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Results for “RNA-Directed DNA Polymerase”

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At least 19 recordsLinked to original sources
PubMed1975

Serological analysis of human deoxyribonucleic acid polymerases. Preparation and properties of antiserum to deoxyribonucleic acid polymerase I from human lymphoid cells.

The preparation and properties of an antiserum to human DNA polymerase I (6 to 8 S) are described. Care was taken in the purification of the antigen to remove certain other DNA polymerases found in human cells. An incubation of antigen and antiserum lasting about 48 hours is necessary to achieve maximal inhibition. About 1 mug of the antipolymerase immunoglobulin G, prepared in rats, neutralizes 60% of the activity present in 54 ng of the enzyme. Tritrations varying both antiserum and enzyme demonstrate clear regions of antigen and antibody excess. Inhibition of enzyme activity is about the same whether the templateprimer is (dA)n-(dT)12-18, or partially digested DNA. An assay was developed which measures the remaining activity in the supernatant after precipitation of enzyme-antibody complexes with goat anti-rat immunoglobulin G. In this assay, 2.2 mug of the antipolymerase immunoglobulin G quantitatively bind 33 ng of DNA polymerase I. With use of the direct neutralization assay and the immuno-precipitation test, we found little, if any, antigenic relationship between DNA polymerase I and DNA polymerase II (3.4 S). Similarly, little, if any, relationship was found to the DNA polymerases from five RNA tumor viruses. The activities of RNA-directed DNA polymerases from the blood leukocytes of two patients with acute myelogenous leukemia and from the placentas of rhesus monkeys were not inhibited in neutralization assays which were shortened because these enzymes were thermolabile. In identically shortened neutralization assays, the antipolymerase immunoglobulin G neutralized up to 76% of the activity of DNA polymerase I. In addition to its utility in distinguishing cellular DNA polymerases, the rat antiserum should be useful reagent for testing of novel DNA polymerases isolated in small quantities from human tumors for contamination with DNA polymerase I. This enzyme is present in abundance in proliferating tissue and often confuses the biochemical characterization of these novel enzymes.

Animals↗
PubMed2026

Functional minigenome system reveals polymerase features of swine orthopneumovirus.

Swine orthopneumovirus (SOV), a recently identified porcine pneumovirus, has been detected in pig farms worldwide; however, its pathogenicity and molecular biology remain poorly understood. To facilitate the study of SOV replication and transcription, we developed a functional minigenome system based on consensus sequences from multiple strains of SOV and related pneumoviruses. Here, we constructed and optimized this system in BSRT7/5 cells, revealing that the RNA-dependent RNA polymerase (RdRp) activity depends on a conserved protein phosphatase 1 (PP1) binding site within the phosphoprotein P, as a single F131A substitution markedly reduced polymerase function. Additionally, we identified and characterized the M2-1 binding site on P, which is essential for viral transcription. These findings provide new insights into SOV polymerase complex requirements and establish a foundation for reverse genetics approaches to rescue infectious viruses, advancing our understanding of SOV biology and its potential role in porcine respiratory disease.IMPORTANCERecently, a newly identified porcine pneumovirus, swine orthopneumovirus (SOV), was detected in pig farms in different countries. Although detected mainly in sick animals, this virus has not been isolated yet and its pathogenicity remains to be determined. We started by setting up a minigenome system with a view to develop reverse genetics and rescue infectious virions. This minigenome system was used to study the functioning of the SOV RNA polymerase and compared it with RSV. Although some similarities exist between SOV and RSV, the RdRp of RSV cannot rescue the SOV minigenome. SOV seems to belong to another genus/genogroup of pneumoviruses, which includes PVM and the canine pneumovirus. Our functional minigenome paves the way for reverse genetics of SOV and determination of its pathogenicity in different host species.

Swine Diseases↗
PubMed2026

Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing.

CRISPR-based prime editors (PEs) install precise edits into genomic DNA without generating double-strand breaks. Their editing efficiency is highly dependent on reverse transcriptases (RTs), but efficient RT candidates remain limited. Here, we identified 19 novel active RTs by screening 558 candidates. Among them, RERV-RT, derived from Rattus norvegicus, exhibited the highest activity. Through structure-guided engineering and deep mutational scanning, we developed an optimized variant, enRERV-RT, which outperforms conventional M-MLV-RT-based PE systems by 1.20-fold in mammalian and plant cells, and by 1.88-fold at hard-to-edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high-throughput platform, TRAP-seq-PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV-RT exhibited higher editing efficiencies than those based on M-MLV-RT. Collectively, our work establishes a versatile, high-efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.

Animals↗
PubMed2025

Enzymes in high-throughput RNA sequencing: Applications and challenges.

High-throughput RNA sequencing provides genome-wide information on the dynamics of RNA in each cell and how the dynamics responds to environmental changes. Next-generation sequencing by the Illumina platform currently provides the highest information output as compared to other platforms. A key component of next generation sequencing of each RNA is the successful end-to-end reverse-transcription into a cDNA strand. This can be highly challenging given the propensity of each RNA to adopt ordered structures and to contain post-transcriptional modifications. While many reverse transcriptase (RT) enzymes have been developed over the years to maximize read-through of an RNA, their processivity and efficiency varies, raising the question of how to select the RT for the experiment at hand. Here, we use tRNA as a model for genome-wide sequencing, as tRNA has a stable secondary and tertiary structure and has a high density and wide variety of post-transcriptional modifications, presenting one of the most challenging problems of sequencing RNA. We compare the efficiency of end-to-end cDNA synthesis of tRNA among several recent RT enzymes and provide a general sequencing workflow that is applicable to most of these enzymes.

High-Throughput Nucleotide Sequencing↗
PubMed2025

MapID-based quantitative mapping of chemical modifications and expression of human transfer RNA.

Detection and quantification of tRNA chemical modifications are critical for understanding their regulatory functions in biology and diseases. However, tRNA-seq-based methods for modification mapping encountered challenges both experimentally (poor processivity of heavily modified tRNAs during reverse transcription or RT) and bioinformatically (frequent reads misalignment to highly similar tRNA genes). Here, we report "MapID-tRNA-seq" where we deployed an evolved reverse transcriptase (RT-1306) into tRNA-seq and developed "MapIDs" that reduce redundancy of the human tRNA genome and explicitly annotate genetic variances. RT-1306 generated robust mutations against m1A and m3C, and RT stops against multiple bulky roadblock modifications. MapID-assisted data processing enabled systematic exclusion of false-positive discoveries of modifications which arise from reads misalignment onto similar genes. We applied MapID-tRNA-seq into mapping m1A, m3C and expression levels of tRNAs in three mammary cell lines, which revealed cell-type dependent modification sites and potential translational regulation of the reduced mitochondrial activities in breast cancer.

Humans↗
PubMed1975

Type C RNA tumor virus isolated from cultured human acute myelogenous leukemia cells.

Previously, type C RNA tumor virus-related components have been described in blood leukocytes from patients with acute myelogenous leukemia. These components, for example, reverse transcriptase, have been shown to be most closely related to those from two oncogenic subhuman primate type C viruses (woolly monkey sarcoma virus and gibbon ape leukemia virus). Now, we report the continuous production of budding type C viruses with the same characteristic reverse transcriptase by three separate culturings of leukocytes from a single bleeding from a patient with acute myelogenous leukemia. These isolations were made possible by the discovery of a source of conditioned media which sustains exponential growth of human myelogenous leukemia cells in liquid suspension culture.

Cell Differentiation↗
PubMed2025

Disassembly activates Retron-Septu for antiphage defense.

Retrons are antiphage defense systems that produce multicopy single-stranded DNA (msDNA) and hold promise for genome engineering. However, the mechanisms of defense remain unclear. The Retron-Septu system integrates retron and Septu antiphage defenses. Cryo-electron microscopy structures reveal asymmetric nucleoprotein complexes comprising a reverse transcriptase, msDNA (a hybrid of msdDNA and msrRNA), and two PtuAB copies. msdDNA and msrRNA are essential for assembling this complex, with msrRNA adopting a conserved lariat-like structure that regulates reverse transcription. Notably, the assembled Retron-Septu complex is inactive, with msdDNA occupying the PtuA DNA binding site. Activation occurs upon disassembly, releasing PtuAB, which degrades single-stranded DNA to restrict phage replication. This "arrest-and-release" mechanism underscores the dynamic regulatory roles of msDNA, advancing our understanding of antiphage defense strategies.

Cryoelectron Microscopy↗
PubMed2026

A UG5 reverse transcriptase-nitrilase antiviral module confers phage immunity in the plant symbiont Sinorhizobium meliloti.

Bacteriophages exert strong selective pressure on soil- and rhizosphere-associated bacteria, including plant-associated symbionts. Reverse transcriptase-associated defense systems of the UG family are widespread across bacterial lineages, yet their ecological roles remain largely undefined. Within this family, UG5 systems are distinguished by reverse transcriptases fused to or associated with a nitrilase domain. Here, we combine phylogenetic, metagenomic, and functional analyses to investigate the evolutionary context and antiviral activity of UG5-associated systems. Phylogenetic analysis of 728 nitrilase domains places UG5-associated nitrilases within a well-supported UG-related radiation encompassing the UG1, UG5, and UG6 families, with UG1 nested within a broader UG5 lineage. Metagenomic analysis further revealed UG5-associated reverse transcriptases in soil- and rhizosphere-derived metagenomes. Based on this observation, we characterized a UG5-large reverse transcriptase (RT)-associated system, here designated DRT11, encoded on the pSymA megaplasmid of Sinorhizobium meliloti RMO17, a nitrogen-fixing symbiont of Medicago sativa. Despite lacking the transmembrane protein typical of canonical UG5-large architectures, DRT11 confers protection against naturally occurring M. sativa rhizosphere phages with podovirus-like morphology. Phage infection assays reveal protection at low multiplicities of infection, consistent with an abortive-infection-like mechanism. Moreover, mutational analyses demonstrate that antiviral activity requires only the RT and its fused C-terminal nitrilase domain, establishing DRT11 as a minimal UG5-associated antiviral system.IMPORTANCEIn this study, we report the functional characterization of a UG5-large reverse transcriptase-associated defense system (DRT11) encoded on the pSymA megaplasmid of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Using a combination of phylogenetic, metagenomic, genomic, and experimental approaches, we demonstrate that DRT11 functions as a bona fide antiviral defense module, providing protection against naturally occurring rhizosphere phages through a minimal reverse transcriptase-nitrilase architecture. This work establishes direct functional evidence for antiviral activity within the UG5 family and clarifies the evolutionary placement of UG5-associated systems within the broader UG radiation.

Phylogeny↗
PubMed2025

New retron systems from environmental bacteria identify triggers of anti-phage defense and expand tools for genome editing.

Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase (RT), a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase (RT), noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we identify seven new retron systems, each isolated from a different host bacterium. We characterize DNA production by these retrons and test their ability to defend against a panel of Escherichia coli phages. We find that two of these retrons are disrupted by other elements, in one case a group II intron and in another a separate defense system, yet both retrons still produce RT-DNA. For two other retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing in E. coli, demonstrating their potential use in a biotechnological application.

Gene Editing↗
PubMed1975

Different hematological diseases induced by type C viruses chemically activated from embryo cells of different mouse strains.

Type C RNA viruses can be induced by certain chemicals from cells of many mouse strains. Both C58 and BALB/c cells have been shown to contain endogenous viruses that are designated N-tropic because they grow preferentially in cells of NIH Swiss mouse origin. While demonstrating many similar biological and immunological properties, the C58-induced virus is around 10-fold more infectious per physical particle than the N-tropic virus of BALB/c cells. In the present studies, inoculation of these viruses into newborn NIH Swiss mice led to the development of diseases associated with splenomegaly and lymphadenopathy at similar frequency in each group. The disease induced by C58-MuLV was histophathologically diagnosed as lymphoblastic leukemia and was highly malignant following transplantation into newborn mice. The histopathological appearance of spleens from BALB/c virus-affected animals was distinguishable, demonstrating instead myeloid metaplasia or myelogenous leukemia. These findings provide evidence that different endogenous mouse type C viruses can induce distinct diseases in the same mouse strain. Furthermore, they implicate the N-tropic virus endogenous to C58 cells as a major factor in the development of lymphoblastic leukemia that occurs at high frequency in that strain.

Animals↗
PubMed1975

Effect of glucocorticoids on activation of leukemia virus in AKR mouse embryo cells.

The effect of glucocorticoids on activation and replication of leukemia virus in AKR mouse embryo cells was analyzed. The number of cells detected as positive by fluorescent antibody techniques as well as the virus production in cells chronically producing virus was doubled at optimal concentrations of glucocorticoids. The effect of the hormones in activated cells was found to be not on the process of activation per se but rather on synthesis of the viral components after activation has occurred. Intracellular reverse transcriptase levels were not changed by hormone treatment. The stimulation of virus synthesis by glucocorticoids requires binding of the steroid to a cytoplasmic receptor protein.

Animals↗
PubMedDate not supplied

Search for C-type particles in human neoplasia.

The salient biologic and morphologic characteristics of RNA tumor (oncornavirus) virus are reviewed. The ultrastructure of replicating oncornaviruses is illustrated in detail. C-type particles wide spread in at least three orders of animals were sighted in human sarcomas and leukemias. One case, an infantile fibrosarcoma, is presented from our cases surveyed for the presence of C-type particles. Tissue cultures derived from this tumor contained viral particles and had an elevated reverse transcriptase activity associated with the presence of 70 S RNA. The particles were larger (125 to 150nm) than those of the murine or avian Type C particles.

Animals↗
PubMed2024

PTGS is dispensable for the initiation of epigenetic silencing of an active transposon in Arabidopsis.

Transposable elements (TEs) are repressed in plants through transcriptional gene silencing (TGS), maintained epigenetic silencing marks such as DNA methylation. However, the mechanisms by which silencing is first installed remain poorly understood in plants. Small interfering (si)RNAs and post-transcriptional gene silencing (PTGS) are believed to mediate the initiation of TGS by guiding the first deposition of DNA methylation. To determine how this silencing installation works, we took advantage of ÉVADÉ (EVD), an endogenous retroelement in Arabidopsis, able to recapitulate true de novo silencing with a sequence of PTGS followed by a TGS. To test whether PTGS is required for TGS, we introduce active EVD into RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) mutants, an essential PTGS component. EVD activity and silencing are monitored across several generations. In the absence of PTGS, silencing of EVD is still achieved through installation of RNA-directed DNA methylation (RdDM). Our study shows that PTGS is dispensable for de novo EVD silencing. Although we cannot rule out that PTGS might facilitate TGS, or control TE activity, initiation of epigenetic silencing can take place in its absence.

Arabidopsis↗