Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

Identification ofArmillaria species from hokkaido by analysis of the intergenic spacer (IGS) region of ribosomal DNA using PCR-RFLP

The intergenic spacer (IGS) region, which is located between the 3′ end of 26S ribosomal DNA (rDNA) and the 5′ end of 5S rDNA, of sixArmillaria species from Hokkaido was investigated using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Restriction with onlyAlu I could distinguishA. mellea subsp.nipponica from the other species. WithAlu I andDde I,A. ostoyae andA. gallica could be distinguished from the other species. Digestion withAlu I resulted in two patterns (types A and B) ofA. singula and three patterns (types A, B, and C) ofA. jezoensis. One pattern (type B) of the former species and two patterns (types B and C) of the latter species were each different from those of the other species.Armillaria sinapina gave only oneAlu I digestion pattern, which was identical to that ofA. jezoensis (type A) andA. singula (type A). However, by digestion withDde I,A. singula (type A) could be distinguished fromA. jezoensis (type A) andA. sinapina.     

Ribosomal RNA genes of Phaseolus coccineus. I

rDNA fragments, including the whole intergenic spacer (IGS) region of P. coccineus, were cloned into dephosphorylated pUC 13 plasmid. Four clones of different insert size were analysed. Restriction patterns and physical maps of these length variants (pPH1, pPH2, pPH5, pPH6) were performed through complete Eco RI cleavage and partial digestion with Hpa II, Hae III, Sau 3AI, Sma I. Evidence was obtained that the length heterogeneity of the four genes was mainly due to a differing number of about 170 bp sub-repeating elements in the IGS. Indeed, there are 16 of these in pPH1, about 34 in pPH2, 10 in pPH5 and about 60 in pPH6. The sequence analysis of pPH6 sub-repeats revealed that there are two types of sub-repeats: short ones (S) of 162 bp and long ones (L) of 176 bp. The homology between S and L is high (93.8%). S and L elements are present in at least three of the four genes investigated, as shown by a restriction pattern obtained with Hae III digestion to completion. The relative frequency of S and L types, however, differs among the four genes. The possible functional meaning of the subrepeat structure is discussed on the basis of the homology between the S and L sequences on the one hand and on the other the ribosomal sequences of: i) Xenopus promoter(s); ii) wheat block A sub-repeats; iii) presumptive promoter(s) of wheat.     

Molecular structure of the Frankia spp. nifD—K intergenic spacer and design of Frankia genus compatible primer

The nifD—K intergenic spacer (IGS) of ArI3 and ACoN24d were found to have a length 265 and 199 nucleotides, respectively. They are markedly less conserved than the two neighbouring genes and have, in some instances, a repeated structure reminiscent of an insertion event The repeated sequence and the IGSs have no detectable homology with sequences in DNA databanks. The IGS has a stem-loop structure with a low folding energy, lower than that between nifH and nifD. No convincing alignment of IGS sequences could be obtained among Frankia strains. Only between ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structure. No promoter could be detected in the IGSs. The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleotides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN₇). The codon usage was similar to that of previously sequenced Frankia genes with a strong bias toward G- and C-ending codons except in the case of glycine where GGT is frequent. Alignment of the three Frankia nifK sequences (EUN1f; ArI3 and ACoN24d) with those of other nitrogen-fixing bacteria permitted detection of a sequence conserved among the three Frankia strains but absent in the other sequences. A primer targeted to that region in combination with FGPD807-85 amplified the nifD—K IGS sequences of all Frankia strains (except the non-nitrogen-fixing Frankia strains CN3 and AgB1-9) and yet failed to amplify DNA of all other nitrogen–fixing bacteria. Conversely, the failure of primer FGPK700′-92 to amplify Alnus-infective strains could be explained by point mutations in the 3′ part of the primer.     

Heterogeneity and organization of the ribosomal RNA genes ofCucurbita maxima

Thirty-six clones were recovered fromCucurbita maxima genomic DNA which had been enriched for rDNA and cleaved at the unique repeat unitHind III site. Twenty-nine of these, which contain complete rDNA units, were compared to a standard whose intergenic spacer (IGS) nucleotide sequence has been determined. Twenty-one are identical in length and restriction site pattern. Eight which differ from the standard in length do so because of addition or deletion of varying numbers of IGS subrepetitive units of two different classes, with four of the length variants being different in both of these classes. Seven clones were isolated which contain incomplete repeat units, six of which are composites of rDNA and non-rDNA material. They have been cleaved at the unique rDNAHind III site at one end and at a non-rDNAHind III site at the other. We consider it most likely that these are derived from the termini of repeat unit tandem arrays, although other explanations are possible. Twelve individual plants of two different cultivars were examined for heterogeneity of IGS length distribution. They all appear to be identical in this regard.     

The intergenic spacer region of the ribosomal RNA gene of Penicillium marneffei shows almost no DNA sequence diversity

The fungus Penicillium marneffei causes fatal systemic infections and is endemic in many parts of South-East Asia, especially Thailand. The intergenic spacer (IGS) region, the most variable region of rRNA genes, was found to be highly conserved among 58 P. marneffei strains. IGS analysis might not be suitable for molecular epidemiological analysis of P. marneffei infections.     

Comparative analysis of the intergenic spacer regions and population structure of the species complex of the pathogenic yeast Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI +5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.     

Inter- and intraspecific genomic variability of the 16S-23S intergenic spacer regions (ISR) in representatives of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans

The complete sequences of 32 intergenic spacer regions (ISR) from Acidithiobacillus strains, including 29 field strains isolated from coal, copper, molybdenum mine wastes or sediment of different geoclimatic regions in China, reference strain ATCC19859 and the type strains of the two species were determined. These data, together with other sequences available in the GenBank database, were used to carry out the first detailed assessment of the inter- and intraspecific genomic variability of the ISR sequences and to infer phylogenetic relationships within the genus. The total length of the 16S-23S rRNA intergenic spacer regions of the Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans strains ranged from 451 to 490 bp, and from 434 to 456 bp, respectively. The degree of intrageneric ISR sequence similarity was higher than the degree of intergeneric similarity, and the overall similarity values of the ISRs varied from 60.49% to 84.71% between representatives of different species of the genus Acidithiobacillus. Sequences from the spacer of the A. thiooxidans and A. ferrooxidans strains ranged from 86.71% to 99.56% and 92.36% to 100% similarity, respectively. All Acidithiobacillus strains were separated into three phylogenetic major clusters and seven phylogenetic groups. ISR may be a potential target for the development of in situ hybridization probe aimed at accurately detecting acidithiobacilli in the various acidic environments.     

Rapid species identification and partial strain differentiation of Clostridium butyricum by PCR using 16S-23S rDNA intergenic spacer regions

Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain-specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S-23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.     

Ribosomal DNA ITS-1 Intergenic Spacer Polymorphism in Triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.     

Molecular systematics of Brassica and allied genera in subtribes Brassicinae, Raphaninae, Moricandiinae, and Cakilinae (Brassicaceae, tribe Brassiceae); the organization and evolution of ribosomal gene families

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of ribosomal DNA (rDNA) of Brassica and allied genera. The total genomic DNA of 95 accessions of 52 species representing 16 genera was restricted with six enzymes, and the restriction fragments were probed with three ribosomal clones (pTA71, Ver 18‐6, and Ver 6‐5). Eleven repeat unit length classes were recognized. The repeat unit size classes of 8.9 kb and 9.5 kb were observed most commonly, being represented in 17 and 14 species, respectively. The restriction enzyme SacI produced three to six (generally three) bands with detectable hybridization to the probe pTA71. This probe–enzyme combination indicated a remarkable uniformity amongst Brassica and allied genera in the coding region of repeat units. By contrast, an extensive size variation in the restriction fragments could be localized in the intergenic spacer (IGS) region. Eleven IGS‐containing length variants were detected. Complex hybridization patterns, resulting from extensive repeat unit heterogeneity and taxon‐specific methylation of one or more cleavage sites, were obtained with the EcoRI + pTA71 combination. The relative homologies between the coding regions were evident from the presence of 1.5 kb in all the taxa, and 0.4‐, 1.3‐, and 1.7‐kb fragments in 33, 27, and 24 species, respectively. The SacI + pTA71 and EcoRI + pTA71 combinations were generally able to distinguish taxa both within and between genera. Three restriction endonuclease digests probed with three ribosomal clones yielded essentially identical fragmentation patterns across all the accessions within the cultivated species Brassica campestris, B. oleracea, and B. juncea. In B. napus, three and seven accessions exhibited restriction profiles similar to one and both diploid progenitor species, respectively. Overall, rDNA repeat unit length polymorphism showed good correlation with the cytodeme‐based classification of Brassica and allied genera. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157 , 545–557.     

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.     

Directed alterations of the X-chromosomal ribosomal gene repeats in mutant sc8 of Drosophila melanogaster

The patterns of the ribosomal DNA (rDNA) repeat units in seven Drosophila melanogaster inversional mutants have been studied. Among them, only the In(1)sc⁸ and its deletional derivative Df(1)mal¹² female rDNAs exibited significant reduction in the size of nearly all units, compared to the wild-type females (Canton S, Oregon R). Further investigation shows that each kind of repeat (insertion-free, insertion-containing) in the Xsc⁸ rDNA array is highly enriched with short (reduced to 4 kilobases) intergenic spacers (IGSs). We revealed two main types of rearrangements. Only part of the 4 kb IGSs display variable length deletions (0.2–0.6 kb) at the 5′ ends, within the so-called ‘1900’ base pair (bp) region, recognizable by restriction endonuclease AluI. The presence of additional 100–150 bp DNA in the start portion of this region has also been demonstrated. In contrast, the 3′ end spacer regions, corresponding to the external transcribed spacer, do not show any changes in size. These data indicate how reductions of approximately 1.1 kb DNAs in sc⁸ IGSs, carrying both the rearranged and non-rearranged ‘1900’ sequences, are achieved: the fixed decrease of a number of 240 bp AluI subrepeats, clustered in the central IGS portion, also contribute. None of the other similar inversional mutants examined has so many IGS variants. Therefore, alterations in the Xsc⁸ rRNA gene cluster seem not to be dependent on its inversional status. This revised version was published online in July 2006 with corrections to the Cover Date.