(1/16045) Transcriptional repression by the Drosophila giant protein: cis element positioning provides an alternative means of interpreting an effector gradient.
Early developmental patterning of the Drosophila embryo is driven by the activities of a diverse set of maternally and zygotically derived transcription factors, including repressors encoded by gap genes such as Kruppel, knirps, giant and the mesoderm-specific snail. The mechanism of repression by gap transcription factors is not well understood at a molecular level. Initial characterization of these transcription factors suggests that they act as short-range repressors, interfering with the activity of enhancer or promoter elements 50 to 100 bp away. To better understand the molecular mechanism of short-range repression, we have investigated the properties of the Giant gap protein. We tested the ability of endogenous Giant to repress when bound close to the transcriptional initiation site and found that Giant effectively represses a heterologous promoter when binding sites are located at -55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites are moved to more distal locations, repression is diminished. Rather than exhibiting a sharp 'step-function' drop-off in activity, however, repression is progressively restricted to areas of highest Giant concentration. Less than a two-fold difference in Giant protein concentration is sufficient to determine a change in transcriptional status of a target gene. This effect demonstrates that Giant protein gradients can be differentially interpreted by target promoters, depending on the exact location of the Giant binding sites within the gene. Thus, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradient. We also demonstrate that a chimeric Gal4-Giant protein lacking the basic/zipper domain can specifically repress reporter genes, suggesting that the Giant effector domain is an autonomous repression domain. (+info)
(2/16045) Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA.
Inheritable mutations in nucleotide excision repair (NER) genes cause cancer-prone human disorders, such as xeroderma pigmentosum, which are also characterized by symptoms of accelerated ageing. To study the impact of NER deficiency on mutation accumulation in vivo, mutant frequencies have been determined in liver and brain of 2-16 month old NER deficient XPA-/-, lacZ hybrid mice. While mutant frequencies in liver of 2-month old XPA-/-, lacZ mice were comparable to XPA+/-, lacZ and the lacZ parental strain animals, by 4 months of age mutant frequencies in the XPA-deficient mice were significantly increased by a factor of two and increased further until the age of 16 months. In brain, mutant frequencies were not found to increase with age. These results show that a deficiency in the NER gene XPA causes an accelerated accumulation of somatic mutations in liver but not in brain. This is in keeping with a higher incidence of spontaneous liver tumors reported earlier for XPA-/- mice after about 15 months of age. (+info)
(3/16045) Inducible genetic suppression of neuronal excitability.
Graded, reversible suppression of neuronal excitability represents a logical goal of therapy for epilepsy and intractable pain. To achieve such suppression, we have developed the means to transfer "electrical silencing" genes into neurons with sensitive control of transgene expression. An ecdysone-inducible promoter drives the expression of inwardly rectifying potassium channels in polycistronic adenoviral vectors. Infection of superior cervical ganglion neurons did not affect normal electrical activity but suppressed excitability after the induction of gene expression. These experiments demonstrate the feasibility of controlled ion channel expression after somatic gene transfer into neurons and serve as the prototype for a novel generalizable approach to modulate excitability. (+info)
(4/16045) An Arabidopsis 14-3-3 protein can act as a transcriptional activator in yeast.
The 14-3-3 proteins are a group of highly conserved and widely distributed eukaryotic proteins with diverse functions. One 14-3-3 protein, AFT1 from Arabidopsis thaliana, was found to be able to activate transcription in yeast. When fused to the DNA-binding domain of a bacterial protein LexA, AFT1 can activate transcription of reporter genes that contain LexA operator sequences in their promoters. Although the in vivo function of AFT1 is not completely known, its similarity to previously identified proteins found in transcription complexes of Arabidopsis and maize suggests that AFT1 and some other 14-3-3 proteins may activate gene expression in other systems as well. (+info)
(5/16045) The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo.
Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules. (+info)
(6/16045) Fibroblast growth factor-8 expression is regulated by intronic engrailed and Pbx1-binding sites.
Fibroblast growth factor-8 (FGF8) plays a critical role in vertebrate development and is expressed normally in temporally and spatially restricted regions of the vertebrate embryo. We now report on the identification of regions of Fgf8 important for its transcriptional regulation in murine ES cell-derived embryoid bodies. Stable transfection of ES cells, using a human growth hormone reporter gene, was employed to identify regions of the Fgf8 gene with promoter/enhancer activity. A 2-kilobase 5' region of Fgf8 was shown to contain promoter activity. A 0.8-kilobase fragment derived from the large intron of Fgf8 was found to enhance human growth hormone expressed from the Fgf8 promoter 3-4-fold in an orientation dependent manner. The intronic fragment contains DNA-binding sites for the AP2, Pbx1, and Engrailed transcription factors. Gel shift and Western blot experiments documented the presence of these transcription factors in nuclear extracts from ES cell embryoid bodies. In vitro mutagenesis of the Engrailed or Pbx1 site demonstrated that these sites modulate the activity of the intronic fragment. In addition, in vitro mutagenesis of both Engrailed and Pbx1 sites indicated that other unidentified sites are responsible for the transcriptional enhancement observed with the intronic fragment. (+info)
(7/16045) Regulation of human hsp90alpha gene expression.
Mammalian HSP90alpha and HSP90beta are encoded by two individual genes. On the basis of the upstream sequences of the human hsp90alpha gene, GenBank accession number U25822, we have constructed CAT reporter plasmids driven by individual fragments of the hsp90alpha gene. We found that (1) the proximal heat shock element complex located at -96/-60 enhances hsp90alpha promoter expression; (2) heat shock induction depends upon the coexistence of distal heat shock element at -1031/-1022 and the proximal heat shock element complex of the hsp90alpha gene; (3) unlike hsp90beta, downstream sequences of the transcription start site inhibit hsp90alpha expression. We conclude that the regulatory mechanisms for the expression of hsp90alpha and hsp90beta genes are different. (+info)
(8/16045) Regulatory sequences of the mouse villin gene that efficiently drive transgenic expression in immature and differentiated epithelial cells of small and large intestines.
Villin is an early marker of epithelial cells from the digestive and urogenital tracts. Indeed villin is expressed in the stem cells and the proliferative cells of the intestinal crypts. To investigate the underlying molecular mechanisms and particularly those responsible for the restricted tissue specificity, a large genomic region of the mouse villin gene has been analyzed. A 9-kilobase (kb) regulatory region of the mouse villin gene (harboring 3.5 kb upstream the transcription start site and 5.5 kb of the first intron) was able to promote transcription of the LacZ reporter gene in the small and large intestines of transgenic mice, in a transmissible manner, and thus efficiently directed subsequent beta-galactosidase expression in epithelial cells along the entire crypt-villus axis. In the kidney, the transgene was also expressed in the epithelial cells of the proximal tubules but is likely sensitive to the site of integration. A construct lacking the first intron restricted beta-galactosidase expression to the small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and differentiated epithelial cells of the small and/or large intestinal mucosa. (+info)