PMID: 11572317;Abstract:
Androgen receptor (AR) and glucocorticoid receptor (GR) influence distinct physiologic responses in steroid-responsive cells despite their shared ability to selectively bind in vitro to the same canonical DNA sequence (TGTTCT). While the DNA-binding domains (DBDs) of these receptors are highly conserved, the amino N-terminal domain (NTD) and hormone-binding domain (HBD) are evolutionarily divergent. To determine the relative contribution of these functional domains to steroid-specific effects in vivo, we constructed a panel of AR/GR gene fusions by interchanging the NTD, DBD, and HBD regions of each receptor and measured transcriptional regulatory activities in transfected kidney and prostate cell lines. We found that GR was approximately 10-fold more active than AR when tested with the mouse mammary tumor virus promoter, and that this difference in activity was primarily owing to sequence divergence in the NTDs. We also tested transcriptional activation of the androgen-dependent rat probasin promoter, and in this case, AR was at least twofold more active than GR. Analysis of the chimeric receptors revealed that this difference mapped to the DBD region of the two receptors. Transcriptional repression functions of the wild-type and chimeric receptors were measured using an activator protein 1 (AP-1) transrepression assay and identified the GR HBD as a more potent transrepressor of AP-1 transcriptional activation than the AR HBD. Taken together, our analyses reveal that evolutionary sequence divergence between AR and GR functional domains results in unique promoter-specific activities within biologic systems in which both AR and GR are normally expressed.
PMID: 6775816;Abstract:
When a cloned 6 kb Eco RI-Sal I fragment of mouse ribosomal gene nontranscribed spacer DNA (rDNA NTS) was used to screen a BALB/c mouse gene library, 25% of the recombinant phage hybridized with it. In situ hybridization experiments and characterization of 12 clones selected using this probe supported the idea that sequences homologous to this rDNA NTS region are scattered throughout the genome. Subsequently, sequences homologous to mouse rDNA NTS were found flanking mouse μ, α and γ2b immunoglobulin C(H) genes. One region was localized 3' to the μ coding sequence, an area which has been identified as an intervening sequence between the secreted C(μ) heavy chain terminus and the C terminal portion of the membrane-bound C(μ) heavy chain.
PMID: 6095060;PMCID: PMC368912;
It has been proposed that cell-specific responses to steroid action are the result of coordinate expression of steroid gene networks. Using three different cell systems, we have performed transcriptional analyses to determine if the observed hormone-induced alterations in gene expression are consistent with a limited number of potential target genes in any one cell type. Our results indicate that greater than 95% of the transcripts in dexamethasone-treated rat hepatoma (HTC), or mouse lymphoma (WEH17) cells, are similar to the mRNAs in untreated cells based on subtraction hybridization. In addition, we find that although the castration-induced expression of androgen-regulated transcripts in the rat ventral prostate (RVP) is significantly different between normal and castrated rats (19%), the majority of these mRNAs are accounted for by the over abundance of sulfated glycoprotein-2 sequences. Specifically, analysis of an RVP subtracted cDNA library revealed that sulfated glycoprotein-2 mRNA masked the presence of less abundant differentially expressed sequences, confirming that the actual size of the RVP androgen gene network is small. We conclude that steroid-mediated changes in transcription accurately reflect the expression of a few cell-specific target genes, and thus support the model of steroid gene networks. The potential to characterize key elements which determine both the time course and magnitude of cell-specific hormone responses is discussed.
PMID: 17257211;Abstract:
We have developed a novel molecular genetic approach to investigating gene regulation in adult mosquitoes called whole body transfection (WBT). This DNA microinjection method allows for both constitutive and regulated expression of plasmid vectors in the fat body and midgut of adult mosquitoes within 24 h of injection. Using a luciferase reporter gene containing the Aedes aegypti heat shock protein 70 (Hsp70) promoter, we optimized the WBT protocol at various times post-injection and used these parameters to measure the expression of a vitellogenin-luciferase reporter gene in response to blood meal feeding. These studies showed that a 843 bp fragment of the Ae. aegypti vitellogenin-C (VgC) promoter caused a greater than 200-fold induction of luciferase activity in a strict tissue-specific manner, and only in response to feeding. Functional mapping of the VgC promoter by WBT identified essential upstream regulatory elements in the region spanning -780 to -182 bp from the transcriptional start site. We also constructed a lipopolysaccharide-regulated expression vector using a 1096 bp genomic fragment of the Ae. aegypti cecropin B (CecB) promoter. Our data show that four days after WBT injection, the CecB-luciferase reporter gene could be induced more than 100-fold in the fat body following lipopolysaccharide injection. Moreover, we found that lipopolysaccharide-induction of the CecB reporter gene occurred up to 28 days post-WBT injection. These data suggest that WBT could provide a novel strategy to express recombinant proteins and RNAi constructs in adult mosquitoes using conventional microinjection methods. © 2006 The Authors Journal compilation © 2006 The Royal Entomological Society.
