We will develop highly specific DNA cleavage tools, based on coupling adenine methylation with the methylation-dependent restriction-endonuclease Dpnl. We have produced DNA fragments of over 1,000,000 base pairs in size with one methylase/Dpnl cleavage system, and separated the resulting pieces by pulsed field gel electrophoresis. These results encourage us to develop the other methylase/Dpnl cleavage specificities based on known restriction modification systems, of which there are at least 22 possible. The cleavage specificities so generated recognize DNA sequences ranging from 8 to 14 base pairs in length and give average fragment sizes of 65,000 (4(8)) to 256,000,000 (4(14)) base pairs on random DNA. We intend to characterize, purify and clone those methylases that have the correct Gm6A specificity for use with Dpnl. This battery of methylase/Dpnl specificities can be applied to three mapping strategies: (1) The production of a physical map of the human genome using restriction fragment length polymorphism probes that have already been ordered genetically. (2) Construction of unique sites in insertion vectors such as retroviruses for the mapping of large portions of the human genome by Smith-Birnstiel indirect end-labelling. (3) Identification of 'linking clones' containing rare cleavage sites for the production of a physical map of these rare sites in the human genome.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI034829-08
Application #
2429412
Study Section
Genome Study Section (GNM)
Project Start
1989-12-15
Project End
1998-05-31
Budget Start
1997-06-01
Budget End
1998-05-31
Support Year
8
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Sidney Kimmel Cancer Center
Department
Type
DUNS #
City
San Diego
State
CA
Country
United States
Zip Code
92121
Achtman, Mark; Hale, James; Murphy, Ronan A et al. (2013) Population structures in the SARA and SARB reference collections of Salmonella enterica according to MLST, MLEE and microarray hybridization. Infect Genet Evol 16:314-25
Evans, Matthew R; Fink, Ryan C; Vazquez-Torres, Andres et al. (2011) Analysis of the ArcA regulon in anaerobically grown Salmonella enterica sv. Typhimurium. BMC Microbiol 11:58
Arrach, Nabil; Cheng, Pui; Zhao, Ming et al. (2010) High-throughput screening for salmonella avirulent mutants that retain targeting of solid tumors. Cancer Res 70:2165-70
Frye, Jonathan G; Lindsey, Rebecca L; Rondeau, Gaelle et al. (2010) Development of a DNA microarray to detect antimicrobial resistance genes identified in the National Center for Biotechnology Information database. Microb Drug Resist 16:9-19
Thijs, Inge M; Zhao, Hui; De Weerdt, Ami et al. (2010) The AI-2-dependent regulator LsrR has a limited regulon in Salmonella Typhimurium. Cell Res 20:966-9
Santiviago, Carlos A; Reynolds, M Megan; Porwollik, Steffen et al. (2009) Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice. PLoS Pathog 5:e1000477
Xia, Xiao-Qin; McClelland, Michael; Porwollik, Steffen et al. (2009) WebArrayDB: cross-platform microarray data analysis and public data repository. Bioinformatics 25:2425-9
Andrews-Polymenis, Helene L; Santiviago, Carlos A; McClelland, Michael (2009) Novel genetic tools for studying food-borne Salmonella. Curr Opin Biotechnol 20:149-57
Merighi, Massimo; Septer, Alecia N; Carroll-Portillo, Amanda et al. (2009) Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium. BMC Microbiol 9:42
Singh, Varsha; Mishra, Shrutkirti; Rao, G R K et al. (2008) Evaluation of nested PCR in detection of Helicobacter pylori targeting a highly conserved gene: HSP60. Helicobacter 13:30-4

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