Non-canonical ribosomal DNA segments in the human genome, and nucleoli functioning.

Ribosomal DNA (rDNA) in the human genome is represented by tandem repeats of 43 kb nucleotide sequences that form nucleoli organizers (NORs) on each of five pairs of acrocentric chromosomes. RDNA-similar segments of different lengths are also present on (NOR)(-) chromosomes. Many of these segments contain nucleotide substitutions, supplementary microsatellite clusters, and extended deletions. Recently, it was shown that, in addition to ribosome biogenesis, nucleoli exhibit additional functions, such as cell-cycle regulation and response to stresses. In particular, several stress-inducible loci located in the ribosomal intergenic spacer (rIGS) produce stimuli-specific noncoding nucleolus RNAs. By mapping the 5'/3' ends of the rIGS segments scattered throughout (NOR)(-) chromosomes, we discovered that the bonds in the rIGS that were most often susceptible to disruption in the rIGS were adjacent to, or overlapped with stimuli-specific inducible loci. This suggests the interconnection of the two phenomena - nucleoli functioning and the scattering of rDNA-like sequences on (NOR)(-) chromosomes.

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2.

In eukaryotes, mature rRNA sequences are produced from single large (45S) precursor (pre-rRNA) as the result of successive removal of spacers through a series of rapid and intricate actions of endo- and exonucleases. The excision of internal transcribed spacer (ITS2), a eukaryotic-specific insertion, remains the most elusive processing step. ITS2 is the element mandatory for all eukaryotic pre-rRNAs that contain at least three processing cleavage sites for precise 5.8S and 28S formation. Conserved core sequences (cis-elements) binding to trans-factors provide for precise rRNA processing, whereas rapidly diverging regions between the core sequences preserve internal complementarity, which guarantees the spatial integrity of ITS2. Characteristic differences in the formation of such insertions during evolution should reflect the relationships between taxa. The phylogeny of the reptiles and the relationships between taxa proposed by scientists are controversial. To delineate the structural and functional features preserved among reptilian ITS2s, we cloned and sequenced 58 ITS2s belonging to four reptile orders: Squamata, Crocodilians, Aves, and Testudines. We studied the subsequent alignment and folding of variable regions. The sizes and packing of the loop-stems between conserved consensus segments in reptiles vary considerably between taxa. Our phylogenetic trees constructed on the basis of the reptile ITS2s primary structural alignments revealed a split between Iguania clade and all other taxa. True lizards (suborder Scleroglossa) and snakes (suborder Serpentes) show sister relationships, as well as the two other reptilian orders, Crocodilia+Aves and Testudines. In summary, our phylogenetic trees exhibit a mix of specific features deduced or, to the contrary, rejected earlier by other authors.

Evolutionary divergence of the pre-promotor region of ribosomal DNA in the great apes.

The human ribosomal intergenic spacer (rIGS) differs considerably on nucleotide sequence and regulatory elements positioning from their counterparts in the mouse, rat and Xenopus laevis. In the present study, we have PCR amplified, cloned and sequenced the rIGS fragments of about 4.5 kb length, located approximately 2 kb upstream of the rRNA transcription start point for the great apes, Pan paniscus, Pan troglodytes, Gorilla gorilla and Pongo pygmaeus. Alignment of the primates' orthologic nucleotide sequences reveals high extent of similarity, with the exception of highly repetitious region between the two Alu repeats, nearest to the onset of transcription. Data obtained have been analyzed for further understanding of the evolution of repetitive sequences. We have also shown, that MARs/SARs distribution patterns in the pre-promoter rIGSs of the great apes and the mouse are surprisingly similar in spite of an absence of similarity in the primary structure and regulatory elements organization in the region under study.