Regulation of gene expression in the chick oviduct. 18. Effect of estrogen on gene expression in the chick oviduct. Regulation of the ovomucoid gene

By using both double-colony hybridization and an in situ immunostaining assay for transformants, 39 cDNA clones (clone p-16a) encoding mouse liver microsomal testosterone 16a-hydroxylase (cytochrome P-4501,6) were isolated from a cDNA library constructed in the cloning vector pUC-9 with poly(A)+ RNA immunoenriched from total liver polysomes of male 129/J mice. mRNA selected by hybridization with clone p-16a translated the P-4501" apoprotein in vitro. Total cellular proteins, which were prepared from immunopositive transformant Escherichia coli cells, were conjugated with Sepharose 4B. Antibody purified with the Sepharose 4B conjugate from mixed antiserum to P-45016. and P45015a specifically inhibited testosterone 16a-hydroxylase activity in microsomes. The cDNA insert of one recombinant plasmid (clone P-16a-1) was 1.75 kilobases in size and contained one or more internal restriction sites for HindIII, BamHI, Bgl I, Pst I, Alu I, HinpI, and Rsa I. 32P-labeled clone p-16a-1 hybridized with a single mRNA (2000 bases) that was 10 times more concentrated in liver cells from male 129/J mice than in female mice. This result was consistent with the finding that poly(A)+ RNA from male mice translated 10 times as much P-4501" in vitro as did the poly(A)+ RNA from females.Thus, the predominant expression of testosterone 16a-hydroxylase in male 129/J mice is regulated pretranslationally, presumably at the transcriptional level of the P-4501" gene. male mice (5). It is not yet understood how the sex-dependent developmental regulation of testosterone hydroxylase occurs at the molecular level.To investigate sexual regulation of testosterone hydroxylase activities in mice, we have previously purified liver microsomal cytochrome P-450s specific for the 16a-or 15a-hydroxylation based on the specific hydroxylase activity in eluates from chromatographic columns (6, 7). Throughout the purification procedure, we demonstrated that sex-dependent differences in testosterone 16a-and 15a-hydroxylase activity in inbred mice (129/J) can be explained by the existence of highly specific cytochrome P-450s for 16a-and 15a-hydroxylation activities in male and female mice, respectively. A specific antibody to purified P-45016a or P-45015a fraction was able to inhibit nearly 100% of either 16a-or 15a-hydroxylase activity in untreated male or female 129/J mice (6, 7).To extend further our knowledge of the mechanism of sexual regulation of testosterone 16a-hydroxylase in 129/J mice, we used the specific antibody to P-45016,, to measure indirectly the level of translatable P-45016, mRNA in male and female mouse livers. In addition, we used the specific antibody to isolate and characterize the cDNA encoding P45016a mRNA. Finally, we quantitated directly the level of P-45016, mRNA in liver of male and female mice by hybridization with the isolated cDNA.Many liver proteins and enzymes such as a2-microglobulin, mouse major urinary protein, steroid hormone and prolactin receptors, monoamine oxidase, aldehyde oxidase, drug oxidase, and steroid hy...

mentioning

confidence: 99%

Sex-dependent expression of mouse testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha): cDNA cloning and pretranslational regulation.

Harada¹,

Negishi²

1985

“…Various genes are silent until some particular stimulus results in the activation of transcription of the gene. Clear examples of such positive transcriptional control include the genes for hemoglobin (1) and the various genes subject to hormonal control (2)(3)(4)(5)(6)(7), as well as the Drosophila heat shock genes (8).…”

mentioning

confidence: 99%

Activation of early adenovirus transcription by the herpesvirus immediate early gene: evidence for a common cellular control factor.

Feldman

Imperiale

Nevins

1982

119

Adenovirus mutants carrying a defective EIA gene, such as d1312, are unable to express any of the early viral genes upon infection of HeLa cells. However, efficient expression of the other early adenovirus genes was obtained when d1312-infected HeLa cells were coinfected with pseudorabies virus, a herpesvirus. By employing a temperature-sensitive pseudorabies mutant (tsGl) it was demonstrated that the herpesvirus function responsible for the induction of adenovirus transcription was the immediate early gene, a gene required for the activation of herpesvirus early gene expression and the maintenance of early and late herpesvirus transcription. Specifically, HeLa cells coinfected with d1312 and tsGl, when shifted to the nonpermissive temperature, lost their capacity to express the early adenovirus genes. Furthermore, activation of early adenovirus gene expression in herpesvirus coinfection occurred earlier and at a higher level than in wild-type adenovirus infection. Therefore, the herpesvirus immediate early protein not only activates the early adenovirus transcription units but apparently does so more efficiently than the adenovirus ElA gene product. Because of this fact, we argue that the activation, either by the EIA protein or the herpesvirus immediately early protein, most likely occurs indirectly through interaction with a cellular protein rather than by a direct recognition of regulatory sequences at the adenovirus promoters.The induction of gene expression through transcriptional activation is surely an important aspect of development and differentiation. Various genes are silent until some particular stimulus results in the activation of transcription of the gene. Clear examples of such positive transcriptional control include the genes for hemoglobin (1) and the various genes subject to hormonal control (2-7), as well as the Drosophila heat shock genes (8).There is also an induction of gene expression through transcriptional activation during the normal course of adenovirus infection of HeLa cells. The activity of the various early viral transcription units depends on the action of the product of the viral EIA gene (9-11). We have previously postulated (11) that the function of the ElA protein may involve the inactivation of a cellular factor rather than a direct positive interaction with the early viral promoters. For example, early viral genes can be expressed in the absence of E1A function ifcellular protein synthesis is blocked (11,12). These results suggest that a short-lived cellular factor that might have a normal role in controlling the expression of some group of cellular genes must be inactivated to allow the expression of the early adenovirus genes. If such a mechanism were indeed operating, then one might expect to find circumstances, independent ofthe action ofthe adenovirus ElA gene product, whereby this cellular factor was rendered inactive or reduced in functional concentration. Such a condition should then allow the growth and expression ofthe adenovirus mutants deficient in th...

“…Transcriptional regulation was previously demonstrated as responsible for the tissue-specific synthesis of two very abundant mRNAs, ovalbumin (20) and ovomucoid (21), and for hemoglobin switching in chicken embryos (22). The temporary relationship between the onset of transcription and the appearance of mature mRNA in the same tissue has not previously been investigated.…”

mentioning

confidence: 99%

Isolation of a cDNA clone for mouse urinary proteins: age- and sex-related expression of mouse urinary protein genes is transcriptionally controlled.

Derman¹

1981

(3,5, 6). Study of the expression of MUP genes thus can probe different facets of regulation of eukaryotic genes-e.g., tissue specificity, commitment ofcells to produce a particular protein, and the basis for hormonal regulation.In order to initiate these studies in the MUP system, a recombinant DNA probe containing the sequences of one MUP mRNA has been obtained, and the level of regulation responsible for the age-and sex-related differences in MUP mRNA synthesis was investigated. Previous experiments (7) suggested that, for a group of 11 liver-specific mRNA molecules, transcriptional control is primarily responsible for the establishment and maintenance ofliver-specific mRNA patterns. The question of the temporal relationship between the onset of transcription and the appearance of mature mRNA during tissue ontogeny, however, was not approached. The present experiments show that there is little or no transcription ofMUP genes in immature liver cells which do not yet produce MUP mRNA. In addition, it appears that the male-specific increase in MUP mRNA concentration (1-3) is also accomplished by an increase in the rate of transcription of MUP genes in the liver nuclei of adult male mice compared to adult female mice.METHODS AND MATERIALS Animals, DNA, and RNA. An inbred strain of Swiss white mice (NCS) raised at the Rockefeller University was used in all experiments. The construction of recombinant plasmids was as described (7) polyacrylamide gels (12) and visualized by autoradiography. Determination ofRNA Size. Formaldehyde-denatured RNA was electrophoresed in 1.5% agarose/1. 1 M formaldehyde gels (13) with 20 mM 3-(N-morpholino)propanesulfonic acid/5 mM Na acetate/i mM EDTA, pH 7.0, for electrophoresis buffer.After transfer to nitrocellulose filters (13), the size ofRNA complementary to plasmid plivS-1 (MUP) was determined by hybridization to nick-translated (14) plivS-1 DNA (7). The RNAladen nitrocellulose filter was first incubated in 30% formamide/ 0.3 M NaCl/50 mM piperazinediethanesulfonic acid, pH 7.0/ 10 mM EDTA/0.2% NaDodSO4 containing denatured salmon sperm DNA at 100 pug/ml and double-strength Denhardt's solution (15) for 10 hr at 45°C. The hybridization solution also contained 10% (wt/vol) dextran sulfate and probe DNA at 105 cpm/ml.Quantitation of RNA Abundance and of Transcription Rates. These measurements were performed exactly as described (7). De-rived from MUP mRNA. Construction and selection ofrecombinant cDNA plasmids complementary to mRNAs present exclusively (or predominantly) in the mouse liver, compared to Abbreviation: MUP, mouse urinary protein. RESULTS Construction and Selection of Recombinant Plasmids