Analysis of fungal inteins

Abstract

Homing endonucleases are highly specific DNases, commonly called HEGs, encoded by self-splicing intein and intron sequences. They recognise long target sequences and confer mobility to their host sequence by introducing a double-stranded DNA break in a cognate allele lacking the element. During the repair process, the element harbouring the endonuclease along with some of its flanking sequences is duplicated through homologous recombination, a process which is referred to as 'homing'. Intein homing leads to super-Mendelian inheritance of the intein distorting the expected 1:1 inheritance ratio. Because of their unique ability to rapidly increase in frequency, an intein life-cycle that follows a cyclical mode of invasion, fixation, degeneration and loss accompanied by frequent horizontal transfer has been proposed. An alternative model suggests that an empty allele can persist in equilibrium with an intein that carries an active HEG. Although some homing endonucleases have been extensively studied in vitro, the activity and specificity of many remain unknown. To investigate the ability of homing endonucleases to recognise, cut and invade target sequences, fungal HEG-carrying inteins were selected for in vivo and in vitro analyses. Natural Botrytis cinerea strains carrying either an intein-encoded homing endonuclease in their Prp8 gene or an empty allele were isolated. Mating experiments demonstrated super-Mendelian inheritance of the B. cinerea intein, all progeny inheriting the element. During intein homing or horizontal transfer, sequences flanking an intein element are transferred to the recipient. Further analysis of the sequences flanking the PRP8 intein elements and/or the expected intein insertion site in other Botrytis species, closely related genera and representative species from the filamentous ascomycetes suggested the following; i) the PRP8 intein in Botrytis has been vertically transmitted from a polymorphic ancestor with no evidence of horizontal transmission, ii) either model of intein life-cycle can be suggested with regards to the PRP8 intein in mycelial fungi, and iii) regardless of the intein life-cycle, there have been several independent events of intein loss. There are also two distinct types of VMA1 inteins in Saccharomyces cerevisiae (referred to as cVMA and mVMA in this work). Previous studies have demonstrated HEG activity and homing of the cVMA intein but not the mVMA intein. In this work the HEG activity of both intein types was illustrated on two different target sequences, one of which demonstrated a much reduced ability to be invaded by both inteins. To analyse HEG activity and homing of inteins that are not easily analysed in their native hosts, an intein-based knock-out knock-in system was developed in S. cerevisiae. This transgenic system allowed the insertion of the Aspergillus fumigatus PRP8 intein (AfuPRP8) and a putative target sequence into the Prp8 gene of S. cerevisiae. Preliminary analyses from mating experiments with such transgenic strains suggest that the AfuPRP8 HEG may introduce a double-stranded DNA break which is repaired through gene conversion. In addition to the intein and HEG studies, a bacterial in vivo splicing assay was developed using the Cryptococcus neoformans PRP8 mini-intein. Intein splicing could quantitatively be measured as the growth rate on minimal media containing lactose as the sole carbon source. As intein splicing inhibitors have been suggested as drugs in therapies of pathogenic fungi such an assay can be useful in large scale screening for intein splicing inhibitors.