Chromosome mapping of H1 histone and 5S rRNA gene clusters in three species of Astyanax (Teleostei, Characiformes)

We report here on the physical mapping of the H1 histone genes (hisDNA) and the 5S ribosomal DNA (rDNA) in 3 Neotropical fish species of the genus Astyanax(A. altiparanae, A. bockmanni and A. fasciatus) and the comparative analysis of the chromosomes bearing these genes. Nucleotide analyses by sequencing of both genes were also performed. The distribution of the H1 histone genes was more conserved than that of the rRNA genes, since these were always located in the pericentromeric regions of 2 chromosome pairs. 5S rDNA was found on one of the pairs that presented an H1 histone cluster; this seems to be a conserved chromosomal feature of the genus Astyanax. In addition, individuals of A. bockmanni and A. fasciatus showed clusters of 5S rDNA on 1 pair of acrocentric chromosomes, not found in A. altiparanae. The results obtained by chromosome mapping as well as by sequencing of both genes showed that A.bockmanni is more closely related to A. fasciatus than to A. altiparanae. The results allow the characterization of cytogenetic markers for improved elucidation of the processes involved in karyotype differentiation of fish genomes.     

Evolutionary dynamics of 5S rDNA location in acridid grasshoppers and its relationship with H3 histone gene and 45S rDNA location

We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers.     

45S rDNA和5S rDNA在南瓜、丝瓜和冬瓜染色体上的比较定位

首次利用荧光原位杂交和双色荧光原位杂交技术对45S和5S rDNA在南瓜(Cucurbita moschata Duch)、丝瓜(Luffa cylindrical Roem)、冬瓜(Benincasa hispida Cogn)的有丝分裂中期染色体上进行了物理定位分析。南瓜有5对45S rDNA位点, 2对5S rDNA位点; 丝瓜具有5对45S rDNA位点, 1对5S rDNA位点; 冬瓜具有2对45S rDNA位点, 1对5S rDNA位点, 5S rDNA位点与其中一对45S rDNA位点都位于7号染色体短臂上, 并在物理位置上紧密相邻。45S rDNA在这3种作物染色体上数目变化较大, 但在染色体上都倾向分布在短臂末端, 其分布模式较为一致。5S rDNA在这3种作物染色体上数目相对保守, 但在染色体上分布的位置变化较大。文中讨论了45S rDNA和5S rDNA在植物基因组中不同的进化趋势。     

Conserved 5S and variable 45S rDNA chromosomal localisation revealed by FISH in <Emphasis Type="Italic">Astyanax scabripinnis</Emphasis> (Pisces,Characidae)

Fluorescence in situhybridisation (FISH) and double FISH experiments were carried out to ascertain the chromosomal distribution pattern of the 45S and 5S ribosomal (r) DNAs in four populations of the characid fish Astyanax scabripinnis – a group considered to be a species complex for its wide karyotypical and morphological diversity. The results regarding the 45S rDNA agreed with this hypothesis, since these sites showed intra- and inter-populational, numerical and positional variations. However, the data obtained with the 5S rDNA probe revealed a highly conserved chromosomal distribution pattern of these sequences among individuals of each population, as well as among the populations analysed. We consider this contrasting situation as a functional divergence between 45S and 5S ribosomal DNAs, which may reflect the localisation of these sequences in distinct nuclear compartments, leading them to undergo differentiated evolutionary processes.     

Cytogenetic characterization of the invasive mussel species Xenostrobus securis Lmk. (Bivalvia: Mytilidae)

The chromosomes of the invasive black-pigmy mussel (Xenostrobus securis (Lmk. 1819)) were analyzed by means of 4',6-diamidino-2-phenylindole (DAPI) / propidium iodide (PI) and chromomycin A3 (CMA) / DAPI fluorescence staining and fluorescent in situ hybridization using major rDNA, 5S rDNA, core histone genes, linker histone genes, and telomeric sequences as probes. The diploid chromosome number in this species is 2n = 30. The karyotype is composed of seven metacentric, one meta/submetacentric, and seven submetacentric chromosome pairs. Telomeric sequences appear at both ends of every single chromosome. Major rDNA clusters appear near the centromeres on chromosome pairs 1 and 3 and are associated with bright CMA fluorescence and dull DAPI fluorescence. This species shows five 5S rDNA clusters close to the centromeres on four chromosome pairs (2, 5, 6, and 8). Three of the four core histone gene clusters map to centromeric positions on chromosome pairs 7, 10, and 13. The fourth core histone gene cluster occupies a terminal position on chromosome pair 8, also bearing a 5S rDNA cluster. The two linker histone gene clusters are close to the centromeres on chromosome pairs 12 and 14. Therefore, the use of these probes allows the unequivocal identification of 11 of the 15 chromosome pairs that compose the karyotype of X. securis.     

Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species

Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.     

Molecular organization of 5S rDNA in fishes of the genus Brycon.

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.     

Gene mapping of 5S rDNA sites in eight fish species from the Paraíba do Sul river basin, Brazil

The use of improved cytogenetic techniques such as fluorescent in situ hybridization (FISH) has offered important methodologies for cytotaxonomic and evolutionary studies. In particular, the mapping of 5S rDNA sites has proved to be an excellent marker in the study of different organisms and, more recently, in fish. In the present work, the FISH technique was used to map the 5S rDNA sites in the chromosomes of eight neotropical fish species from the Paraíba do Sul river basin, four of these belonging to the order Characiformes, family Characidae, genus Astyanax (A. scabripinnis, A. parahybae, A. giton and A. intermedius) and four to the order Siluriformes, family Loricariidae (Neoplecostomus microps, Harttia loricariformis, Hypostomus affinis and Upsilodus sp.). Karyotype evolution aspects of the analyzed groups are discussed.     

Chromosomal localization of 5S and 18S–5.8S–25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents ( P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.     

Colocalization of repetitive DNAs and silencing of major rRNA genes. A case report of the fish Astyanax janeiroensis

The heterochromatin composition and loca- tion in the genome of the fish Astyanax janeiroensis was investigated using Chromomycin A(3) and DAPI fluorochromes and fluorescence in situ hybridization (FISH) with 18S rDNA and As51 satellite DNA probes, respectively. Distinct repetitive DNA classes were found, namely: (1) C-positive centromeric/telomeric heterochromatin, (2) NOR-associated GC-rich heterochromatin (18S(+)/GC(+)) and (3) As51(+)/18S(+) heterochromatin colocalized on 14 distinct heterochromatic domains with attenuated fluorescence of DAPI staining (As51(+)/18S(+)/DAPI attenuated signal). Besides these fourteen associated repetitive DNAs, another eight sites with only 18S rDNA were also found, comprising altogether 22 18S rDNA sites in the genome of the species under study. Up to seven 18S rDNA sites were found to be active, i.e., were characterized as positive after silver staining (Ag-NORs). It was noteworthy that in all As51(+)/18S(+) domains the 18S rDNA were not found to be active sites due to the silencing of these genes when associated with the As51 satellite DNA in the same heterochromatic domain. The dispersion of the As51 sites in the genome of the species is hypothesized to probably originate from a transposable element. Several chromosomal and karyotype markers are similar between A. janeiroensis and A. scabripinnis, indicating a close relationship between these species.     

Physical mapping of 5S rDNA in two species of Knifefishes: Gymnotus pantanal and Gymnotus paraguensis (Gymnotiformes)

Physical mapping of 5S rDNA in 2 species of knifefishes, Gymnotuspantanal and G. paraguensis (Gymnotiformes), was performed using fluorescence in situ hybridization with a 5S rDNA probe. The 5S rDNA PCR product from the genomes of both species was also sequenced and aligned to determine non-transcribed spacer sequences (NTS). Both species under study had different patterns of 5S rDNA gene cluster distribution. While in the karyotype of G. pantanal two 5S rDNA-bearing pairs were observed, the karyotype of G. paraguensis possessed as many as 19 such pairs. Such multiplication of 5S rDNA gene clusters might be caused by the involvement of transposable elements because the NTS of G. paraguensis was 400 bp long with high identity (90%) with a mobile transposable element called Tc1-like transposon, described from the cyprinid fish Labeo rohita.     

Variability in rDNA loci in Iberian species of the genus Zabrus (Coleoptera: Carabidae) detected by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with a PCR-amplified 18S ribosomal probe was used to map rDNA loci in 19 taxa of the ground beetle genus Zabrus (2n = 47-63) from the Iberian Peninsula. A quantitative and qualitative variation has been observed among related species, subspecies, populations, and even individuals. The number of rDNA-carrying chromosomes varies from 2 to 12, and the extent of the signal from small dots to entire arms. Changes altering the number of rDNA clusters seem to be uncoupled from the variation found in the chromosome number. Mechanisms that explain the numerical variation and spreading of rDNA clusters throughout the genome within the genus Zabrus are briefly discussed. No concordance between the pattern of rDNA sites and the phylogenetic relationships as based on morphological characters has been found.     

Molecular Organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural Features and Evolution of Piscine 5S rRNA Genes and Nontranscribed Intergenic Spacers

The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S-5.8S-26S rDNA and a C genome specific DNA sequence in the genus Avena.

A physical map of the locations of the 5S rDNA genes and their relative positions with respect to 18S-5.8S-26S rDNA genes and a C genome specific repetitive DNA sequence was produced for the chromosomes of diploid, tetraploid, and hexaploid oat species using in situ hybridization. The A genome diploid species showed two pairs of rDNA loci and two pairs of 5S loci located on both arms of one pair of satellited chromosomes. The C genome diploid species showed two major pairs and one minor pair of rDNA loci. One pair of subtelocentric chromosomes carried rDNA and 5S loci physically separated on the long arm. The tetraploid species (AACC genomes) arising from these diploid ancestors showed two pairs of rDNA loci and three pairs of 5S loci. Two pairs of rDNA loci and 2 pairs of 5S loci were arranged as in the A genome diploid species. The third pair of 5S loci was located on one pair of A-C translocated chromosomes using simultaneous in situ hybridization with 5S rDNA genes and a C genome specific repetitive DNA sequence. The hexaploid species (AACCDD genomes) showed three pairs of rDNA loci and six pairs of 5S loci. One pair of 5S loci was located on each of two pairs of C-A/D translocated chromosomes. Comparative studies of the physical arrangement of rDNA and 5S loci in polyploid oats and the putative A and C genome progenitor species suggests that A genome diploid species could be the donor of both A and D genomes of polyploid oats. Key words : oats, 5S rDNA genes, 18S-5.8S-26S rDNA genes, C genome specific repetitive DNA sequence, in situ hybridization, genome evolution.     

Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes

In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae). The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes). This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes) and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.     

Ordered arrangement and rearrangement of chromosomes during spermatogenesis in two species of planarians (Plathelminthes)

The pattern of distribution of telomeric DNA (TTAGGG), 28S rDNA, and 5S rDNA has been studied using fluorescence in situ hybridization (FISH) and primed in situ labelling during spermatogenesis and sperm formation in the filiform spermatozoa of two species of planarians, Dendrocoelum lacteum and Polycelis tenuis (Turbellaria, Plathelminthes). In both species, the positions of FISH signals found with each probe sequence are constant from cell to cell in the nuclei of mature sperm. Chromosome regions containing 5S and 28S rDNA genes are gathered in distinct bundles of spiral form. In early spermatids with roundish nuclei, the sites of a given sequence on different chromosomes remain separate. Centromeres (marked by 5S rDNA) gather into a single cluster in the central region of the slightly elongated sperm nucleus. During spermatid maturation, this cluster migrates to the distal pole of the nucleus. In Polycelis, telomeric sites gather into three distinct clusters at both ends and in the middle of the moderately elongated nucleus. These clusters retain their relative positions as the spermatid matures. All the chromosome ends bearing 28S rDNA gather only into the proximal cluster. Our data suggest that structures in the nucleus selectively recognise chromosome regions containing specific DNA sequences, which helps these regions to find their regular places in the mature sperm nucleus and causes clustering of the sites of these sequences located on different chromosomes. This hypothesis is supported by observations on elongated sperm of other animals in which a correlation exists between ordered arrangement of chromosomes in the mature sperm nucleus and clustering of sites of the same sequence from different chromosomes during spermiogenesis. Received: 15 December 1997; in revised form: 24 March 1998 / Accepted: 14 April 1998     

大麦45S和5S rDNA定位及5S rDNA伸展纤维的FISH分析

用荧光原位杂交技术对45S和5SrDNA在大麦(Hordeum vulgare L．)有丝分裂中期染色体进行了确定分析,较强的45SrDNA信号共有2对,分别分布在大麦的第1染色体的短臂和第2染色体的长臂。而5SrDNA则只有1对杂交信号,位于第3染色体的长臂,但信号较弱。用伸展DNA纤维的荧光原位杂交(Fiber—FISH)技术测定了5SrDNA在大麦的基因组中的拷贝数,计算出5SrDNA的拷贝数约为408～416。对大麦品种中rDNA位点数目的可变性进行了讨论。     

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers.

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.     

Ribosomal DNAs: an exception to the conservation of gene order in rice genomes

rDNA (18S-5.8S-25S rDNA) and 5S rDNA loci were visualized on the chromosomes of six species of the genus Oryza by fluorescence in situ hybridization (FISH) and the labeled rice chromosomes were identified based on their condensation patterns. As a result, the chromosomes harboring rDNA and/or 5S rDNA loci were determined in the complement for all the known rice genomes. Variation in the location of the rDNA loci indicated the transpositional nature of the rDNAs in the genus Oryza, as also suggested in Triticeae and Allium. Comparative analysis of the locations of rDNA loci among rice, maize and wheat revealed that variability in the physical location of the rDNA loci was characteristic of the genus Oryza and also of the genera of Gramineae. This variability in the location of the rDNA loci between evolutionarily related species is in sharp contrast to the conservation of the general order of genes in their genomes.     

Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.