The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.
The internal transcribed spacers (ITS) of nuclear ribosomal DNA of 33 species of genus Paeonui (Paeoniaceae) were sequenced. In section Paeonia, different patterns of nucleotide additivity were detected in 14 diploid and tetraploid species at sites that are variable in the other 12 species of the section, suggesting that reticulate evolution has occurred. Phylogenetic relationships of species that do not show additivity, and thus ostensibly were not derived through hybridization, were reconstructed by parsimony analysis. The taxa presumably derived through reticulate evolution were then added to the phylogenetic tree according to additivity from putative parents. The study provides an example of successfully using ITS sequences to reconstruct reticulate evolution in plants and further demonstrates that the sequence data could be highly informative and accurate for detecting hybridization. Maintenance ofparental sequences in
The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to mRNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multi-wavelength fluoresence microscopy to follow assembly of single spliceosomes in real time in whole cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes, and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.
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