Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.

Rapid isolation of high molecular weight plant DNA

The use of some multiple-sequence alignments in phylogenetic analysis, particularly those that are not very well conserved, requires the elimination of poorly aligned positions and divergent regions, since they may not be homologous or may have been saturated by multiple substitutions. A computerized method that eliminates such positions and at the same time tries to minimize the loss of informative sites is presented here. The method is based on the selection of blocks of positions that fulfill a simple set of requirements with respect to the number of contiguous conserved positions, lack of gaps, and high conservation of flanking positions, making the final alignment more suitable for phylogenetic analysis. To illustrate the efficiency of this method, alignments of 10 mitochondrial proteins from several completely sequenced mitochondrial genomes belonging to diverse eukaryotes were used as examples. The percentages of removed positions were higher in the most divergent alignments. After removing divergent segments, the amino acid composition of the different sequences was more uniform, and pairwise distances became much smaller. Phylogenetic trees show that topologies can be different after removing conserved blocks, particularly when there are several poorly resolved nodes. Strong support was found for the grouping of animals and fungi but not for the position of more basal eukaryotes. The use of a computerized method such as the one presented here reduces to a certain extent the necessity of manually editing multiple alignments, makes the automation of phylogenetic analysis of large data sets feasible, and facilitates the reproduction of the final alignment by other researchers.

/pdf/selection-of-conserved-blocks-from-multiple-alignments-for-i0qfazjwh5.pdf

Selection of Conserved Blocks from Multiple Alignments for Their Use in Phylogenetic Analysis

The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans--the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.

/pdf/analysis-of-the-genome-sequence-of-the-flowering-plant-3vizsb52m2.pdf

Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.

Several plant mitochondrial genomes contain repeated sequences that are postulated to be sites of homologous intragenomic recombination (1-3). In this report, we have used filter hybridizations to investigate sequence relationships between the cloned mitochondrial DNA (mtDNA) recombination repeats from turnip, spinach and maize and total mtDNA isolated from thirteen species of angiosperms. We find that strong sequence homologies exist between the spinach and turnip recombination repeats and essentially all other mitochondrial genomes tested, whereas a major maize recombination repeat does not hybridize to any other mtDNA. The sequences homologous to the turnip repeat do not appear to function in recombination in any other genome, whereas the spinach repeat hybridizes to reiterated sequences within the mitochondrial genomes of wheat and two species of pokeweed that do appear to be sites of recombination. Thus, although intragenomic recombination is a widespread phenomenon in plant mitochondria, it appears that different sequences either serve as substrates for this function in different species, or else surround a relatively short common recombination site which does not cross-hybridize under our experimental conditions. Identified gene sequences from maize mtDNA were used in heterologous hybridizations to show that the repeated sequences implicated in recombination in turnip and spinach/pokeweed/wheat mitochondria include, or are closely linked to genes for subunit II of cytochrome c oxidase and 26S rRNA, respectively. Together with previous studies indicating that the 18S rRNA gene in wheat mtDNA is contained within a recombination repeat (3), these results imply an unexpectedly frequent association between recombination repeats and plant mitochondrial genes.

/pdf/recombination-sequences-in-plant-mitochondrial-genomes-3t16cerojk.pdf

Recombination sequences in plant mitochondrial genomes: diversity and homologies to known mitochondrial genes

We have surveyed the distribution and reconstructed the phylogeny of the group-I intron that is positioned in the anticodon loop of the tRNALeu gene in cyanobacteria and several plastid genomes. Southern-blot and PCR analyses showed that the tRNALeu intron is found in all 330 land plants that were examined. The intron was also found, and sequenced, in all but one of nine charophycean algae examined. Conversely, PCR analyses showed that the tRNALeu group-I intron is absent from the red, cryptophyte and haptophyte algae, although it is present in three members of the heterokont lineage. Phylogenetic analyses of the intron indicate that it was present in the cyanobacterial ancestor of the three primary plastid lineages, the Rhodophyta, Chlorophyta, and Glaucocystophyta. Its present-day distribution in plastids is consistent with a history of strictly vertical transmission, with no losses in land plants, several losses among green algae, and nearly pervasive loss in the Rhodophyta and its secondary derivatives.

The cyanobacterial origin and vertical transmission of the plastid tRNA(Leu) group-I intron.

A group I intron has recently been shown to have invaded mitochondrial cox1 genes by horizontal transfer many times during the broad course of angiosperm evolution. To investigate the frequency of acquisition of this intron within a more closely related group of plants, we determined its distribution and inferred its evolutionary history among 14 genera of the monocot family Araceae. Southern blot hybridizations showed that 6 of the 14 genera contain this intron in their cox1 genes. Nucleotide sequencing showed that these six introns are highly similar in sequence (97.7%-99.4% identity) and identical in length (966 nt). Phylogenetic evidence from parsimony reconstructions of intron distribution and phylogenetic analyses of intron sequences is consistent with a largely vertical history of intron transmission in the family; the simplest scenarios posit but one intron gain and two losses. Despite this, however, striking differences in lengths of exonic co-conversion tracts, coupled with the absence of co-conversion in intron-lacking taxa, indicate that the six intron-containing Araceae probably acquired their introns by at least three and quite possibly five separate horizontal transfers. The highly similar nature of these independently acquired introns implies a closely related set of donor organisms.

/pdf/multiple-acquisitions-via-horizontal-transfer-of-a-group-i-3rv334e6yh.pdf

Multiple acquisitions via horizontal transfer of a group I intron in the mitochondrial cox1 gene during evolution of the Araceae family.

Origin of introns--early or late?

We used the chloroplast gene rbcL as a model to study the frequency and relative timing of transfer of chloroplast sequences to the mitochondrial genome. Southern blot survey of 20 mitochondrial DNAs confirmed three previously reported groups of plants containing rbcL in their mitochondrion, while PCR studies identified a new mitochondrial rbcL. Published and newly determined mitochondrial and chloroplast rbcL sequences were used to reconstruct rbcL phylogeny. The results imply five or six separate interorganellar transfers of rbcL among the angiosperms examined, and hundreds of successful transfers across all flowering plants. By taxonomic criteria, the crucifer transfer is the most ancient, two separate transfers within the grass family are of intermediate ancestry, and the morning-glory transfer is most recent. All five mitochondrial copies of rbcL examined exhibit insertion and/or deletion events that disrupt the reading frame (three are grossly truncated); and all are elevated in the proportion of nonsynonymous substitutions, providing clear evidence that these sequences are pseudogenes.

/pdf/phylogenetic-analysis-reveals-five-independent-transfers-of-3zzq08m6zy.pdf

Jeffrey D. Palmer

Papers

Recombination sequences in plant mitochondrial genomes: diversity and homologies to known mitochondrial genes

The cyanobacterial origin and vertical transmission of the plastid tRNA(Leu) group-I intron.

Multiple acquisitions via horizontal transfer of a group I intron in the mitochondrial cox1 gene during evolution of the Araceae family.

Origin of introns--early or late?

Phylogenetic analysis reveals five independent transfers of the chloroplast gene rbcL to the mitochondrial genome in angiosperms