Site-Specific Self-Catalyzed DNA Depurination, the Basis of a Spontaneous Mutagenic Mechanism of Wide Evolutionary Significance

The integron-gene cassette system contributes to multiple antibiotic resistance in bacteria and is likely to be of broader evolutionary significance. However, the majority of integron diversity consists of chromosomal integrons (CIs), with mostly unknown phenotypes, which are poorly characterized. A pUC-based reporter plasmid (pUS23) was developed containing a recombination site [aadB 59 base element (59-be)] upstream of promoterless aadB [gentamicin (Gm) resistance] and gfp (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI in Pseudomonas stutzeri strain Q. Electroporation of pUS23 into P. stutzeri Q gave ampicillin-resistant transformants, which yielded GmR green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at attI was detected by PCR in 8 % of GmR colonies and the frequency of attI integration was estimated as 2·0×10−8 per P. stutzeri Q(pUS23) cell. RT-PCR confirmed integron-mediated expression of aadB in one recombinant strain (Q23-17) and a promoter (Pc) was localized to the 5′ end of the intI gene. The integrated pUS23 and flanking integron DNA were cloned from genomic DNA of strain Q23-17 and sequenced, confirming that site-specific integration of the entire reporter plasmid had occurred at the attI site. An insertion sequence (ISPst5; IS5 family) was discovered in the vector backbone of the reporter plasmid integrated at attI and also in a pUS23 derivative recovered as a plasmid in Escherichia coli JM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.

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Adaptive Mutation: Has the Unicorn Landed?

Genetics ◽

10.1093/genetics/148.4.1453 ◽

1998 ◽

Vol 148 (4) ◽

pp. 1453-1459 ◽

Cited By ~ 2

Author(s):

Patricia L Foster

Keyword(s):

Adaptive Evolution ◽

Evolutionary Significance ◽

Adaptive Mutation ◽

Original Sense ◽

Mutagenic Mechanism

AbstractReversion of an episomal Lac− allele during lactose selection has been studied as a model for adaptive mutation. Although recent results show that the mutations that arise during selection are not “adaptive” in the original sense, the mutagenic mechanism that produces these mutations may nonetheless be of evolutionary significance. In addition, a transient mutational state induced in a subpopulation of starving cells could provide a species with a mechanism for adaptive evolution.

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Large-cluster and combined fluorescent and gold immunoprobes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166920 ◽

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.

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The challenge of detecting modifications on proteins

Essays in Biochemistry ◽

10.1042/ebc20190055 ◽

2020 ◽

Vol 64 (1) ◽

pp. 135-153 ◽

Cited By ~ 1

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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