Analysis of the chloroplast large subunit ribosomal RNA gene from 17 Chlamydomonas taxa: three internal transcribed spacers and 12 groups I intron insertion sites

Previous reports on the chloroplast large subunit rRNA genes of the two distantly related green algae Chlamydomonas eugametos and Chlamydomonas reinhardtii indicate differences in the distribution of group I introns and suggest a different arrangement of internal transcribed spacers. To provide insights into the origin of these two types of intervening sequences, we have undertaken the sequencing of the chloroplast rrnL genes of 15 additional Chlamydomonas taxa and have characterized the mature large subunit rRNA species they encode in addition to those specified by the C. reinhardtii rrnL. These analyses disclosed the presence of three internal transcribed spacers sharing the same positions in all of the 17 taxa as well as the presence of a total of 39 group I introns representing 12 insertion sites. Of these insertion sites, only one has been identified in non-chlamydomonas taxa. The distribution of Chlamydomonas introns is highly variable and, in many respects, is not consistent with the phylogeny deduced from chloroplast rRNA sequence comparisons. This phylogeny features two main lineages of Chlamydomonas taxa forming sister groups. Because earlier branching organisms in the green algal/land plant lineage display no chloroplast rDNA introns. It appears that all of the intron insertion positions in Chlamydomonas are of recent origins, with some of the positions having arisen subsequent to the divergence of the two main Chlamydomonas lineages. Remarkably, the rRNA regions corresponding to most of the group I intron insertion positions in rRNA genes have been assigned functional roles suggesting that they lie in exposed regions of the ribosome. On the basis of this striking correlation between exposed rRNA regions and intron insertion sites. We speculate that the reversal of the self-splicing reaction has played a major role in the creation of the multiple intron insertion positions found in rRNA genes as well as in the proliferation of group I introns elsewhere in the Chlamydomonas chloroplast genome. 0388-44160 Matrix-generator and Monte Carlo methods have been employed to study the influence of thermal fluctuations on the overall sizes and shapes of curved pieces of DNA. The DNA model involves the independent angular parameters relating successive base-pair steps: the sequence-dependent equilibrium values and fluctuations of the twist, tilt, and roll angles. The curved sequence under study is the [A(5)X(5)](n) repeating polymer, the AA and XX steps having different equilibrium roll and twist values. Both planar circles and superhelices are analysed. Detailed comparison is made between the rigorous statistical mechanical representation of the DNA and simplified static models currently used in the literature. That is, a more realistic "flexible wedge" model is contrasted with the existing "static wedge" model of DNA curvature, which is demonstrated to be inadequate. The size of the coils is described by the unperturbed root-mean-square end-to-end distance and the shape by a ratio of the principal moments of the radius of gyration. The moment ratios indicate that when DNA is relatively short (e.g. its length is shorter than half a turn of the static superhelix), the flexible chains are more "short and thick" than the static structure. The end-to-end distances, however, are practically the same in the two models. For longer DNA fragments, the flexible chain is more extended in terms of the end-to-end distance and more globular in terms of the moment ratio. Thus, fluctuations "blur" the curvature of longer DNA fragments compared with static models. Furthermore, the overall average shape of slightly curved DNA subject to natural and twisting fluctuations is essentially indistinguishable from that of the corresponding "straight" DNA. Such configurational similarities are apparently responsible for the relative insensitivity of the polyacrylamide gel matrix to small degrees of DNA curvature. These findings raise serious questions regarding the quantitative estimation of wedge angles in DNA from electrophoretic experiments, based on static models. Comparison between planar circles and superhelices shows that when fluctuations are considered, the flexible circles are more spherical than the superhelices. The results imply that when DNA bending is exactly "in phase" with the helical repeat (i.e. when the DNA loop is exactly planar at 0 K), the DNA coil is packed more tightly than when bending and twisting are "out of phase" (and a superhelix is formed at 0 K). This finding is consistent with polyacrylamide gel electrophoresis data testifying to an increase in DNA retardation when twisting is more precisely "tuned". Finally, it is found that asymmetry of bending in the roll coordinate can produce significant macroscopic curvature of DNA. "Skewed" bending of AA steps in the A(5)X(5) polymer is indistinguishable from symmetric bending with the same mean value and fluctuation of roll. Asymmetric rolling in the AA dimer thus can introduce substantial curvature in DNAs containing A(n)T(n) blocks,even if the lowest energy stacked

arrangement of AA base-pair steps is nearly flat, thereby resolving apparent contradictions between the crystallographic and solution structures of chains containing A(n)T(n) tracts.