Inteins and introns within the<i> prp8</i> ‐gene of four <i>Eupenicillium</i> species

Elleuche, Skander; Pelikan, Constantin; Nolting, Nicole; Pöggeler, Stefanie

doi:10.1002/jobm.200800168

Cited by 7 publications

(3 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Intron acquisition might thus have served to prevent intein homing by disrupting the HE recognition sites. This has been proposed in the case of Prp8 inteins in Eupenicillium species ( 39 ), and intron-intein mutual exclusivity was described in Prp8 in strains of Bathycoccus ( 27 ). The case of DNApol paralogs in Anaeramoeba is particularly intriguing by its symmetry.…”

Section: Resultsmentioning

confidence: 81%

Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes

Gallot-Lavallée,

Jerlström-Hultqvist,

Zegarra-Vidarte

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Inteins are self-splicing protein elements found in viruses and all three domains of life. How the DNA encoding these selfish elements spreads within and between genomes is poorly understood, particularly in eukaryotes where inteins are scarce. Here, we show that the nuclear genomes of three strains of Anaeramoeba encode between 45 and 103 inteins, in stark contrast to four found in the most intein-rich eukaryotic genome described previously. The Anaeramoeba inteins reside in a wide range of proteins, only some of which correspond to intein-containing proteins in other eukaryotes, prokaryotes, and viruses. Our data also suggest that viruses have contributed to the spread of inteins in Anaeramoeba and the colonization of new alleles. The persistence of Anaeramoeba inteins might be partly explained by intragenomic movement of intein-encoding regions from gene to gene. Our intein dataset greatly expands the spectrum of intein-containing proteins and provides insights into the evolution of inteins in eukaryotes.

show abstract

Section: Resultsmentioning

confidence: 81%

Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes

Gallot-Lavallée,

Jerlström-Hultqvist,

Zegarra-Vidarte

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…For comparison the S. cerevisiae HIS3 and the S. pombe his5 + genes share 58% sequence identity and yet do not recombine during integrative transformation [41]. The sequence divergence, the fact that the Pch PRP8 intein is well characterized [9,19,20,39], splices at high efficiency across a broad range of temperatures [19] and lacks a DNA binding domain led to its choice in our studies.…”

Section: Discussionmentioning

confidence: 99%

An intein with genetically selectable markers provides a new approach to internally label proteins with GFP

et al. 2011

View full text Add to dashboard Cite

BackgroundInteins are proteins that catalyze their own removal from within larger precursor proteins. In the process they splice the flanking protein sequences, termed the N-and C-terminal exteins. Large inteins frequently have a homing endonuclease that is involved in maintaining the intein in the host. Splicing and nuclease activity are independent and distinct domains in the folded structure. We show here that other biochemical activities can be incorporated into an intein in place of the endonuclease without affecting splicing and that these activities can provide genetic selection for the intein. We have coupled such a genetically marked intein with GFP as the N-terminal extein to create a cassette to introduce GFP within the interior of a targeted protein.ResultsThe Pch PRP8 mini-intein of Penicillium chrysogenum was modified to include: 1) aminoglycoside phosphotransferase; 2) imidazoleglycerol-phosphate dehydratase, His5 from S. pombe ; 3) hygromycin B phosphotransferase; and 4) the transcriptional activator LexA-VP16. The proteins were inserted at the site of the lost endonuclease. When expressed in E. coli, all of the modified inteins spliced at high efficiency. Splicing efficiency was also greater than 96% when expressed from a plasmid in S. cerevisiae. In addition the inteins conferred either G418 or hygromycin resistance, or histidine or leucine prototropy, depending on the inserted marker and the yeast genetic background. DNA encoding the marked inteins coupled to GFP as the N-terminal extein was PCR amplified with ends homologous to an internal site in the yeast calmodulin gene CMD1. The DNA was transformed into yeast and integrants obtained by direct selection for the intein's marker. The His5-marked intein yielded a fully functional calmodulin that was tagged with GFP within its central linker.ConclusionsInteins continue to show their flexibility as tools in molecular biology. The Pch PRP8 intein can successfully tolerate a variety of genetic markers and still retain high splicing efficiency. We have shown that a genetically marked intein can be used to insert GFP in one-step within a target protein in vivo.

show abstract

“…The intein was fused to an N-terminal GST-tag and a C-terminal HIS-peptide, a system that has been used in previous studies for the investigation of PRP8 inteins (Elleuche et al 2008;Elleuche et al 2006;Elleuche et al 2009;Elleuche and Pöggeler 2007). To elucidate optimal native amino acid sequences at the splice site junctions, 25 different variants were constructed and tested (Fig.…”

Section: Analysis Of Protein Splicing Using Gst-his Reporter Systemmentioning

confidence: 99%

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

Heyde

Lockhauserbäumer

Uetrecht

et al. 2015

Appl Microbiol Biotechnol

Self Cite

View full text Add to dashboard Cite

Extein amino acid residues around the splice site junctions affect the functionality of inteins. To identify an optimal sequence context for efficient protein splicing of an intein from the thermoacidophilic archaeon Picrophilus torridus, single extein amino acid residues at the splice site junctions were continuously deleted. The construction of a set of different truncated extein variants showed that this intein tolerates multiple amino acid variations near the excision sites and exhibits full activity when -1 and +1 extein amino acid residues are conserved in an artificial GST-intein-HIS fusion construct. Moreover, splicing of the recombinant intein took place at temperatures between 4 and 42 °C with high efficiency, when produced in Escherichia coli. Therefore, structural model predictions were used to identify optimal insertion sites for the intein to be embedded within a hemicellulase from the psychrophilic bacterium Pseudoalteromonas arctica. The P. torridus intein inserted before amino acid residue Thr75 of the reporter enzyme retained catalytic activity. Moreover, the catalytic activity of the xylan-degrading hydrolase could be easily monitored in routine plate assays and in liquid test measurements at room temperature when produced in recombinant form in E. coli. This tool allows the indirect detection of the intein's catalytic activity to be used in screenings.

show abstract

Inteins and introns within the prp8 ‐gene of four Eupenicillium species

Cited by 7 publications

References 31 publications

Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes

Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes

An intein with genetically selectable markers provides a new approach to internally label proteins with GFP

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

Contact Info

Product

Resources

About