Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo

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 Ll.LtrB intron (ΔORF 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.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.Group II introns are catalytic RNAs that are also mobile genetic elements. Although their primary sequences vary considerably, the intron RNAs fold into well-conserved structures (Figure 1). The generalized group II intron secondary structure consists of six helical domains emerging from a central wheel [1,2]. Some group II introns are capable of self-splicing; however for efficient splicing in vivo, the intron-encoded ORF or host-encoded splicing factors are required [3]. Mobile group II introns encode a reverse transcriptase/maturase, with the coding region generally located in domain IV.Group II introns were first identified in fungal mitochondria and grouped on the basis of their secondary structures [2,4,5]. They have been found in organelle genomes of fungi and plants and also in a number of bacteria [6]. None have been found in animals, although it has been suggested that group II intr