Donald L. Riddle - US grants

The proposed research is a continued investigation of the genetic control of development in a simple animal model. The approach is to apply genetic, microscopic, ultrastructural and biochemical methods to a detailed analysis of a postembryonic "developmental switch" in the life cycle of the nematode, Caenorhabditis elegans. The switch is manifested at the second larval molt, when starvation or overcrowded conditions may result in the formation of dauer larvae, a developmentally arrested, non-feeding larval stage. The overall aim is to determine the genetic basis of the developmental program for dauer larva formation and recovery, and the molecular and physiological mechanisms for the implementation of the developmental program. Specific goals are proposed which fall into six complementary areas of investigation: (a) biochemical and physical analysis of the biologically active substances, including a newly discovered pheromone, which act as environmental cues influencing the decision made between the two alternate developmental pathways, (b) selection and genetic analysis of mutants, either affected in the developmental decision preceding the L2 molt, affected in the timing of the developmental sequence leading to the dauer larvae, or affected in specific aspects of dauer larva morphogenesis, (c) electron microscopic characterization of the sensory neuroanatomy, or glandular anatomy, of specific mutants suspected to have sensory defects or secretory abnormalities, (d) developmental studies designed to test the response of developing larvae to specific environmental stimuli, and determine the timing of commitment to that to that response, (e) neurological studies designed to identify the specific cells which function in the process of entry into, or exit from, the dauer stage, and (f) molecular studies to determine the extent and timing of transcriptional controls associated with the alternate developmental sequences. New methods will be used to simplify genetic selection, and permit more rapid genetic analysis of mutants. The analysis of wild-type development at the physiological level, and at the level of developmentally regulated gene transcripts, will provide new frames of reference for the analysis of mutant phenotypes.

genetic models; genetic registry /resource /referral center; Caenorhabditis elegans; aging; literature survey; information dissemination; genetic strain; genetic mapping; mutant; cryopreservation;

The nematode, Caenorhabditis elegans, is currently utilized as a laboratory model for a variety of biomedical research applications, including the study of metazoan development. The enzyme RNA polymerase II, which is responsible for the synthesis of messenger RNA, plays such a central role in cellular activity that its expression, control and activity must be examined if the genetic and cellular mechanisms that underlie development and aging are to be understood. The goal of the genetic work is to characterize genes encoding subunits of the enzyme, select lethal and conditional-lethal alleles of these genes, and determine the effect of such mutations on normal development and on the expression of selected mutant phenotypes. Two genes already have been identified by selection of mutants resistant to the RNA polymerase II inhibitor, alpha-amanitin(amanitin). One dominant mutant, ama-1(m118)IV, produces an RNA polymerase II that is 100-fold more resistant to the toxin in vitro than is the wild-type enzyme. Six lethal alleles of ama-1 have been characterized. Additional lethal alleles will be isolated and a fine-structure genetic map of the ama-1 gene will be generated. Revertants of lethal alleles will be selected as a means to obtain additional variants with altered activity, and as a means to identify suppressor mutations that may define structural genes for other RNA polymerase subunits, transcription factors, or for regulatory elements controlling the level of RNA polymerase synthesis. The in vitro activity and amanitin sensitivity of RNA polymerase isolated from strains carrying mutations in ama-2 and let-276 (two genes that influence sensitivity to amanitin) will be determined. Also, the properties of RNA polymerase extracted from dauer larvae (a non-feeding, developmentally arrested dispersal stage formed in response to starvation or overcrowding) will be examined, since this unique larval stage does not appear to contain significant amounts of mRNA that can be translated in vitro. The DNA sequence of the entire ama-1 gene will be determined. Coding regions and introns will be defined, and promoter structure determined. Mutational alterations in certain ama-1 mutants will be characterized to correlate the DNA sequence with the fine-structure genetic map. The size and amount of mRNA complementary to the ama-1 gene will be determined. ama-1 mRNA from revertants of ama-1 lethal and conditional-lethal alleles will be quantitated to detect any promoter mutants with abnormally high levels of ama-1 mRNA.

The proposed research is a continued investigation of the genetic control of development in a simple animal model. The approach is to apply genetic, microscopic, ultrastructural, molecular and biochemical methods to a detailed analysis of a postembryonic "developmental switch" in the life cycle of the nematode, Caenorhabditis elegans. The switch is manifested at the second larval molt, when starvation or overcrowded conditions may result in the formation of the dauer larva, a developmentally arrested, non-feeding dispersal stage. Alternate developmental fates may be expressed (either formation of a growing third-stage larva, or formation of a dauer larva) based on environmental conditions. When the environment becomes favorable for growth, the dauer larva resumes feeding and development. The overall aim is to determine the genetic basis for the behavioral and developmental response to the environment, and the molecular and physiological mechanisms for implementation of the developmental program. Specific goals are proposed, which fall into complementary areas of investigation: (a) cloning and molecular characterization of selected genes involved in processing the response to environmental stimuli, (b) continued genetic studies, including the analysis of somatic mosaics to determine the developmental foci for action of specific genes that define steps in the genetic pathway for dauer larva formation, (c) electron microscopic and biochemical analysis of a mutant deficient in the synthesis of the dauer-inducing pheromone, (d) cloning and classification of sequences expressed specifically in animals destined to become dauer larvae, and not expressed in larvae committed to growth and (e) biochemical analysis of the physiological changes associated with entry into (and exit from) the dauer stage, including changes in protein phosphorylation. The analysis of wild-type developmental at the physiological level, and at the level of developmentally regulated gene transcripts, will provide new frames of reference for the analysis of mutant phenotypes. Finally, continued molecular characterization of dpy- 13 ("dumpy"), a collagen-like gene that effects body length, will be aimed at determining its role in specifying cuticle development and structure. It will be determined other dumpy genes are homologous to dpy-13.

The goal is to determine the molecular and genetic mechanisms controlling a developmental "switch" in a simple animal model. The approach utilizes the advantages that the nematode C. elegans offers for molecular genetic studies. The switch is manifested at the second larval molt when alternate developmental fates may be expressed (either formation of a growing third-stage larva, or formation of a developmentally arrested, non-feeding dauer larva) based on environmental conditions. Starvation and overcrowding during the first larval stage induces formation of dauer larvae, which may survive for months, and when they find conditions favorable for growth, they resume development. The developmental decisions governing entry into, or exit from, the dauer stage are made in response to the ratio of food to the C. elegans dauer-inducing pheromone. Mutants affected in the decision to form dauer larvae are either dauer- constitutive, which form dauer larvae in abundant food, or dauer- defective, which cannot form dauer larvae when starved. Interactions between specific mutants have been used to construct pathways for gene action, now including more than 20 genes. Three genes have been cloned by transposon-tagging, and they encode molecules. Intermediate steps in the pathway are mediated by the daf-1 and daf-4 genes, which encode transmembrane receptor serine kinases. The daf-1 receptor was the first receptor serine kinase reported, and mammalian activin and TGF-beta receptors subsequently were found to be related to it. Activin and TGF- beta are growth factors of profound importance in vertebrate development. If the signal transduction pathway that regulates the C. elegans dauer larva is the nematode analogue of a TGF-beta or activin signalling system, C. elegans genetics may provide the means to identify the missing links between signal generation and control of gene expression in these vertebrate systems. The daf-12 gene specifies what we believe to be the last step in signal transduction. It encodes a member of the steroid-thyroid hormone receptor superfamily. Whereas daf-1 and daf-4 are required for normal non-dauer development, daf-12 activity is required for dauer larva morphogenesis. We propose that the daf kinases phosphorylate proteins that promote growth, and directly or indirectly inactivate the daf-12 receptor, possibly by preventing synthesis of a dauer-inducing hormone. Specific goals fall into complementary areas aimed at (1) understanding the relationship between the dauer and activin/TGF-beta signalling system, (2) identifying the ligands for the daf-1 and daf-4 receptors, (3) identifying the downstream targets for phosphorylation, (4) understanding how mutational changes affect daf-1 and 4 structure and function, (5) determining whether the daf-12 receptor is a ligand- activated transcription factor, (6) identifying DNA target sites for daf- 12 action, (7) genetically identifying additional genes involved in the dauer pathway, including those which may have essential functions in development, and (8) characterizing daf gene homologs in a parasitic nematode.

A selected group of outstanding University of Missouri (MU) faculty (15 from Biochemistry, 11 from Biological Sciences, ten from Molecular Microbiology and Immunology, and four from Pharmacology) propose to continue developing an interdisciplinary program in molecular and cellular biology with six NIH-sponsored predoctoral traineeships. Each of the NIH stipends will be supplemented with $5,000 in State funds to bring trainees to the $15,000 stipend level of all 24 planned MU Molecular Biology Predoctoral Fellows. Our Molecular Biology Program provides an ideal framework by which trainees have broad access to Ph.D. thesis research opportunities across disciplinary, departmental, and even college lines. Cooperative involvement of faculty from different departments on Ph.D. committees, and as research mentors, is a natural aspect of our program. Training will consist of the following integrated elements: (1) formal courses in genetics, molecular biology, biochemistry, cell biology and developmental biology; (2) practical research experience under the supervision of at least three different faculty members prior to thesis research; (3) seminars presented by distinguished scientists; (4) student seminars and journal clubs, including instruction in the responsible conduct of research; (5) participation in Molecular Biology Week; and (6) yearly participation in national or international meetings. Our faculty received their own training in atmospheres that encouraged multidisciplinary experimental approaches to research problems, and their current research bridges traditional scientific disciplines. Thus, our training program is designed to allow the student great flexibility in choice of research problems. Candidates for traineeships are drawn from the four participating departments by referral to the Program's Fellowships and Awards Committee. The Program itself supplements vigorous departmental recruiting efforts, and recruitment of underrepresented minorities is a high priority. Selection is based on academic record, letters of recommendation, graduate record examinations, personal interviews, and other evidence of research promise. We select exceptional candidates who are committed to a research career. The professional skills and insights they obtain in our Program enable them to perform to our mutual credit. Primary facilities are in the School of Medicine and in the Colleges of Agriculture and Arts and Sciences, all within 5 to 10 minute walks. These facilities are well equipped, and recent hiring of new Molecular Biology faculty with newly renovated laboratory space provides a great opportunity for new students to join expanding research groups. The Program also operates five expertly staffed Core Facilities with multi- user equipment to support molecular technologies in research, and to enrich the training environment.

animal population genetics; developmental genetics; longevity; larva; Caenorhabditis elegans; alternatives to animals in research; gene mutation; gene expression; animal genetic material tag;

The goal is to determine the molecular and genetic mechanisms controlling a developmental "switch" in a simple animal model. The approach utilizes the advantages that the nematode C. elegans offers for molecular genetic studies. The switch is manifested at the second larval molt when alternate developmental fates may be expressed (either formation of a growing third-stage larva, or formation of a developmentally arrested, non-feeding dauer larva) based on environmental conditions. Starvation and overcrowding during the first larval stage induces formation of dauer larvae, which may survive for months, and when they find conditions favorable for growth, they resume development. The developmental decisions governing entry into, or exit from, the dauer stage are made in response to the ratio of food to the C. elegans dauer-inducing pheromone. Mutants affected in the decision to form dauer larvae are either dauer- constitutive, which form dauer larvae in abundant food, or dauer- defective, which cannot form dauer larvae when starved. Interactions between specific mutants have been used to construct pathways for gene action, now including more than 20 genes. Three genes have been cloned by transposon-tagging, and they encode molecules. Intermediate steps in the pathway are mediated by the daf-1 and daf-4 genes, which encode transmembrane receptor serine kinases. The daf-1 receptor was the first receptor serine kinase reported, and mammalian activin and TGF-beta receptors subsequently were found to be related to it. Activin and TGF- beta are growth factors of profound importance in vertebrate development. If the signal transduction pathway that regulates the C. elegans dauer larva is the nematode analogue of a TGF-beta or activin signalling system, C. elegans genetics may provide the means to identify the missing links between signal generation and control of gene expression in these vertebrate systems. The daf-12 gene specifies what we believe to be the last step in signal transduction. It encodes a member of the steroid-thyroid hormone receptor superfamily. Whereas daf-1 and daf-4 are required for normal non-dauer development, daf-12 activity is required for dauer larva morphogenesis. We propose that the daf kinases phosphorylate proteins that promote growth, and directly or indirectly inactivate the daf-12 receptor, possibly by preventing synthesis of a dauer-inducing hormone. Specific goals fall into complementary areas aimed at (1) understanding the relationship between the dauer and activin/TGF-beta signalling system, (2) identifying the ligands for the daf-1 and daf-4 receptors, (3) identifying the downstream targets for phosphorylation, (4) understanding how mutational changes affect daf-1 and 4 structure and function, (5) determining whether the daf-12 receptor is a ligand- activated transcription factor, (6) identifying DNA target sites for daf- 12 action, (7) genetically identifying additional genes involved in the dauer pathway, including those which may have essential functions in development, and (8) characterizing daf gene homologs in a parasitic nematode.