James H. Thomas - US grants

The goal of the proposed research is to understand how cell interactions contribute to the development of the nematode Caenorhabditis elegans. This study will use genetic, classical, and molecular methods to probe cell interactions at the single cell level. The research will focus on a set of cell interactions that are controlled by the homeotic gene lin-12. lin-12 is thought to be directly involved in cell communication: it encodes a protein that is probably located on the cell surface, and whose extracellular domain has sequence homology with mammalian LDL receptor and epidermal growth factor (EGF). Other genes that act in this process of cell interaction will be sought by identifying mutations that specifically suppress lin-12 defects, or by identifying mutations in other genes that cause lin-12 like phenotypes. Six such sup genes have already been identified. Loss-of-function mutations of the sup genes will be identified, and their effects on development studied. Temperature-shift experiments using temperature-sensitive mutants will define the time of gene action. Genetic mosaic analysis will determine in which cells the sup genes function. These results, together with epistasis interactions, will be used to infer a pathway of gene interactions. In corollary experiments, biochemical purification of the signal molecule used in one of the lin-12 controlled cell interactions will be attempted. From the analysis of the genetic pathway, key genes will be chosen to subject to molecular analysis. The ultimate goal will be to completely understand the role of these genes in mediating cell interaction. Study of cell interactions is directly relevant to an understanding of at least certain cancers. The intercellular growth signals PDGF, TGFa, and EGF (to which lin-12 is homologous) have been implicated in growth of cancer cells. Oncogenes themselves can encode mutant growth factors or receptors. Studies in C. elegans should provide important insights into the role of cell interactions in the regulation of growth and development.

The goal of the proposed research is to investigate neuron and muscle excitability in the nematode C. elegans. We have identified over 30 genes that regulate excitation of defecation, egg-laying, and body-wall muscles, and we have molecularly cloned five of these genes. The genetics of four of these is strikingly similar: each has dominant gain-of-function mutations (gf) that cause strong defects in muscle excitation, and loss-of-function (lf) mutations cause little or no obvious phenotype. All four genes encode K+ channels. We think that the gf mutations in each K+ channel cause channel activation in vivo, accounting for their strong excitation defects. The fact that lf mutations cause relatively minor defects suggests that the many K+ channels have overlapping functions in vivo. We will continue analysis of these genes and other similar genes identified in genetic screens. The K+ channels will be expressed in cultured cells to study their electrophysiological properties. It will be particularly interesting to study how the gf mutations affect channel properties. Two of these K+ channels are related to the human HERG channel, defects in which cause a cardiac malfunction called long-QT. Long-QT can also be caused by tricyclic antidepressants and certain cardiac anti-arrhythmic drugs. We have evidence that these drugs also block one (but not the other) of the C. elegans HERG-related channels in vivo and in vitro. We will study the C. elegans channels and their human equivalents to understand the basis for this specificity and its implications for long-QT disorder. We have shown that the fifth muscle excitation gene, called unc- 43, encodes the nematode homologue of calcium-calmodulin dependent protein kinase II (CaMKII). CaMKII is implicated as a key regulator of synaptic activity, particularly of synaptic plasticity that underlies learning and memory. We have many lf mutations in unc-43, including null mutations. These mutants are viable and have complex behavioral abnormalities. There is also one gf mutation in unc-43, and we think that this mutant CaMKII is partially Ca++ independent (activated). This gf mutant is also viable and confers complex defects that are the opposite of those in null mutants. We have begun to use the unc-43 activated mutation to identify extragenic suppressors of its various phenotypes, some of which we expect to encode direct CaMKII substrates. We propose to use a combination of genetic analysis, molecular cloning, and biochemical analysis to characterize these potential targets and to determine whether they are directly phosphorylated by the unc-43 CaMKII. We will also continue genetic screens to identify additional potential targets.

The goal of the proposed research is to understand the cellular, genetic, and molecular mechanisms of chemosensory response to the dauer-inducing pheromone of Caenorhabditis elegans. The dauer larva is a developmentally distinct alternative third-larval stage (L3) that is specialized for long- term survival under harsh conditions. The choice between the normal L3 and the dauer larva is controlled primarily by chemosensory assessment of the environmental concentration of a secreted dauer-inducing pheromone. More than 25 genes have been identified that control the process of dauer formation and they have been placed into a complex epistasis pathway. Some of these genes are implicated in the chemosensory process per se, while others probably act downstream of chemosensation to activate the dauer- larval developmental program. This proposal concentrates on the genes implicated in the function and development of the dauer pheromone chemosensory cells. One aim is to rigorously test the role of sensory neurons that have been implicated in controlling pheromone response. Pheromone responsiveness will be measured in animals in which individual identified neurons have been eliminated with a laser microbeam. Another aim is to complete a thorough search for mutations in genes important for the function and development of these sensory cells. Mutations in genes already implicated in pheromone chemosensation will be used to identify other such genes using a variety of classical genetic approaches. One gene, daf-11, already implicated specifically in the process of pheromone chemosensation, will be cloned by transposon tagging. A variety of molecular biological and genetic approaches will be taken to defining the function of daf-11, including DNA sequence analysis, gene product localization, and genetic mosaic analysis. Another gene, daf-19, is implicated in the development of a large set of sensory neurons, including those controlling dauer formation. daf-19 will be cloned using detailed genetic and physical mapping and DNA transformation. The cloned gene will be used to test the involvement of daf-19 in the development of the sensory cells by DNA sequence analysis and expression studies. Sensory modulation of dauer formation in C. elegans provides an excellent model for the investigation of sensory and environmental influences on development. The opportunity to both apply extensive genetic analysis to such a problem and to define the sensory pathway at the single neuron level is unique. In addition, response to dauer pheromone is a model for the general process of chemosensation, which is currently poorly understood.

The objective of this project is to define the reaction mechanisms of human placental 3beta-hydroxysteroid dehydrogenase/steroid 5->4-ene- isomerase (3beta-HSD/isomerase) by relating function to structure. In placenta, 3beta-HSD/isomerase catalyzes the conversion of maternal pregnenolone to progesterone, a hormone that promotes uterine quiescence during pregnancy. The enzyme competitively utilizes dehydroepiandrosterone, the primary steroid product of the fetal adrenal gland near term, to produce androstenedione that is further metabolized to 17beta-estradiol. Thus, placental 3beta-HSD/isomerase bridges hormonal communication between the mother and fetus to mediate the locally increased estrogen/progesterone balance that has been associated with the onset of labor. Characterization of 3beta-HSD/isomerase may ultimately allow pharmacologic control of the placenta enzyme, independent of the different gonadal/adrenal isoenzyme, to prevent premature births. Homogeneous enzyme purified from human placenta is in-hand. Wild-type enzyme as been overexpressed by baculovirus in insect cells and found to be kinetically identical to native placenta enzyme. The order of substrate and coenzyme binding for the 3beta-HSD and isomerase activities is studied using both classic isotopic ligand exchange and novel affinity labeling/ligand protection experiments. The placenta isomerase reaction mechanism and activation by essential cofactor are compared to the known bacterial isomerase mechanism (with no cofactor requirement) by measuring spectral changes in 19-nortestosterone and 17beta-estradiol upon binding to the enzyme in the presence or absence of NADH. Stopped-flow spectroscopy experiments address our hypothesis that a time-dependent conformational change mediates the NADH-induced activation of isomerase. Affinity radiolabeling and ligand protection experiments map the binding sites for 3beta-HSD substrate, isomerase substrate, and cofactor in the known primary structure of this single, multifunctional protein. Using our cDNA that encodes placental 3beta-HSD/isomerase, probable catalytic amino acids in these identified regions are mutated using modified synthetic oligonucleotides. Probable cofactor and membrane anchoring regions are deleted. The mutated and wild-type cDNA are over-expressed by baculovirus in suspensions of insect Sf-9 cells. The functional significance of each expressed, purified mutant enzyme is determined by rigorous kinetic analyses previously applied to the native enzyme. These analyses include the measurement of Michaelis-Menton constants for 3beta- HSD substrates, isomerase substrates, and cofactors, inhibition kinetics for product steroids and NADH, and inactivation/protection profiles using affinity alkylators that are specific for the modified binding site. These structure/function studies localize the catalytic amino acids for the enzyme activities in the primary structure and test our unique hypothesis that the sequential 3beta-HSD and isomerase reactions are catalyzed at contiguous sites in a single steroid binding region.

neural degeneration; mutant; molecular genetics; apoptosis; phenotype; gene mutation; autosomal recessive trait; autosomal dominant trait; larva; detergents; neurons; lasers; Caenorhabditis elegans; site directed mutagenesis; molecular cloning;

The objective of this project is to define the reaction mechanisms of human placental 3beta-hydroxysteroid dehydrogenase/steroid 5->4-ene- isomerase (3beta-HSD/isomerase) by relating function to structure. In placenta, 3beta-HSD/isomerase catalyzes the conversion of maternal pregnenolone to progesterone, a hormone that promotes uterine quiescence during pregnancy. The enzyme competitively utilizes dehydroepiandrosterone, the primary steroid product of the fetal adrenal gland near term, to produce androstenedione that is further metabolized to 17beta-estradiol. Thus, placental 3beta-HSD/isomerase bridges hormonal communication between the mother and fetus to mediate the locally increased estrogen/progesterone balance that has been associated with the onset of labor. Characterization of 3beta-HSD/isomerase may ultimately allow pharmacologic control of the placenta enzyme, independent of the different gonadal/adrenal isoenzyme, to prevent premature births. Homogeneous enzyme purified from human placenta is in-hand. Wild-type enzyme as been overexpressed by baculovirus in insect cells and found to be kinetically identical to native placenta enzyme. The order of substrate and coenzyme binding for the 3beta-HSD and isomerase activities is studied using both classic isotopic ligand exchange and novel affinity labeling/ligand protection experiments. The placenta isomerase reaction mechanism and activation by essential cofactor are compared to the known bacterial isomerase mechanism (with no cofactor requirement) by measuring spectral changes in 19-nortestosterone and 17beta-estradiol upon binding to the enzyme in the presence or absence of NADH. Stopped-flow spectroscopy experiments address our hypothesis that a time-dependent conformational change mediates the NADH-induced activation of isomerase. Affinity radiolabeling and ligand protection experiments map the binding sites for 3beta-HSD substrate, isomerase substrate, and cofactor in the known primary structure of this single, multifunctional protein. Using our cDNA that encodes placental 3beta-HSD/isomerase, probable catalytic amino acids in these identified regions are mutated using modified synthetic oligonucleotides. Probable cofactor and membrane anchoring regions are deleted. The mutated and wild-type cDNA are over-expressed by baculovirus in suspensions of insect Sf-9 cells. The functional significance of each expressed, purified mutant enzyme is determined by rigorous kinetic analyses previously applied to the native enzyme. These analyses include the measurement of Michaelis-Menton constants for 3beta- HSD substrates, isomerase substrates, and cofactors, inhibition kinetics for product steroids and NADH, and inactivation/protection profiles using affinity alkylators that are specific for the modified binding site. These structure/function studies localize the catalytic amino acids for the enzyme activities in the primary structure and test our unique hypothesis that the sequential 3beta-HSD and isomerase reactions are catalyzed at contiguous sites in a single steroid binding region.

DESCRIPTION (Adapted from applicant's abstract): The SSRI antidepressants, including fluoxetine (Prozac), are the drugs most commonly prescribed for treatment of depressive disorders. The in vivo targets of fluoxetine that are responsible for its antidepressive effects and its side effects are not well established. Systematically establishing such targets in the human or in mammalian models of depression is not possible. We are using the nematode Caenorhabditis elegans to identify mechanisms of fluoxetine action. In preliminary studies we have established that fluoxetine has three distinct effects in C. elegans. One of these effects is probably due to blocking serotonin reuptake, as expected from one known target in mammals. The other two effects appear not to involve serotonin. We have isolated mutations in seven genes that confer resistance to one of the non-serotonergic effects of fluoxetine (Nrf mutants). We have molecularly cloned one of these genes, called nrf-6. nrf-6 encodes the defining member of a family of multi-pass transmembrane proteins. We propose to investigate nrf-6 by completing this initial molecular analysis and by determining its expression pattern, subcellular localization, and site of function in conferring fluoxetine sensitivity. We propose to clone two additional fluoxetine resistance genes that we have identified and to perform similar analyses as for nrf-6. We propose to continue mutagenesis screens to identify other genes that mediate the nose contraction and other fluoxetine responses. Finally, we propose to identify in mammals members of the new nrf-6 family as a first step toward determining whether the nematode-fluoxetine interaction might have direct relevance to humans.

[unreadable] DESCRIPTION (provided by applicant): The principal objective of this program is to train graduate students to function effectively as geneticists, enabling them to apply the rigor of genetic methods to pertinent issues in contemporary biology and medicine. The variety of approaches taken by the training faculty ensures a broad scope of training that encompasses microbial genetics, human and clinical genetics, yeast, plant, nematode, insect, fish, and mouse genetics, aging and population genetics, and genomics and computational genetics. The predoctoral trainees supported by this program gain specific expertise from thesis research in one of these subdisciplines while gaining a broader intellectual outlook from formal coursework, first-year research rotations, and journal club. A well-established program of seminars and other forums for exchange of research findings lends further perspective and provides valuable interaction with postdoctoral fellows, whose research training is also integrated into the program. Access to medical and clinical aspects of genetics derives both from formal coursework and from association with the Division of Medical Genetics in the Department of Medicine. The training program draws faculty with genetic expertise from a number of departments at the University, including the Department of Biology in the College of Arts and Sciences, the Departments of Genome Sciences, Biochemistry, Microbiology, Pathology, and Pediatrics in the School of Medicine, as well as with the Basic Sciences Division of the Fred Hutchinson Cancer Research Center. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Robust genome sequencing technology has resulted in over 180 completed genomes, with sequencing projects for an additional 700+ organisms in progress. The difficult and important problem of experimentally determining the proteins encoded by these genomes lags far behind. We propose to complement existing messenger-RNA based approaches with high-throughput mass spectrometry of the entire protein complement of a complex animal, the nematode Caenorhabditis elegans. Our approach combines open- reading-frame (ORF) analysis of the fully sequenced C. elegans genome with high-throughput mass spectrometry, using multidimensional protein identification technology (MudPIT). Our long-term goal is development of these methods to the point that at least 80% of all proteins in a newly sequenced organism can be identified in a few months of concerted effort by a small group of investigators. This goal requires development of the following tools: 1) efficient evolutionary analysis of genomic ORFs to identify a computationally manageable set of candidate peptides for mass spectrum matching; 2) a robust method for biochemical fractionation of intact proteins from whole organisms or tissues; and 3) analytical approaches to assessing the significance of MudPIT matches to specific candidate peptides. Peptide cleavage, fractionation, and 2-dimensional (2D) mass spectrometry methods are established in our labs and are currently sufficient to achieve our goal with the addition of these tools. Our specific milestone for this 2-year grant period is the identification of at least 10,000 unique proteins (>50% of all predicted proteins in C. elegans) and validation by orthogonal methods of at least 50 of the proteins that are not yet supported by other data. The end result will be both an extensive map of the C. elegans proteome and a high-throughput pipeline that will allow similar analysis of any complex animal or plant proteome whose genome sequence is available. [unreadable] [unreadable] [unreadable]