvate carboxykinase, and major urinary protein. Testosterone markedly increased the synthesis of the androgen-inducible mRNAs, but not the control mRNAs. Induction was not seen in mutant mice lacking functional androgen receptor protein. For GUS, ODC, and RP-2 mRNAs, the fold induction of synthesis was less than the fold induction of concentration, suggesting that mRNA stabilization also plays a part in the response to androgen. For GUS, ODC, and RP-2 mRNAs, but not KAP mRNA, induction of synthesis was rapidly reversed after testosterone removal. KAP mRNA was also exceptional in that its concentration was disproportionately high compared with its rate of synthesis, implying that it is a particularly stable mRNA.     

DNA determinants of structural and regulatory variation within the murine beta-glucuronidase gene complex.

The murine beta-glucuronidase (GUS) gene complex, [Gus], encompasses the GUS structural element, Gus-s, and a set of regulatory elements which serve to modulate Gus-s expression. Three common GUS haplotypes representing virtually all inbred strains of laboratory mice have been compared with respect to GUS mRNA sequence. Results of such comparisons revealed sequence variations which target the location of one of the GUS regulatory elements to sequences within Gus-s and which account for known electrophoretic and heat stability differences among GUS allozymes of the three common GUS haplotypes.     

Tissue-preferential expression of a rice alpha-tubulin gene, OsTubA1, mediated by the first intron

The genomic clone encoding an alpha-tubulin, OsTubA1, has been isolated from rice (Oryza sativa L.). The gene consists of four exons and three introns. RNA-blot analysis showed that the gene is strongly expressed in actively dividing tissues, including root tips, young leaves, and young flowers. Analysis of chimeric fusions between OsTubA1 and beta-glucuronidase (GUS) revealed that the intron 1 was required for high-level GUS expression in actively dividing tissues, corresponding with normal expression pattern of OsTubA1. Fusion constructs lacking the intron 1 showed more GUS staining in mature tissues rather than young tissues. When the intron 1 was placed at the distal region from 5'-upstream region or at the 3'-untranslated region, no enhancement of GUS expression was observed. Sequential deletions of the OsTubA1 intron 1 brought about a gradual reduction of GUS activity in calli. These results suggest that tissue-preferential expression of the OsTubA1 gene is mediated by the intron 1 and that it may be involved in a mechanism for an efficient RNA splicing that is position dependent.     

A floxed allele of the androgen receptor gene causes hyperandrogenization in male mice.

We previously generated a conditional floxed mouse line to study androgen action, in which exon 3 of the androgen receptor (AR) gene is flanked by loxP sites, with the neomycin resistance gene present in intron 3. Deletion of exon 3 in global AR knockout mice causes androgen insensitivity syndrome, characterized by genotypic males lacking normal masculinization. We now report that male mice carrying the floxed allele (AR(lox)) have the reverse phenotype, termed hyperandrogenization. AR(lox) mice have increased mass of androgen-dependent tissues, including kidney, (P < 0.001), seminal vesicle (P < 0.001), levator ani muscle (P = 0.001), and heart (P < 0.05). Serum testosterone is not significantly different. Testis mass is normal, histology shows normal spermatogenesis, and AR(lox) males are fertile. AR(lox) males also have normal AR mRNA levels in kidney, brain, levator ani, liver, and testis. This study reaffirms the need to investigate the potential phenotypic effects of floxed alleles in the absence of cre in tissue-specific knockout studies. In addition, this androgen hypersensitivity model may be useful to further investigate the effects of subtle perturbations of androgen action in a range of androgen-responsive systems in the male.     

The developmental appearance of androgen receptor protein and androgen inducibility of the gus gene complex in mouse kidney

During postnatal development of mouse kidney the androgen responsiveness of epithelial cells for β-glucuronidase induction, cellular hypertrophy, and other enzyme inductions appears coincidentally with a rise in androgen receptor protein. Initially, a low level of receptor is present but no response is seen. Beginning at about 12 days of age responsiveness begins to increase, reaches a half-maximal level at 18–20 days, and full responsiveness by 28–30 days. The limiting factor appears to be levels of androgen receptor protein. Our experiments shed no light on the question of why each androgen responsive cell type in the organism differentiates the capacity to induce a different array of proteins. However, they do suggest that responsiveness of the β-glucuronidase gene does not appear until a minimum threshold level of receptor is exceeded, and that the response of the gene may not be saturated even at the highest levels of receptor reached.     

The first intron of rice EPSP synthase enhances expression of foreign gene

Translatable exon sequences in pre-mRNA often are separated by non-coding introns in eu-karyotic genomes. The removal of non-coding introns from pre-mRNA and the splicing together of translatable exons sequence is an essential requirement of gene expression. DNA size of introns in a gene is 5—10 times larger than that of exon, which can store more information and is helpful for a gene during evolution[1]. In many experiments on gene expression, it is indispensable for a gene to be expresse…     

The first intron of rice EPSP synthase enhances expression of foreign gene

The first intron (EPI) of rice 5-enolpyruvylshikimate 3-phosphate synthase gene was isolated by PCR from one clone with genomic EPSP synthase gene. Sequence analysis showed that the first intron is 704 bp in length with 36.2% G+C content. To investigate its effect on expression of foreign gene, we inserted the first intron between CaMV35S promoter and β-glucuronidase (GUS) gene. The transient expression results showed that GUS could be expressed effectively with EPI. The GUS activity in transgenic tobacco shows that the EPI can greatly enhance the expression level of β-glucuronidase (P < 0.01) compared with transgenic tobacco without the first intron, and 3-to 6-fold increase in GUS activity in some transgenic tobaccos. Northern blot indicated the first intron was spliced from GUS pre-mRNA, and the steady-state mRNA levels of GUS with EPI in transgenic tobaccos were higher than that in transgenic tobacco without EPI, which suggested that the first intron of EPSP was a non-translated intron.     

Expression of Tubb3, a beta-tubulin isotype, is regulated by androgens in mouse and rat Sertoli cells

Our previous analysis of Sertoli cell androgen receptor (AR) knockout (SCARKO) mice revealed that several cytoskeletal components are a potential target of androgen action. Here, we found that one of these components, the beta-tubulin isotype Tubb3, is differentially regulated in testes from SCARKO mice (relative to littermate controls) from Postnatal Day 10 to adulthood. The Tubb3 gene is unique in this respect, as at Day 10, no other beta-tubulin genes are significantly regulated by AR. We further characterized androgen regulation of Tubb3 in vivo and in vitro and demonstrated that it is a conserved feature in both mice and rats. To investigate whether androgens directly regulate Tubb3 expression, we screened for androgen response elements (AREs) in the Tubb3 gene. In silico analysis revealed the presence of four ARE motifs in Tubb3 intron 1, two of which bind to AR in vitro. Mutation of one of these (ARE1) strongly reduced androgen-dependent reporter gene expression. These results, coupled with the finding that the AR binds to the Tubb3 ARE region in vivo, suggest that Tubb3 is a direct target of AR. Our data strengthen the contention that androgens exert their effects on spermatogenesis, in part, through modulation of the Sertoli cell cytoskeleton. Androgen regulation of beta-tubulin has also been described in neurons, fortifying the already known similarity in microtubule organization in Sertoli cell processes and neurons, the only other cell type in which Tubb3 is known to be expressed.     

转基因小鼠中外源基因部分内含子序列的缺失及其对转录的影响

利用PCR扩增及PCR测序在显微注射法产生的转基因小鼠中发现,整合在小鼠染色体上的肌球蛋白轻链2启动子(myosinlightchain2promoter,MLC2)-糜酶(chymase)外源融合基因存在两种形式,一种为全长的融合基因,另一种在糜酶结构基因的第一内含子中缺失了213bp的序列。RT-PCR结果表明,缺失了部分内含子序列的外源融合基因不能在转基因小鼠心脏中表达.而全长的外源融合基因则能较高水平地表达,竞争性PCR定量实验表明在200ng心脏总RNA反转录产物中约含5.05(±1.38)×106个糜酶cDNA分子。上述结果表明,糜酶结构基因的第一内含子可能对MLC2-糜酶基因的表达具有调控作用。