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Eukaryotic Cell, April 2008, p. 721-726, Vol. 7, No. 4
1535-9778/08/$08.00+0     doi:10.1128/EC.00441-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High-Frequency Intragenomic Heterogeneity of the Ribosomal DNA Intergenic Spacer Region in Trichophyton violaceum

Jen-Chyi Chang,^1* Mark Ming-Long Hsu,² Richard C. Barton,³ and Colin J. Jackson⁴

Department of Medical Laboratory Science and Biotechnology,¹ Department of Dermatology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan,² Mycology Reference Centre, The General Infirmary at Leeds, Leeds LS1 3EX, United Kingdom,³ School of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, United Kingdom⁴

Received 3 December 2007/ Accepted 13 February 2008

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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.

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Dermatophytes are a group of fungi specializing in the infection of keratinous tissues such as skin, hair, and nails. Trichophyton violaceum is an anthropophilic dermatophyte (transmitted between humans) and is the major causal agent of tinea capitis (scalp ringworm) in many parts of Africa (2, 21, 29) and Asia (12, 18, 22).

A lack of adequate methods for strain differentiation in T. violaceum has prevented efforts to address epidemiological questions in T. violaceum infections. DNA-based strain typing methods have long been recognized as the best approach to differentiating individual strains of pathogenic fungi, but a strain typing method for T. violaceum has not been described. Molecular strain differentiation making use of polymorphisms in the rRNA gene (rDNA) repeat region of the closely related species Trichophyton rubrum has been reported previously (13, 14). In T. rubrum, there are two different types of subrepeat element (TrS1 and TrS2) in the rDNA intergenic spacer (IGS) region. Analysis of variations in the copy number of the two subrepeats was successfully applied in the strain differentiation of T. rubrum. The homogeneous nature of IGS sequences within genomes of any one strain made PCR-based typing amenable. The aim of this study was to identify and characterize subrepeats in the IGS of T. violaceum and to determine whether they would provide a useful method of molecular strain differentiation in this species. The actual outcome of this study was the revelation of a high degree of heterogeneity of repeat sequences in the IGS region of T. violaceum, raising questions about the evolution of rRNA in fungi.

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Culture and storage of T. violaceum isolates. Thirty-two randomly selected Taiwanese isolates of T. violaceum, isolated from hair samples submitted to the Mycology Laboratory of the Department of Dermatology, National Cheng Kung University Hospital, Tainan, Taiwan, from October 1989 to February 1999, were recovered from stored cultures in sterile distilled water or Sabouraud's dextrose (SAB) agar (Difco Ltd., Oxford, United Kingdom) tubes overlaid with liquid paraffin for this study. All these isolates were identified to species level on the basis of colony characteristics and standard microscopy.

These isolates were labeled as JC1 to JC32. The 32 isolates were cultured from 29 patients, with three pairs of isolates from the same patient.

As T. violaceum does not produce single-celled conidia during routine culture in vitro, it was necessary to use the single celled arthroconidia present in the black-dot lesions of patients with tinea capitis. In order to investigate single conidial isolates, two black-dot samples taken from within two follicles on 28 January 2002 were selected randomly from a patient with T. violaceum tinea capitis infection and ground together with 500 µl sterile distilled water in a 2-ml-working-capacity sterile glass tissue grinder (Kimble/Kontes, Vineland, NJ) until the material was separated into individual arthroconidia as much as possible. The separated arthroconidia were then enumerated in a counting chamber. Suspensions of single and clumped arthroconidia were plated onto four Mycosel agar plates (BBL, Becton Dickinson). The strains cultured by this method were labeled with the prefix S.

In order to investigate single conidial isolates exclusively, single arthroconidial cultures were obtained by micromanipulation of the black-dot material from another sample of black-dot material from within a follicle, from a patient with T. violaceum tinea capitis. The black-dot material was spread out on a SAB plate containing 300 mg/liter cycloheximide using a sterile needle, and 23 individual arthroconidia were separated out using a customized Prior microscope with long-working-distance lenses (40x and 20x with 12.5x eyepieces) and a custom-engineered micromanipulation device. The single arthroconidium strains from the micromanipulation technique were cultured on SAB agar plates.

Four isolates of T. violaceum from cases of tinea capitis in Iran were also included in the study. These Iranian isolates were stored at –80°C in 1 ml sterile distilled water with 10% (vol/vol) dimethyl sulfoxide in the culture collection at the Mycology Reference Centre, Leeds, United Kingdom.

The rDNA internal transcribed spacer I (ITSI) and ITSII regions of five randomly selected strains (S1, JC1, JC7, JC16, and JC32) were sequenced using primers ITS1 and ITS4 (13) and compared to sequences at the NCBI; they were similar to sequences labeled as being derived from Trichophyton violaceum, Trichophyton glabrum, Trichophyton gourvilii, Trichophyton soudanense, or Trichophyton yaoundei, all at 99% or greater similarity (the latter four species are considered by many authors as being conspecific with T. violaceum). Differences were seen only in the number of TA repeats in a region of the ITSII.

Genomic DNA extraction. The method for extraction of fungal genomic DNA was as described previously (13) with modification to the preparation of fungal biomass. T. violaceum from a SAB agar plate was inoculated into 100 ml of SAB broth in a 250- or 300-ml Erlenmeyer flask and incubated with shaking (180 to 200 rpm/min) for 1 to 2 weeks at 30°C. When there was sufficient growth by visual inspection, hyphal biomass was harvested by negative pressure filtration and washed twice with 100 ml of sterile distilled water through a Whatman no. 1 filter paper (Brentford, Middlesex, United Kingdom) in a Buchner funnel. This biomass was stored at –75°C overnight or longer before extraction of nucleic acids. Small amounts of the hyphae grown in broth were subcultured onto a SAB agar slant before filtration and incubated at 25°C for 1 week to assess purity. The concentration of the extracted DNA was adjusted to 1 mg/ml, and the extract was stored at –20°C.

Detection of rDNA polymorphisms in T. violaceum. The restriction fragment length polymorphism (RFLP) Southern blotting methods were as described previously (13) with minor modifications. Ten micrograms of each DNA sample was digested with 25 U of restriction endonuclease EcoRI (Takara Biotechnology, Japan) for 24 h in a total volume of 20 µl. The rDNA probe was amplified from template DNA of T. violaceum JC16 by using universal primers ITS1 and ITS4 (28). The 700-bp probe consisted of a 30-bp fragment from the 3' end of the small-subunit (SSU) rDNA plus the adjacent ITSI, 5.8S rDNA, and ITSII regions (see Fig. 2).

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FIG. 2. EcoRI restriction map of the Trichophyton violaceum rDNA showing how an EcoRI digest generates two fragments from each rDNA repeat unit including a fragment containing the IGS region where putative subrepeat elements are proposed to be present.

DAPI staining of arthroconidia. One black-dot sample from a patient with T. violaceum tinea capitis was suspended in 20 µl of sterile distilled water and was teased apart with inoculating needles into small pieces. The suspension was then mounted in 2 µg/ml 4',6'-diamidino-2-phenylindole (DAPI). Arthroconidia were then observed at x400 magnification by bright-field and UV fluorescence microscopy (Olympus BX51 microscope) and photographed.

Sequence determination of the IGS region. The method of sequence determination was adapted from a previous method with slight changes (14). Genomic DNAs extracted from T. violaceum strains S1, S6, S8, and S10 were selected as templates from which to amplify the IGS regions using primers 25SCON2 and NS1-R (14). The commercial Expand Long Template PCR System kit was used (Roche Applied Science, Mannheim, Germany). Master Mix 1 contained 250 nmol of each deoxynucleoside triphosphate and 2.5 pmol of each upstream (25SCON2) and downstream (NS1-R) primer and was made up in a total volume of 24.0 µl with sterile water. One microliter of 10-fold-diluted stock T. violaceum genomic DNA was added to give approximately 100 ng of template.

Master Mix 2 contained 5 µl of 10x Expand Long Template PCR System buffer 3, 1 µl of enzyme mix, and 19 µl of sterile distilled water. Immediately prior to thermal cycling, the Master Mix 2 was added to Master Mix 1. The amplification reaction was carried out in a Hybaid thermocycler (Thermo Scientific, Waltham, MA) with an initial denaturation for 2 min at 93°C, followed by 35 cycles of primer annealing at 51°C for 0.5 min, extension at 68°C for 12 min, and denaturation at 93°C for 0.5 min. A terminal extension step of 68°C for 7 min completed the PCR.

The PCR product was purified using a High Pure PCR product purification kit (Roche Applied Science). It was then subcloned into the yT&A vector system (Yeastern Biotech Co., Ltd., Taiwan) and transformed into the Escherichia coli host strain ECOS101 (Yeastern Biotech Co., Ltd., Taiwan). The presence of cloned IGS inserts in the vector-transformed E. coli was confirmed by PCR using primers 25SCON2 and NS1-R on the extracted plasmid DNA. Sequencing of the IGS inserts was carried out using a primer-walking strategy, on an Applied Biosystems 3730 automated sequencer by Mission Biotech, Taipei, Taiwan.

Nucleotide sequence accession number. The nucleic acid sequence data for the IGS region of plasmid S8-1 have been deposited in the GenBank/EMBL/DDBJ databases with the accession number EF363337.

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RFLPs in the rDNA of T. violaceum isolates. Hybridization of an rDNA probe to immobilized EcoRI restriction fragments of genomic DNA identified extensive RFLPs in the rDNA of 32 Taiwanese isolates of T. violaceum (Fig. 1). A conserved DNA fragment, approximately 3.2 kb in length, was present in all 32 isolates. In addition to this, each individual strain showed multiple polymorphic bands ranging in size from 4 to 10 kb. In many strains the pattern of bands was characterized by a number of fragments of incrementally increasing sizes from 5 to 10 kb. No two isolates showed identical patterns of rDNA RFLPs. The patterns of four Iranian isolates were similar to those of Taiwanese isolates, with the conserved 3.2-kb fragment and abundant higher-molecular-weight bands (data not shown). From the results, a provisional restriction map of the T. violaceum rDNA repeat, similar to that of T. rubrum (13), was constructed (Fig. 2). As with T. rubrum it was proposed that the 3.2-kb constant band represented an EcoRI restriction fragment containing part of the 5.8S rRNA gene, the ITS2 spacer, and most of the large-subunit (LSU) rRNA gene. The variable higher-molecular-weight bands derived from fragments containing the entire IGS region, together with the SSU rRNA gene. The variability seen in these bands was predicted to derive from subrepeat elements within the IGS region (Fig. 2).

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FIG. 1. The rDNA RFLP patterns of 32 randomly selected Taiwanese Trichophyton violaceum isolates from patients with tinea capitis. Genomic DNA was digested with EcoRI and electrophoresed, and the blotted gel was hybridized with a probe derived from the ITSI, 5.8S rRNA, and ITSII regions.

rDNA RFLP of T. violaceum single conidial strains. Thirteen strains were grown from preparations of arthroconidia (95% single arthroconidia) from two black-dot samples from one patient with T. violaceum tinea capitis. The IGS profiles of these 13 cultures (S1 to S13) are shown in Fig. 3. The pattern of bands in these strains was similar to that seen in RFLP assays of previous strains, with a 3.2-kb constant fragment and multiple high-molecular-weight bands, often increasing incrementally in size. When the profiles were compared by visual inspection, there were nine different patterns in the 13 strains, with strains S2, S3, S7, S9, and S13 appearing identical, while the other eight strains were unique. The results suggested that there were mixed T. violaceum strains within a single infection, as multiple patterns were seen from strains isolated from a single patient, but also revealed that different IGS lengths existed within strains derived from a single T. violaceum arthroconidium.

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FIG. 3. The rDNA RFLP patterns of single conidial strains of Trichophyton violaceum S1 to S13 from a black-dot lesion from a single patient with tinea capitis analyzed as described for Fig. 1. The results show nine band patterns differing by at least one band in the 13 strains.

In order to determine the source of the variation in the IGS in T. violaceum, it was important to analyze strains deriving unequivocally from a single arthroconidium. Strains produced from plating out arthroconidial suspensions may have derived from the small numbers of clumps of more than one conidium present in suspensions. Thus, seven strains were grown from single arthroconidia separated by micromanipulation from a black-dot sample. When digests of genomic DNA were hybridized with the rDNA probe, the RFLP profiles of these seven strains were similar to previous results, and multiple high-molecular-weight fragments were present in all strains (data not shown). The results strongly suggested that different IGS lengths exist within an arthroconidium. The in vitro stability of the rDNA IGS RFLP patterns was assessed by subculturing these seven strains 10 times each over a period of 3 months. The rDNA profiles were unchanged after 10 rounds of subculture (data not shown).

Detection of nuclear number in a conidium. A single nucleus was observed in DAPI-stained arthroconidia examined from a black-dot specimen (Fig. 4). This was also confirmed by transmission electron microscopy (data not shown). This strongly suggested that the different IGS lengths seen by IGS RFLP in single conidial isolates must exist within a nucleus. Furthermore, this suggested that variable copy numbers of subrepeat elements must exist in different copies of the IGS region within a T. violaceum genome.

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FIG. 4. Micrograph of T. violaceum arthroconidia from a black-dot lesion from a patient with tinea capitis. (Left) UV fluorescence microscopy; (right) bright-field microscopy. Note that there is a single nucleus in each arthroconidial cell.

Sequence determination of the IGS regions. PCR across the entire IGS region using primers 25SCON2 and NS1-R on genomic DNA from single conidial cultures produced multiple banding patterns; however, these were smeary and difficult to interpret (data not shown). Therefore, 13 IGS regions derived from PCR products from five different single conidial cultures of T. violaceum (S1, S4, S6, S8, and S10) were cloned. Recombinant plasmids from each strain contained inserts of different sizes (Fig. 5). Four of the clones (S1-1, S1-4, S6-5, and S8-1) were sequenced and contained inserts with lengths of 2,601 bp, 2,941 bp, 2,600 bp, and 2,942 bp, respectively. S1-1 and S1-4 were both from the same single conidial strain, while S6-5 and S8-1 were each from different strains. In addition to the entire IGS region, all DNA inserts contained 418 bp from the 3' end of the LSU rDNA and 38 bp from the 5' end of the SSU rDNA.

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FIG. 5. Ethidium bromide-stained agarose gel of PCR products from 13 E. coli plasmid preparations representing potential clones of the total Trichophyton violaceum IGSs from four different T. violaceum strains, S1, S6, S8, and S10, showing variations in the size of this region between strains and between clones from the same strain.

The IGS maps of clones S1-1, S1-4, S6-5, and S8-1 are shown in Fig. 6. Two repetitive regions were identified in the sequence. The first subrepeat locus was located 451 bp downstream from the predicted 3' end of the LSU rDNA in clone S1-1. Two copies (plasmids S1-1 and S6-5) or four copies (plasmids S1-4 and S8-1) of a tandemly repetitive 171-bp element (TvS1) were identified, with an adjacent 67-bp partial repeat. These results strongly suggested that variable copy numbers of the TvS1 repeat could be present in different copies of the IGS within a single T. violaceum genome. A second set of shorter subrepeat elements (TvS2) was located 703 bp upstream from the predicted 5' end of the SSU rDNA. TvS2 was present as one full (77-bp) and one partial tandem copy of a 38-bp repeat sequence, with sequence similarity to the partial S2 repeats of T. rubrum and Trichophyton interdigitale (14, 15). The TvS2 copy number did not vary among the four IGS sequences. All four clones contained a polydeoxythymidine microsatellite, 17 to 19 bp in length, which was located 135 bp downstream from the predicted 3' end of the LSU rDNA. Overall, the sequences of the four IGSs were very similar except in the copy number of TvS1 and in the length of the poly(T) tract (17 residues in S1-4, 18 in S6-5 and S8-1, and 19 in S1-1). In addition, a number of single nucleotide polymorphisms were identified. Counting from the 3' end of the LSU sequence in S8-1, at positions 771, 1013, and 1369, the bases are T, T, and C, but in the other clones, they are C, C, and T, respectively. In S1-4, at position 1650 there is a G but in the other clones an A. Attempts to design primers from this sequence to amplify TvS1 from genomic DNA again produced smears of bands that were difficult to interpret (data not shown).

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FIG. 6. The IGS map of four clones from three different T. violaceum strains, S1, S6, and S8, including two clones from strain S1, S1-1 and S1-4, showing differences in the number of TvS1 repeats between strains and between clones from the same strain (upper map, S1-1 and S6-5; lower map, S1-4 and S8-1). (T)17 to (T)19 indicate a polydeoxythymidine microsatellite containing 17 to 19 T residues, respectively. TvS1 and TvS2 represent the large (variable) and small (conserved) subrepeat elements, respectively. Numerical values show the sizes in base pairs of the repeat and interrepeat regions.

The molecular weights of bands in the 5- to 10-kb range in Fig. 3 were estimated by ONE-Dscan 1-D gel analysis software, and the differences between adjacent band sizes were calculated. The results showed that there is a good correlation between the estimated length differences and the size of the 171-bp repeat or multiples of this size (data not shown).

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When 32 randomly selected Taiwanese T. violaceum isolates were analyzed by hybridization of EcoRI-digested genomic DNA with an rDNA probe, all 36 isolates showed abundant bands and every individual pattern was distinct (Fig. 1). This high level of variation in the rRNA IGS in T. violaceum isolates is unique in the genus Trichophyton. Analysis of a small number of Iranian isolates suggested that this variation was independent of the geographical site of isolation of the T. violaceum strains. Although T. violaceum and T. rubrum are very closely related (9), most T. rubrum strains (41 of 50, 82%) when analyzed in the same way produced a single high-molecular-weight band, which varied in size between strains (13). In this report and similar studies of the IGS of Trichophyton tonsurans (4) and Trichophyton interdigitale (15, 20), the majority of strains studied shared a small number of simple pattern types. The question of why there are so many polymorphic bands and unique pattern types in T. violaceum was therefore an important one to address. The presence of mixed-strain infections in onychomycosis was demonstrated using rDNA IGS analysis (10, 30). Our results show that mixed T. violaceum strain infection in tinea capitis can also occur, though a clear picture of the extent of mixed infection will be difficult to obtain due to the complexity of the banding pattern produced and the lack of knowledge of the frequency changes in this DNA region during infection. The IGS sequences of T. rubrum (14) and T. violaceum are very similar (>85% identity), and both contain large (TrS1 and TvS1) and small (TrS2 and TvS2) subrepeats. The large subrepeat in T. violaceum is 171 bp, and that in T. rubrum is 200 bp. The difference of 29 bp in TvS1 compared to the T. rubrum equivalent repeat TrS1 is the loss of a palindromic sequence seen only in the T. rubrum large subrepeat (14). The results of a sequence similarity search using the 5S rRNA gene of Aspergillus nidulans revealed that the 5S RNA gene of T. violaceum is not located in the IGS. This is similar to the situation in T. rubrum (14), and as with most eukaryotes (25) the 5S rRNA genes are probably dispersed throughout the genome in dermatophytes.

The number of copies of the rDNA repeats in T. violaceum or other dermatophytes has not been defined, but in Candida albicans there are approximately 120 copies (16) and in other fungi the copy number varies between 45 and 180 (5).

We propose that the size variation seen in DNA fragments that include the IGS region can be accounted for at least in part by differences in the copy number of the TvS1 repeat unit within different IGS regions within the genome of a strain. However, we cannot rule out the presence of other genetic variations within the IGS or elsewhere in the rDNA that may account for some of the variation seen, particularly in strains where there are high-molecular-weight fragments observed.

Comparison of the results seen here in T. violaceum to the results of similar analysis in T. rubrum highlights an important difference, in that most strains of T. rubrum (82%) were homogeneous in their IGS regions (13), while all T. violaceum strains were shown to have heterogeneous IGSs. Although apparent IGS heterogeneity has been suggested for a few strains of T. rubrum (10, 13, 14, 30), Trichophyton mentagrophytes (19), and Microsporum canis (31), prior to this study it had never been proven by cloning and sequencing of multiple IGSs from a strain derived from a single, mononucleate conidium. Heterogeneity within the rRNA repeats is relatively rare in eukaryotes but has been reported in Fusarium (23), Neotyphodium lolii (a grass endophyte) (6), Xanthophyllomyces dendrorhous (a basidomycete yeast) (3), and Pythium helicoides (17). In other eukaryotes intragenomic variation has been observed in organisms as diverse as crayfish (11), ladybird beetles (26), and larches (27), in each case involving ITS heterogeneity. In Xanthophyllomyces and Pythium intragenomic heterogeneity was ascribed to indel variation in the ITS or the ITS and IGS regions (3, 17). Interestingly in Neotyphodium lolii variable numbers of 111- and 119-bp repeats were seen in the IGS regions within individual strains (6), very similar to the situation seen in T. violaceum.

In the present study, the rDNA RFLP Southern blotting method was initially evaluated for its ability to differentiate T. violaceum strains cultured from patients with tinea capitis in order to address questions of the epidemiology of the disease. However, the unexpected variation and complexity seen in different isolates and strains, together with a lack of knowledge of the details of the infection process and the frequency of changes in the IGS during infection, are likely to limit its use in epidemiologic studies.

It has been suggested that variation in the IGS of rDNA may have a considerable effect on development, evolution, and ecology through the effects on growth rate regulation, resulting from the role of the IGS in production of rRNA (7). Although the T. violaceum IGS has been completely sequenced in this study, the precise locations of the external transcribed spacer or equivalent and any promoters, enhancers, or terminators of rDNA transcription have not been determined. The chromosomal location or locations of the tandemly repeated rDNAs in T. violaceum or T. rubrum are not known, though in many fungi they are on a single chromosome. If the rDNA was distributed on multiple chromosomes in T. violaceum, this may account for the IGS heterogeneity seen. In most eukaryotic organisms, the ribosomal genes are tandemly arrayed and undergo concerted evolution (5). This is a universal biological phenomenon and is responsible for the homogenization of rDNA repeats within a repeat array, among arrays dispersed in an individual genome, and throughout a recombining population (1). It has been shown that the rRNA repeats within the genomes of several fungi are highly conserved including the IGS regions (5). In this study, it appears that the IGS of T. violaceum is partially excluded from the concerted evolution of the rRNA gene locus, especially in the TvS1 region. Recently, the relaxation of concerted evolution in the different ITS regions within the genome of the fungus Glomus etunicatum was demonstrated (24). Furthermore, variation seen in the 5S rRNA multigene family of four filamentous fungi, Aspergillus nidulans, Fusarium graminearum, Magnaporthe grisea, and Neurospora crassa, indicates "birth-and-death" evolution (25). The formation of IGS heterogeneity in T. violaceum may be similar or may reflect differences in, for example, the rates of mitotic recombination in contrast with the relative homogeneity of the closely related T. rubrum. Recently, Graser et al. (8) have analyzed differences in microsatellite markers in isolates of T. rubrum from around the world and shown that there are two broad population groups, one of which derived predominantly from Africa and was largely associated with tinea capitis and corporis. Isolates of T. violaceum had similar marker loci (unlike the other dermatophytes examined) but were clearly distinct as a species. It would be of interest to examine the IGS regions of these "population 2" isolates of T. rubrum, which may shed light on how IGS heterogeneity evolved in T. violaceum.

 
This work was partly supported by the National Science Council, Taiwan (NSC 89-2314-B-006-181).

We especially thank Keith H. Holland for critical comments and helpful advice on the study. Our thanks also go to David Walker for his guidance in transmission electron microscopy work, to Richard Warmseley for the use of the micromanipulation device, and to Shu-Ying Li for her support in sequencing work.

 
* Corresponding author. Mailing address: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan. Phone: (886) 6 2353535, ext. 5765. Fax: (886) 6 2363956. E-mail: jcjc{at}mail.ncku.edu.tw

Published ahead of print on 22 February 2008.

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Eukaryotic Cell, April 2008, p. 721-726, Vol. 7, No. 4
1535-9778/08/$08.00+0     doi:10.1128/EC.00441-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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