No abstract

Familial multiple endocrine neoplasia type 1 (MEN-1) is an autosomal dominant disorder characterized by the combined occurrence of tumors of the parathyroid glands, the pancreas, and the pituitary gland. Pancreatic tumors have previously been shown to be associated with the loss of alleles on chromosome 11; we therefore looked for similar genetic alterations in specimens of parathyroid tumors, which are the most common feature of MEN-1. We obtained parathyroid tumors and peripheral-blood leukocytes from six patients with MEN-1; 18 cloned human DNA sequences from chromosome 11 were then used to identify restriction-fragment-length polymorphisms. A loss of heterozygosity was detected in parathyroid tumors from three of the six patients with MEN-1; this finding demonstrated that allelic deletions on chromosome 11 are involved in the monoclonal development of parathyroid tumors in patients with MEN-1. In addition, studies of three affected families (with 17 affected members and 51 unaffected members) established linkage with the oncogene INT2 (peak lod score, 3.30, at 0 percent recombination); the MEN-1 gene was thus mapped to the pericentromeric region of the long arm of chromosome 11 (11q13). Our location of the MEN-1 gene at 11q13 is close to the location previously reported. We conclude that a single inherited locus on chromosome 11, band q13, causes MEN-1 and that the monoclonal development of parathyroid and pancreatic tumors in patients with MEN-1 involves similar allelic deletions on chromosome 11.

The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we studied the pheromone-response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. Here, we describe the development of yeast strains containing ura4 and lacZ reporter genes under the control of the pheromone-regulated sxa2 promoter and the use of these strains to isolate mutants defective in their ability to regulate signalling. Several different types of mutant were identified. Some mutants were defective in proteins already known to regulate the pheromone-signalling pathway (Rgs1, Map1, Map2). Our approach also identified the MAP kinase phosphatase Pmp1 as a regulator of the pheromone-response pathway. Although previously shown to regulate other MAP kinase pathways in Sz. pombe, this is the first demonstration of a role for Pmp1 in pheromone signalling.

No abstract

ATP is one of the substrates of luciferase. ATP concentrations can be measured by quantitating the light output from a luciferase reaction. As kinases also use ATP, it is possible to assay kinase activity through the loss of luminescence in a coupled luciferase reaction. We have applied this luminescence-based ATP depletion approach to a model serine/threonine kinase. We find that the method may be run as an endpoint assay, in which ATP detection reagents (containing luciferase and luciferin) are added at the end of the reaction, or in a kinetic mode, where the ATP detection reagents are present throughout the reaction. The ATP depletion approach is capable of detecting kinase inhibitors. Six inhibitors of the model kinase, previously identified using other screening methods, are also active in the luminescence-based approach and display a similar rank order of potency. An advantage of the method is that kinase inhibitors, because they increase luminescence (by reversing the enzyme-dependent loss of signal), are immediately distinguishable from compounds such as luciferase inhibitors and luminescence quenchers, which further reduce the luminescence. The compound collections that we screened were rich in compounds that reduced luminescence. Compounds that have dual kinase and luciferase inhibitory activity, or kinase inhibitory activity combined with luminescence quenching, might be missed by being classified as false negatives. We show that the kinetic form of the assay can be used to minimize this possibility.