C. Thomas Caskey - US grants

The release factor (RF) proteins 1 and 2 are involved in codon specific bacterial peptide chain termination. The mechanism of the codon recognition and subsequent peptidyl-tRNA hydrolysis is not understood at a molecular level. We propose to use recombinant DNA techniques to identify the regions of release factor molecules involved in these reactions. This will be accomplished by determining the in vivo and in vitro behavior of mutant RF1 and RF2 molecules generated by in vitro mutagenesis of these cloned genes. We will determine the translational and transcriptional regulation of release factor genes. We propose to clone and characterize the mammalian release factor gene using the Lambdagtll expression cloning vector. These studies will elucidate the mechanism of peptide chain termination and compare E. coli and mammalian release factors for structure, regulation, and functional domains.

The detailed study of mammalian genes and their regulation was markedly stimulated by developments in somatic cell genetic techniques. Availability of selective methods, molecular cloning, gene transfer vectors, and embryonic cellular manipulations has widened the technical approaches to the study of mammalian genes, structural and regulatory. This conference will draw together investigators who share the interests of the study of eukaryotic genes. Each of the topics is in the forefront of active investigation and is chaired by a distinguished lecturer who has been instrumental in a technologic development. Through the mechanisms of lecture, posters, informal discussions and workshops, we propose to facilitate knowledge and technique exchange in the field of mammalian gene study.

The Lesch-Nyhan (L-N) syndrome is an X-linked recessively inherited disease characterized by mental retardation, chorioathetosis, self-mutilative behavior and hyperuricemia. The disorder is genetically lethal, and since the affected males do not reproduce, it arises frequently by new mutation. We propose to study the molecular basis of new mutation in man by examining the types of mutation as well as the paternal gametic origin. The basis of the high level of hypoxanthine-guanine phosphoriboro-syltransferase (HPRT) expression is the central nervous system and the effect of its deficiency state (HPRT-) on neural development is unresolved. We propose to study both cell culture and transgenic mice in an effort to develop a model for L-N pathophysiologic studies. Finally, an attempt to develop HPRT- mice which correspond to the L-N disease of man will be made towards the objective of a model animal system for study and correction of the disease in man.

Adenosine deaminase (ADA) deficiency, a rare autosomal recessive condition causing a form of severe combined immunodeficiency, provides an optimal model disease to develop strategies for gene therapy of diseases which may be ameliorated by gene transfer into hematopoietic tissue. Our recent success in obtaining stable long term expression of human ADA in mice, via retroviral gene transfer in hematopoietic cells, offers encouragement that the techniques can be further improved and applied to gene therapy of inherited human diseases. Human hematopoietic stem cell infection will be attempted with the vector that was successful in transducing long term expression in mice (delta N2ADA) and with new vectors which will be constructed. Enrichment for primitive stem cells from hum bone marrow will allow us to examine the infectability of those cells with retroviral vectors. For this purpose, positive selection with anti-CD34 monoclonal antibodies will be combined with negative selection using anti-CD33 and anti-Ia antibodies to enrich the target population for primitive hematopoietic progenitor cells. Infection of primitive stem cells will be evaluated in long term bone marrow culture and through transplantation of human hematopoietic progenitors in immuno-deficient mice. Questions regarding issues of safety and expansion of the technology to the number of cells required for hematology reconstitution will be addressed. Ornithine transcarbamylase (OTC) deficiency, the most common and severe human defect in ureagenesis, is an excellent model disease to develop and test gene therapy strategies aimed at correcting inborn errors of liver cell metabolism. We have recently identified the molecular basis of OTC deficiency in the sparse fur (spf) mouse, the mouse model for the human defect. Construction of retroviral and adeno-associated viral vectors will be undertaken and the vectors tested for their ability to infect and transduce OTC activity into spf hepatocytes in vitro and in vivo. The creation of transgenic spf mice which express human OTC cDNA in small bowel and are biochemically and phenotypically correct for OTC deficiency points to a new target tissue for gene therapy of this disease. Recombinant retrovirus and adeno-associated virus which encode for OTC will be used to infect spf small intestinal epithelial cells to determine if gene transfer and expression of OTC in this tissue is possible and if successful transduction of OTC activity in this tissue will correct the inborn error of these mice.

DNA sequence analysis is currently provided at the Institute for Molecular Genetics (IMG) at Baylor College of Medicine by a core facility. The sequencing core provides a mechanism whereby investigators can obtain DNA sequence information without having to dedicate their own laboratory resources. Fragments are provided to the facility in a variety of forms and in different DNA doning vectors, and the data is returned via a computer network. The sequencing capacity of the facility is almost saturated with the current 373A Applied Biosystems automated DNA sequencer operated on every working day and approximately 50% of weekend days. The methods for DNA template preparation and for manipulating the sequencing reaction components are now being simplified, and the demands from individual projects are increasing. We estimate a minimum of a 100% increase in demand for the facilities throughput within 12 months. To cater for this additional capacity an Applied Biosystems 373 Plus DNA Sequencing system is requested. The instrument consists of a robot for generating DNA templates and a fluorescent DNA sequencer to analyze the reaction products. The instrument will more than double the capacity of the DNA sequencing facility as the robot can be used to supply products for both the new and existing fluorescence analyzers. The wide interest in defining and manipulating individual DNA fragments means the increase in rate and capacity for the generation of DNA sequence will benefit all IMG investigators and their staff.

The X chromosome of man comprises approximately 5% of the genome or 150 Mb. As of HGM 10, there were 44 cloned genes, 175 mapped genes and disease loci, and 212 polymorphic DNA markers (the largest number of any human chromosome). Intense investigation of the X chromosome relates primarily to the high frequency of genetic diseases manifested by hemizygous males and its role in sex development. Additional features which make the X chromosome a high priority for genome analysis include: 1) its high degree of homology across mammalian species (Ohno's law), 2) its regulated inactivation and reactivation in mammalian females (Lyon hypothesis), 3) its partial homology with the Y chromosome and the unique features of recombination between distal Xp and Yp, and 4) the presence of the HPRT gene, which makes the X chromosome amenable to a variety of somatic cell genetic strategies for mapping using forward or back selection at this locus. Choice of the x chromosome for complete mapping and sequencing is logical one for a molecular genetics program based in a medical school (Baylor College of Medicine) where significant strengths exist in medical genetics and molecular genetic basis of human disease. The college has made a strong commitment to the proposed Human Genome Program Center by dedicating over 5000 sq. ft. of additional space to its cores and research projects. To complement the existing nucleus of human molecular genetics research activity, we are adding to our Center basic research expertise in yeast genetics for vector development, mouse mapping for comparative studies, and a substantial computational expertise from Baylor and Rice University to address problems of data assembly and analysis. The two primary goals of the Center for the first five years of funding are: 1) Development of a YAC vector contig map of the entire X-chromosome within 2- 5 years and 2) development of sequencing technology capable of up to 5 megabases of contig sequencing annually.

We request funding for the Genome Sequencing and Analysis Conference IV, to be held September 26-29, 1992, at the Hyatt Regency Hilton Head, Hilton Head Island, South Carolina. This conference is the fourth of it's type and the only scientific conference devoted solely to work in genomic sequencing. Our purpose is to bring together key researchers in this field. We will add an emphasis on the analysis of genomic sequence this year. Invited speakers will present their latest research projects and discuss future goals. Discussion periods, informal workshops and breaks will provide time for interaction between attendees. A poster session will be held to allow laboratories to present their ongoing work. A limited number of exhibit booths will be set up to allow suppliers to introduce their technology to the attendees. Afterwards, a summary of the proceedings will be published if an appropriate publication expresses interest. This conference has been supported by NIH, the DOE and by corporate contributions. The funding requested in this proposal covers the cost of reimbursing 25 invited domestic speakers for travel, per diem, ground transportation, and registration expenses. We are also requesting a part-time salary for a secretary to process reimbursements and a part-time salary for a conference administrator. There are no "for profit" aspects to this conference. All support from the combined source goes to funding the conference.

protein kinase; myotonic dystrophy; enzyme substrate; protein sequence; genetic markers; immobilized enzymes; complementary DNA; nucleic acid repetitive sequence; SDS polyacrylamide gel electrophoresis; molecular cloning; affinity chromatography; human tissue; laboratory mouse; genetic library; human genetic material tag;

DESCRIPTION (provided by applicant): Support is requested for predoctoral positions to establish a novel interdisciplinary training program in pharmacological sciences. The objective is to train leaders for academia, biotechnology/industrial firms, and regulatory agencies who will develop new innovations, approaches, and strategies for drug development and accelerate the rate of bringing new therapeutics to the market. Such leaders are essential if we are to reduce the enormous cost and length of time it currently takes to bring safe, effective agents to the public. The proposed training program is unique in several ways: (1) it spans the entire range of drug development from initial target identification and validation through FDA approval, (2) it is led by a PI and leadership team with extensive, first-hand experience in the pharmaceutical industry as well as academia, (3) it introduces a systems biology approach to achieving efficacy and safety by identifying and selectively modulating relevant pathways, rather than individual targets and addresses all major phases of the drug development process, and (4) it includes analyses of major successes and failures in the pharmaceutical industry and regulatory agencies. This proposal builds on a current Roadmap Training Grant that has established an effective organizational foundation for training in drug discovery and development at the Gulf Coast Consortia's Keck Center, an educational cooperative of 6 neighboring institutions - Baylor College of Medicine, M. D. Anderson Cancer Center, Rice University, University of Houston, The University of Texas Health Science Center at Houston, and The University of Texas Medical Branch at Galveston. The training faculty includes 55 members with broad research expertise and extensive training experience. Trainees will matriculate and receive first year support from their home institutions, and training grant stipends will be awarded on a competitive basis for years 2-3 of graduate training. Training will involve didactic courses, seminars, drug development team tutorials, retreats, and career development activities. These will be open to all pre- and postdoctoral trainees at the 6 institutions, and they may obtain a Certificate in Pharmacological Sciences by participating, which will greatly increase the program's impact. The home institution will award the Ph.D. PUBLIC HEALTH RELEVANCE: This program will train future leaders for universities, the pharmaceutical industry, and government regulatory agencies in the entire process - from basic laboratory discoveries through government approval - of providing safe, effective new drugs to the public. These leaders will decrease the current unacceptably high cost, length of time, and number of side effects now associated with developing new drugs.