Mutations in the Lactococcus lactis Ll. LtrB group II intron that retain mobility in vivo -: art. no. 17

Background: Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron ( LI. LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested. Results: The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb( 1 2). This mutant had a mobility frequency comparable to the wild-type LI. LtrB intron (DeltaORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II ( nts 404 459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent. Conclusions: The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb( 1 2) and DII( ii) a, b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.