van Kessel, JC and Hatfull, GF
(2007)
Recombineering in Mycobacterium tuberculosis.
Nature Methods, 4 (2).
147 - 152.
ISSN 1548-7091
![[img]](http://d-scholarship.pitt.edu/style/images/fileicons/text_plain.png) |
Plain Text (licence)
Available under License : See the attached license file.
Download (1kB)
|
Abstract
Genetic dissection of M. tuberculosis is complicated by its slow growth and its high rate of illegitimate recombination relative to homologous DNA exchange. We report here the development of a facile allelic exchange system by identification and expression of mycobacteriophage-encoded recombination proteins, adapting a strategy developed previously for recombineering in Escherichia coli. Identifiable recombination proteins are rare in mycobacteriophages, and only 1 of 30 genomically characterized mycobacteriophages (Che9c) encodes homologs of both RecE and RecT. Expression and biochemical characterization show that Che9c gp60 and gp61 encode exonuclease and DNA-binding activities, respectively, and expression of these proteins substantially elevates recombination facilitating allelic exchange in both M. smegmatis and M. tuberculosis. Mycobacterial recombineering thus provides a simple approach for the construction of gene replacement mutants in both slow- and fast-growing mycobacteria.
Share
Citation/Export: |
|
Social Networking: |
|
Details
Item Type: |
Article
|
Status: |
Published |
Creators/Authors: |
Creators | Email | Pitt Username | ORCID  |
---|
van Kessel, JC | | | | Hatfull, GF | gfh@pitt.edu | GFH | |
|
Date: |
1 February 2007 |
Date Type: |
Publication |
Journal or Publication Title: |
Nature Methods |
Volume: |
4 |
Number: |
2 |
Page Range: |
147 - 152 |
DOI or Unique Handle: |
10.1038/nmeth996 |
Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
Refereed: |
Yes |
ISSN: |
1548-7091 |
MeSH Headings: |
DNA-Binding Proteins--genetics; Exonucleases--genetics; Gene Targeting--methods; Mycobacteriophages--chemistry; Mycobacteriophages--genetics; Mycobacterium smegmatis--genetics; Mycobacterium smegmatis--growth & development; Mycobacterium tuberculosis--genetics; Mycobacterium tuberculosis--growth & development; Mycobacterium tuberculosis--virology; Recombination, Genetic; Transformation, Genetic; Viral Proteins--genetics |
PubMed ID: |
17179933 |
Date Deposited: |
14 Jan 2013 16:19 |
Last Modified: |
02 Feb 2019 15:57 |
URI: |
http://d-scholarship.pitt.edu/id/eprint/16733 |
Metrics
Monthly Views for the past 3 years
Plum Analytics
Altmetric.com
Actions (login required)
 |
View Item |