This application proposes to devise improved methods for directed mutagenesis, and to apply these methods to define regulatory sequences in DNA. We will exploit a new method for producing """"""""complete mutant libraries"""""""", which allows the isolation of all possible single base substitution mutations within a region of DNA. The target sequence is synthesized by a slight modification of the usual automated synthetic procedure, in which chains are built stepwise from the 3' end by the addition of nucleoside phosphoramidite monomeric units. Before synthesis each of the four monomer reservoirs is """"""""doped"""""""" with a small amount of each of the other three, to produce a level of contamination sufficient to produce a small number of mutations per molecule synthesized. Following synthesis, the mutagenized sequences are amplified by biological cloning, to produce a mutant library containing all possible point substitution mutations of the original sequence. It is practical to identify mutants directly by sequencing random isolates from the library. We are exploring phenotypes of mutants in three libraries which we have already constructed and characterized: 1) the glucocorticoid response element (GRE) of mouse mammary tumor virus, 2) an enhancer-like element in the Tyl transposon of yeast, and 3) the RNA polymerase III promoter of the VAI gene of adenovirus. We propose to construct and analyze a library of mutations in the alpha I domain of the H-2DP gene from the major histocompatibility complex of the mouse. We propose to construct complete mutant libraries for genes E and J of phage PhiX174. This project will also serve to evaluate the feasibility and usefulness of a complete chemical synthesis of the PhiX174 genome. We propose to use mutations of defined DNA sequences from a prokaryotic source as a """"""""shuttle target"""""""" to assay in vivo mutagenesis in mammals. Our objective is to develop a relatively rapid and economical method for measurement of mutation rates in whole animals. The phage PhiX174 genome, carrying mutations of defined sequence, has been stably introduced into mouse L cells in tissue culture, and we are able to rescue it in E. coli by transfection of spheroplasts. We propose to use this system to measure reversion rates of PhiX174 mutations following mutagenic treatment of the L cells. We propose to use DNA microinjection to produce mice carrying integrated copies of the PhiX174 genome, for use in whole animal mutagenesis assays.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI008998-26
Application #
2058961
Study Section
Special Emphasis Panel (NSS)
Project Start
1977-03-01
Project End
1997-02-28
Budget Start
1994-03-01
Budget End
1995-02-28
Support Year
26
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Speake, Cate; Presnell, Scott; Domico, Kelly et al. (2015) An interactive web application for the dissemination of human systems immunology data. J Transl Med 13:196
Chaussabel, Damien; Baldwin, Nicole (2014) Democratizing systems immunology with modular transcriptional repertoire analyses. Nat Rev Immunol 14:271-80
Obermoser, Gerlinde; Presnell, Scott; Domico, Kelly et al. (2013) Systems scale interactive exploration reveals quantitative and qualitative differences in response to influenza and pneumococcal vaccines. Immunity 38:831-44