Abstract

The major thrust of this proposal is a genetic and biochemical analysis of the regulation of plasmid DNA replication and the extrachromosomal state of plasmid elements in bacteria with particular emphasis on the nature and mechanism of action of plasmid specified proteins and plasmid nucleotide sequences involved in the regulated initiation of plasmid DNA replication and the segregation of plasmid elements during cell division. A related problem, the genetic and biochemical mechanisms of plasmic incompatibility (i.e., the failure of certain plasmid elements to co-exist stably in a bacterial cell) also will be stressed. Efforts will be concentrated on plasmids R6K (multi-copy antibiotic resistance plasmid of narrow host range) and RD2 (limited copy number per cell and broad host range). Studies will be carried out in vitro and in vivo with Escherichia coli cells and E. coli extracts. In the case of RK2 the in vivo plasmid replication and maintenance studies will be extended to bacteria distantly related to E. coli. An analysis of the incompatibility regions of plasmid mini-F also will be continued. Finally, plasmid Co1E1 and its derivatives will be used extensively in the construction by recombinant DNA techniques of joint replicons with R6K, RK2 and mini-F and their derivatives in the analysis of regulatory mechanisms for initiation of replication, segregation of plasmids and plasmid incompatibility. Extensive analyses of regions essential for replication and incompatibility properties will be carried out at the levels of plasmid DNA reconstructions using recombinant DNA techniques, isolation and characterization of plasmid replication mutants, nucleotide sequence analysis, characterization of plasmid specified proteins involved in the regulation of plasmid replication and incompatability. A broad host range, plasmid cloning vehicle derived from plasmid RD2 will be further developed using recombinant DNA procedures. Finally, efforts will be made to develop autonomous circular DNA replicons in animal cells grown in tissue culture.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI007194-20
Application #
3124346
Study Section
(MG)
Project Start
1978-02-01
Project End
1986-01-31
Budget Start
1985-02-01
Budget End
1986-01-31
Support Year
20
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093

  Publications

Verheust, Celine; Helinski, Donald R (2007) The incC korB region of RK2 repositions a mini-RK2 replicon in Escherichia coli. Plasmid 58:195-204
Konieczny, I; Helinski, D R (1997) The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone. Proc Natl Acad Sci U S A 94:14378-82
Cereghino, J L; Helinski, D R; Toukdarian, A E (1994) Isolation and characterization of DNA-binding mutants of a plasmid replication initiation protein utilizing an in vivo binding assay. Plasmid 31:89-99
Roberts, R C; Strom, A R; Helinski, D R (1994) The parDE operon of the broad-host-range plasmid RK2 specifies growth inhibition associated with plasmid loss. J Mol Biol 237:35-51
Perri, S; Helinski, D R (1993) DNA sequence requirements for interaction of the RK2 replication initiation protein with plasmid origin repeats. J Biol Chem 268:3662-9
Cereghino, J L; Helinski, D R (1993) Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria. J Biol Chem 268:24926-32
Fang, F C; Durland, R H; Helinski, D R (1993) Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria. Gene 133:1-8
Roberts, R C; Spangler, C; Helinski, D R (1993) Characteristics and significance of DNA binding activity of plasmid stabilization protein ParD from the broad host-range plasmid RK2. J Biol Chem 268:27109-17
Lin, J; Helinski, D R (1992) Analysis of mutations in trfA, the replication initiation gene of the broad-host-range plasmid RK2. J Bacteriol 174:4110-9
Greener, A; Lehman, S M; Helinski, D R (1992) Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria. Genetics 130:27-36

Showing the most recent 10 out of 29 publications