Fluorescence Detection of Specific Sequence of Nucleic Acids by Oxazole Yellow-Linked Oligonucleotides. Homogeneous Quantitative Monitoring of In Vitro Transcription

Nopp44/46 is a phosphoprotein of the protozoan parasite Trypanosoma brucei that is localized to the nucleolus. Based on the primary sequence, Nopp44/46 appears to be a protein composed of distinct domains. This communication describes the relationship of these domains to the known functional interactions of the molecule and suggests that the amino-terminal region defines a novel homology region that functions in nucleolar targeting. We have previously shown that Nopp44/46 is capable of interacting with nucleic acids and associating with a protein kinase. Using in vitro transcription and translation, we now demonstrate that the nucleic acid binding function maps to the carboxy-terminal domain of the molecule, a region rich in arginine-glycine-glycine motifs. Our experiments reveal that a central region containing a high proportion of acidic residues is required for association with the protein kinase. Analysis of transfectants expressing epitope-tagged Nopp44/46 deletion constructs showed that the amino-terminal 96 amino acids allowed nuclear and nucleolar accumulation of the protein. This region of the molecule shows homology to several recently described nucleolar proteins. Deletion of a 27-amino-acid region within this domain abrogated nucleolar, but not nuclear, localization. These studies show that Nopp44/46 is composed of distinct modules, each of which plays a different role in molecular interactions. We suggest that this protein could facilitate interactions between sets of nucleolar molecules.

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A Motif within SET-Domain Proteins Binds Single-Stranded Nucleic Acids and Transcribed and Supercoiled DNAs and Can Interfere with Assembly of Nucleosomes

Molecular and Cellular Biology ◽

10.1128/mcb.25.5.1891-1899.2005 ◽

2005 ◽

Vol 25 (5) ◽

pp. 1891-1899 ◽

Cited By ~ 58

Author(s):

Wladyslaw A. Krajewski ◽

Tatsuya Nakamura ◽

Alexander Mazo ◽

Eli Canaani

Keyword(s):

Nucleic Acids ◽

Target Genes ◽

Binding Capacity ◽

Position Effect ◽

In Vitro Transcription ◽

Position Effect Variegation ◽

Nucleosome Assembly ◽

Set Domain ◽

Eukaryotic Chromatin

ABSTRACT The evolutionary conserved SET domain is present in many eukaryotic chromatin-associated proteins, including some members of the trithorax (TrxG) group and the polycomb (PcG) group of epigenetic transcriptional regulators and modifiers of position effect variegation. All SET domains examined exhibited histone lysine methyltransferase activity, implicating these proteins in the generation of epigenetic marks. However, the mode of the initial recruitment of SET proteins to target genes and the way that their association with the genes is maintained after replication are not known. We found that SET-containing proteins of the SET1 and SET2 families contain motifs in the pre-SET region or at the pre-SET-SET and SET-post-SET boundaries which very tightly bind single-stranded DNA (ssDNA) and RNA. These motifs also bind stretches of ssDNA generated by superhelical tension or during the in vitro transcription of duplex DNA. Importantly, such binding withstands nucleosome assembly, interfering with the formation of regular nucleosomal arrays. Two representatives of the SUV39 SET family, SU(VAR)3-9 and G9a, did not bind ssDNA. The trx Z11 homeotic point mutation, which is located within TRX SET and disrupts embryonic development, impairs the ssDNA binding capacity of the protein. We suggest that the motifs described here may be directly involved in the biological function(s) of SET-containing proteins. The binding of single-stranded nucleic acids might play a role in the initial recruitment of the proteins to target genes, in the maintenance of their association after DNA replication, or in sustaining DNA stretches in a single-stranded configuration to allow for continuous transcription.

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A Novel In Vitro Transcription Method for Producing siRNAs Without Specific Sequence Requirements

Molecular Biotechnology ◽

10.1385/mb:31:3:187 ◽

2005 ◽

Vol 31 (3) ◽

pp. 187-192 ◽

Cited By ~ 6

Author(s):

Xudong Zhu ◽

Tao Li ◽

Ying Dang ◽

Yi Feng ◽

Peitang Huang

Keyword(s):

In Vitro Transcription ◽

Specific Sequence ◽

Sequence Requirements

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Inhibition of HIV-1 LTR-driven in vitro transcription by molecular hybrids based on peptide nucleic acids mimicking the NF-κB binding site

International Journal of Molecular Medicine ◽

10.3892/ijmm.9.6.633 ◽

2002 ◽

Author(s):

Carlo Mischiati ◽

Monica Borgatti ◽

Giordana Feriotto ◽

Cristina Rutigliano ◽

Laura Breda ◽

...

Keyword(s):

Nucleic Acids ◽

Binding Site ◽

Peptide Nucleic Acids ◽

In Vitro Transcription ◽

Molecular Hybrids ◽

Hiv 1

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Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells

eLife ◽

10.7554/elife.01535 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 72

Author(s):

Safia Deddouche ◽

Delphine Goubau ◽

Jan Rehwinkel ◽

Probir Chakravarty ◽

Sharmin Begum ◽

...

Keyword(s):

Antisense Rna ◽

Rna Synthesis ◽

In Vitro Transcription ◽

Encephalomyocarditis Virus ◽

Type I Interferons ◽

Type I ◽

Specific Sequence ◽

Infected Cells ◽

Rna Complexes

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells.

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Preparation of Labeled DNA, RNA, and Oligonucleotide Probes

Cold Spring Harbor Protocols ◽

10.1101/pdb.top100578 ◽

2022 ◽

Vol 2022 (1) ◽

pp. pdb.top100578

Author(s):

Michael R. Green ◽

Joseph Sambrook

Keyword(s):

Nucleic Acids ◽

In Vitro Transcription ◽

Heterogeneous Population ◽

Oligonucleotide Probes ◽

Nick Translation ◽

Chain Reaction ◽

Random Priming ◽

Polymerase Chain ◽

Enzymatic Methods

Labeled nucleic acids and oligonucleotides are typically generated by enzymatic methods such as end-labeling, random priming, nick translation, in vitro transcription, and variations of the polymerase chain reaction (PCR). Some of these methods place the label in specific locations within the nucleic acid (e.g., at the 5′ or 3′ terminus); others generate molecules that are labeled internally at multiple sites. Some methods yield labeled single-stranded products, whereas others generate double-stranded nucleic acids. Finally, some generate probes of defined length, whereas others yield a heterogeneous population of labeled molecules. Options available for generating and detecting labeled nucleic acids, as well as advice on designing oligonucleotides for use as probes, is included here.

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