The research here proposed has as its objective the improvement of methods of site-specific mutagenesis which are based on a common strategy--the generation of a short, single-stranded gap at a defined site on a circular DNA molecule, followed by the use of such a gap as a target for efficient in vitro mutagenic reactions. This research will involve refinement of the enzymatic reactions used to introduce nicks at unique sites and to convert such site-specific nicks into short gaps. In addition, a new mutagenic reaction, which induces mutations by stimulating DNA polymerase to misincorporate nucleotides during repair (misrepair) of single-stranded gaps, will be extensively evaluated. Initially, this reaction will be applied to several unique restriction sites on the plasmid pBR322 in order to optimize the efficiency of mutagenesis and to analyze the pattern of induced base substitution mutations. Once these properties have been defined, site-specific mutagenesis with this method will be used in the analysis of two cloned genes. First, mutations causing amino acid substitutions in the signal peptide of the TEM beta-lactamase will be constructed, and their effects on the transport of this enzyme across the bacterial cell membrane will be studied. Secondly, a long-term mutational analysis of the actin gene of Saccharomyces cerevisiae will be conducted. This project will involve the development of methods for efficiently replacing the chromosomal actin gene with mutant alleles constructed in vitro. In addition to providing improved methods for site-specific mutagenesis which should be applicable to any cloned segment of DNA, this research can be expected to yield new information on the biochemistry of protein secretion in prokaryotes. Furthermore, once a collection of conditional lethal mutant alleles of the yeast actin gene have been obtained, many basic questions dealing with the molecular biology of actin--its roles in maintaining cell structure and normal physiology in eukaryotic cells--should be answerable.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM034171-02
Application #
3284722
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-05-01
Project End
1990-04-30
Budget Start
1985-05-01
Budget End
1986-04-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Shortle, D (1994) Assignment of amino acid type in 1H-15N correlation spectra by labeling with 14N-amino acids. J Magn Reson B 105:88-90
Stites, W E; Meeker, A K; Shortle, D (1994) Evidence for strained interactions between side-chains and the polypeptide backbone. J Mol Biol 235:27-32
Alexandrescu, A T; Abeygunawardana, C; Shortle, D (1994) Structure and dynamics of a denatured 131-residue fragment of staphylococcal nuclease: a heteronuclear NMR study. Biochemistry 33:1063-72
Alexandrescu, A T; Shortle, D (1994) Backbone dynamics of a highly disordered 131 residue fragment of staphylococcal nuclease. J Mol Biol 242:527-46
Shortle, D; Abeygunawardana, C (1993) NMR analysis of the residual structure in the denatured state of an unusual mutant of staphylococcal nuclease. Structure 1:121-34
Gittis, A G; Stites, W E; Lattman, E E (1993) The phase transition between a compact denatured state and a random coil state in staphylococcal nuclease is first-order. J Mol Biol 232:718-24
Li, Y K; Kuliopulos, A; Mildvan, A S et al. (1993) Environments and mechanistic roles of the tyrosine residues of delta 5-3-ketosteroid isomerase. Biochemistry 32:1816-24
Green, S M; Shortle, D (1993) Patterns of nonadditivity between pairs of stability mutations in staphylococcal nuclease. Biochemistry 32:10131-9
Sondek, J; Shortle, D (1992) A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites. Proc Natl Acad Sci U S A 89:3581-5
Green, S M; Meeker, A K; Shortle, D (1992) Contributions of the polar, uncharged amino acids to the stability of staphylococcal nuclease: evidence for mutational effects on the free energy of the denatured state. Biochemistry 31:5717-28

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