Comparative cytogenetic study of two sister species of Iberian ground voles, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus (rodentia, cricetidae)

The two Iberian species of pine voles, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus of the subfamily Arvicolinae (Cricetidae, Rodentia), were compared after G- and C-banding and chromosomal mapping of ribosomal RNA genes (rDNA), telomeric repeats, and satellite DNA Msat-160. Notwithstanding their close relationship (one sister group in phylogenetic analyses) and sharing of the diploid and fundamental chromosome numbers, the 2 species show notable differences in the sex chromosome morphology, the number and distribution of rDNA sites, constitutive heterochromatin and satDNA patterns. The only telomeric repeats showed normal, all-telomeric, distribution in karyotypes of both species. The data are discussed with regard to interspecific and intrageneric variation of the analyzed characters and the chromosomal evolution in the genus Microtus.     

Variable arrangement of 5S ribosomal genes within the ribosomal DNA repeats of arthropods.

The polymerase chain reaction and hybridization to genomic blots were used to investigate whether the previously observed inclusion of 5S ribosomal RNA genes in the 28S-18S ribosomal DNA intergenic regions of some crustacean species (copepods) could also be detected in other arthropods. Such an arrangement was found not only in other calanoid copepod species but also in a cirriped, an euphausid, and a spider. It is interesting that species from two different calanoid copepod genera do not have this type of arrangement. We conclude that the inclusion of 5S ribosomal RNA genes within the ribosomal DNA repeats has probably occurred repeatedly during the evolution of arthropod species and that the mechanism(s) responsible for these insertions could also be responsible for their loss.     

Comparative Molecular Cytogenetic Analysis of Two Congeneric Species, <Emphasis Type="Italic">Mugil Curema</Emphasis> and <Emphasis Type="Italic">M. Liza</Emphasis> (Pisces,Mugiliformes), Characterized by Significant Karyotype Diversity

Two congeneric mullet species, Mugil liza and M. curema, respectively with an all-uniarmed and an all-biarmed karyotype, were cytogenetically studied by base-specific fluorochrome staining and FISH-mapping of 45S and 5S ribosomal RNA genes (rDNA) and the (TTAGGG)_n telomeric repeats. Whereas 45S rDNA sites might be homeologus in the two species, 5S rDNA sites are not, as they are localized on chromosome arms of different size. In both species, the (TTAGGG)_n telomeric probe hybridized to natural telomeres and was found scattered along the NORs. In metacentric chromosomes of M. curema, no pericentromeric signals of the telomeric probe were detected. Data are discussed in relation to the karyotype evolution in Mugilidae and to the mechanisms and the evolutionary implications of Robertsonian rearrangements in M. curema.     

Oligonucleotide sequences of pancreatic and T1 ribonuclease digests of 5S ribosomal RNA from mouse cells.

Oligonucleotides produced by complete pancreatic and T1 RNase digestion of 5S ribosomal RNA from a mouse hepatoma, MH 134, have been separated with two-dimensional electrophoresis and their nucleotide sequences determined. Except for the presence of a 5'-terminal diphosphate, these nucleotide sequences were identical with those of KB cells, confirming the identity of the primary structure of 5S RNA between these animals. Both oligonucleotide patterns produced with these enzymes from 5S RNA of the liver were also identical with those of the hepatoma. All these agree with the strong conservation of 5S RNA genes in animal species. However, when 5S ribosomal RNA was extracted from ribosomes which were prepared from microsomal pellet, pancreatic RNase digest contained two trinucleotides (A-G-Cp and G-A-Cp) that were not found in 5S RNA prepared with a one-step procedure. It was concluded that different isolation procedure might indeed cause artifactual fragments on enzymatic digestion due to internal nicks produced during isolation. The significance of 5'-terminal diphosphate in relation to the biosynthesis of 5S ribosomal RNA is also discussed.     

Karyotype structure, supernumerary chromosomes and heterochromatin distribution suggest a pathway of karyotype evolution in Dactylorhiza (Orchidaceae)

The relationships between Dactylorhiza romana and D. saccifera from southern Italy were analysed. These two species, both with 2 n = 2 x = 40 chromosomes and belonging to different sections of the genus, were distinguishable on the basis of karyotype structure and heterochromatin amounts and distribution. Their C-banded karyotypes differed considerably. D. saccifera showed most chromosomes with banded regions in the short arms, whereas in D. romana the bands were located mostly at telomeric regions of longer arms. Several individuals of D. romana had one or two very large heterochromatic supernumerary chromosomes. Based on evidence resulting from karyotype structure and heterochromatin distribution in the two species and on the genetic distances derived from the comparison of ITS sequences, it is suggested that D. romana represents a primitive form with respect to D. saccifera and is a possible intermediate step in the evolution of the genus Dactylorhiza from the 42-chromosome Orchis group. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 138 , 85–91.     

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.     

3D organization of interphase fibroblast nuclei in two closely related shrew species, Sorex granarius and S. araneus, differed by the structure of chromosome termini

To study 3D organization of fibroblast interphase nuclei in two sibling shrew species, Sorex araneus from Cordon race and S. granarius, FISH with probe to telomeric and rDNA repeats, and immunofluorescence with ANA CREST and antibodies to nucleolus protein B23 were used. Karyotypes of studied species are composed of near identical chromosomal arms and differ by the number of metacentrics and the structure of terminal chromosome regions. The large telomeres containing on the average 218 kbp of telomere repeats characterize the short arms in all of 32 S. granarius acrocentrics. Telomere repeats in them alternate with nbosomal repeats. These regions also contain active NORs. In contrast, active NORs in S. araneus are localized at the terminal regions of 8 chromosomal arms (Zhdanova et al., 2005, 2007b). We have shown that telomere associations of chromosomes and contacts of a part of telomere clusters with inner nuclear membrane and nucleolus characterize interphase nuclei of both S. granarius and S. araneus. Moreover, the partial colocalization of telomere and ribosomal clusters, and spatial nearness of centomeric and telomeric regions were revealed in the interphase nuclei of S. granarius. Evidently, only those ribosomal clusters that contain a number of active ribosomal genes display connection with nucleolus. The stripping of nucleolus materials during transition of fibroblasts to mitosis and the role of B23 protein in this process has been studied.     

The complete mitochondrial genome of Solen strictus (Bivalvia: Solenidae)

In this paper, we determined the complete mitochondrial genome of Solen strictus (Bivalvia: Solenidae). The whole mitogenome of S. strictus is 16,535?bp in length with a base composition of 21.7% A, 41.0% T, 25.6% C, and 11.7% G and contains 12 protein-coding genes (atp8 is missing), 2 ribosomal RNA genes, 22 transfer RNA genes, and a major non-coding region (MNR). Some peculiar patterns including tandem repeats and microsatellite-like elements are found in the MNR of S. strictus.     

Evaluation of ribosomal RNA gene restriction patterns for the classification of Bacillus species and related genera

AIMS: To identify Bacillus species and related genera by fingerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classification of Bacillus species and related genera. METHODS AND RESULTS: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from five newly created Bacillus-related genera, and five species from five phenotypically related genera were tested. A total of 77 distinct 16S rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene fingerprinting and the 23S and 5S rRNA genes fingerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. CONCLUSIONS: The Bacillus species along with the species from related genera were identified successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, but does not duplicate, phylogenies based on 16S rRNA gene sequences.     

Unusual sequences,homologous to 5S RNA,in ribosomal DNA repeats of the nematodeMeloidogyne arenaria

Summary There are sequences homologous to 5S ribosomal RNA in the ribosomal DNA (rDNA) repeats of the plant-parasitic nematodeMeloidogyne arenaria. This is surprising, because in all other higher eukaryotes studied to date, the genes for 5S RNA are unlinked to and distinct from a tandem rDNA repeat containing the genes for 18S, 5.8S, and 28S ribosomal RNA. Previously, only prokaryotes and certain lower eukaryotes (protozoa and fungi) had been found to have both the larger rRNAs and 5S rRNA represented within a single DNA repeat. This has raised questions on the organization of these repeats in the earliest cell (progenote), and on subsequent evolutionary relationships between pro- and eukaryotes.Evidence is presented for rearrangements and deletions withinMeloidogyne rDNA. The unusual life cycles (different levels of ploidy, reproduction by meiotic and mitotic parthenogenesis) of members of this genus might allow rapid fixation of any variants with introduced 5S RNA sequences. The 5S RNA sequences inMeloidogyne rDNA may not be expressed, but their presence raises important questions as to the evolutionary origins and stability of repeat gene families.     

Cytogenetic and molecular characterization of the Abies alba genome and its relationship with other members of the Pinaceae

Genome size, karyotype structure, heterochromatin distribution, position and number of ribosomal genes, as well as the ITS2 sequence of the internal transcribed spacer (ITS) were analysed in silver fir (Abies alba Mill.). The analysis also included characterization of the Arabidopsis-type of telomeric repeats in silver fir and in related species. The results were compared with results from other species of the Pinaceae, to evaluate phylogeny and chromosomal and molecular evolution in the Pinaceae. Integrated chromosomal data provided insights into chromosome and karyotype evolution in the Pinaceae. The evolutionary trend for GC-rich heterochromatic blocks seems to involve loss of blocks that are not associated with rDNA. Similarly, numerous large blocks of interstitial plant telomeric repeats that are typical for all analysed species of the genus Pinus were not observed in the evolutionarily younger genera, such as Abies, Picea and Larix. On the contrary, the majority of telomeric sequences in these three genera appeared confined to the chromosome ends. We confirmed the current position of Abies and Tsuga in subfamily Abietoideae and the position of Pinus in the subfamily Pinoideae based on ITS2 sequences. Pseudotsuga is placed together with Larix into the subfamily Laricoideae. We conclude that the current position of the genus Picea in the subfamily Abietoideae should be reconsidered and, possibly, the genus Picea should be reclassified as a separate subfamily, Piceoideae, as recently proposed.     

Ribosomal Gene Structure, Variation and Inheritance in Maize and Its Ancestors

We have examined the structure of nuclear genes coding for ribosomal RNAs in maize and its wild relatives, the teosintes and Tripsacum. Digestion of the rDNA (genes coding for 18S, 5.8S and 26S RNAs) with 15 restriction endonucleases (with six base pair recognition sites) yields essentially a single map for the approximately 10,000 repeat units within an individual plant or species. Both length and site variation were detected among species and were concentrated in the intergenic spacer region of the rDNA repeat unit. This result is in agreement with patterns of rDNA change observed among wheat and its relatives (Triticeae), and among vertebrate species. Digestion of these nuclear DNAs with BamHI and subsequent hybridization with a 5S RNA gene-specific probe allowed determination of the size of the 5S gene repeat unit in maize, teosintes, and Tripsacum. Groupings in the genus Zea were characterized by distinct repeat unit types five Tripsacum species examined shared a 260 base pair major repeat unit type. Additionally, several other restriction endonuclease cleavage patterns differentiated among the 5S DNAs within the genus Zea. The rDNA and 5S DNA restriction site variation among the species can be interpreted phylogenetically and agrees with biochemical, karyotypic, and morphological evidence that places maize closest to the Mexican teosintes. For both gene arrays, contributions from each parental genome can be detected by restriction enzyme analysis of progeny from crosses between maize and two distantly related teosintes, Zea luxurians or Zea diploperennis, but certain teosinte arrays were underrepresented in some of the hybrids.     

Patterns of rDNA and telomeric sequences diversification: contribution to repetitive DNA organization in Phyllostomidae bats

Chromosomal organization and the evolution of genome architecture can be investigated by physical mapping of the genes for 45S and 5S ribosomal DNAs (rDNAs) and by the analysis of telomeric sequences. We studied 12 species of bats belonging to four subfamilies of the family Phyllostomidae in order to correlate patterns of distribution of heterochromatin and the multigene families for rDNA. The number of clusters for 45S gene ranged from one to three pairs, with exclusively location in autosomes, except for Carollia perspicillata that had in X chromosome. The 5S gene all the species studied had only one site located on an autosomal pair. In no species the 45S and 5S genes collocated. The fluorescence in situ hybridization (FISH) probe for telomeric sequences revealed fluorescence on all telomeres in all species, except in Carollia perspicillata. Non-telomeric sites in the pericentromeric region of the chromosomes were observed in most species, ranged from one to 12 pairs. Most interstitial telomeric sequences were coincident with heterochromatic regions. The results obtained in the present work indicate that different evolutionary mechanisms are acting in Phyllostomidae genome architecture, as well as the occurrence of Robertsonian fusion during the chromosomal evolution of bats without a loss of telomeric sequences. These data contribute to understanding the organization of multigene families and telomeric sequences on bat genome as well as the chromosomal evolutionary history of Phyllostomidae bats.     

Divergence of primate ribosomal RNA genes as assayed by restriction enzyme analysis

Primate ribosomal RNA (rRNA) genes have been compared by restriction endonuclease mapping. In all species examined, the restriction map of the reiterated ribosomal DNA is simple (within the limits of detection by hybridization with rRNA) and is consistent with a high degree of homogeneity among the repeats. Within a species, all members have similar rDNA restriction patterns. However, different species of primates have distinctly different rDNA restriction maps; even chimpanzee and man can be discerned by their rDNA restriction patterns. Possible mechanisms for maintenance of homogeneity of the rDNA repeats within a species, while allowing divergence among closely related species, are discussed.     

Comparative cytogenetic analysis of the “Sylvaemus” group of Apodemus (Rodentia, Muridae): A. sylvaticus from Sicily and A. flavicollis from the central Apennines

Chromosome mapping of three common markers, ie major and 5S rRNA genes (rDNA) and telomeric repeats, and conventional chromosome bandings were applied to two sibling species, Apodemus sylvaticus Linnaeus, 1758 and A. flavicollis Melchior, 1834, to further investigate intra- and interspecific karyological differentiation in the genus Apodemus. A slight variation of the rDNA-patterns was detected between the two Apodemus species. In both of them, the major NORs were located on autosome pairs 8, 11, 12 and 22, while the two other rDNA sites detected on chromosomes 7 and 21 were variable in, respectively, A. sylvaticus and A. flavicollis. Several tiny rDNA sites were present on the sex chromosomes in both species, but their incidence was lower in A. flavicollis. Single 5S rDNA chromosomal sites were conserved on chromosome pair 20. No interstitial sites of telomeric repeats were present in either species. In the Sicilian population of A. sylvaticus, the constitutive heterochromatin pattern corresponded to the “sylvaticus-E1” cytotype, while A. flavicollis had a species-specific pattern restricted to centromeres of all chromosomes. The results are discussed in relation to cytogenetic data available for the genus, with emphasis on the Sylvaemus group/subgenus.     

Evolution of rRNA gene clusters and telomeric repeats during explosive genome repatterning in TATERILLUS X (Rodentia, Gerbillinae)

A survey of 28S and 5S rRNA gene clusters, and telomeric repeats was performed using single and double FISH in the Taterillus genus (Rodentia, Muridae, Gerbillinae). Taterillus was previously demonstrated to have undergone a very recent and extensive chromosomal evolution. Our FISH results demonstrate that rRNA genes can vary in location and number irrespective of the phylogenetic relationships. Telomeric repeats were detected in pericentromeric and interstitial regions of several chromosomes, thus providing nonambiguous evolutionary footprints of Robertsonian and tandem translocation events. These footprints are discussed in reference to the molecular process of these karyotypical changes. Also, examples of colocation of rDNA clusters and telomeric repeats lend support to their possible involvement in nucleolus formation. Finally, the presence of rRNA genes, and the extensive amplification of telomeric repeats at specific loci within a double X-autosome translocated element which were not observed on the homologous Y1 and Y2, served as basis for an epigenomic hypothesis on X-autosome translocation viability in mammals.     

Tandemly arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces cerevisiae.

Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA. These genes are arranged in a single tandem array of 100 repeats. Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene. Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA. The coding sequence of this gene is different from that of the "normal" 5S gene in three positions located at the 3' end of the gene.     

Three-dimensional organization of interphase fibroblast nuclei in two closely related shrew species (<Emphasis Type="Italic">Sorex granarius</Emphasis> and <Emphasis Type="Italic">Sorex araneus</Emphasis>) differing in the structures of their chromosome termini

A FISH with a probe for telomeric and rDNA repeats and immunofluorescence with ANA CREST and antibodies to nucleolae protein B23 were used to study the three-dimensional (3D) organization of fibroblast interphase nuclei in two shrew twin species, Sorex granarius and Sorex araneus, of the Cordon race. Karyotypes of these species are composed of nearly identical chromosomal arms and differ in the number of their metacentrics and the structures of their terminal chromosome regions. In the short arms of S. granarius, 32 of the acrocentrics have large telomeres that contain an average of 218 kb telomere repeats, which alternate with ribosomal repeats. These regions also contain active nucleolar organizing regions (NORs). In contrast, in active NORs in S. araneus are localized at the terminal regions of 8 chromosomal arms (Zhdanova et al., 2005; 2007b). Here, we show that associations of chromosomes by telomeres and the contact of a part of the telomere clusters with the inner nuclear membrane and nucleolus characterize the interphase nuclei of both Sorex granarius and Sorex araneus. We also reveal the partial colocalization of telomere and ribosomal clusters and the spatial proximity of centomeric and telomeric regions in the interphase nuclei of S. granarius. It appears that only ribosomal clusters containing a sufficient number of active ribosomal genes exhibit a connection with the nucleolus. Nucleolus disassembly during the fibroblastís transition to mitosis and the role of the B23 protein in this process have been studied.