Intergenomic gene transfer in diploid and allopolyploid Gossypium

Background Intergenomic gene transfer (IGT) between nuclear and organellar genomes is a common phenomenon during plant evolution. Gossypium is a useful model to evaluate the genomic consequences of IGT for both diploid and polyploid species. Here, we explore IGT among nuclear, mitochondrial, and plastid genomes of four cotton species, including two allopolyploids and their model diploid progenitors (genome donors, G. arboreum: A2 and G. raimondii: D5). Results Extensive IGT events exist for both diploid and allotetraploid cotton (Gossypium) species, with the nuclear genome being the predominant recipient of transferred DNA followed by the mitochondrial genome. The nuclear genome has integrated 100 times more foreign sequences than the mitochondrial genome has in total length. In the nucleus, the integrated length of chloroplast DNA (cpDNA) was between 1.87 times (in diploids) to nearly four times (in allopolyploids) greater than that of mitochondrial DNA (mtDNA). In the mitochondrion, the length of nuclear DNA (nuDNA) was typically three times than that of cpDNA. Gossypium mitochondrial genomes integrated three nuclear retrotransposons and eight chloroplast tRNA genes, and incorporated chloroplast DNA prior to divergence between the diploids and allopolyploid formation. For mitochondrial chloroplast-tRNA genes, there were 2-6 bp conserved microhomologies flanking their insertion sites across distantly related genera, which increased to 10 bp microhomologies for the four cotton species studied. For organellar DNA sequences, there are source hotspots, e.g., the atp6-trnW intergenic region in the mitochondrion and the inverted repeat region in the chloroplast. Organellar DNAs in the nucleus were rarely expressed, and at low levels. Surprisingly, there was asymmetry in the survivorship of ancestral insertions following allopolyploidy, with most numts (nuclear mitochondrial insertions) decaying or being lost whereas most nupts (nuclear plastidial insertions) were retained. Conclusions This study characterized and compared intracellular transfer among nuclear and organellar genomes within two cultivated allopolyploids and their ancestral diploid cotton species. A striking asymmetry in the fate of IGTs in allopolyploid cotton was discovered, with numts being preferentially lost relative to nupts. Our results connect intergenomic gene transfer with allotetraploidy and provide new insight into intracellular genome evolution.

Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases.

tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.

Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases

tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.