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Alan M. Lambowitz – One of the best experts on this subject based on the ideXlab platform.

  • Biotechnological applications of mobile group II introns and their reverse Transcriptases: Gene targeting, RNA-seq, and non-coding RNA analysis
    Mobile DNA, 2014
    Co-Authors: Peter J. Enyeart, Georg Mohr, Andrew D Ellington, Alan M. Lambowitz
    Abstract:

    Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse Transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse Transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse Transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse Transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

  • Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
    Journal of Molecular Biology, 1999
    Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. Lambowitz
    Abstract:

    Group II introns encode reverse Transcriptases that function in both intron mobility and RNA splicing. The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription. Here, immunoblotting and UV cross-linking experiments show that the reverse Transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62). p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse Transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion. Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse Transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing. Although the reverse Transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided. Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer. Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3 0 exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1. Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62. # 1999 Academic Press

  • Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
    Journal of molecular biology, 1999
    Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. Lambowitz
    Abstract:

    Group II introns encode reverse Transcriptases that function in both intron mobility and RNA splicing. The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription. Here, immunoblotting and UV cross-linking experiments show that the reverse Transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62). p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse Transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion. Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse Transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing. Although the reverse Transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided. Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer. Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3′ exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1. Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62.

Amnon Hizi – One of the best experts on this subject based on the ideXlab platform.

  • Mode of inhibition of HIV reverse Transcriptase by 2-hexaprenylhydroquinone, a novel general inhibitor of RNA-and DNA-directed DNA polymerases.
    Biochemical Journal, 1997
    Co-Authors: Shoshana Loya, Amira Rudi, Yoel Kashman, Amnon Hizi
    Abstract:

    A natural compound from the Red Sea sponge Ircinia sp., 2hexaprenylhydroquinone (HPH), has been shown to be a general inhibitor of retroviral reverse Transcriptases (from HIV-1, HIV-2 and murine leukaemia virus) as well as of cellular DNA polymerases (Escherichia coli DNA polypolymerase I, and DNA polymerases a and b). The pattern of inhibition was found to be similar for all DNA polymerases tested. Thus the mode of inhibition was studied in detail for HIV-1 reverse Transcriptase. HPH is a non-competitive inhiinhibitor and binds the enzyme irreversibly with high anity (K i fl 0.62 lM). The polar hydroxy groups have been shown to be of key importance. A methylated derivative, mHPH, which is devoid of these polar moieties, showed a significantly decreased capacity to inhibit all DNA polymerases tested. Like the natural product, mHPH binds the

  • Expression and mutational analysis of the reverse Transcriptase of the lentivirus equine infectious anemia virus.
    Biochemical and Biophysical Research Communications, 1993
    Co-Authors: M. Shaharabany, N.r. Rice, Amnon Hizi
    Abstract:

    The reverse Transcriptase of equine infectious anemia virus (EIAV) shows sequence similarity with the reverse Transcriptases of other lentiviruses, particularly with those of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2). We have constructed a plasmid that when introduced into E. coli induces the synthesis of substantial quantities of the nearly authentic EIAV reverse Transcriptase. The viral and bacterially expressed reverse Transcriptases are similar in their molecular weights. The bacterial expression clone was used to generate deletion mutants of the protein. Mutations in both amino and carboxyl terminal regions of the polypeptide strongly affect the DNA polypolymerase activity of the enzyme. Thus, EIAV reverse Transcriptase resembles the reverse Transcriptases of HIV-1 and HIV-2 and can serve as a suitable enzyme for studying the structure-function relationship in lentiviral reverse Transcriptase.

Steven Zimmerly – One of the best experts on this subject based on the ideXlab platform.

  • Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
    Journal of Molecular Biology, 1999
    Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. Lambowitz
    Abstract:

    Group II introns encode reverse Transcriptases that function in both intron mobility and RNA splicing. The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription. Here, immunoblotting and UV cross-linking experiments show that the reverse Transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62). p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse Transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion. Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse Transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing. Although the reverse Transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided. Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer. Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3 0 exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1. Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62. # 1999 Academic Press

  • Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
    Journal of molecular biology, 1999
    Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. Lambowitz
    Abstract:

    Group II introns encode reverse Transcriptases that function in both intron mobility and RNA splicing. The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription. Here, immunoblotting and UV cross-linking experiments show that the reverse Transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62). p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse Transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion. Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse Transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing. Although the reverse Transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided. Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer. Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3′ exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1. Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62.

  • efficient integration of an intron rna into double stranded dna by reverse splicing
    Nature, 1996
    Co-Authors: Jian Yang, Philip S. Perlman, Steven Zimmerly, Alan M. Lambowitz
    Abstract:

    SOME group II introns are mobile elements as well as catalytic RNAs1,2. Introns aI1 and aI2 found in the gene COX1 in yeast mitochondria encode reverse Transcriptases which promote site-specific insertion of the intron into intronless alleles (‘homing’)3–6. For aI2 this predominantly occurs by reverse transcription of unspliced precursor RNA at a break in double-strand DNA made by an endonuclease encoded by the intron7. The aI2 endonuclease involves both the excised intron RNA, which cleaves the DNA’s sense strand by partial reverse splicing; and the intron-encoded reverse Transcriptase which cleaves the antisense strand8. Here we show that aI1 encodes an analogous endonuclease specific for a different target site compatible with the different exon-binding sequences of the intron RNA. Over half of aI1 undergoes complete reverse splicing in vitro, thus integrating linear intron RNA directly into the DNA. This unprecedented reaction has implications for both intron mobility and evolution, and potential genetic engineering applications.