In previous years we had disrupted the genes encoding the pertussis toxin sensitive G proteins Gi2, Gi1, Gi3 and Go. Conditional knockouts for Gi2 and Go were also generated. Double knockouts involving Gi2 and Go are lethal. We expect to learn from combining Gi2 with Gi3 and enhanced survival of Go KO mice by removing the floxed genes at various times after birth. Breeding programs have been set up to add cre recombinase under several specific promoters so as to remove the genes both generally in all issues or in specific cell types such as in dopaminergic neurons to remove Go or lymphocytes to remove Gi from Gi3 KO mice. Most phenotypic studies are done in collaboration with out side investigators, The locus encoding the Gs alpha subunit (termed GNAS complex locus is imprinted, i.e. its transcription proceeds from only one of the two alleles, making us and mice haploid for these gene products. The locus comprises five transcription units of which Gs-alpha is one and XXLas, Nespas, Nesp and 1A (a non-transcribed RNA) are the others. For Gs-alpha this occurs in only five tissues or so, but is generalized for the other transcripts. Imprinting is the result of differential methylation of promoters. This led us to become interested in DNA methylation and to the development of a high resolution method to survey DNA methylation at a genome wide scale. In this method we cut genomic DNA with methylation sensitive restriction enzymes, snip off with EcoP15I 25-nt tags from the newly created ends and subject these tags to massively parallel sequencing by any one of three commercially available technologies. The method is now ready to be used to explore differences between tissues, and effects of development, age and environmental exposures on DNA methylation. We are now, finally, in position to assess the methylation pattern of blood borne neutrophils and test the hypothesis that changes in this patterns can be used to report on environmental exposures leaving a footprint in the stem cell niche of the hematopoietic compartment of the bone marrow. Additional information to be gathered will contribute to the description of differentially methylated domains or regions (DMRs) and whether the methylation status can inform about allelic exclusion phenomena of which imprinting is but one of many forms in which allelic exclusion is manifested. A second study focused on properties of Gs-alpha mutants that may inform on the molecular mechanism by which receptors activate Gs. We previously analyzed the Gs-alpha R265E mutant. This year we concentrated on mutants that affect binding of Mg: Gs-alpha T204A, T204Q and T204E. Combined, they revealed that the conformational change that confers the ability of Gs-alpha to activate adenylyl cyclase (effector-activating function) is independent of the conformational change responsible for activation of its GTPase activity (auto-turnoff function). We attempted to crystallize the cognate mutation in a transducin alpha subunit to better understand the atomic basis of the changed properties, but this was not possible, very likely because the loss of the Mg binding function of T204 relaxes the structure creating a rather disorganized molecule. Finally, we are continuing under the guidance of Dr. Yanshun Liu (staff scientist) to work on the co-crystalization of rhodopsin with its cognate G protein transducin. Rhodopsin is both extracted from bovine retinas and and from stably transfected tissue culture cells. Transducin will be made using recombinant DNA techniques that allow us to express the alpha subunit in bacteria, and the beta-gamma subunit in insect cells. Alpha and beta-gamma dimers will then by purified and assembled into transducin (alpha-beta-gamma trimer). We continue collaborating with extramural scientists in the analysis of the phenotypes that arise in G protein deficient mice. The most notable finding this year has been the discovery that the Go G protein is a negative regulator of insulin secretion and does so by diminishing the readily releasable pool of insulin granules in the pancreatic beta cell.
Showing the most recent 10 out of 12 publications