Browsing by Subject "Group II introns"
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Item The C-terminal DNA endonuclease region and biotechnology applications of a group II intron reverse transcriptase from Thermosynechoccus elongatus(2011-08) Smith, Whitney Gail; Lambowitz, Alan; Stevens, ScottGroup II introns insert site-specifically into DNA target sites through a process termed retrohoming. They consist of a structured, catalytically active intron RNA and its encoded protein. The protein contains several domains, including a reverse transcriptase domain and a DNA endonuclease domain used for bottom-strand cleavage. Recently, the thermophile Thermosynechococcus elongatus BP-1 was found to contain eight functional group II intron-encoded proteins. The proteins are thermostable and active at temperatures up to 65°C. The intron-encoded protein, TeI4c displays the greatest reverse transcriptase activity of these eight proteins, as well as high fidelity and processivity; ideal qualities for a commercial reverse transcriptase. This work explores the possibility of using TeI4c for biotechnology applications, and specifically examines the C-terminal endonuclease domain of TeI4c and its effect on reverse transcription. Additionally, this work investigates the retrohoming activity of a TeI4c truncation that deletes the endonuclease domain.Item DNA target site recognition and toward gene targeting in mammalian cells by the Ll.LtrB group II intron RNP(2013-05) Hanson, Joseph Haskell; Lambowitz, AlanMobile group II introns insert site-specifically into DNA target sites through a mechanism ("retrohoming") that involves reverse splicing of the intron RNA into the DNA and its subsequent reverse transcription by an intron-encoded protein (IEP) that is associated with the RNA in a ribonucleoprotein (RNP) complex. Characterization of this RNP complex and its retrohoming activities have enabled the development of programmable mobile group II intron gene targeting vectors routinely used in prokaryotic organisms. Building upon recent research by our lab to develop gene targeting in Xenopus laevis and Drosophila melanogaster using the group II intron Ll.LtrB from Lactococcus lactis, I describe work to extend this system to mammalian cells. I demonstrate that group II intron RNPs can be delivered to mammalian cells efficiently and produced in vivo via a CMV/T7 hybrid expression system. Using a robust single-strand annealing assay to detect homologous recombination induced by double-strand breaks (DSBs), I found that group II intron-mediated DSBs are efficiently repaired by mammalian cells. Despite varied approaches, I failed to detect endogenous group II intron-mediated gene targeting in human and mouse cells in culture. Gene expression microarray analysis and in vivo imaging of RNP molecules indicated that group II intron RNPs are sequestered away from the genome and induce host innate immune responses. I also investigated how the C-terminal DNA-binding domain of the Ll.LtrB IEP contributes to DNA target site recognition. Building upon previous mass spectrophotometric analysis of site-specific UV-crosslinking, I used genetic and biochemical analyses to identify potential protein contacts for key target site residues T-23 and T+5. Genetic selection of mutants in a region contacting T+5 led to identification of LtrA variants with increased retrohoming efficiency. My results provide evidence that the DNA-binding domain of a group II intron reverse transcriptase functions in DNA target site recognition and suggest new methods for changing its DNA target specificity and targeting efficiency.Item DNA target site recognition by the Ll.LtrB group II intron RNP(2011-08) Whitt, Jacob Tinsley; Lambowitz, Alan; Browning, Karen S.; Iverson, Brent L.; Paull, Tanya T.; Yin, WhitneyMobile group II introns are retroelements that site-specifically insert into DNA target sequences. The group II intron mobility pathway is mediated by a ribonucleoprotein particle (RNP) composed of excised intron RNA and an intron-encoded protein (IEP). The intron lariat inserts at a specific DNA target sequence and is then reverse transcribed by the IEP. Both the intron RNA and IEP are required for DNA target site recognition. I have identified the contact sites within the IEP responsible for recognition of two key positions in the DNA target, T+5 and T-23. IEP recognition of T+5 in the 3'-exon is required for endonuclease cleavage of the bottom-strand of the DNA target site, which generates a primer used for initiation of reverse transcription of the intron. The T+5 base is contacted by G498 in the LtrA DNA-binding domain and nearby residues, particularly K499, potentially bolster this interaction. Recognition of T-23 in the distal 5'-exon is required for initial recognition of the DNA target site by the RNP. The T533 side-chain contacts the T-23 base and the L534 side-chain may also contribute to recognition through hydrophobic interactions with the C5 methyl group. A mutant, L534H, that switches target site specificity to T-23G has been characterized. In order for the RNP to make these and other contacts in the 5'- and 3'-exons simultaneously, the DNA must be bent. I have dissected the role of DNA bending in the intron mobility pathway and found that the DNA is bent at two progressively larger angles as the reaction proceeds. The predominant bend angle at earlier time points places the bottom-strand DNA cleavage site at the protein endonuclease active site. The predominant bend angle of later time points places the cleaved DNA site at the RT domain active site for initiation of reverse transcription of intron cDNA. Finally, in a practical application of group II intron mobility, I have used reprogrammed group II introns ("targetrons") to target two genes in Bacillus subtilis to demonstrate the suitability of targetron technology for gene targeting in the Gram-positive Bacillus genus.Item Group II intron and gene targeting reactions in Drosophila melanogaster(2011-08) White, Travis Brandon; Lambowitz, Alan; Bull, James J.; Macdonald, Paul M.; Paull, Tanya T.; Stevens, Scott W.Mobile group II introns are retroelements that insert site-specifically into double-stranded DNA sites by a process called retrohoming. Retrohoming activity rests in a ribonucleoprotein (RNP) complex that contains an intron-encoded protein (IEP) and the excised intron RNA. The intron RNA uses its ribozyme activity to reverse splice into the top strand of the DNA target site, while the IEP cleaves the bottom DNA strand and reverse transcribes the inserted intron. My dissertation focuses on the Lactococcus lactis Ll.LtrB group II intron and its IEP, denoted LtrA. First, I investigated the ability of microinjected Ll.LtrB RNPs to retrohome into plasmid target sites in Drosophila melanogaster precellular blastoderm stage embryos. I found that injection of extra Mg2+ into the embryo was crucial for efficient retrohoming. Next, I compared retrohoming of linear and lariat forms of the intron RNP. Unlike lariat RNPs, retrohoming products of linear intron RNPs displayed heterogeneity at the 5’-intron insertion junction, including 5’-exon resection, intron truncation, and/or repair at regions of microhomology. To investigate whether these junctions result from cDNA ligation by non-homologous end-joining (NHEJ), I analyzed retrohoming of linear and lariat intron RNPs in D. melanogaster embryos with null mutations in the NHEJ genes lig4 and ku70, as well as the DNA repair polymerase polQ. I found that null mutations in each gene decreased retrohoming of linear compared to lariat intron RNPs. To determine whether novel activities of the LtrA protein contributed to the linear intron retrohoming 5’ junctions, I assayed the polymerase, non-templated nucleotide addition and template-switching activities of LtrA on oligonucleotide substrates mimicking the 5’-intron insertion junction in vitro. Although LtrA efficiently template switched to 5’-exon DNA substrates, the junctions produced differed from those observed in vivo, indicating that template switching is not a significant alternative to NHEJ in vivo. Finally, I designed and constructed retargeted Ll.LtrB RNPs to site-specifically insert into endogenous chromosomal DNA sites in D. melanogaster. I obtained intron integration efficiencies into chromosomal targets up to 0.4% in embryos and 0.021% in adult flies. These studies expand the utility of group II intron RNPs as gene targeting tools in model eukaryotic organisms.Item Toward group II intron-based genome targeting in eukaryotic cells(2009-12) Vernon, Jamie Lee; Lambowitz, AlanMobile group II introns consist of a self-splicing RNA molecule and an intron-encoded protein with reverse transcriptase activity that function together in an RNP and catalyze the insertion of the intron into specific DNA target sites by a process known as retrohoming. The mechanism of insertion requires the intron RNA to bind and reverse splice into one strand of the DNA target site, while the intron-associated protein cleaves the opposite DNA strand and reverse transcribes the intron RNA. DNA target site recognition and binding are dependent upon base pairing between the intron RNA and the target DNA molecule. By modifying the recognition sequences in the intron RNA, group II introns can be engineered to insert into virtually any desired target DNA. Based on this technology, a novel class of commercially available group II intron-based gene targeting vectors, called targetrons, has been developed. Targetrons have been used successfully for gene targeting in a broad range of bacteria. Previously, our laboratory demonstrated that group II introns retain controllable retrohoming activity in mammalian cells, albeit with very low targeting efficiency. However, the gene targeting capability of group II introns is not limited to direct insertion of the intron. Group II introns can also create double-strand breaks that stimulate homologous recombination. By virtue of these attributes, mobile group II introns offer great promise for applications in genetic engineering, functional genomics and gene therapy. Here I present the results of experiments in which I tested group II introns for gene targeting activities in eukaryotic cells. First, I demonstrated that group II introns injected into zebrafish (Danio rerio) embryos retain in vivo plasmid targeting activity that is enhanced by the addition of magnesium chloride and deoxynucleotides. I also verified that similar in vivo targeting activity is retained in Drosophila melanogaster embryos. Further, I describe repeated experiments in zebrafish embryos designed to target the zebrafish genome with inconclusive results. Group II introns were also delivered to cultured human cells for genome targeting. Here I present promising evidence for the ability of group II introns to stimulate homologous recombination between an exogenously introduced donor DNA molecule and the chromosome. The donor DNA was delivered either as a linearized double-stranded plasmid by electroporation or as a single stranded genome of a recombinant adeno-associated virus (AAV). In both cases, cells receiving both the group II intron RNP and the donor DNA showed more efficient integration of the donor DNA than introduction of the donor DNA alone. The studies presented here provide insight into the potential of using group II introns for future applications in gene targeting in eukaryotes.