Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.     

Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: repeat units,inverted sequences,pseudogenes and chromosome loci

In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish, Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were determined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence IN SITU hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively. An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA, the presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements.     

Chromosomal localization of a novel repetitive sequence in theChenopodium quinoa genome

In this study, a novel repetitive sequence pTaq10 was isolated from theTaq I digest of the genomic DNA of the pseudocerealChenopodium quinoa. Sequence analysis indicated that this 286-bp monomer is not homologous to any known retroelement sequence. FISH and Southern blot analysis showed that this sequence is characterized by an interspersed genomic organization. After FISH, hybridization signals were observed as small dots spread throughout all of the chromosomes. pTaq hybridization signals were excluded from 45S rRNA gene loci, but they partly overlapped with 5S rDNA loci. pTaq10 is not a species-specific sequence, as it was also detected inC. berlandieri.     

Analysis of 5S rDNA changes in synthetic allopolyploids Triticum x Aegilops

By the example of three synthetic allopolyploids: Aegilops sharonensis x Ae. umbellulata (2n =28), Triticum urartu x Ae. tauschii (2n =28), T. dicoccoides x Ae. tauschii (2n =42) the 5S rDNA changes at the early stage of allopolyploidization were investigated. Using fluorescent in situ hybridization (FISH), the quantitative changes affecting the separate loci of one of the parental genomes were revealed in plants of S3 generation of each hybrid combination. Souther hybridization with genomic DNA of allopolyploid T. urartu x Ae. tauschii (TMU38 x TQ27) revealed lower intensity of the fragments from Ae. tauschii compared with the T. urartu fragments. It may be confirmation of the reduction of signal on 1D chromosome that was revealed in this hybrid using FISH. Both appearance of a new 5S rDNA fragments and full disappearance of fragments from parental species were not showed by Southern hybridization, as well as PCR-analysis of 5-15 plants of S2-S3 generations. The changes were not found under comparison of primary structure of nine 5S rDNA sequences of allopolyploid TMU38 x TQ27 with analogous sequences from parental species genomes. The observable similarity by FISH results of one of the studied synthetic allopolyploids with natural allopolyploid of similar genome composition indicates the early formation of unique for each allopolyploid 5S rDNA organization.     

The Close Relationship Between the A and B Genomes in Avena L. (Poaceae) Determined by Molecular Cytogenetic Analysis of Total Genomic, Tandemly and Dispersed Repetitive DNA Sequences

The genusAvena L. (Poaceae) consists of diploid, tetraploid,and hexaploid species, with the B genome known only in tetraploidspecies and the D genome in the hexaploid species. DNA:DNAinsitu hybridization, using total genomic DNA from diploidAvenastrigosa Schreb. (A_sgenome) as a probe, labelled all 28 chromosomesof the AB tetraploidAvena vaviloviana (Malz.) Mordv. stronglyand uniformly, revealing the close relationship between thesetwo genomes. Comparison of patterns of size-separated DNA restrictionfragments between the diploidA. strigosa and the tetraploidA.vaviloviana , using 32 different restriction enzymes, revealedno differences. Southern hybridization using total AB genomicDNA as a probe also gave no differences in banding patternsbetween the two genomes, even when a large excess of A genomicDNA was used as a block. From anA. vaviloviana genomic library,1800 colonies were blotted and probed sequentially with A andAB genomic DNA, but no colony was identified to be B genomespecific. DNA digests of AB genome tetraploids with restrictionenzymeHae III gave a strong band at 4.2 kb. Clone pAbKB3, derivedfrom the 4.2 kb band, was found to be part of aTy1-copia -likeretrotransposon present in A and B genome chromosomes. ClonedrRNA genes were used forin situ hybridization and showed thatdiploidA. strigosa has four major sites for 18S-25S rDNA andtwo pairs of sites for 5S rDNA (pairs on the same satellitedchromosome, on different chromosome arms), while 4xA. vavilovianahas eight major sites for 18S-25S rDNA and four pairs of sitesfor 5S rDNA (pairs on the same satellited chromosome, on differentchromosome arms). A repetitive sequence from rye pSc119.2, showeddispersed hybridization, while the telomeric sequence in clonepLT11 hybridized to telomeres. Again no discrimination was possiblebetween A and B genome chromosomes. The molecular similaritiesbetween the diploidA. strigosa and thebarbata group tetraploidsclearly indicate that thebarbata group of tetraploids arosefrom A_sdiploids through autotetraploidization. Avena ; evolution; repetitive sequences; in situ hybridization; retrotransposons; genome organization     

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.     

DNA hybridization probe for the Pseudomonas fluorescens group.

Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group. The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms. pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P. fluorescens group and separated them clearly from all other bacteria tested in the present study. Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P. aeruginosa. It was compared with the published 23S RNA sequence from Escherichia coli.     

DNA hybridization probe for the Pseudomonas fluorescens group

Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group. The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms. pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P. fluorescens group and separated them clearly from all other bacteria tested in the present study. Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P. aeruginosa. It was compared with the published 23S RNA sequence from Escherichia coli.     

Systematic analysis and evolution of 5S ribosomal DNA in metazoans

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12 766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.     

Chromosomal location and reorganization of the 45S and 5S rDNA in theBrachyscome lineariloba complex (Asteraceae)

The chromosomal locations of the 45S (18S-5.8S-26S) and 5S ribosomal DNA in theBrachyscome lineariloba complex and two related species have been determined by the use of multicolor fluorescencein situ hybridization (McFISH). TheBrachyscome lineariloba complex includes five cytodemes with 2n=4, 8, 10, 12 and 16. Each of the 5S and 45S rDNA loci occurs at two sites on chromosomes in cytodemes with 2n=4. While in cytodemes with 2n=8, 10, 12 and 16, the number of 5S rDNA sites increases from four to eight paralleled to the genomic addition of diploid (4 chromosomes) or haploid (2 chromosomes) dosage. Of the 5S rDNA sites, only one pair is major, except for the cytodeme with 2n=10. The remaining 5S rDNA sites are minor and seem to have reduced the unit number of the 5S rDNA during the successive genomic additions. The 45S rDNA site is detected only at two nucleolar organizing regions in all cytodemes regardless of successive genomic addition. The loss or diminution of 45S rDNA sequences seem to have proceeded more rapidly than 5S rDNA sequences in theB. lineariloba complex.     

垃圾填埋场渗滤液中古细菌群落16S rRNA基因的ARDRA分析

利用特异性的引物对,选择性扩增垃圾填埋场渗滤液中古细菌群落的18S rRNA基因片断,在此基础上建立16S rDNA克隆文库,经古细菌通用寡核苷酸探针的原位杂交筛选后,克隆文库内古细菌16S rDNA扩增片断的多样性通过ARDRA分析（amplified rDNA restriction analysis)而获得,利用PCR将各组重克隆子内的16S rDNA外源片断再扩增出来后,两种限制性内切酶－Hha I和HaeⅢ－被分别用于16S rDNA克隆片断的限制酶切分析,结果表明,随机选出的70个古细菌16S rDNA克隆片断被妥为21个不同的ARDRA型（组）,其中的两个优势型总共占了所有被分析克隆子的60％,而其余19个型的相对丰度均处于较低的水平,当中的14个型更仅含有1个克隆子,通过对16S rRNA基因的PCR扩增,克隆及其ARDRA分析,能快速地获得有关填埋场渗滤液中古细菌群落的结构及其多样性的初步信息。     

Satellite DNA derived from 5S rDNA in Physalaemus cuvieri (Anura, Leiuperidae)

In the present study, we describe for the first time a family of 190-bp satellite DNA related to 5S rDNA in anurans and the existence of 2 forms of 5S rDNA, type I (201 bp) and type II (690 bp). The sequences were obtained from genomic DNA of Physalaemus cuvieri from Palmeiras, State of Bahia, Brazil. Analysis of the nucleotide sequence revealed that the satellite DNA obtained by digestion with EcoRI, called PcP190EcoRI, is 70% similar to the coding region of type I 5S rDNA and 66% similar to the coding region of type II 5S rDNA. Membrane hybridization and PCR amplification of the sequence showed that PcP190EcoRI is tandemly repeated. The satellite DNA as well as type I and type II 5S rDNA were localized in P. cuvieri chromosomes by fluorescent in situ hybridization. The PcP190EcoRI sequence was found in the centromeres of chromosomes 1-5 and in the pericentromeric region of chromosome 3. Type I 5S rDNA was detected in chromosome 3, coincident with the site of PcP190EcoRI. Type II 5S rDNA was located interstitially in the long arm of chromosome 5. None of these sequences co-localized with nucleolar organizer regions. Our data suggests that this satellite DNA originates from the 5S ribosomal multigene family, probably by gene duplication, nucleotide divergence and sequence dispersion in the genome.     

Cloning of tomato nuclear ribosomal DNA. rDNA organization in leaves and suspension-cultured cells

The rDNA fragments of EcoRI digested tomato nuclear DNA were detected by cross-hybridization with cloned Aspergillus nidulans rDNA. Two fragments coding for 18S and 25S rRNA, respectively were cloned and characterized. They were used to study structural organization of rRNA genes of a tomato cell suspension culture by hybridization with specifically cleaved nuclear DNA. The repeating units found were variable in restriction sites and in size with a basic unit of 8.6 kbp. Additionally, the analysis clearly demonstrates that nuclear DNA isolated from tomato leaves (Lycopersicon esculentum cv. Lukullus) and cultured cells derived therefrom bear significant differences in the structural organization of their ribosomal DNA.     

白菜rDNA及C0t-1DNA的荧光原位杂交及其核型分析

以早熟白菜苔为实验材料,从其基因组DNA中分离出C0t-1DNA并用生物素标记作探针,25SrDNA用地高辛标记作探针,对有丝分裂中期相染色体进行双色荧光原位杂交。每对染色体上均显示出了特定的C0t-1DNA荧光原位杂交带型,5对染色体上显示出了25SrDNA荧光原位杂交带型。双色荧光原位杂交证实了C0t-1DNA与25SrDNA二者具有一致的染色体位置特征,表明基于rDNA及C0t-1 DNA的荧光原位杂交核型分析技术,优于目前普遍采用的只基于rDNA的荧光原位杂交核型分析方法。结合C0t-1 DNA与25SrDNA的荧光原位杂交带型和传统的染色体的形态学标记分析方法及白菜已公布的基于rDNA分布的核型分析结果,创建了一个精确的白菜核型。     

The Molecular through Ecological Genetics of Abnormal Abdomen. II. Ribosomal DNA Polymorphism Is Associated with the Abnormal Abdomen Syndrome in DROSOPHILA MERCATORUM

Restriction endonuclease cleavage analyses of cloned and genomic DNA samples indicate that the structure of the DNA encoding the large cytoplasmic RNAs (rDNAs) is altered in Drosophila mercatorum lines which exhibit an abnormal abdomen (aa) phenotype. In a majority of the rDNA repeat units from aa flies, the 28S coding sequence is interrupted by a large [5-6 kilobase pairs (kbp)] insert. A subclone containing this inserted DNA (ins 3) hybridizes primarily to rDNA-containing sequences in in situ and genomic blot hybridization experiments. Additionally, genomic nitrocellulose blot hybridization analyses show that ins- containing rDNA repeat units are clustered in a spontaneously arising aa mutant. This rDNA alteration in D. mercatorum flies with the aa phenotype more closely resembles the bobbed (bb) defect of D. hydei than the bb defect of D. melanogaster, which involves alterations in rDNA copy number. By analogy with the other Drosophila systems, we propose that the altered D. mercatorum rDNA repeat units are defective in rRNA production at a critical stage. The lowered levels of rRNA ultimately would limit the concentration of ribosomes needed to produce large quantities of a protein (in these cases, juvenile hormone esterase) needed for normal development.     

Oligo-chip based detection of tick-borne bacteria

We have developed an oligonucleotide-chip based assay for detection of 16S ribosomal PCR products from tick-borne bacteria. This chip contains 14 specific probes, which target variable regions of 16S rDNA of tick-borne bacteria including Borrellia spp., Rickettsia spp., Anaplasma spp., Coxiella burnetii and Francisella tularensis. The specificity of these probes was tested by hybridization of the chip with fluorescently labeled PCR products amplified from the genomic DNA of selected tick-borne bacteria. The assay was also tested for detection of tick-borne bacteria in single ticks.     

Characterization of the two rRNA gene operons present in Thiobacillus ferrooxidans

The organization of rRNA genes from the autotrophic, acidophilic bacterium Thiobacillus ferrooxidans has been examined. Two rRNA operons were found in this microorganism by means of genomic hybridization studies. Recombinant plasmids, pTR-3 and pTR-1 that carry a portion of 16/23 S rDNA from one operon and the 5'-flanking region of the second operon, respectively, were identified and characterized.     

Genomic organization and phylogenetic relationships in the genus Dasypyrum analysed by Southern and in situ hybridization of total genomic and cloned DNA probes

Molecular cytogenetic methods have been used to study the controversial phylogenetic relationships between the species Dasypyrum villosum (L.) Candargy (2n=2x=14) and D. breviaristatum (Lindb. f.) Frederiksen (2n=4x=28). Using total genomic DNA from the two species as probes for in situ hybridization to chromosomes, we found that the pericentromeric regions of the chromosome arms of both species are similar, while distal regions show substantial differences. Two dispersed repetitive DNA sequences were isolated: pDbKB45 is distributed along the chromosomes but amplified in the subtelomeric regions of D. breviaristatum chromosomes, while pDbKB49, in both species, is less amplified in terminal regions. Size-separated restriction enzyme digests of DNA showed many repetitive fragments, but few in common between the two species. After probing Southern transfers with D. breviaristatum genomic DNA, all lanes showed similar hybridization patterns although one extra small band was evident in the D. breviaristatum lanes. In contrast, probing with D. villosum DNA showed very substantial differences between the two species. Genomic in situ hybridization to meiotic metaphases from an interspecific hybrid showed seven bivalents of D. breviaristatum origin and seven univalents from D. villosum. We also analysed the physical organization of 5S rDNA, 18S-25S rDNA and a tandemly repeated sequence from rye. Our data support an autotetraploid origin for D. breviaristatum, but its genome and that of D. villosum show extensive differences, so the tetraploid is unlikely to be directly derived from D. villosum. Received: 29 March 1996; in revised form: 28 December 1996 / Accepted: 2 February 1997