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Alan M. Lambowitz - One of the best experts on this subject based on the ideXlab platform.
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Biotechnological applications of mobile group II introns and their reverse Transcriptases: Gene targeting, RNA-seq, and non-coding RNA analysis
Mobile DNA, 2014Co-Authors: Peter J. Enyeart, Georg Mohr, Andrew D Ellington, Alan M. LambowitzAbstract: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.
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Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
Journal of Molecular Biology, 1999Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. LambowitzAbstract: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
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Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
Journal of molecular biology, 1999Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. LambowitzAbstract: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.
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efficient integration of an intron rna into double stranded dna by reverse splicing
Nature, 1996Co-Authors: Jian Yang, Philip S. Perlman, Steven Zimmerly, Alan M. LambowitzAbstract: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.
Amnon Hizi - One of the best experts on this subject based on the ideXlab platform.
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Mode of inhibition of HIV reverse Transcriptase by 2-hexaprenylhydroquinone, a novel general inhibitor of RNA-and DNA-directed DNA polymerases.
Biochemical Journal, 1997Co-Authors: Shoshana Loya, Amira Rudi, Yoel Kashman, Amnon HiziAbstract: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 polymerase 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 inhibitor 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
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Expression and mutational analysis of the reverse Transcriptase of the lentivirus equine infectious anemia virus.
Biochemical and Biophysical Research Communications, 1993Co-Authors: M. Shaharabany, N.r. Rice, Amnon HiziAbstract: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 polymerase 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.
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Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
Journal of Molecular Biology, 1999Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. LambowitzAbstract: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
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Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
Journal of molecular biology, 1999Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. LambowitzAbstract: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.
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efficient integration of an intron rna into double stranded dna by reverse splicing
Nature, 1996Co-Authors: Jian Yang, Philip S. Perlman, Steven Zimmerly, Alan M. LambowitzAbstract: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.
Philip S. Perlman - One of the best experts on this subject based on the ideXlab platform.
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Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
Journal of Molecular Biology, 1999Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. LambowitzAbstract: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
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Group II intron reverse Transcriptase in yeast mitochondria. Stabilization and regulation of reverse Transcriptase activity by the intron RNA.
Journal of molecular biology, 1999Co-Authors: Steven Zimmerly, John V. Moran, Philip S. Perlman, Alan M. LambowitzAbstract: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.
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efficient integration of an intron rna into double stranded dna by reverse splicing
Nature, 1996Co-Authors: Jian Yang, Philip S. Perlman, Steven Zimmerly, Alan M. LambowitzAbstract: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.
Lawrence A. Loeb - One of the best experts on this subject based on the ideXlab platform.
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A screen in Escherichia coli for nucleoside analogs that target human immunodeficiency virus (HIV) reverse Transcriptase: coexpression of HIV reverse Transcriptase and herpes simplex virus thymidine kinase.
Journal of Virology, 1995Co-Authors: Baek Kim, Lawrence A. LoebAbstract:Human immunodeficiency virus (HIV) reverse Transcriptase substitutes for temperature-sensitive DNA polymerase I (Pol Its) in Escherichia coli, providing a screen for anti-HIV reverse Transcriptase nucleoside analogs in bacteria. Since phosphorylation of nucleosides in E. coli is limited to thymidine and its derivatives, we coexpressed herpes simplex virus thymidine kinase, an enzyme that phosphorylates a wide variety of nucleoside analogs, together with HIV reverse Transcriptase. Coexpression of herpes simplex virus thymidine kinase and HIV reverse Transcriptase rendered Pol Its cells sensitive to dideoxycytidine. Studies with different nucleoside analogs indicate that this bacterial screening system is able to select and identify nucleoside analogs that specifically target HIV reverse Transcriptase.
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Fidelity of HIV-1 reverse Transcriptase copying RNA in vitro.
Biochemistry, 1992Co-Authors: Lawrence A. LoebAbstract:The genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse Transcriptase. We developed an assay for reverse Transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro. A lacZ alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription. The corresponding DNA template was used to examine errors by reverse Transcriptase during second-strand DNA synthesis. With both templates, the mutations introduced by reverse Transcriptase were identified by their mutant phenotypes in an M13 lacZ alpha-complementation assay. We found that the reverse Transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse Transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates. The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template. The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28,000 and 37,000, respectively. The most frequent mutations produced by HIV-1 RT copying the RNA template were C----T transitions and G----T transversions resulting from misincorporation of dAMP.
