Evolving Together: Cassandra Retrotransposons Gradually Mirror Promoter Mutations of the 5S rRNA Genes.

The 5S rRNA genes are among the most conserved nucleotide sequences across all species. Similar to the 5S preservation we observe the occurrence of 5S-related nonautonomous retrotransposons, so-called Cassandras. Cassandras harbor highly conserved 5S rDNA-related sequences within their long terminal repeats, advantageously providing them with the 5S internal promoter. However, the dynamics of Cassandra retrotransposon evolution in the context of 5S rRNA gene sequence information and structural arrangement are still unclear, especially: (1) do we observe repeated or gradual domestication of the highly conserved 5S promoter by Cassandras and (2) do changes in 5S organization such as in the linked 35S-5S rDNA arrangements impact Cassandra evolution? Here, we show evidence for gradual co-evolution of Cassandra sequences with their corresponding 5S rDNAs. To follow the impact of 5S rDNA variability on Cassandra TEs, we investigate the Asteraceae family where highly variable 5S rDNAs, including 5S promoter shifts and both linked and separated 35S-5S rDNA arrangements have been reported. Cassandras within the Asteraceae mirror 5S rDNA promoter mutations of their host genome, likely as an adaptation to the host's specific 5S transcription factors and hence compensating for evolutionary changes in the 5S rDNA sequence. Changes in the 5S rDNA sequence and in Cassandras seem uncorrelated with linked/separated rDNA arrangements. We place all these observations into the context of angiosperm 5S rDNA-Cassandra evolution, discuss Cassandra's origin hypotheses (single or multiple) and Cassandra's possible impact on rDNA and plant genome organization, giving new insights into the interplay of ribosomal genes and transposable elements.

Analysis of 5S rDNA Genomic Organization Through the RepeatExplorer2 Pipeline: A Simplified Protocol.

The ribosomal RNA genes (rDNA) are universal genome components with a housekeeping function, given the crucial role of ribosomal RNA in the synthesis of ribosomes and thus for life-on-Earth. Therefore, their genomic organization is of considerable interest for biologists, in general. Ribosomal RNA genes have also been largely used to establish phylogenetic relationships, and to identify allopolyploid or homoploid hybridization.Here, we demonstrate how high-throughput sequencing data, through graph clustering implemented in RepeatExplorer2 pipeline ( https://repeatexplorer-elixir.cerit-sc.cz/galaxy/ ), can be helpful to decipher the genomic organization of 5S rRNA genes. We show that the linear shapes of cluster graphs are reminiscent to the linked organization of 5S and 35S rDNA (L-type arrangement) while the circular graphs correspond to their separate arrangement (S-type). We further present a simplified protocol based on the paper by (Garcia et al., Front Plant Sci 11:41, 2020) about the use of graph clustering of 5S rDNA homoeologs (S-type) to identify hybridization events in the species history. We found that the graph complexity (i.e., graph circularity in this case) is related to ploidy and genome complexity, with diploids typically showing circular-shaped graphs while allopolyploids and other interspecific hybrids display more complex graphs, with usually two or more interconnected loops representing intergenic spacers. When a three-genomic comparative clustering analysis from a given hybrid (homoploid/allopolyploid) and its putative progenitor species (diploids) is performed, it is possible to identify the corresponding homoeologous 5S rRNA gene families, and to elucidate the contribution of each putative parental genome to the 5S rDNA pool of the hybrid. Thus, the analysis of 5S rDNA cluster graphs by RepeatExplorer, together with information coming from other sources (e.g., morphology, cytogenetics) is a complementary approach for the determination of allopolyploid or homoploid hybridization and even ancient introgression events.

Mechanism of 5S RNP recruitment and helicase-surveilled rRNA maturation during pre-60S biogenesis.

Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ribosomal RNA is incorporated into the developing pre-60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53-MDM2, is poorly understood. Here, we report nine nucleolar pre-60S cryo-EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre-60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A-loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.

Single Copies of the 5S rRNA Inserted into 45S rDNA Intergenic Spacers in the Genomes of Nototheniidae (Perciformes, Actinopterygii).

In the vast majority of Animalia genomes, the 5S rRNA gene repeats are located on chromosomes outside of the 45S rDNA arrays of the nucleolar organiser (NOR). We analysed the genomic databases available and found that a 5S rDNA sequence is inserted into the intergenic spacer (IGS) between the 45S rDNA repeats in ten species of the family Nototheniidae (Perciformes, Actinopterigii). We call this sequence the NOR-5S rRNA gene. Along with Testudines and Crocodilia, this is the second case of a close association between four rRNA genes within one repetitive unit in deuterostomes. In both cases, NOR-5S is oriented opposite the 45S rDNA. None of the three nucleotide substitutions compared to the canonical 5S rRNA gene influenced the 5S rRNA secondary structure. In transcriptomes of the Patagonian toothfish, we only found NOR-5S rRNA reads in ovaries and early embryos, but not in testis or somatic tissues of adults. Thus, we consider the NOR-5S gene to be a maternal-type 5S rRNA template. The colocalization of the 5S and 45S ribosomal genes appears to be essential for the equimolar production of all four rRNAs in the species that show rDNA amplification during oogenesis. Most likely, the integration of 5S and NOR rRNA genes occurred prior to Nototheniidae lineage diversification.

High production of transfer RNAs identifies the presence of developing oocytes in ovaries and intersex testes of teleost fish.

5S rRNA is highly transcribed in fish oocytes and this transcription levels can be used to identify the presence of oocytes in the intersex testes of fish exposed to xenoestrogens. Similar to 5S rRNA, tRNAs are transcribed by RNA polymerase III (Pol-III) in eukaryotes, so this study focuses in the analysis of the levels of expression of tRNAs in the gonads (ovaries and testes) of eight teleost species as a possible new oocyte molecular marker. Total RNA extracted from gonads of six commercial teleost species in the Biscay Bay, from the pollution sentinel species thicklip grey mullet (Chelon labrosus) known present intersex testes in response to xenoestrogens in Gernika estuary and from the laboratory model species Danio rerio were analysed through capillary electrophoresis. Bioanalyzer electropherograms were used to quantify the concentrations of tRNAs, 5S and 5.8S rRNA. All studied ovaries expressed significantly higher levels of tRNAs and 5S rRNA than testes. A tRNA to 5.8S rRNA index was calculated which differentiates ovaries from testes, and identifies some intersex testes in between testes and ovaries in mullets. The tRNA/5.8S ratio was highest in ovaries in previtellogenic stage, decreasing towards maturity. Thus, strong oocyte expression of tRNAs is an additional proof of high activity levels of Pol-III during early stages of oocyte development in teleost ovaries. Incidentally, we observed that miRNA concentrations were always higher in testes than ovaries. The indexing approach developed in the present study could have multiple applications in teleost reproduction research and in the development of early molecular markers of intersex condition.

5S rRNA pseudogene transcripts are associated with interferon production and inflammatory responses in alcohol-associated hepatitis.

BACKGROUND AND AIMS: Interferon (IFN) signaling is critical to the pathogenesis of alcohol-associated hepatitis (AH), yet the mechanisms for activation of this system are elusive. We hypothesize that host-derived 5S rRNA pseudogene (RNA5SP) transcripts regulate IFN production and modify immunity in AH. APPROACH AND RESULTS: Mining of transcriptomic datasets revealed that in patients with severe alcohol-associated hepatitis (sAH), hepatic expression of genes regulated by IFNs was perturbed and gene sets involved in IFN production were enriched. RNA5SP transcripts were also increased and correlated with expression of type I IFNs. Interestingly, inflammatory mediators upregulated in sAH, but not in other liver diseases, were positively correlated with certain RNA5SP transcripts. Real-time quantitative PCR demonstrated that RNA5SP transcripts were upregulated in peripheral blood mononuclear cells (PBMCs) from patients with sAH. In sAH livers, increased 5S rRNA and reduced nuclear MAF1 (MAF1 homolog, negative regulator of RNA polymerase III) protein suggested a higher activity of RNA polymerase III (Pol III); inhibition of Pol III reduced RNA5SP expression in monocytic THP-1 cells. Expression of several RNA5SP transcript-interacting proteins was downregulated in sAH, potentially unmasking transcripts to immunosensors. Indeed, siRNA knockdown of interacting proteins potentiated the immunostimulatory activity of RNA5SP transcripts. Molecular interaction and cell viability assays demonstrated that RNA5SP transcripts adopted Z-conformation and contributed to ZBP1-mediated caspase-independent cell death. CONCLUSIONS: Increased expression and binding availability of RNA5SP transcripts was associated with hepatic IFN production and inflammation in sAH. These data identify RNA5SP transcripts as a potential target to mitigate inflammation and hepatocellular injury in AH.

High diversity of 5S ribosomal DNA and evidence of recombination with the satellite DNA PcP190 in frogs.

Distinct types of 5S rDNA repeats, differing by size and composition of its non-transcribed spacer (NTS), have been found in diverse taxa. Both concerted evolution and birth-and-death evolution have proven to play important roles in the evolution of the 5S rDNA family. In anurans, however, this subject has been underexplored as only a few anuran species had their 5S rDNA characterized and evolutionary analyzed to date. In the present study, we characterized the 5S rDNA sequences from species of two anuran families using classical molecular biology techniques and bioinformatic approaches. Based on the NTS, more than one type of 5S rDNA was identified in each analyzed species, which suggests that birth-and-death processes take part in the evolution of these sequences along the Anura tree of life. In addition, closely related species shared the same types of sequences, in accordance with the model of concerted evolution. We also found evidence of recombination between 5S rDNA and PcP190 satellite DNA, a repetitive sequence that is derived from the 5S rDNA. The interplay between 5S rDNA and PcP190 satellite DNA might favor the maintenance of the latter in the genome and respond to its presence in several species of frogs. The analysis of 5S rRNA transcripts confirmed the type I 5S rDNA of leiuperine as a functional 5S rRNA gene. Finally, the chromosome mapping of 5S rDNA sequences allowed some inferences of chromosome homology in Leiuperinae. In conclusion, our study provides additional information about the organization, differentiation and functionality of 5S rDNA in anuran species, revealing the potential participation of satellite DNA in the evolution of this family of rDNA.

Five Species of Taxus Karyotype Based on Oligo-FISH for 5S rDNA and (AG3T3)3.

As a relict plant, Taxus is used in a variety of medicinal ingredients, for instance to treat a variety of cancers. Taxus plants are difficult to distinguish from one another due to their similar morphology; indeed, some species of Taxus cytogenetic data still are unclear. Oligo-FISH can rapidly and efficiently provide insight into the genetic composition and karyotype. This is important for understanding the organization and evolution of chromosomes in Taxus species. We analysed five Taxus species using two oligonucleotide probes. (AG3T3)3 signals were distributed at the chromosome ends and the centromere of five species of Taxus. The 5S rDNA signal was displayed on two chromosomes of five species of Taxus. In addition to Taxus wallichiana var. mairei, 5S rDNA signals were found proximal in the remaining four species, which signals a difference in its location. The karyotype formula of Taxus wallichiana was 2n = 2x = 24m, its karyotype asymmetry index was 55.56%, and its arm ratio was 3.0087. Taxus × media's karyotype formula was 2n = 2x = 24m, its karyotype asymmetry index was 55.09%, and its arm ratio was 3.4198. The karyotype formula of Taxus yunnanensis was 2n = 2x = 24m, its karyotype asymmetry index was 55.56%, and its arm ratio was 2.6402. The karyotype formula of Taxus cuspidate was 2n = 2x = 24m, its karyotype asymmetry index was 54.67%, its arm ratio was 3.0135, and two chromosomes exhibited the 5S rDNA signal. The karyotype formula of T. wallichiana var. mairei was 2n= 2x = 22m + 2sm, its karyotype asymmetry index was 54.33%, and its arm ratio was 2.8716. Our results provide the karyotype analysis and physical genetic map of five species of Taxus, which contributes to providing molecular cytogenetics data for Taxus.

Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs.

To synthesize a chirally inverted ribosome with the goal of building mirror-image biology systems requires the preparation of kilobase-long mirror-image ribosomal RNAs that make up the structural and catalytic core and about two-thirds of the molecular mass of the mirror-image ribosome. Here, we chemically synthesized a 100-kilodalton mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of the full-length mirror-image 5S, 16S, and 23S ribosomal RNAs from enzymatically assembled long mirror-image genes. We further exploited the versatile mirror-image T7 transcription system for practical applications such as biostable mirror-image riboswitch sensor, long-term storage of unprotected kilobase-long l-RNA in water, and l-ribozyme-catalyzed l-RNA polymerization to serve as a model system for basic RNA research.

Enriched tandem repeats in chromosomal fusion points of Rineloricaria latirostris (Boulenger, 1900) (Siluriformes: Loricariidae).

Cytogenetic data showed the enrichment of repetitive DNAs in chromosomal rearrangement points between closely related species in armored catfishes. Still, few studies integrated cytogenetic and genomic data aiming to identify their prone-to-break DNA sites. Here, we aimed to obtain the repetitive fraction in Rineloricaria latirostris to recognize the microsatellite and homopolymers flanking the regions previously described as chromosomal fusion points. The results indicated that repetitive DNAs in R. latirostris are predominantly DNA transposons, and considering the microsatellite and homopolymers, A/T-rich expansions were the most abundant. The in situ localization demonstrated the A/T-rich repetitive sequences were scattered on the chromosomes, while A/G-rich microsatellite units were accumulated in some regions. The DNA transposon hAT, the 5S rDNA, and 45S rDNA (previously identified in Robertsonian fusion points in R. latirostris) were clusterized with some microsatellites, especially (CA)n, (GA)n, and poly-A, which were also enriched in regions of chromosomal fusions. Our findings demonstrated that repetitive sequences such as rDNAs, hAT transposons, and microsatellite units flank probable evolutionary breakpoint regions in R. latirostris. However, due to the sequence unit homologies in different chromosomal sites, these repeat DNAs only may facilitate chromosome fusion events in R. latirostris rather than working as a double-strand breakpoint site.

Karyological and nuclear DNA content variation of the genus Asparagus.

Asparagus wild relatives could be a promising possibility to extent the genetic variability of garden asparagus and for new cultivars with favorable traits such as high yield stability, disease resistance and stress tolerance. In order to achieve an efficient use in breeding, a detailed cytogenetic characterization of the accessions is necessary. This study worked on 35 Asparagus accessions, including A. officinalis cultivars ('Darlise', 'Ravel' and 'Steiners Violetta') and Asparagus wild relatives, for which the number of chromosomes, their size, the nuclear DNA content, and the genomic distribution of 5S and 45S rDNA were analyzed. Different ploidy levels (diploid, triploid, tetraploid, pentaploid and hexaploid) were found. Furthermore, the size of the chromosomes of all diploid Asparagus accessions was determined which led to differences in the karyotypic formula. A. plocamoides harbors the smallest chromosome with 1.21 µm, whereas the largest chromosome with 5.43 µm was found in A. officinalis. In all accessions one 5S rDNA locus per genome was observed, while the number of 45S rDNA loci varied between one (A. albus, A. plumosus, A. stipularis) to four (A. setaceus). In most Asparagus accessions, the 5S and 45S rDNA signals were located on different chromosomes. In contrast, the genomes of A. africanus, A. plocamoides, A. sp. (a taxonomically unclassified Asparagus species from Asia) and A. verticillatus (diploid accessions) have one 5S and one 45S rDNA signal on the same chromosome. The measured 2C DNA content ranges from 1.43 pg (A. plocamoides, diploid) to 8.24 pg (A. amarus, hexaploid). Intraspecific variations for chromosome number, karyotypic formula, signal pattern with 5S and 45s rDNA probes and DNA content were observed. Interspecific variations were also recognized in the genus Asparagus.

Genomic architecture of 5S rDNA cluster and its variations within and between species.

BACKGROUND: Ribosomal DNAs (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosomes during generation of genome assembly. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. RESULTS: Here we generate ultra-long Oxford Nanopore Technologies (ONT) and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA's repeating units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform assembly of the cluster by taking advantage of the long reads that carry these units, which led to an assembly of 5S rDNA cluster consisting of up to 167 consecutive 5S rDNA units in the N2 strain. The ordering and copy number of various rDNA units are consistent with the separation time between strains. Surprisingly, we observed a drastically reduced level of variation in the unit composition in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than in the C. elegans N2 strain, suggesting that N2, a widely used reference strain, is likely to be defective in maintaining the 5S rDNA cluster stability compared with other wild isolates of C. elegans or C. briggsae. CONCLUSIONS: The results demonstrate that Nanopore DNA sequencing reads are capable of generating assembly of highly repetitive sequences, and rDNA units are highly dynamic both within and between population(s) of the same species in terms of sequence and copy number. The detailed structure and variation of the 5S rDNA units within the rDNA cluster pave the way for functional and evolutionary studies.

Oligo-FISH Can Identify Chromosomes and Distinguish Hippophaë rhamnoides L. Taxa.

Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of Hippophaë rhamnoides has not been achieved using oligonucleotide sequences. Here, the chromosomes of five H. rhamnoides taxa in the mitotic metaphase and mitotic metaphase to anaphase were detected using the oligo-FISH probes (AG3T3)3, 5S rDNA, and (TTG)6. In total, 24 small chromosomes were clearly observed in the mitotic metaphase (0.89-3.03 µm), whereas 24-48 small chromosomes were observed in the mitotic metaphase to anaphase (0.94-3.10 µm). The signal number and intensity of (AG3T3)3, 5S rDNA, and (TTG)6 in the mitotic metaphase to anaphase chromosomes were nearly consistent with those in the mitotic metaphase chromosomes when the two split chromosomes were integrated as one unit. Of note, 14 chromosomes (there is a high chance that sex chromosomes are included) were exclusively identified by (AG3T3)3, 5S rDNA, and (TTG)6. The other 10 also showed a terminal signal with (AG3T3)3. Moreover, these oligo-probes were able to distinguish one wild H. rhamnoides taxon from four H. rhamnoides taxa. These chromosome identification and taxa differentiation data will help in elucidating visual and elaborate physical mapping and guide breeders' utilization of wild resources of H. rhamnoides.

Widespread association of ERα with RMRP and tRNA genes in MCF-7 cells and breast cancers.

tRNA gene transcription by RNA polymerase III (Pol III) is a tightly regulated process, but dysregulated Pol III transcription is widely observed in cancers. Approximately 75% of all breast cancers are positive for expression of Estrogen Receptor alpha (ERα), which acts as a key driver of disease. MCF-7 cells rapidly upregulate tRNA gene transcription in response to estrogen and ChIP-PCR demonstrated ERα enrichment at tRNALeu and 5S rRNA genes in this breast cancer cell line. While these data implicate the ERα as a Pol III transcriptional regulator, how widespread this regulation is across the 631 tRNA genes has yet to be revealed. Through analyses of ERα ChIP-seq datasets, we show that ERα interacts with hundreds of tRNA genes, not only in MCF-7 cells, but also in primary human breast tumours and distant metastases. The extent of ERα association with tRNA genes varies between breast cancer cell lines and does not correlate with levels of ERα binding to its canonical target gene GREB1. Amongst other Pol III-transcribed genes, ERα is consistently enriched at the long non-coding RNA gene RMRP, a positive regulator of cell cycle progression that is subject to focal amplification in tumours. Another Pol III template targeted by ERα is the RN7SL1 gene, which is strongly implicated in breast cancer pathology by inducing inflammatory responses in tumours. Our data indicate that Pol III-transcribed non-coding genes should be added to the list of ERα targets in breast cancer.

45S rDNA Repeats of Turtles and Crocodiles Harbor a Functional 5S rRNA Gene Specifically Expressed in Oocytes.

In most eukaryotic genomes, tandemly repeated copies of 5S rRNA genes are clustered outside the nucleolus organizer region (NOR), which normally encodes three other major rRNAs: 18S, 5.8S, and 28S. Our analysis of turtle rDNA sequences has revealed a 5S rDNA insertion into the NOR intergenic spacer in antisense orientation. The insertion (hereafter called NOR-5S rRNA gene) has a length of 119 bp and coexists with the canonical 5S rDNA clusters outside the NOR. Despite the ∼20% nucleotide difference between the two 5S gene sequences, their internal control regions for RNA polymerase III are similar. Using the turtle Trachemys scripta as a model species, we showed the NOR-5S rDNA specific expression in oocytes. This expression is concurrent with the NOR rDNA amplification during oocyte growth. We show that in vitellogenic oocytes, the NOR-5S rRNA prevails over the canonical 5S rRNA in the ribosomes, suggesting a role of modified ribosomes in oocyte-specific translation. The orders Testudines and Crocodilia seem to be the only taxa of vertebrates with such a peculiar rDNA organization. We speculate that the amplification of the 5S rRNA genes as a part of the NOR DNA during oogenesis provides a dosage balance between transcription of all the four ribosomal RNAs while producing a maternal pool of extra ribosomes. We further hypothesize that the NOR-5S rDNA insertion appeared in the Archelosauria clade during the Permian period and was lost later in the ancestors of Aves.

5S-IGS rDNA in wind-pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species.

Standard models of plant speciation assume strictly dichotomous genealogies in which a species, the ancestor, is replaced by two offspring species. The reality in wind-pollinated trees with long evolutionary histories is more complex: species evolve from other species through isolation when genetic drift exceeds gene flow; lineage mixing can give rise to new species (hybrid taxa such as nothospecies and allopolyploids). The multi-copy, potentially multi-locus 5S rDNA is one of few gene regions conserving signal from dichotomous and reticulate evolutionary processes down to the level of intra-genomic recombination. Therefore, it can provide unique insights into the dynamic speciation processes of lineages that diversified tens of millions of years ago. Here, we provide the first high-throughput sequencing (HTS) of the 5S intergenic spacers (5S-IGS) for a lineage of wind-pollinated subtropical to temperate trees, the Fagus crenata - F. sylvatica s.l. lineage, and its distant relative F. japonica. The observed 4963 unique 5S-IGS variants reflect a complex history of hybrid origins, lineage sorting, mixing via secondary gene flow, and intra-genomic competition between two or more paralogous-homoeologous 5S rDNA lineages. We show that modern species are genetic mosaics and represent a striking case of ongoing reticulate evolution during the past 55 million years.

Physical localization of 45S rDNA in Cymbopogon and the analysis of differential distribution of rDNA in homologous chromosomes of Cymbopogon winterianus.

Cymbopogon, commonly known as lemon grass, is one of the most important aromatic grasses having therapeutic and medicinal values. FISH signals on somatic chromosome spreads off Cymbopogon species indicated the localization of 45S rDNA on the terminal region of short arms of a chromosome pair. A considerable interspecific variation in the intensity of 45S rDNA hybridization signals was observed in the cultivars of Cymbopogon winterianus and Cymbopogon flexuosus. Furthermore, in all the varieties of C. winterianus namely Bio-13, Manjari and Medini, a differential distribution of 45S rDNA was observed in a heterologous pair of chromosomes 1. The development of C. winterianus var. Manjari through gamma radiation may be responsible for breakage of fragile rDNA site from one of the chromosomes of this heterologous chromosome pair. While, in other two varieties of C. winterianus (Bio-13 and Medini), this variability may be because of evolutionary speciation due to natural cross among two species of Cymbopogon which was fixed through clonal propagation. However, in both the situations these changes were fixed by vegetative method of propagation which is general mode of reproduction in the case of C. winterianus.

Analyses of the Updated "Animal rDNA Loci Database" with an Emphasis on Its New Features.

We report on a major update to the animal rDNA loci database, which now contains cytogenetic information for 45S and 5S rDNA loci in more than 2600 and 1000 species, respectively.The data analyses show the following: (i) A high variability in 5S and 45S loci numbers, with both showing 50-fold or higher variability. However, karyotypes with an extremely high number of loci were rare, and medians generally converged to two 5S sites and two 45S rDNA sites per diploid genome. No relationship was observed between the number of 5S and 45S loci. (ii) The position of 45S rDNA on sex chromosomes was relatively frequent in some groups, particularly in arthropods (14% of karyotypes). Furthermore, 45S rDNA was almost exclusively located in microchromosomes when these were present (in birds and reptiles). (iii) The proportion of active NORs (positively stained with silver staining methods) progressively decreased with an increasing number of 45S rDNA loci, and karyotypes with more than 12 loci showed, on average, less than 40% of active loci. In conclusion, the updated version of the database provides some new insights into the organization of rRNA genes in chromosomes. We expect that its updated content will be useful for taxonomists, comparative cytogeneticists, and evolutionary biologists. .

Comparative cytogenetics of Serrasalmidae (Teleostei: Characiformes): The relationship between chromosomal evolution and molecular phylogenies.

Serrasalmidae has high morphological and chromosomal diversity. Based on molecular hypotheses, the family is currently divided into two subfamilies, Colossomatinae and Serrasalminae, with Serrasalminae composed of two tribes: Myleini (comprising most of pacus species) and Serrasalmini (represented by Metynnis, Catoprion, and remaining piranha's genera). This study aimed to analyze species of the tribes Myleini (Myloplus asterias, M. lobatus, M. rubripinnis, M. schomburgki, and Tometes camunani) and Serrasalmini (Metynnis cuiaba, M. hypsauchen, and M. longipinnis) using classical and molecular cytogenetic techniques in order to understand the chromosomal evolution of the family. The four species of the genus Myloplus and T. camunani presented 2n = 58 chromosomes, while the species of Metynnis presented 2n = 62 chromosomes. The distribution of heterochromatin occurred predominantly in pericentromeric regions in all species. Tometes camunani and Myloplus spp. presented only one site with 5S rDNA. Multiple markers of 18S rDNA were observed in T. camunani, M. asterias, M. lobatus, M. rubripinnis, and M. schomburgkii. For Metynnis, however, synteny of the 18S and 5S rDNA was observed in the three species, in addition to an additional 5S marker in M. longipinnis. These data, when superimposed on the phylogeny of the family, suggest a tendency to increase the diploid chromosome number from 54 to 62 chromosomes, which occurred in a nonlinear manner and is the result of several chromosomal rearrangements. In addition, the different karyotype formulas and locations of ribosomal sequences can be used as cytotaxonomic markers and assist in the identification of species.

FISH-Based Karyotype Analyses of Four Dracaena Species.

The genus Dracaena is the main source of dragon's blood, which is a plant resin and has been used as traditional medicine since ancient times in different civilizations. However, the chromosome numbers and karyotypes present in this genus remain poorly understood. In this study, fluorescence in situ hybridization (FISH) using oligonucleotide probes for ribosomal DNAs (5S and 45S rDNA) and telomeric repeats (TTTAGGG)3 was applied to analyze 4 related species: Dracaena terniflora Roxb., Dracaena cambodiana Pierre ex Gagnep., Aizong (Dracaena sp.), and Dracaena cochinchinensis (Lour.) S.C. Chen. In all 4 species, both 5S and 45S rDNA showed hybridization signals in the paracentromeric region of a pair of chromosomes; the sizes of the 45S rDNA signals were larger than those of the 5S rDNA. Importantly, the telomeric repeat signals were located in the telomeric regions of almost all chromosomes. The results indicated that the chromosome number of all 4 Dracaena species is 2n = 40, and the lengths of the mitotic metaphase chromosomes range from 0.99 to 2.98 µm. Our results provide useful cytogenetic information, which will be beneficial to future studies in genome structure of the genus Dracaena.