The pattern of structures on most of the adult cuticle of Drosophila is determined in the larval imaginal disks. The Drosophila growth factor homolog decapentaplegic [dpp] is believed to participate in pattern formation in imaginal disks, primarily along what will become the proximal-to-distal axis of adult appendages. We report that dpp expression in wing, leg, and eye-antennal imaginal disks is localized to a band of cells along the presumptive proximal-to-distal axis. The pattern and level of dpp expression in imaginal disks is affected by mutant lesions that remove 3' cis-regulatory sequences. We demonstrate that one portion of the 3' cisregulatory region contains regulatory elements sufficient to activate gene expression in a subset of the cells that normally express dpp in the imaginal disks, allowing rescue of dpp mutant phenotypes. We propose that the complete dpp expression pattern is generated by an array of 3' regulatory elements that differ in their potency in specific disks and in certain positions within a disk. The identification of the factors that activate these elements should provide clues as to how positional information is encoded in imaginal disks.
To better understand the signaling pathways which lead to DNA synthesis in mammalian cells, we have studied the transcriptional activation of genes needed during the S phase of the cell cycle. Transcription of the gene encoding a pyrimidine biosynthetic enzyme, carbamoyl-phosphate synthase (glutamine-hydrolyzing)/ aspartate carbamoyltransferase/dihydroorotase (cad), increases at the G 1 /S-phase boundary. We have mapped the growth-dependent response element in the hamster cad gene to the extended palindromic E-box sequence, CCACGTGG, which is centered at ؉65 in the 5 untranslated sequence. Mutation of the E box abolished growth-dependent transcription, and an oligonucleotide corresponding to the cad sequence at ؉55 to ؉75 (؉55/؉75) restored growth-dependent regulation to nonresponsive cad promoter mutants when placed downstream of the transcription start site. The same oligonucleotide conferred less G 1 /S-phase induction when placed upstream of basal promoter elements. An analogous oligonucleotide containing the mutant E box had no effect in either location. Nuclear proteins bound the cad ؉55/؉75 element in a cell cycle-dependent manner in electromobility shift assays; antibodies specific to USF and Max blocked the DNA-binding activity of different growth-regulated protein-DNA complexes. Expression of c-Myc mutants which have been shown to dominantly interfere with the function of c-Myc and Max significantly inhibited cad transcription during S phase but had no effect on transcription from another G 1 /S-phase-activated promoter, dhfr. These data support a model whereby E-box-binding proteins activate serum-induced transcription from the cad promoter at the G 1 /S-phase boundary and suggest that a Max-associated protein complex contributes to the serum response.Cellular responses to external growth stimuli involve complex signal transduction pathways which lead to changes in the expression of genes needed for DNA synthesis and cell division. The carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (cad) gene encodes a trifunctional enzyme which catalyzes the first three steps in the de novo synthesis of pyrimidines (21). In mammalian cells, the levels of both CAD enzymatic activity and cad mRNA correlate with the proliferative state of the cell. For example, the endogenous cad mRNA level increased approximately 10-fold at the G 1 /S transition following serum stimulation of quiescent cells (58). Nuclear run-on assays demonstrate a 3-to 10-fold increase in the rate of cad transcription after serum stimulation, suggesting that a significant portion of growth-dependent regulation occurs at the transcriptional level (44,58). In accordance with a model of transcriptional regulation, the activity of a 164-bp region of the cad promoter fused to a reporter gene increases 15-fold at the G 1 /S-phase boundary in a serum starvation/stimulation assay (50). To identify the factor(s) which coordinates cad expression with the growth responsiveness of the cell, we have analyzed the...
The yellow obese syndrome in mice encompasses many pleiotropic effects including yellow fur, maturity-onset obesity, hyperinsulinemia, insulin resistance, hyperglycemia, increased skeletal length and lean body mass, and increased susceptibility to neoplasia. The molecular basis of this syndrome is beginning to be unraveled and may have implications for human obesity and diabetes. Normally, the agouti gene is expressed during the hair-growth cycle in the neonatal skin where it functions as a paracrine regulator of pigmentation. The secreted agouti protein antagonizes the binding of the alpha-melanocyte-stimulating hormone to its receptor (melanocortin 1 receptor) on the surface of hair bulb melanocytes, causing alterations in intracellular cAMP levels. Widespread, ectopic expression of the mouse agouti gene is central to the yellow obese phenotype, as demonstrated by the molecular cloning of several dominant agouti mutations and the ubiquitous expression of the wild-type agouti gene in transgenic mice. Recent experiments have revealed that the hypothalamus and adipose tissue are biologically active target sites for agouti in the yellow obese mutant lines.
The agouti gene product is a secreted protein that acts in a paracrine manner to regulate coat color in mammals. Several dominant mutations at the agouti locus in mice cause the ectopic, ubiquitous expression of agouti, resulting in a condition similar to adult-onset obesity and non-insulin-dependent diabetes mellitus. The human agouti protein is 85% homologous to mouse agouti; however, unlike the mouse agouti gene, human agouti is normally expressed in adipose tissue. To address whether expression of agouti in human adipose tissue is physiologically relevant, transgenic mice were generated that express agouti in adipose tissue. Similar to most humans, these mice do not become obese or diabetic. However, we found that daily insulin injections significantly increased weight gain in the transgenic lines expressing agouti in adipose tissue, but not in nontransgenic mice. These results suggest that insulin triggers the onset of obesity and that agouti expression in adipose tissue potentiates this effect. Accordingly, the investigation of agouti's role in obesity and non-insulin-dependent diabetes mellitus in mice holds significant promise for understanding the pathophysiology of human obesity.
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