DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Evolution of primate gene expression: drift and corrective sweeps?

Changes in gene expression play an important role in species' evolution. Earlier studies uncovered evidence that the effect of mutations on expression levels within the primate order is skewed, with many small downregulations balanced by fewer but larger upregulations. In addition, brain-expressed genes appeared to show an increased rate of evolution on the branch leading to human. However, the lack of a mathematical model adequately describing the evolution of gene expression precluded the rigorous establishment of these observations. Here, we develop mathematical tools that allow us to revisit these earlier observations in a model-testing and inference framework. We introduce a model for skewed gene-expression evolution within a phylogenetic tree and use a separate model to account for biological or experimental outliers. A Bayesian Markov chain Monte Carlo inference procedure allows us to infer the phylogeny and other evolutionary parameters, while quantifying the confidence in these inferences. Our results support previous observations; in particular, we find strong evidence for a sustained positive skew in the distribution of gene-expression changes in primate evolution. We propose a "corrective sweep" scenario to explain this phenomenon.

Effect of antibiotics on large ribosomal subunit assembly reveals possible function of 5 S rRNA.

Functional large ribosomal subunits of Thermus aquaticus can be reconstituted from ribosomal proteins and either natural or in vitro transcribed 23 S and 5 S rRNA. Omission of 5 S rRNA during subunit reconstitution results in dramatic decrease of the peptidyl transferase activity of the assembled subunits. However, the presence of some ribosome-targeted antibiotics of the macrolide, ketolide or streptogramin B groups during 50 S subunit reconstitution can partly restore the activity of ribosomal subunits assembled without 5 S rRNA. Among tested antibiotics, macrolide RU69874 was the most active: activity of the subunits assembled in the absence of 5 S rRNA was increased more than 30-fold if antibiotic was present during reconstitution procedure. Activity of the subunits assembled with 5 S rRNA was also slightly stimulated by RU69874, but to a much lesser extent, approximately 1.5-fold. Activity of the native T. aquaticus 50 S subunits incubated in the reconstitution conditions in the presence of RU69874 was, in contrast, slightly decreased. The presence of antibiotics was essential during the last incubation step of the in vitro assembly, indicating that drugs affect one of the last assembly steps. The 5 S rRNA was previously shown to form contacts with segments of domains II and V of 23 S rRNA. All the antibiotics which can functionally compensate for the lack of 5 S rRNA during subunit reconstitution interact simultaneously with the central loop in domain V (which is known to be a component of peptidyl transferase center) and a loop of the helix 35 in domain II of 23 S rRNA. It is proposed that simultaneous interaction of 5 S rRNA or of antibiotics with the two domains of 23 S rRNA is essential for the successful assembly of ribosomal peptidyl transferase center. Consequently, one of the functions of 5 S rRNA in the ribosome can be that of assisting the assembly of ribosomal peptidyl transferase by correctly positioning functionally important segments of domains II and V of 23 S rRNA.

Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA.

Functionally active large ribosomal subunits of thermophilic bacterium Thermus aquaticus have been assembled in vitro from ribosomal proteins and either natural or in vitro-transcribed 23S rRNA and 5S rRNA. Sedimentation properties of reconstituted subunits were similar to those of native ribosomal 50S subunits. Subunits reconstituted with in vitro-transcribed rRNAs exhibited high activity in the peptidyl transferase assay and in a poly(U)-dependent cell-free translation system (22 and 30%, respectively, compared to that of native 50S subunits). Catalytic activity of reconstituted subunits critically depended on the presence of 5S rRNA. rRNA mutations known to affect functions of the native ribosome produced similar effects in reconstituted T. aquaticus 50S subunits. Subunits assembled with in vitro-transcribed T. aquaticus 23S rRNA containing the G2267A mutation (G2252A in Escherichia coli), which interferes with binding of peptidyl-tRNA in the ribosomal P-site, showed drastically reduced peptidyl transferase activity, whereas clindamycin resistance mutation A2084G (A2058G in E. coli) rendered assembled subunits tolerant to clindamycin inhibition. Thus, reconstitution of functional subunits with in vitro-transcribed rRNA makes possible the use of in vitro genetics for mutational analysis of 23S rRNA functions in translation. In addition, the ability to assemble catalytically active 50S subunits from the rRNA transcript lacking any posttranscriptional modifications clearly demonstrates that modified nucleotides in 23S rRNA are dispensable for the principal activities of the ribosome.

Characterization of functionally active subribosomal particles from Thermus aquaticus.

Peptidyl transferase activity of Thermus aquaticus ribosomes is resistant to the removal of a significant number of ribosomal proteins by protease digestion, SDS, and phenol extraction. To define the upper limit for the number of macromolecular components required for peptidyl transferase, particles obtained by extraction of T. aquaticus large ribosomal subunits were isolated and their RNA and protein composition was characterized. Active subribosomal particles contained both 23S and 5S rRNA associated with notable amounts of eight ribosomal proteins. N-terminal sequencing of the proteins identified them as L2, L3, L13, L15, L17, L18, L21, and L22. Ribosomal protein L4, which previously was thought to be essential for the reconstitution of particles active in peptide bond formation, was not found. These findings, together with the results of previous reconstitution experiments, reduce the number of possible essential macromolecular components of the peptidyl transferase center to 23S rRNA and ribosomal proteins L2 and L3. Complete removal of ribosomal proteins from T. aquaticus rRNA resulted in loss of tertiary folding of the particles and inactivation of peptidyl transferase. The accessibility of proteins in active subribosomal particles to proteinase hydrolysis was increased significantly after RNase treatment. These results and the observation that 50S ribosomal subunits exhibited much higher resistance to SDS extraction than 30S subunits are compatible with a proposed structural organization of the 50S subunit involving an RNA "cage" surrounding a core of a subset of ribosomal proteins.