scholarly journals Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression

2009 ◽  
Vol 83 (17) ◽  
pp. 8409-8417 ◽  
Author(s):  
P. Joel Ross ◽  
Michael A. Kennedy ◽  
Robin J. Parks
Keyword(s):  
Transgene Expression ◽  
Great Promise ◽  
Cell Detection ◽  
Nuclear Factors ◽  
Insulator Element ◽  
Pml Bodies ◽  
Eukaryotic Dna ◽  
Foreign Dna ◽  
Mechanistic Basis ◽  
Epigenetic Repression

ABSTRACT Helper-dependent adenovirus (hdAd) vectors have shown great promise as therapeutic gene delivery vehicles in gene therapy applications. However, the level and duration of gene expression from hdAd can differ considerably depending on the nature of the noncoding stuffer DNA contained within the vector. For example, an hdAd containing 22 kb of prokaryotic DNA (hdAd-prok) expresses its transgene 60-fold less efficiently than a similar vector containing eukaryotic DNA (hdAd-euk). Here we have determined the mechanistic basis of this phenomenon. Although neither vector was subjected to CpG methylation and both genomes associated with cellular histones to similar degrees, hdAd-prok chromatin was actively deacetylated. Insertion of an insulator element between the transgene and the bacterial DNA derepressed hdAd-prok, suggesting that foreign DNA nucleates repressive chromatin structures that spread to the transgene. We found that Sp100B/Sp100HMG and Daxx play a role in repressing transgene expression from hdAd and act independently of PML bodies. Thus, we have identified nuclear factors involved in recognizing foreign DNA and have determined the mechanism by which associated genes are repressed.

scholarly journals Systemic Delivery of Tyrosine-Mutant AAV Vectors Results in Robust Transduction of Neurons in Adult Mice

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Asako Iida ◽  
Naomi Takino ◽  
Hitomi Miyauchi ◽  
Kuniko Shimazaki ◽  
Shin-ichi Muramatsu
Keyword(s):  
Transgene Expression ◽  
Neurological Diseases ◽  
Systemic Delivery ◽  
Adeno Associated Virus ◽  
Gene Therapies ◽  
Adult Mice ◽  
The Central Nervous System ◽  
Foreign Dna ◽  
The Brain ◽  
Limited Usefulness

Recombinant adeno-associated virus (AAV) vectors are powerful tools for both basic neuroscience experiments and clinical gene therapies for neurological diseases. Intravascularly administered self-complementary AAV9 vectors can cross the blood-brain barrier. However, AAV9 vectors are of limited usefulness because they mainly transduce astrocytes in adult animal brains and have restrictions on foreign DNA package sizes. In this study, we show that intracardiac injections of tyrosine-mutant pseudotype AAV9/3 vectors resulted in extensive and widespread transgene expression in the brains and spinal cords of adult mice. Furthermore, the usage of neuron-specific promoters achieved selective transduction of neurons. These results suggest that tyrosine-mutant AAV9/3 vectors may be effective vehicles for delivery of therapeutic genes, including miRNAs, into the brain and for treating diseases that affect broad areas of the central nervous system.

scholarly journals The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Charanya Kumar ◽  
Sahil Batra ◽  
Jack D Griffith ◽  
Dirk Remus
Keyword(s):  
Genome Instability ◽  
Hybrid Structure ◽  
Replication Fork Progression ◽  
Rna:Dna Hybrid ◽  
Eukaryotic Dna ◽  
Intrinsic Capacity ◽  
Leading Strand ◽  
Rna:Dna Hybrids ◽  
Mechanistic Basis ◽  
Fork Stalling

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

scholarly journals The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

2021 ◽  
Author(s):  
Charanya Kumar ◽  
Sahil Batra ◽  
Jack Griffith ◽  
Dirk Remus
Keyword(s):  
Genome Instability ◽  
Hybrid Structure ◽  
Replication Fork Progression ◽  
Rna:Dna Hybrid ◽  
Eukaryotic Dna ◽  
Intrinsic Capacity ◽  
Leading Strand ◽  
Rna:Dna Hybrids ◽  
Mechanistic Basis ◽  
Fork Stalling

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

scholarly journals An siRNA-guided Argonaute protein directs RNA Polymerase V to initiate DNA methylation

2021 ◽  
Author(s):  
Meredith J Sigman ◽  
Kaushik Panda ◽  
Rachel Kirchner ◽  
Lauren L McLain ◽  
Hayden Payne ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Argonaute Protein ◽  
Unanswered Question ◽  
Small Interfering Rnas ◽  
Pol V ◽  
Target Locus ◽  
Target Sites ◽  
Foreign Dna ◽  
Epigenetic Repression

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA Polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without preexisting DNA methylation. We find that Pol V follows and is dependent upon the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of preexisting DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

scholarly journals An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation

Nature Plants ◽  
2021 ◽  
Author(s):  
Meredith J. Sigman ◽  
Kaushik Panda ◽  
Rachel Kirchner ◽  
Lauren L. McLain ◽  
Hayden Payne ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Argonaute Protein ◽  
Unanswered Question ◽  
Small Interfering Rnas ◽  
Pol V ◽  
Target Locus ◽  
Target Sites ◽  
Foreign Dna ◽  
Epigenetic Repression

AbstractIn mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

scholarly journals Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken β-Globin Locus HS4 Insulator Element

2009 ◽  
Vol 17 (4) ◽  
pp. 667-674 ◽  
Author(s):  
Hideki Hanawa ◽  
Motoko Yamamoto ◽  
Huifen Zhao ◽  
Takashi Shimada ◽  
Derek A Persons
Keyword(s):  
Transgene Expression ◽  
Lentiviral Vector ◽  
Insulator Element

Are HOLZ lines kinematic in off-zone-axis orientation?

1993 ◽  
Vol 51 ◽  
pp. 692-693
Author(s):  
J. M. Zuo ◽  
A. L. Weickenmeier ◽  
R. Holmestad ◽  
J. C. H. Spence
Keyword(s):  
Structure Determination ◽  
Standard Procedure ◽  
High Order ◽  
Zone Axis ◽  
Great Promise ◽  
Line Position ◽  
Measured Intensity ◽  
Constant Intensity ◽  
Axis Orientation ◽  
Diffraction Condition

The application of high order reflections in a weak diffraction condition off the zone axis center, including those in high order laue zones (HOLZ), holds great promise for structure determination using convergent beam electron diffraction (CBED). It is believed that in this case the intensities of high order reflections are kinematic or two-beam like. Hence, the measured intensity can be related to the structure factor amplitude. Then the standard procedure of structure determination in crystallography may be used for solving unknown structures. The dynamic effect on HOLZ line position and intensity in a strongly diffracting zone axis is well known. In a weak diffraction condition, the HOLZ line position may be approximated by the kinematic position, however, it is not clear whether this is also true for HOLZ intensities. The HOLZ lines, as they appear in CBED patterns, do show strong intensity variations along the line especially near the crossing of two lines, rather than constant intensity along the Bragg condition as predicted by kinematic or two beam theory.

Development of the UHV-STM

1990 ◽  
Vol 48 (1) ◽  
pp. 322-323
Author(s):  
M. Iwatsuki ◽  
S. Kitamura ◽  
A. Mogami
Keyword(s):  
Surface Science ◽  
Real Space ◽  
Atomic Scale ◽  
Scanning Tunneling ◽  
Great Promise ◽  
Force Microscopy ◽  
Atomic Force ◽  
Stm Image ◽  
Surface Construction ◽  
Very High

Since Binnig, Rohrer and associates observed real-space topographic images of Si(111)-7×7 and invented the scanning tunneling microscope (STM),1) the STM has been accepted as a powerful surface science instrument.Recently, many application areas for the STM have been opened up, such as atomic force microscopy (AFM), magnetic force microscopy (MFM) and others. So, the STM technology holds a great promise for the future.The great advantages of the STM are its high spatial resolution in the lateral and vertical directions on the atomic scale. However, the STM has difficulty in identifying atomic images in a desired area because it uses piezoelectric (PZT) elements as a scanner.On the other hand, the demand to observe specimens under UHV condition has grown, along with the advent of the STM technology. The requirment of UHV-STM is especially very high in to study of surface construction of semiconductors and superconducting materials on the atomic scale. In order to improve the STM image quality by keeping the specimen and tip surfaces clean, we have built a new UHV-STM (JSTM-4000XV) system which is provided with other surface analysis capability.

Behavioral Genetics: Concepts for Research and Practice in Language Development and Disorders

1995 ◽  
Vol 38 (5) ◽  
pp. 1126-1142 ◽  
Author(s):  
Jeffrey W. Gilger
Keyword(s):  
Language Development ◽  
Behavioral Genetics ◽  
State Of The Art ◽  
Genetic Research ◽  
Great Promise ◽  
Behavioral Genetic ◽  
Fine Grained ◽  
Future Goals ◽  
Current State ◽  
Research Designs

This paper is an introduction to behavioral genetics for researchers and practioners in language development and disorders. The specific aims are to illustrate some essential concepts and to show how behavioral genetic research can be applied to the language sciences. Past genetic research on language-related traits has tended to focus on simple etiology (i.e., the heritability or familiality of language skills). The current state of the art, however, suggests that great promise lies in addressing more complex questions through behavioral genetic paradigms. In terms of future goals it is suggested that: (a) more behavioral genetic work of all types should be done—including replications and expansions of preliminary studies already in print; (b) work should focus on fine-grained, theory-based phenotypes with research designs that can address complex questions in language development; and (c) work in this area should utilize a variety of samples and methods (e.g., twin and family samples, heritability and segregation analyses, linkage and association tests, etc.).

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