An Evolutionary and Biochemical Characterization of a Self-splicing Group II Intron and Its Encoded LAGLIDADG Homing Endonuclease in Leptographium Truncatum - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook An Evolutionary and Biochemical Characterization of a Self-splicing Group II Intron and Its Encoded LAGLIDADG Homing Endonuclease in Leptographium Truncatum write by Sahra-Taylor Mullineux. This book was released on 2010. An Evolutionary and Biochemical Characterization of a Self-splicing Group II Intron and Its Encoded LAGLIDADG Homing Endonuclease in Leptographium Truncatum available in PDF, EPUB and Kindle. Evolutionary relationships amongst strains of the fungal genus Leptographium and related taxa were inferred using the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat. To generate robust sequence alignments for phylogenetic analysis the relationship between DNA sequence variability and RNA structural conservation of ITS segments was examined. The results demonstrate that structural conservation of helical regions is facilitated by compensatory base changes, compensating insertions/deletions, and, possibly, RNA strand slippage. A high mol % G+C bias for ITS1 and ITS2 and structural constraints at the RNA level appear to limit the types of changes observed. Fifty strains of Leptographium were screened for the presence of introns within mitochondrial genes. Superimposing intron survey data onto the ITS-derived phylogenetic tree reveals that introns are absent from the small ribosomal RNA (rns) gene of all strains of L. procerum yet are found in all strains of L. lundbergii. Amongst members of L. wingfieldii, L. terebrantis, and L. truncatum intron distribution is stochastic and is not correlated to the evolutionary relationships amongst strains. A group II intron/LAGLIDADG homing endonuclease gene (HEG) composite element from the mt rns gene of L. truncatum strain CBS929.85 was characterized. Intron-catalyzed splicing was tested using ORF-less and ORF-containing precursor transcripts, and both versions of the intron readily self-splice under moderate temperature and ionic conditions (37 °C and 6 mM MgCl2). Cleavage activity of the intron-encoded protein (I-LtrII) was tested using an N-terminal His6-tagged and near native protein. The homing endonuclease cleaves double-stranded DNA 2 nucleotides upstream of the intron insertion site within the exon, generating 4 nucleotide 3' OH overhangs. Intron splicing is not enhanced by the addition of I-LtrII and RNA-binding assays indicate that the His6-tagged protein does not bind to the intron. Phylogenetic relationships amongst the rns gene, intron, and amino acid sequences were inferred. An evolutionary model of the composite element is proposed in which the HEG invaded a group II intron and mobilized it. The mobile genetic element may be transmitted vertically amongst L. lundbergii strains and horizontally through lateral gene transfer amongst strains of L. wingfieldii, L. terebrantis, and L. truncatum.
Biochemical Characterization of Homing Endonucleases Encoded by Fungal Mitochondrial Genomes
Biochemical Characterization of Homing Endonucleases Encoded by Fungal Mitochondrial Genomes - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Biochemical Characterization of Homing Endonucleases Encoded by Fungal Mitochondrial Genomes write by Tuhin Guha. This book was released on 2014. Biochemical Characterization of Homing Endonucleases Encoded by Fungal Mitochondrial Genomes available in PDF, EPUB and Kindle. The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential 'on/off switch' for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies.
