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For the last few decades a wealth of evidence have been obtained (including new fossils, and new nucleotide sequences) which provide a way of revising the current evolutionary theories. The primary structures of a number of orthological regions from a variety of genomes have been evaluated. Full sized genomic sequences for some organisms, have been detected. This review is devoted to modern concepts of the tetrapod's rise and diversification, and of the contemporary vertebrates' class formation.

A first report on structural organization of ribosomal DNA arrays in some members of the order Squamata is presented. The data obtained point to unusually small (for vertebrates) size of the rDNA repetitive unit (approximately, 10 to 15 kb) in the lizard species examined. Analysis of BAC library of Uta stansburiana (Iguania) showed that haploid genome of this lizard contained a single cluster, consisting of about ten rDNA repeats. Determination of the extent of rDNA unit repetition in some other representatives of the order Squamata, using the method of comparative real-time PCR, showed that the number of rDNA units varied from one or several dozens in Iguanina to several hundred repeats in Scincomorpha and Varonoidea. The results are discussed in terms of an ambiguous position of the family Iguania on the evolutionary trees constructed based on morphological and molecular data.

During the pre-rRNA cleavage pathway, the excision of ITS2, a eukaryotic specific insertion, remains the most elusive processing step, even in yeast. Comparison of the ITS2 sequences in different organisms permits to reveal conservative, presumably functionally important elements as well as obtain new information about ITS2 divergence in evolution. We have cloned and sequenced the ITS2 of three lizard species, Agama caucasia (Agamidae), Darevskia armeniaca and Lacerta strigata (Lacertidae) and detected in them a set of specific and conservative structural elements employing secondary structure consensus for vertebrate ITS2. Furthermore, we have performed an alignment and comparative analysis of the ITS2 sequences for the two lizards families. It enables us to propose that modern lizard species formation in evolution was accompanied by ITS2 duplication in the rDNA of their common progenitors.

It is a common point of view today that tandem ribosomal genes are subject to concerted evolution, a process that promotes homogeneity among the many copies through the mechanisms of unequal homologous exchange and gene conversion. These mechanisms can lead to opposite results: they can correct and eliminate new variants and they also can promote the spread of new gene variants throughout individual gene clusters among homologous and non homologous chromosomes. A number of experiments performed to decide which of these mechanisms is more important have yielded contradictory answers to this question. In this work we have cloned and partially sequenced 36 PCR-amplified copies of the human rIGS segment 22763-23523 apart from the transcription start point, obtained from the individual genome. This segment is enriched by (G)n and (AG)n clusters and enters into 2kb LR2 element of the human rIGS. Comparative analysis showed that absolutely identical sequences are absent among 36 inserts sequenced. The 26 clones differed only on a number of repeating elements in the paralogous (G)n and (AG)n clusters. The last ten clones differed not only on a number of cluster repeating elements, but contained insertions and deletions of distinct sizes, which do not correspond to the human rIGS polymorphism variants described earlier.

Owing to a great progress in studying the human genome, its euchromatic portion is almost completely sequenced; the complete sequence is still unknown only for pericentric and telomeric regions and short arms of acrocentric chromosomes. Extended satellite blocks and segment duplications located in these regions substantially hinder the joining of the sequenced fragments and construction of the full-length genome map. The sequence was established for a 1.5-kb human chromosome 13 subtelomeric region, which is about 10 kb away from the rDNA cluster, and deposited in GenBank under accession no. AF478540. The region showed 83-84% homology to the pericentric region of human chromosome 19, and contained short fragments homologous to the pericentric region of human chromosome 13. The results may contribute to the current revision of genome evolution concepts in view of numerous segment duplications revealed.

Investigation of randomly cloned genomic and chromosome-specific sequences of ribosomal DNA (rDNA) from different organisms show that different regions of this long repeat unit evolve at different rates. This proves to be true not only with regard to evolutionary variability of transcribed and nontranscribed intergenic (spacer) regions of rDNA. The intergenic spacer (rIGS) of human ribosomal DNA contains both highly variable and more conservative regions with putative regulatory functions. In the present study a comparative analysis of some segments of the rIGS pre-promoter (regulatory) region in human and pygmy chimpanzee (Pan paniscus) was carried out. For these purposes, the corresponding DNA fragments were amplified in PCR with oligonucleotide primers specific to human rIGS and sequenced. Our results show that at the background of substantial structural similarity of these regions in man and chimpanzee, i.e., the presence of highly homologous sequences and similar repetitive units, there are substantial differences between them. These differences are associated with point mutations, insertions, deletions, and complex structural rearrangements.

Due to the complete absence of ribosomal DNA (genetic symbol bb-), the Xbb- chromosome of Drosophila is lethal both in homozygous conditions and in compound with the Xbb- chromosome. However, in the cross between the C(1)RM/Ybb- females and the Xbb-/BSYbb+ males, characterized by the development of lethal Xbb-/Ybb- zygotes, two fertile males were detected. These males possessed all the markers of the Xbb- chromosome but lacked the Y chromosome BS marker. Genetic analysis of their progeny showed that genes responsible for restoration of viability and fertility of these exceptional males were associated with the X chromosome. The crossover tests showed that in one case these genes were tightly linked to the w locus (the bbAM1 allele), and in the second case they were located 12.6 map units to the right of the Tu locus (the bbAM7 allele). It has also been shown that the bb locus was transposed to the X chromosome within the short arm of Y chromosome. Transposition of the BSYbb+ chromosome-specific rDNA sequences to the X chromosome was confirmed by means of Southern blotting. These data indicate that replacement of the bb locus is realized by transposition rather than recombination.

Fragments overlapping the tPA gene and its 5'- and 3'-flanking regions were isolated from human liver DNA library cloned in lambda Charon4A vector. A BglII fragment comprising the 3' end and the adjacent genomic region (total length 3.7 kb) was subcloned in plasmid pUC19 and its restriction map was determined. The nucleotide sequence of the 5' region of this fragment was compared with the 3' end region of the tPA gene and the corresponding regions of five published variants of tPA mRNA cDNA from different tissues; discrepancies in seven positions were revealed, which might be caused by intragenomic polymorphism.

The frequency of specific mini- and micro-satellites known also as short tandem repeated sequences (STR) in the human 13 chromosome was estimated by hybridization of STR core oligonucleotides to recombinant cosmid clones transferred to a grid from a human 13 chromosome specific cosmid library ICRF Lawrist 4 C108 (DN L4/HS 13). Oligonucleotides: M13 and Jeffreys minisatellite core sequences and micro-satellite core sequences (TCC)5, (CAC)5, and (GACA)4 were [gamma-32P] end labeled and hybridized to membrane filters carrying good ordered cosmid clones. It was shown that great number of all these mini- and micro-satellite copies (besides of Jeffreys minisatellite) are spread independently along the 13th chromosome. It was also estimated that two or more (GACA)n blocks present in the same cosmid (i.e. on the stretch of 40-50 kb) forming similar groups of clustered micro-satellites. The interesting peculiarity has been recorded that some (GACA)n+ cosmids are also hybridizable to conservative 28SrDNA 3'-fragment that indicates that (GACA)n localization in the nucleoli area. As the result of it we began the creation of a new highly polymorphic markers collections for these chromosome.

The restriction map of bovine 28S rRNA gene and adjacent 5'-spacer region was determined. The high level of intragenomic and population length polymorphism of EcoRI-BamHI restriction fragment was demonstrated to originated from the 3'-end of 28S rDNA and 5'-spacer of rDNA repeat. This polymorphism is more pronounced than the ones revealed in human and murine rDNA repeats and could be compared with genomic fingerprints obtained by M13 or minisatellite DNA hybridization probes. From family blot-panel analysis we concluded that in progeny only parental sets of length variants were inherited and that the copy number of definite variants does not change in the progeny as well. From these results it was proposed that the definite sets of linked in genome length variants are inherited independently from each other.

The fraction of low molecular weight RNAs from the unfertilized eggs and liver of the loach (Misgurnus fossilis) was analyzed by electrophoresis in polyacrylamide gel. The 5S rRNA from unfertilized eggs differs in its electrophoretic mobility from liver 5S rRNA in the presence of 7 M urea at 20 degree C. The electrophoretic mobilities of these two rRNAs are identical at 50 degree C. These data indicate that the loach, just as the amphibians and other teleost fishes, has two types of 5S rRNA, viz. the oocyte and somatic types. Since these molecules are of identical size, their different electrophoretic mobility should be ascribed to variations in their structure. The unfertilized eggs and liver of Misgurnus fossilis contain, apart from the major fraction, a minor fraction of 5S rRNA belonging to another type. The content of 5S rRNA in preparations of the ribosomes from unfertilized eggs is much lower comparing to tht of 5.8s rRNA than in preparations of the ribosomes from somatic tissues (muscles). A possible functional significance of different 5S rRNA types (oocyte and somatic) is discussed.

About 5% of the loach (Misgurnus fossillis L.) DNA reassociates at Cot values virtually equal to zero. 50% of the reassociate are resistant to nuclease S1 treatment and reveal the properties of double-stranded structure when chromatographed on hydroxyapatite. Some proofs of palindromic (hair-pin) nature of this fraction have been obtained. An introduction of nicked scissions into the palindromic DNA by pancreatic DNAse treatment under pessimal conditions made it possible to investigated reassociation kinetics of the nucleotide sequences forming palindromes. Two different types of nucleotide sequences appeared to exist in the palindromic fraction with repetition frequencies (ni) equal to 3 X 10(2) and about 1. Homologies were revealed between these sequences and the fraction of corresponding repetition frequency of the main part of the genome. Adjacent sequences contain repetitive regions with ni equal to 10(5) and 5 X 10(3). On the basis of the data obtained some conclusions were made about the distribution of usual and inverted repetitions in the loach genome.

DNA renaturation kinetics was measured for the genomes of normal (spleen) and malignant (plasmacytoma) mouse tissues and for DNA from liver, sperm and developing embryos of the loach. It has been shown that the measuring of DNA renaturation kinetics makes it possible to reveal differences in the content of certain fractions of the repetitions in the genomes of different species. Moreover, differences in the distribution of the repetitions between hetero- and euchromatine can be identified. Loach embryo DNA (blastula stage) was shown to contain larger amount of the fraction renaturing at Cot less than 10(-2) as compared to liver and sperm DNAs (by 5%). An enrichment with respect to the intermediate repetitions (10(-2) less than Cot less than 10(2)) was found in the mouse plasmacytoma DNA as compared to the spleen DNA. The nature of these distinctions is discussed.

The organization of 5S rRNA genes in the loach Misgurnus fossilis L. was studied. 5S rDNA was cloned in Escherichia coli using the HindIII-fragments of loach genome DNA fused into the plasmid pBR322 restricted at the same site. The recombinant clones were tested by colony hybridization. The presence of the 5S rDNA structural sequences in cloned fragments was determined by the Southern procedure of hybridization with 5S [32P]rRNA of the loach and transcription in the oocyte test system. It was found that the size of 5S rDNA repetitive units corresponds to 240-250 bp and 450-460 bp. By the CsCl centrifugation and restriction analysis it was shown that the 5S genes in the loach genome are arranged in clusters (5-30 repeats per cluster), the smaller repeat was found to contain one coding sequence while the larger repeat contains two coding sequences of 5S rDNA.

The ribosomal DNA of the teleost fish--loach has been isolated from sperm DNA by CsCl density gradient centrifugation. The rDNA sediments on density gradients by two heavy satellites beta = 1.715 and greater than 1.720. The DNA of the first satellite (1.715) was separated and treated by restrictases EcoRI and BamHI. It was shown that there are two EcoRI-sites in rDNA of loach, locating in 18S and 28S rRNA coding sequences. From tandem of repeating ribosomal genes EcoRI cuts out the fragment with homogeneous length-3 megadaltons (constant fragment) and heterogeneous population of fragments 11-13 megadaltons (major) and 7-8 megadaltons (minor fraction). The constant fragment contains mostly 28S coding sequence, and the heterogeneous fragment--18S coding sequence. The data indicate that the ribosomal genes of the loach as well as other higher eucaryotes were organized in genome as tandem of repeating units with heterogeneous length (10-16 megadaltons, 14.5-24 kb) due to heterogeneity of the length of nontranscribed spacer.

The mechanism of pre-rRNA processing is still unknown. In this paper we discuss a possible role of the secondary structure of the internal transcribed spacer region (ITS-1 and ITS-2) in processing of 41S pre-rRNA. Potential possibilities for double strand structure formation in ITS-1 and ITS-2 and its rearrangement in the course of processing of 41S pre-rRNA with involvement of U3 RNA were analyzed for a number of species. We have concluded that this rearrangement is the necessary stage in 41S pre-rRNA processing when every functionally designated area (for mature 18S, 28S and 5,8S rRNAs, for ITS-1, ITS-2 and 21S pre-rRNA) has to be organized into an individual domain. In these strongly structured domains 5'- and 3'-ends are spatially brought together whereas processing sites are localized between these compactised areas.

A fragment of the loach (Misgurnus fossilis L.) ribosomal operon containing 5.8S rDNA and adjacent regions of the internal transcribed spacer (ITS-1, and ITS-2) was sequenced. The 5'-terminal sequencing in 5.8S rDNA was corrected by analysing the primary structure of the loach 5.8S rRNA. This RNA was shown to be presented by three types of molecules; one of these was shorter by 4 nucleotides at the 5'-end because of the processing site being shifted in the rRNA precursor. The two other types differed in the 5'-terminal nucleotide (UMP or AMP). In the cloned fragment under study, the sequence of 5.8S rDNA has TMP at the 5'-terminus. The known nucleotide sequences of 5.8S rRNAs were compared in eukaryotes; as a result, conservative regions were revealed at the sites of molecule modification. All the 5.8S rRNAs of the vertebrates studied were found to have coincidences in the localization of nucleotide substitutions and other mutations (inversions and deletions). The authors propose a model for the secondary structure of ITS-1 and ITS-2 in the region of 5.8S rRNA processing.

Human ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene was used for detecting standard restriction fragments' length polymorphism (RFLPs) in the non-transcribed spacer. The conditions for hybridization of rDNA probe which eliminate cross hybridization of parts of 28S rRNA gene were developed. A test for detecting incompletely restricted DNA was also developed which may be used in experiments for detecting new RFLPs. It was found that a set of standard RFLPs was identical in various human tissues for one individual. Frequency of standard RFLPs in the non-transcribed spacer of human rRNA gene clusters was calculated.