Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Molecular & General Genetics : MGG
M Itaya, T Tanaka

Abstract

We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB::cat, leuB-, and pro::neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB+ pro+)----(leuB::cat pro+)----(leuB- pro::neo)----(leuB- pro+) resulted in a leuB- single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD- and pro-, as well as the double mutation leuB- pro- can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure shoul...Continue Reading

References

Mar 1, 1989·Trends in Genetics : TIG·M R Capecchi
Jan 15, 1982·The Biochemical Journal·J Rosamond
Aug 20, 1963·Biochimica Et Biophysica Acta·H SAITO, K I MIURA
Oct 15, 1958·Proceedings of the National Academy of Sciences of the United States of America·J Spizizen

❮ Previous
Next ❯

Citations

Jul 26, 1996·Biochemical Pharmacology·T StachelhausM A Marahiel
Mar 17, 2004·Diagnostic Microbiology and Infectious Disease·Jiunn Jong WuTsung Chain Chang
May 13, 1997·Proceedings of the National Academy of Sciences of the United States of America·M Itaya, T Tanaka
Jan 22, 2010·PLoS Genetics·Anjana SrivatsanJue D Wang
Jan 11, 2012·Bioscience, Biotechnology, and Biochemistry·Mitsuhiro Itaya
Jun 9, 2004·Microbiology·Eduardo P C Rocha
Apr 10, 2012·FEBS Letters·François KépèsIvan Junier
May 26, 2005·Bioscience, Biotechnology, and Biochemistry·Rie Uotsu-TomitaMitsuhiro Itaya

❮ Previous
Next ❯

Related Concepts

Related Feeds

Allergy & Infectious Diseases (ASM)

Allergies result from the hyperreactivity of the immune system to some environmental substance and can be life-threatening. Infectious diseases are caused by organisms including bacteria, viruses, fungi and parasites. They can be transmitted different ways, such as person-to-person. Here is the latest research on allergy and infectious diseases.

Antimicrobial Resistance (ASM)

Antimicrobial resistance poses a significant threat to the continued successful use of antimicrobial agents for the treatment of bacterial infections.

Antimicrobial Resistance

Antimicrobial resistance poses a significant threat to the continued successful use of antimicrobial agents for the treatment of bacterial infections.

Allergy & Infectious Diseases

Allergies result from the hyperreactivity of the immune system to some environmental substance and can be life-threatening. Infectious diseases are caused by organisms including bacteria, viruses, fungi and parasites. They can be transmitted different ways, such as person-to-person. Here is the latest research on allergy and infectious diseases.