Parameters controlling the gene-targeting frequency at the Sphingomonas species rrn site and expression of the methyl parathion hydrolase gene.
OBJECTIVE
To investigate the key parameters controlling the exogenous methyl parathion hydrolase (MPH) gene mpd-targeting frequency at the ribosomal RNA operon (rrn) site of Sphingomonas species which has a wide range of biotechnological applications.


RESULTS
Targeting vectors with different homology lengths and recipient target DNA with different homology identities were used to investigate the parameters controlling the targeting frequency at the Sphingomonas species rrn site. Targeting frequency decreased with the reduction of homology length, and the minimal size for normal homologous recombination was >100 bp. Homologous recombination could succeed even if there were 3-4% mismatches; however, targeting frequency decreased with increasing sequence divergence. The Red recombination system could increase the targeting frequency to some extent. Targeting of the mpd gene to the rrn site did not affect cell viability and resulted in an increase of MPH-specific activity in recombinants.


CONCLUSIONS
Targeting frequency was affected by homology length, identity and the Red recombination system. The rrn site is a good target site for the expression of exogenous genes.


CONCLUSIONS
This work is useful as a foundation for a better understanding of recombination events involving homologous sequences and for the improved manipulation of Sphingomonas genes in biotechnological applications.
Jiang
J
J
Department of Microbiology, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China.
Zhang
R
R
Cui
Z
Z
He
J
J
Gu
L
L
Li
S
S
D003434:Crossing Over, Genetic / Q000235:genetics; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D020578:Sphingomonas / Q000235:genetics* / Q000254:growth & development; D014169:Transformation, Bacterial / Q000235:genetics
D003470:Culture Media; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; C524863:methyl parathion hydrolase, Sphingomonas; D010744:Phosphoric Monoester Hydrolases