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The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

ABSTRACT: The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of ?-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

SUBMITTER: Valle-Garcia D

PROVIDER: S-EPMC4021028 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

Valle-García David D Griffiths Lyra M LM Dyer Michael A MA Bernstein Emily E Recillas-Targa Félix F

SpringerPlus 20140502

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of AT ...[more]

PMID: 24834375

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Project description:BackgroundIn protein evolution, the mechanism of the emergence of novel protein domain is still an open question. The incremental growth of protein variable regions, which was produced by stochastic insertions, has the potential to generate large and complex sub-structures. In this study, a deterministic methodology is proposed to reconstruct phylogenies from protein structures, and to infer insertion events in protein evolution. The analysis was performed on a broad range of SCOP domain families.ResultsPhylogenies were reconstructed from protein 3D structural data. The phylogenetic trees were used to infer ancestral structures with a consensus method. From these ancestral reconstructions, 42.7% of the observed insertions are nested insertions, which locate in previous insert regions. The average size of inserts tends to increase with the insert rank or total number of insertions in the variable regions. We found that the structures of some nested inserts show complex or even domain-like fold patterns with helices, strands and loops. Furthermore, a basal level of structural innovation was found in inserts which displayed a significant structural similarity exclusively to themselves. The beta-Lactamase/D-ala carboxypeptidase domain family is provided as an example to illustrate the inference of insertion events, and how the incremental growth of a variable region is capable to generate novel structural patterns.ConclusionUsing 3D data, we proposed a method to reconstruct phylogenies. We applied the method to reconstruct the sequences of insertion events leading to the emergence of potentially novel structural elements within existing protein domains. The results suggest that structural innovation is possible via the stochastic process of insertions and rapid evolution within variable regions where inserts tend to be nested. We also demonstrate that the structure-based phylogeny enables the study of new questions relating to the evolution of protein domain and biological function.

Dataset Information

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

Publications

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

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