Novel class of rDNA repeat units in somatic hybrids between Nicotiana and Atropa

Summary Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to ³²P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled ³²P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.     

Cloning of ribosomal RNA genes from an Indian isolate ofGiardia lamblia and the use of intergenic nontranscribing spacer regions in the differentiation ofGiardia from other enteric pathogens

The ribosomal RNA genes from an Indian isolate ofGiardia lamblia have been cloned and characterized with respect to size, composition and copy number. Southern blotting and rDNA cloning ofGiardia lamblia revealed that genes coding for ribosomal RNA (rRNA) are exceptionally small and are encoded within a 5.6 kb genome fragment repeat. The rDNA repeat unit of this isolate was found to be highly G-C rich like other human isolates and the physical map showed severalSmaI sites. There are 132 copies of the rDNA repeat unit per cell in a head to tail arrangement. Two fragments corresponding to intergenic (0.2 kb and 0.3 kb) region and one (0.8 kb) containing both an intergenic region and a small part of the small subunit ribosomal RNA (SS rRNA) have been identified within the rDNA. These were used in heterogeneity studies ofGiardia isolated from two geographic locations as well as in the analysis of cross reactivity with other enteric organisms. In Southern blots, all the three fragments were found to be highly specific for the differential diagnosis ofGiardia spp. from the other enteric pathogens. These findings should help in developing a sensitive and more specific method for the diagnosis of giardiasis over currently available techniques.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

Conservation of rDNA inPolystichum (Dryopteridaceae)

Restriction site variation in the nuclear 18S–25S ribosomal RNA genes (rDNA) was analyzed hierarchically in a species complex in the fern genusPolystichum. Two distinct rDNA repeat types were present in all individuals ofPolystichum examined. No variation was detected among individuals within a population ofP. munitum, among populations ofP. munitum orP. imbricans, or among the six diploid species ofPolystichum from North America, including the circumborealP. lonchitis. The identity of rDNA repeats across all six North American species ofPolystichum may reflect an overall similarity of the nuclear genomes of these species, an observation supported by isozyme data as well. However, this nuclear similarity contrasts sharply with the highly divergent chloroplast genomes of these six species. The conservative nature of the rDNA inPolystichum also is in contrast to the much more variable rDNAs of most angiosperms investigated. Perhaps the tempo and mode of evolution of rDNA in ferns differ from those of angiosperms; however, the data base for fern rDNA is very small. Furthermore, the number of repeat types per individual is consistent with a diploid, rather than polyploid, condition despite the high chromosome number (n = 41) of these plants, although homogenization of multiple, divergent rRNA genes cannot be disproven.     

Ribosomal RNA genes in species of theCynareae tribe (Compositae). I.

Summary By means of Southern blot hybridization, the structure of the ribosomal DNA in six species of theCynareae tribe has been analyzed. Artichoke and wild artichoke possess only one type of ribosomal genes 13 kb long;Onopordum acanthium has at least two types of rDNA repeats 9.9 kb and 10.3 kb long andO. illyricum has a third gene type 9.7 kb long; inGalactites tomentosa there are at least four ribosomal gene types of 10.9, 11.6, 11.5, and 10kb;Carduus nutans possesses two ribosomal gene types of the same length of 12.5 kb that vary in the nucleotide sequence of the external spacer. The rRNA genes of all the species studied have an identical restriction mapping in the 18 S and 25 S regions, differences in length and/or nucleotide sequence are present in the external spacer.     

Ribosomal RNA genes in species of theCynareae tribe (Compositae). II

Summary The structure of the ribosomal DNA has been analyzed in three species of theCynareae tribe using Southern blot hybridization.Silybum marianum possesses two types of ribosomal genes 12.3 and 13.4 kb long;Cirsium vulgare has at least four types of rDNA repeats 10.6, 10.5, 11.7 and 11.9 kb long;Carlina acaulis only one type of ribosomal genes 9.7 kb long. The rRNA genes of the three species studied showed an identical restriction mapping in the 18 S and 25 S regions. However species differentiation in length and/or nucleotide sequences are present in the external spacer and very probably in the internal transcribed spacer.By cytophotometric studies and byin vitro rRNA/DNA filter hybridization, the DNA amount/4 C nucleus and the rDNA percentage were calculated in nine species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Cynara cardunculus subsp.cardunculus (wild artichoke),Onopordon acanthium, O. illyricum, Galactites tomentosa, Carduus nutans, Silybum marianum, Cirsium vulgare andCarlina acaulis. The DNA amount/4 C nucleus in eight species are similar, ranging from 4.24 pg inGalactites tomentosa to 5.96 pg inCirsium vulgare, whileCarlina acaulis has a DNA amount/ 4C nucleus of 11.8 pg. The rDNA percentages range from 0.192% inOnopordon acanthium to 1.022% inSilybum marianum, whileCarlina acaulis has 0.038% of rDNA. This latter species is clearly distinct within theCynareae tribe.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Individual quantitative rDNA variation in three species of the Cucurbitaceae family

Individual quantitative variation in rDNA content within three species of the Cucurbitaceae family has been studied by rRNA/DNA filter hybridization experiments. The results showed a 2.3-fold range of variation in the number of ribosomal cistrons per diploid cell in an Ecballium elaterium natural population. This range of variation is compared with the smaller range observed in three Cucumis sativus and in two Cucurbita pepo varieties obtained as F₁ hybrids between pure lines.This work was supported by CNR Contract No. 74/0267.     

Repeats and subrepeats in the intergenic spacer of rDNA from the nematode Meloidogyne arenaria

Summary Ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria are heterogeneous in size and appear to contain 5S rRNA gene sequences. Moreover, in a recA ⁺ bacterial host, plasmid clones of a 9 kb rDNA repeat show deletion events within a 2 kb intergenic spacer (IGS), between 28S and 5S DNA sequences. These deletions appear to result from a reduction in the number of tandem 129 by repeats in the IGS. The loss of such repeats might explain how rDNA length heterogeneity, observed in the Meloidogyne genome, could have arisen. Each 129 by repeat also contains three copies of an 8 by subrepeat, which has sequence similarity to an element found in the IGS repeats of some plant rDNAs.     

Length polymorphism and homologies of microsatellites in several Cucurbitaceae species

The objectives of this research were to assess (1) the degree of Simple Sequence Repeats (SSR) DNA length polymorphism in melon (Cucumis melo L.) and other species within the Cucurbitaceae family and (2) the possibility of utilizing SSRs flanking primers from single species to other genera or species of Cucurbitaceae. Five melon (CT/GA) _n SSRs were isolated from a genomic library. Two cucumber (Cucumis sativus L.) SSRs were detected through a search of DNA sequence databases, one contained a (CT)₈ repeat, the other a (AT)₁₃ repeat. The seven SSRs were used to test a diverse sample of Cucurbitaceae, including 8 melon, 11 cucumber, 5 squash, 1 pumpkin, and 3 watermelon genotypes. Five of the seven SSRs detected length polymorphism among the 8 melon genotypes. PCR amplification revealed between three and five length variants (alleles) for each SSR locus, with gene diversity values ranging from 0.53 to 0.75. Codominant segregation of the alleles among F₂ progeny was demonstrated for each of the five SSR loci. Four of the seven SSRs detected polymorphism among the 11 cucumber genotypes, with gene diversity values ranging between 0.18 and 0.64. Primers specific to SSRs of C. melo and C. sativus also amplified DNA extracted from genotypes belonging to other genera of the Cucurbitaceae family.     

The construction and characteristics of a BAC library for <Emphasis Type="Italic">Cucumis sativus</Emphasis> L. ‘B10’

Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, which are used for the physical mapping, identification and isolation of genes, and for gene sequencing. A BAC genomic library was constructed from high molecular weight DNA (HMW DNA) obtained from nuclei of the cucumber (Cucumis sativus L. cv. Borszczagowski; B10 line). The DNA was digested with the HindIII restriction enzyme and ligated into the pCC1BAC vector. The library consists of 34,560 BAC clones with an average insert size of 135 kb, and 12.7x genome coverage. Screening the library for chloroplast and mitochondrial DNA content indicated an exceptionally low 0.26% contamination with chloroplast DNA and 0.3% with mitochondrial DNA.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

Insertion and amplification of a DNA sequence in satellite DNA of Cucumis sativus L. (cucumber)

Summary Another satellite DNA repeat (type IV) in the genome of Cucumis sativus (cucumber) was found and investigated with respect to DNA sequence, methylation, and evolution. This satellite shows a repeat length of 360 bp and a GC-content of 47%. The repeats of type IV are highly conserved among each other. Evidence for CG and CNG methylation is presented. By comparison to the previously described satellites (type I/II and type III) from cucumber, it is evident that this repeat is created by an insertion of a 180 bp DNA sequence similar to type I–III into another DNA sequence (or vice versa), and subsequent amplification forming a new satellite repeat. The different satellites of the type I/II, type III, and the 180 bp insert of type IV show a sequence homology of 60%–70%, indicating that the complex satellite DNA of cucumber is originated from a common progenitor by mutation, additional insertion, and amplification events. Copies of a sequence similar to a part of type IV are present in the genome of the related species Cucumis melo (melon).     

Identification of a dispersed MboI repeat family in five higher plant genomes

Digestion of nuclear DNAs of five plants, namelyCucurbita maxima (red gourd),Trichosanthes anguina (snake gourd),Cucumis sativus (cucumber),Cajanus cajan (pigeon pea) andPhaseolus vulgaris (french bean) with the restriction endonucleaseMboI yielded discrete size classes with molecular weights in the range of 0.5 to 5 kbp. TheMboI digestion pattern of Cot 0.1 DNA in french bean is comparable with that of total DNA, indicating that these bands represented highly repeated DNA sequences. Cleavage of the DNAs with varying amounts ofMboI indicated the dispersed nature of the repeat families. Southern hybridization studies using french bean highly repetitive DNA as a probe indicated more homology with repeats of pigeon pea and less homology with red gourd, snake gourd and cucumber repeats.     

Genomic organization of ribosomal DNA and related sequences inTriturus

The ribosomal RNA genes ofTriturus vulgaris meridionalis are clustered at variable and often multiple chromosomal loci. The rDNA repeats exhibit only a limited and discrete length heterogeneity which is accounted for by the non-transcribed spacer (NTS). Interestingly, sequences homologous to the NTS are clustered outside the ribosomal loci. Clones containing such non ribosomal sequences have been isolated from a genomic library ofT. v. meridionalis and analyzed by restriction mapping. These sequences appear to consist mostly of repetitive Bam HI fragments ranging from 500 bp to 1000 bp. The Bam HI fragments are internally repetitious and highly homologous to each other.     

Ribosomal gene variation in soybean (Glycine) and its relatives

Summary The genes encoding the 18S25S ribosomal RNA gene repeat in soybean (Glycine max) and its relatives in the genus Glycine are surveyed for variation in repeat length and restriction enzyme site locations. Within the wild species of subgenus Glycine, considerable differences in repeat size occur, with a maximum observed in G. falcata. Repeat length and site polymorphisms occur in several species, but within individual plants only single repeat types are observed. The rDNA of the cultivated soybean and its wild progenitor, G. soja are identical at the level of this study, and no variation is found in over 40 accessions of the two species. Data from rDNA mapping studies are congruent with those of previous biosystematic studies, and in some instances give evidence of divergences not seen with other approaches.     

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed (DQ119058). The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.