Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

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.     

Repeated DNA sequences found in the large spacer of Vicia faba rDNA

In the large spacer of the rDNA of Vicia faba, multiples of a 0.32 kilobasepair (kb) sequence reiterate to various degrees. We sequenced the repetitious region consisting of the repeating sequences and its flanking regions using two cloned plasmids, which contain V. faba rDNA segments encompassing the whole region of the large spacer. The repetitious region was found to consist of multiple complete copies and one truncated copy of a 325 bp repeat unit and to be flanked by direct repeat sequences of about 150 bp. The set of direct repeats located at either side of the repetitious region differed from each other with about 10% sequence heterogeneity. However, nucleotide sequences of the direct repeats were well conserved between the two clones examined. Southern blot hybridization indicated a widespread distribution within the whole V. faba genome of some related sequences with high homologies to the 325 bp repeat unit and to the direct repeats.     

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.     

Sequence comparison of distal and proximal ribosomal DNA arrays in rice (Oryza sativa L.) chromosome 9S and analysis of their flanking regions

Rice (Oryza sativa ssp. japonica cv. Nipponbare) harbors a ribosomal RNA gene (rDNA) cluster in the nucleolar-organizing region at the telomeric end of the short arm of chromosome 9. We isolated and sequenced two genomic clones carrying rice rDNA fragments from this region. The rice rDNA repeat units could be classified into three types based on length, which ranged from 7,928 to 8,934 bp. This variation was due to polymorphism in the number of 254-bp subrepeats in the intergenic spacer (IGS). Polymerase chain reaction (PCR) analysis suggested that the rDNA units in rice vary widely in length and that the copy number of the subrepeats in the IGS ranges from 1 to 12 in the rice genome. PCR and Southern blot analyses showed that most rDNA units have three intact and one truncated copies of the subrepeats in the IGS, and distal (telomere-side) rDNA units have more subrepeats than do proximal (centromere-side) ones. Both genomic clones we studied contained rDNA-flanking DNA sequences of either telomeric repeats (5′-TTTAGGG-3′) or a chromosome-specific region, suggesting that they were derived from the distal or proximal end, respectively, of the rDNA cluster. A similarity search indicated that retrotransposons appeared more frequently in a 500-kb portion of the proximal rDNA-flanking region than in other subtelomeric regions or sequenced regions of the genome. This study reveals the repetitive nature of the telomeric end of the short arm of chromosome 9, which consists of telomeric repeats, an rDNA array, and a retrotransposon-rich chromosomal region.Sequence accession numbers in DDBJ assigned for OSJNOa063K24 and OSJNBb0013K10 are AP009051 and AP008245, respectively.     

Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus

Summary The technique of chromosome walking was used to isolate approximately 60 kb of DNA from the region containing the complementation group uncoordinated of Drosophila melanogaster, located in that part of the X chromosome which spans the euchromatin-heterochromatin junction. The cloned DNA can be divided into two distinct regions. The first contains sequences that are low copy number or unique and are largely conserved between strains. The second region is characterized by units repeated in tandem arrays and is polymorphic within, and between, strains. Each repetitive unit is separated by a member of an abundant sequence family, part of which is homologous to the ribosomal type 1 insertion sequence of D. melanogaster. The molecular organization of the cloned DNA was compared with that of sequences isolated from regions of intercalary heterochromatin and also with genes which have been characterized from more conventional euchromatic regions.     

Phylogenetic and evolutionary implications of nuclear ribosomal DNA variation in dwarf dandelions (Krigia,Lactuceae, Asteraceae)

Restriction site and length variations of nrDNA were examined for 51 populations of seven species ofKrigia. The nrDNA repeat ranged in size from 8.7 to 9.6 kilobase (kb). The transcribed region, including the two ITSs, was 5.35 kb long in all examinedKrigia populations. In contrast, the size of the nontranscribed IGS varied from 3.35 to 4.25 kb. Eight different types of length-variations were identified among the 51 populations, including distinct nrDNA lengths in the tetraploid and diploid populations of bothK. biflora andK. virginica. However, a few variations were detected among populations of the same species or within a cytotype. All populations ofKrigia sect.Cymbia share a 600 bp insertion in IGS near the 18 S gene, and this feature suggests monophyly of the section. AllKrigia spp. had a conjugated type of subrepeat composed of approximately 75 basepairs (bp) and 125 bp. Base modifications in the gene coding regions were highly conserved among species. Forty-five restriction sites from 15 enzymes were mapped, 24 of which were variable among populations. Only four of the variable sites occurred in the rRNA coding region while 20 variable sites were detected in the noncoding regions. Collectively, 25 enzymes generated about 66 restriction sites in each nrDNA; this amounts to about 4.3% of the nrDNA repeat. A total of 50 restriction sites was variable, 28 of which were phylogenetically informative. Phylogenetic analyses of site mutations indicated that two sections ofKrigia, sect.Cymbia and sect.Krigia, are monophyletic. In addition, relationships among several species were congruent with other sources of data, such as cpDNA restriction site variation and morphology. Both length and restriction site variation supported an allopolyploid origin of the hexaploidK. montana. The average sequence divergence value inKrigia nrDNA was 40 times greater than that of the chloroplast DNA. The rapid evolution of nrDNA sequences was primarily due to changes of the IGS sequences.     

Studies on the diversity of the distinct phylogenetic lineage encompassing Glomus claroideum and Glomus etunicatum

Morphological and molecular characters were analysed to investigate diversity within isolates of the Glomus claroideum/Glomus etunicatum species group in the genus Glomus. The inter- and intra-isolate sequence diversity of the large subunit (LSU) rRNA gene D2 region of eight isolates of G. claroideum and G. etunicatum was studied using PCR-single strand conformational polymorphism (SSCP)-sequencing. In addition, two isolates recently obtained from Southern China were included in the analysis to allow for a wider geographic screening. Single spore DNA isolation confirmed the magnitude of gene diversity found in multispore DNA extractions. An apparent overlap of spore morphological characters was found between G. claroideum and G. etunicatum in some isolates. Analysis of the sequence frequencies in all G. etunicatum and G. claroideum isolates (ten) showed that four LSU D2 sequences, representing 32.1% of the clones analysed for multispore extraction (564) were found to be common to both species, and those sequences were the most abundant in four of the ten isolates analysed. The frequency of these sequences ranged between 23.2% and 87.5% of the clones analysed in each isolate. The implications for the use of phenotypic characters to define species in arbuscular mycorrhizal fungi are discussed. The current position of G. claroideum/G.etunicatum in the taxonomy of the Glomeromycota is also discussed.     

Sequence subfamilies of satellite repeats related to rDNA intergenic spacer are differentially amplified on<Emphasis Type="Italic"> Vicia sativa</Emphasis> chromosomes

We cloned and sequenced the Vicia sativa 25S-18S rDNA intergenic spacer (IGS) and the satellite repeat S12, thought to be related to the spacer sequence. The spacer was shown to contain three types of subrepeats (A, B, and C) with monomers of 173 bp (A), 10 bp (B), and 66 bp (C), separated by unique or partially duplicated sequences. Two spacer variants were detected in V. sativa that differed in length (2990 and 3168 bp) owing to an extra copy of the subrepeat A. The A subrepeats were also shown to be highly homologous to the satellite repeat S12, which is located in large clusters on chromosomes 4, 5, and 6, and is not associated with the rDNA loci. Sequencing of additional S12 clones retrieved from a shotgun genomic library allowed definition of three subfamilies of this repeat based on minor differences in their nucleotide sequences. Two of these subfamilies could be discriminated from the rest of the S12 sequences as well as from the IGS A subrepeats using specific oligonucleotide primers that labeled only a subset of the S12 loci when used in the primed in situ DNA labeling (PRINS) reaction on mitotic chromosomes. These experiments showed that, in spite of the high overall similarity of the IGS A subrepeats and the S12 satellite repeats, there are S12 subfamilies that are divergent from the common consensus and are present at only some of the chromosomes containing the S12 loci. Thus, the subfamilies may have evolved at these loci following the spreading of the A subrepeats from the IGS to genomic regions outside the rDNA clusters.Electronic Supplementary Material Supplementary material is available in the online version of this article at Accession numbers: GenBank AY234364–AY234374. The monomer sequences and additional information about the family of IGS-like repeat S12 will also appear in the PlantSat database (Macas et al. 2002, ) under Accession name Vicia_sativa_IGS-like     

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.     

Intraspecific Diversity within Diaporthe Helianthi: Evidence from Rdna Intergenic Spacer (IGS) Sequence Analysis

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe but recorded sporadically in Italy. The genetic diversity of D. helianthi isolates from different geographic origins (Argentina, France, Italy, Yugoslavia, Romania) was investigated using IGS sequences. A 400 bp fragment of the portion of the IGS region flanking the 5' end of the 18S gene was amplified from each isolate. The aligned nucleotide sequences showed intraspecific sequence homology from 99-100% among French/Yugoslavian isolates to 95-100% among Italian isolates. French/Yugoslavian isolates shared 90-92% sequence homology with Italian isolates. The phylogenetic tree obtained from the aligned data revealed three separate groups. Group 1 included all isolates from France and former Yugoslavia and one isolate from Argentina; Group 2 included all Italian isolates and one isolate from Argentina. The most distantly related isolate was that from Romania (Group 3). The average genetic distances among isolates within Group 1 and within Group 2 were 0.22 and 3.29 respectively. The analysis showed that all isolates originating from countries where severe outbreaks of the disease are reported annually (France and former Yugoslavia) form a well defined taxon characterized by relatively low variability. This group is distinct from the group formed by isolates originating from Italy, whose variability is relatively much higher. Results obtained revealed a marked differentiation among pathogen isolates, and members of Group 1 seem not yet to have spread into Italian sunflower-growing areas.     

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.     

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: Intraspecific heterogeneity within the intergenic spacer region. Fungal Genetics and Biology 29, 19-27. The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.     

The Cucurbita maxima ribosomal DNA intergenic spacer has a complex structure

The nucleotide (nt) sequence of the 5508-nt intergenic spacer (IGS), between the 25S- and the 18S-coding regions of Cucurbita maxima rDNA, was determined. The fragment sequenced is 6142 nt long and includes 472 nt of 25S- and 162 nt of 18S-coding regions. The IGS has a complex primary structure, composed of five repetitive families (A-E) and three unique domains. It is dominated by the presence of nine, tandemly-repeating units of approximately 250 nt (repeat D), each unit containing four copies of an internal subrepeat (repeat E). The repetitive units show sequence variability consisting of nt changes, insertions and deletions. Upstream of the nine D repeats and between two copies of the B repeat is a 575-nt region, highly G + C rich (83%) and heavily biased toward C (58%) in the sense strand. Within this region are six repetitive units, averaging 42 nt (repeat C) each, containing but a single A nt. Downstream from the terminus of the 25S-coding sequence, are two tandem copies of the 103-nt A repeat. The IGS of C. maxima is longer and more complex than that of other plant IGSs described to date. The 600 nt at the 5' portion of cucurbit IGS is more conserved in evolution than the remainder, as revealed by comparison of C. maxima and C. pepo IGS restriction maps and by nucleotide sequence comparison of C. maxima and Cucumis sativa IGSs.     

Intra-isolate Heterogeneity and Reproducibility of PCR-based Genotyping of Cryptosporidium parvum Using the β-tubulin Gene

Cryptosporidium parvum is a common contaminant in surface waters and presents significant problems for the water industry, public health and agriculture. Consequently, ascertaining the contaminating source of waterborne oocysts is of paramount importance. Based on currently available information, isolates of C. parvum can be differentiated into at least two genotypes using polymorphic genetic markers: genotype 1, to date isolated almost exclusively from humans, and genotype 2 isolates from humans and many other animals. Differentiation into these two genotypes has been based on either restriction fragment length polymorphisms or sequencing of PCR amplified gene fragments. The objective of this study was to evaluate the reproducibility of genotyping methods using a single isolate of C. parvum. A 620 bp fragment of the C. parvum -tubulin gene, generated by PCR from multiple aliquots of a single preparation of oocysts of the Iowa isolate, was sequenced. Significant sequence heterogeneity was detected within this single isolate; there was more sequence variation between clones originating from the Iowa isolate (up to 0.9 %) than between individual clones originating from different isolates of C. parvum. Over 6 % of the -tubulin gene sequence positions (38 out of 620 bp) were variable when comparing multiple clones from the one isolate. The results indicated that while the various procedures used for genotyping isolates may introduce some sequence errors, the Iowa isolate used for this investigation appeared to be composed of multiple sub-genotypes. While none of the sequence variations resulted in clones of the Iowa isolate (genotype 2) being mis-identified as genotype 1, the results have important implications if minor sequence variations are to be used for subtyping isolates and drawing conclusions regarding the origin of, or relationships between, C. parvum oocysts in water and the community.     

Sequence analysis of rDNA intergenic spacer (IGS) of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The intergenic spacer region (IGS) has been used for the first time to analyze the genetic variability of Porphyra haitanensis from different areas. In order to determine that whether the IGS sequences could be used for classification and identification in intraspecies of Porphyra, the partial IGS sequences of cultivated strains of P. haitanensis (isolated from Putian-Fujian Province, Shantou-Guangdong Province and Ningbo-Zhejiang Province), were amplified, sequenced and analyzed. The sequence analysis indicated that the partial IGS sequences from the three stains were the external transcribed spacers (ETS) of 3′ end of the IGS gene. In the three stains, the length of IGS sequences ranged from 1,085 to 1,100 bp and the G + C content varied from 50.88% to 51.27%. There were 55 variable sites which occupied approximately 5% of the ETS sequences. Similarity analysis and multisequencing alignment of sequences indicated that the partial IGS sequences of the three stains of P. haitanensis had notable variabilities. Therefore, the IGS sequence could be used as the critical genetic marker in intraspecies of P. haitanensis. Furthermore, IGS sequence analysis will be a powerful tool for genetic diversity and classification in intraspecies of other Porphyra species.     

High-frequency intragenomic heterogeneity of the ribosomal DNA intergenic spacer region in Trichophyton violaceum

The intergenic spacer (IGS) of the rRNA genes was analyzed from the dermatophyte Trichophyton violaceum isolated from cases of tinea capitis in Taiwan and Iran. T. violaceum strains were cultured from different colonies, from single conidial colonies derived by dilution plating, and from micromanipulation of single conidia from clinical samples. A ribosomal DNA probe hybridizing to multiple EcoRI fragments was used to compare restriction fragment length polymorphisms in different T. violaceum isolates. The arthroconidia of T. violaceum that form in vivo during infection were shown to contain a single nucleus by 4',6'-diamidino-2-phenylindole staining. IGS regions from an isolate cultured from a single conidium were amplified, cloned, and sequenced. The results identified that heterogeneity exists between IGS regions within a single T. violaceum genome due to different copy numbers of a 171-bp tandem repeat. This suggests that the IGS of T. violaceum is partially excluded from the concerted evolution of the rRNA gene locus. The heterogeneous character of the IGS regions in T. violaceum contrasts with the closely related dermatophyte Trichophyton rubrum, posing further questions on the phylogeny and the evolution of dermatophyte fungi.