Use of RmInt1, a group IIB intron lacking the intron-encoded protein endonuclease domain, in gene targeting.
The group IIA intron Ll.LtrB from Lactococcus lactis and the group IIB intron EcI5 from Escherichia coli have intron-encoded proteins (IEP) with a DNA-binding domain (D) and an endonuclease domain (En). Both have been successfully retargeted to invade target DNAs other than their wild-type target sites. RmInt1, a subclass IIB3/D intron with an IEP lacking D and En domains, is highly active in retrohoming in its host, Sinorhizobium meliloti. We found that RmInt1 was also mobile in E. coli and that retrohoming in this heterologous host depended on temperature, being more efficient at 28°C than at 37°C. Furthermore, we programmed RmInt1 to recognize target sites other than its wild-type site. These retargeted introns efficiently and specifically retrohome into a recipient plasmid target site or a target site present as a single copy in the chromosome, generating a mutation in the targeted gene. Our results extend the range of group II introns available for gene targeting.
Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científica, C/Profesor Albareda 1, 18008 Granada, Spain.
Fernando M
FM
García-Rodríguez
Antonio
A
Barrientos-Durán
Vanessa
V
Díaz-Prado
Manuel
M
Fernández-López
Nicolás
N
Toro
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004720:Endonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting; D047668:Inteins / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D017434:Protein Structure, Tertiary / Q000235:genetics*; D016962:Sinorhizobium meliloti / Q000235:genetics
D004269:DNA, Bacterial; D004720:Endonucleases