Site-Specific Covalent Modification of RNA Guided by Functionality-Transfer Oligodeoxynucleotides

2009 ◽  
Vol 20 (4) ◽  
pp. 799-803 ◽  
Author(s):  
Kazumitsu Onizuka ◽  
Yosuke Taniguchi ◽  
Shigeki Sasaki
Keyword(s):  
Covalent Modification ◽  
Site Specific

scholarly journals Site-Specific Recombination of TemperateMyxococcus xanthus Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification

1999 ◽  
Vol 181 (13) ◽  
pp. 4062-4070 ◽  
Author(s):  
Vincent Magrini ◽  
Michael L. Storms ◽  
Philip Youderian
Keyword(s):  
Specific Activity ◽  
Active Form ◽  
Attachment Site ◽  
Covalent Modification ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Site Specific Integration ◽  
C Terminus ◽  
Primary Amino ◽  
Reversible Covalent

ABSTRACT Temperate Myxococcus xanthus phage Mx8 integrates into the attB locus of the M. xanthus genome. The phage attachment site, attP, is required in cisfor integration and lies within the int (integrase) coding sequence. Site-specific integration of Mx8 alters the 3′ end ofint to generate the modified intX gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes attP × attB recombination more efficiently than attR × attB, attL × attB, or attB × attB recombination. The attP and attBsites share a common core. Sequences flanking both sides of theattP core within the int gene are necessary forattP function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the attP core. Expression of theuoi gene immediately upstream of int inhibits integrative (attP × attB) recombination, supporting the idea that uoi encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.

scholarly journals Site-specific covalent modification of monoclonal antibodies: in vitro and in vivo evaluations.

1986 ◽  
Vol 83 (8) ◽  
pp. 2632-2636 ◽  
Author(s):  
J. D. Rodwell ◽  
V. L. Alvarez ◽  
C. Lee ◽  
A. D. Lopes ◽  
J. W. Goers ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Covalent Modification ◽  
Site Specific

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

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