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Nuclear-encoded pentatricopeptide repeat (PPR) proteins are site-specific factors for C-to-U RNA editing in plant organelles coevolving with their targets. Losing an editing target by C-to-T conversion allows for eventual loss of its editing factor, as recently confirmed for editing factors CLB19, CRR28, and RARE1 targeting ancient chloroplast editing sites in flowering plants. Here, we report on alternative evolutionary pathways for DOT4 addressing rpoC1eU488SL, a chloroplast editing site in the RNA polymerase β' subunit mRNA. Upon loss of rpoC1eU488SL by C-to-T conversion, DOT4 got lost multiple times independently in angiosperm evolution with intermediate states of DOT4 orthologs in various stages of degeneration. Surprisingly, we now also observe degeneration and loss of DOT4 despite retention of a C in the editing position (in Carica, Coffea, Vicia, and Spirodela). We find that the cytidine remains unedited, proving that DOT4 was not replaced by another editing factor. Yet another pathway of DOT4 evolution is observed among the Poaceae. Although the rpoC1eU488SL edit has been lost through C-to-T conversion, DOT4 orthologs not only remain conserved but also have their array of PPRs extended by six additional repeats. Here, the loss of the ancient target has likely allowed DOT4 to adapt for a new function. We suggest rps3 antisense transcripts as previously demonstrated in barley (Hordeum vulgare) arising from promotor sequences newly emerging in the rpl16 intron of Poaceae as a new candidate target for the extended PPR stretch of DOT4. Altogether, DOT4 and its target show more flexible pathways for evolution than the previously explored editing factors CLB19, CRR28, and RARE1. Certain plant clades (e.g., Amaranthus, Vaccinium, Carica, the Poaceae, Fabales, and Caryophyllales) show pronounced dynamics in the evolution of editing sites and corresponding factors.
Pubmed ID: 30753430
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A second-generation retrovirus producer lines for the generation of helper free ecotropic and amphotropic retroviruses. The lines are based on the 293T cell line (a human embryonic kidney line transformed with adenovirus E1a and carrying a temperature sensitive T antigen co-selected with neomycin). The unique feature of this cell line is that it is highly transfectable with either calcium phosphate mediated transfection or lipid-based transfection protocols-- up to 50% or higher of cells can be transiently transfected. The lines were created by placing into 293T cells constructs capable of producing gag-pol, and envelope protein for ecotropic and amphotropic viruses. The lines offered advantages over previous stable systems in that virus can be produced in just a few days. Academic and non-profit laboratories may obtain the Phoenix cells from either Allele Biotechnology or the National Gene Vector Bank. The vectors may be obtained from Addgene. They are no longer distributing these reagents from the lab.
View all literature mentionsWeb search tool to find regions of similarity between biological sequences. Program compares nucleotide or protein sequences to sequence databases and calculates statistical significance. Used for identifying homologous sequences.
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View all literature mentionsTool to search translated nucleotide databases using a protein query.
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