Department of Biological Sciences, 440 BB, University of Iowa, Iowa City, IA 52242, USA1
Author for correspondence: David R. Soll. Tel: +1 319 335 1117. Fax: +1 319 335 2772. e-mail: david-soll{at}uiowa.edu
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ABSTRACT |
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Keywords: Candida albicans, RPS repetitive units, microevolution, Ca3 fingerprinting
The GenBank accession numbers for the sequences reported in this paper are AF121119, AF121120, AF121121, AF121122, AF121123, AF121124 and AF121125.
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INTRODUCTION |
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The Ca3 probe is composed of seven EcoRI fragments: A, B, C, D1, D2, E and F (Anderson et al., 1993 ). The C fragment alone identifies hypervariable sequences in Southern blots of EcoRI-digested genomic DNA (Anderson et al., 1993
), and for that reason the C fragment has been used effectively to monitor microevolution in colonizing strains (Schroeppel et al., 1994
; Lockhart et al., 1995
, 1996
). Recently, we demonstrated (Lockhart et al., 1995
) that the C fragment contains a portion of the C. albicans repetitive element RPS (Iwaguchi et al., 1992
). RPS elements are present on seven of the eight C. albicans chromosomes (Iwaguchi et al., 1992
; Chindamporn et al., 1995
), and those RPS elements sequenced to date contain three to four tandem repeats, each approximately 172 bp in length, referred to as the alt element (Iwaguchi et al., 1992
; Chibana et al., 1994
). The 29 bp at the end of each alt element represent a common palindromic sequence referred to as COM29. Because of structural similarities between COM29 and both the
attachment site and the bacterial DNA inversion/cross-over sequence (Ehrlich, 1989
), Iwaguchi et al. (1992)
have suggested that COM29 sequences may represent specific recombination sites in the C. albicans genome. The possibility therefore exists that the microevolutionary changes that are identified in clonal populations by the C1 fragment of the Ca3 probe (Lockhart et al., 1995
) are due to genomic reorganizations involving RPS elements. Southern analysis and DNA sequencing experiments described here demonstrate that high-frequency changes in the size of genomic sequences that hybridize to the C fragment of Ca3 involve the insertion and deletion of full-length RPS elements, providing the first estimate of the rate of RPS reorganization, and lead to two alternative models for intrachromosomal RPS recombination.
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METHODS |
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DNA fingerprinting and Southern-blot analysis.
Cells from stored agar slants were plated on YPD agar and harvested after 2 d. DNA was extracted and then digested with one or more selected restriction enzymes following the recommendations of the manufacturer (Promega). Digested DNA (3 µg per lane) was electrophoresed through a 0·6% (w/v) or 0·8% (w/v) agarose gel overnight at 35 V. The gel was stained with ethidium bromide to assess loading and separation, then the DNA was transferred by capillary blotting to a nylon Hybond-N+ membrane (Amersham). For sequential hybridization, the membrane was first prehybridized for 7 h at 65 °C with 10 mg denatured calf thymus DNA ml-1 and then hybridized overnight at 65 °C with the first random-primer-labelled ([32P]dCTP) probe. These hybridization steps were performed in a solution of 5xSSPE (1xSSPE contains 10 mM NaH2PO4, pH 7·5, 10 mM EDTA and 0·18 M NaCl) containing 5% (w/v) dextran sulfate and 0·3% (w/v) SDS. The membrane was washed at 45 °C with a solution of 2xSSPE containing 0·2% (w/v) SDS and exposed to XAR-S film (Eastman Kodak) with a Cronex Lightning-Plus intensifying screen (Du Pont). The blot was then stripped of the first probe by incubating it in 0·4 M NaOH for 30 min at 45 °C, then incubated for 15 min in a solution of 1xSSPE containing 0·1% (w/v) SDS and 0·2 M Tris/HCl, pH 7·5. The blot was then hybridized with the second random primer-labelled probe and again exposed to XAR-S film. This procedure was repeated for subsequent hybridizations.
Cloning hypervariable band fragments.
Subgenomic libraries were constructed for C. albicans isolates RP5.1, RP10.3, RP39.1 and RP39.4. To accomplish this, total genomic DNA of each strain was digested with EcoRI and fractionated in a sucrose gradient. Fractions were analysed by Southern blot hybridization with the C1 subfragment of the Ca3 probe (Lockhart et al., 1995 ), and those containing hypervariable band fragments were selected. Subgenomic libraries were constructed from the selected fractions in phage
EMBL3 (Promega) between EcoRI sites, according to established protocols (Sambrook et al., 1989
). Libraries were plated at a density of 104 recombinant phage per plate and screened with the C1 probe (Church & Gilbert, 1984
). The EcoRI fragments of interest were then subcloned into the pGEM-7Zf(+) plasmid vector (Promega). When cloning fragments into E. coli JM109, we found that the larger the insert, the less stable the clone. Therefore, the integrity of each clone was tested by Southern analysis after each passage in E. coli. Cloned fragments and the genomic DNA of the isolate used to obtain each clone were digested with EcoRI, and Southern blots of these preparations were probed with C1. This provided a direct comparison between each clone and the original genomic fragment. In cases in which the clone showed evidence of recombination, the preparation was abandoned and prepared again.
Nucleotide sequence of hypervariable band fragments.
Plasmid DNA was isolated using a CsClethidium bromide protocol (Sambrook et al., 1989 ). Prior to sequencing, each clone was compared to the original fragment by Southern analysis to ensure it had maintained its integrity. The nucleotide sequence of each plasmid insert was determined in both directions with the ABI model 373A Auto Sequence system (Perkin Elmer/Applied Biosystems) using the PCR cycle-sequencing protocol and fluorescent dye terminator dideoxynucleotides (Perkin Elmer/Applied Biosystems). Homology and alignment of nucleotide sequences to gene databases were performed with MacDNASIS Pro v3.6 software (Hitachi Software Engineering). The 5' and 3' ends of each clone were sequenced using customized synthetic primers. Both ends of the clones were sequenced until a region located in the centre of the fragments and displaying RPS sequence was reached. To sequence the RPS-containing regions of clones RP39.49 kb, RP39.113·7 kb and RP39.16·5 kb, a sequential series of deletions were prepared with the Erase-a-Base System kit (Promega). H3, a HindIII/EcoRI subfragment of RP39.113·7 kb, and RPS39, a Csp45I subfragment of RP39.113·7 kb, were both subcloned in the pGEM-7Zf(+) plasmid vector for further analysis.
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RESULTS |
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Hypervariable genomic fragments contain similar non-RPS 3' and 5' ends
The results obtained from the experiment described in Fig. 3 suggested not only that the hypervariability of high-molecular-mass fragments was due to the reorganization of sequences that hybridize to the RPS-containing C1 subfragment, but also that these hypervariable genomic fragments contained similar upstream C2 regions. To assess directly sequence homologies between hypervariable genomic fragments, several were cloned and sequenced from the 5' and 3' ends until an RPS sequence was penetrated. Those cloned included fragment RP5.118·2 kb and RP5.17·5 kb (both from isolate RP5.1), fragment RP10.318·2 kb (from isolate RP10.3), fragment RP39.113·7 kb (from isolate RP39.1) and fragment RP39.49 kb (from isolate RP39.4). The 3' terminal 2·74 kb sequences of all clones except RP5.17·5 kb were 100% identical. RP5.17·5 kb was truncated at the 3' end due to a transversion from A to T. This resulted in the genesis of an EcoRI restriction site unique to RP5.17·5 kb, 864 bp upstream from the consensus 3' EcoRI site shared by the four other clones (noted by a vertical dashed E site in Fig. 1b
).
The 1·72 kb sequences upstream of the RPS sequence of the five clones were also highly homologous. In addition, they were highly homologous to the region upstream of the RPS sequence in Ca3 that included a portion of the C1 sequence (Lockhart et al., 1995 ) and the C2 sequence (sequenced here). The average homology between the five clones and the homologous Ca3 sequence was 91·5%. The sequence of the C1 portion of this region has been published by Lockhart et al. (1995)
and the C1 and C2 portions by Chindamporn et al. (1998)
in a sequence analysis of the HOK fragment that overlaps a portion of the Ca3 probe. Physical maps comparing the Ca3 probe, the hypervariable fragments and the HOK fragment (Chindamporn et al., 1998
), which contains a partial RPS sequence similar to the one in C, are presented in Fig. 1
.
Differences in the size of hypervariable fragments are due almost exclusively to insertions and deletions of full-size RPS units
Since the 5' and 3' ends of the five sequenced hypervariable genomic fragments were similar in size and highly homologous, the differences in the total lengths of these fragments had to involve sequences within the RPS borders. To investigate the molecular basis of size variation, variable fragments that differed in size in two isolates of the same strain were cloned, sequenced and compared. The Ca3, C1 and RPS39 hybridization patterns of the two selected isolates from patient RP39 differed by a 13·7 kb band unique to the RP 39.1 pattern and a 9 kb band unique to the RP39.4 pattern (Fig. 2), suggesting that the two bands represented altered forms of the same genomic site. This interpretation was supported by the observation that the 5' and 3' terminal sequences of the two clones were identical. Clone RP39.49 kb was 8406 bp in length and contained one full-length RPS unit. Clone 39.113·7 kb was 12792 bp in length and contained three full-length tandem RPS units. The only difference, therefore, between RP39.49 kb and RP39.113·7 kb was the number of full-length RPS units in the RPS cluster.
The three tandem RPS units in RP39.113·7 kb defined by four PstI recognition sites (RPS 39-1, RPS 39-2 and RPS 39-3) were not identical (Fig. 4a). They differed by single base differences and by the number of alt sequences, small tandem repeat units of approximately 172 bp that have previously been demonstrated to differ in number between RPS units (Chibana et al., 1994
). While RPS 39-1 and RPS 39-2 contained the four alt sequences a, b, c and d, RPS 39-3 contained alt sequences a, c and d (Fig. 4a
). When compared to published RPS sequences (Chibana et al., 1994
), RPS 39-1 and RPS 39-2 exhibited the highest homology with RPS 116 (99·5 and 99·1%, respectively), and RPS 39-3 exhibited the highest homology with RPS 620 (99·1%).
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RPS-containing loci dispersed throughout the C. albicans genome share similar upstream and downstream sequences
Chindamporn et al. (1998) presented evidence suggesting that RPS sequences were located between similar 5' and 3' sequences. In addition, we demonstrated that the RPS clusters in the hypervariable clones that were sequenced were located between similar or identical 5' and 3' sequences. However, there are a number of lower-molecular-mass bands that contain RPS elements but that are not hypervariable within a strain. The following experiment was performed to test whether all RPS units throughout the genome are located between similar 3' and 5' sequences. A unique NsiI restriction site was identified in the C2 region and a unique SalI restriction site was identified in the non-RPS 3' region bordering RPS elements in the cloned and sequenced hypervariable fragments. NsiI/SalI-digested DNA preparations of 13 unrelated C. albicans isolates were probed with C2, the 5' end that borders RPS elements in hypervariable fragments, H3, the 3' end that borders RPS elements in hypervariable fragments, and the RPS element RPS39 (Figs 1
and 5
). Strain relatedness was assessed by Ca3 fingerprinting (data not shown). The genetic diversity observed for this collection was comparable to that observed for a previously studied collection of unrelated isolates (Pujol et al., 1997
). If all partial, single and tandem-repeat RPS elements dispersed throughout the genome are flanked by the same non-RPS 5' and 3' sequences, all bands identified by the RPS39 probe should also be identified by the C2 and H3 probes. This was the case for 12 out of the 13 tested isolates (Fig. 5
). The exception was isolate RP41.1 (Fig. 5
). In the Southern blot of this isolate, a 6·7 kb fragment hybridized to the RPS39 probe (Fig. 5b
) and to the C2 probe (Fig. 5a
) but not to the H3 probe (Fig. 5c
). Conversely, a 6·1 kb fragment hybridized to the RPS39 probe (Fig. 5b
) and to the H3 probe (Fig. 5c
), but not to the C2 probe (Fig. 5a
). This result is explained by the presence of a recognition site for either NsiI or SalI in an RPS unit of RP41.1 that separates a 12·8 kb fragment into a 6·1 and a 6·7 kb fragment. Together, these results demonstrate that in all cases, RPS sequences on different chromosomes are located between the same non-RPS 5' and 3' sequences.
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Deletion and insertion of RPS units occur in vitro
By comparing isolates of a strain infecting an individual, we have demonstrated that reorganization of RPS-containing sequences occurs in vivo. To demonstrate that deletions and insertions of RPS-containing sequences occur in vitro and to obtain an estimate of frequency, four C. albicans isolates were grown for 3000 generations at 37 °C. A liquid growth culture of each strain was initiated from a single colony and, therefore, represented a clone. Every 200 generations, cells were plated and a single colony was used to initiate a new liquid growth culture, and was also grown and stored on agar slants for further analysis. Genomic DNA was digested with EcoRI and probed with the C1 fragment (Fig. 6). Each of the four test isolates exhibited microevolution within 3000 generations. In every case, the disappearance of a band at one molecular size was accompanied by the appearance of a band at another molecular size, suggesting a molecular size change at the same genomic locus. This conclusion is further supported by the relative stability of each band after a size change. The only example in which two bands changed at the same time was in isolate RP5.1, between 600 and 800 generations. In every case, the change in Ca3 pattern was observed in the C1 pattern (data not shown). In every case, increases or decreases in the size of a band over time occurred in multiples of approximately 22·5 kb, the approximate size range of the full-length RPS units so far sequenced. These results demonstrate that spontaneous reorganization of sequences with tandemly repeated RPS units occurs at high frequency in vitro. The combined results of the four C. albicans isolates each monitored over 3000 generations provide a rough estimate of one reorganizational event per 1000 generations. These results also demonstrate that microevolutionary changes involving RPS-containing sequences can lead to divergence, convergence and reversion of patterns.
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DISCUSSION |
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Microevolution involves the deletion/insertion of full-length RPS elements at specific genomic sites
The microevolutionary changes identified by Ca3 and the C1 fragment that commonly take place in infecting strains of C. albicans are restricted primarily to high-molecular-mass bands. We have demonstrated that the great majority of the genomic fragments that represent the high-molecular-mass hypervariable bands of the Ca3 pattern possess the same 3' and 5' non-RPS sequences bordering a single RPS unit or tandem sequence of RPS units. To test the molecular basis of size variability of these RPS-containing bands, we cloned and sequenced a fragment from two isolates of a single infecting strain that had undergone a size change, and demonstrated that the difference in size was due exclusively to a difference in the number of full-length tandem RPS units. By Southern analysis, we further demonstrated that over time, changes that occur in vitro in the size of RPS-containing fragments involve the deletion or addition of full-length RPS units. Additions and deletions occurred at equal frequencies, and the mean frequency of change was estimated to be one per 1000 cell divisions. This provides us with the first piece of information that can be used for weighting bands in a more complex computation of similarity between Ca3 fingerprint patterns.
Models for RPS reorganization
We have presented evidence that the majority of reorganizational events involving RPS units result in a single band change. If an unequal recombinational event occurred between homologous RPS-containing loci on chromatids of homologous chromosomes, the probabilities of generating daughter cells with a change in the molecular mass of two, one or no bands in the Southern blot hybridization pattern would be 25%, 50% and 25%, respectively. Since the daughter cells with no change cannot be discriminated, the proportions of band changes one would expect are 67% for single-band changes and 33% for double-band changes. We observed frequencies of 92% and 8%, respectively. Our results, therefore, suggest that reorganization during growth does not involve unequal recombination between chromatids of homologous chromosomes. It has been suggested that RPS elements may play a role in ectopic chromosomal reorganization (Chibana et al., 1994 ; Chu et al., 1993
). The electrophoretic karyotypes of each of the four strains (39.2, 39.1, 10.3 and 5.1) that were monitored in vitro for RPS reorganization events were compared at zero time and after 3000 generations (data not shown). The electrophoretic karyotypes were found in all cases to be stable, demonstrating that within the limited number of generations monitored, reciprocal ectopic recombination between RPS-containing sequences did not occur. This does not, however, rule out the possibility that they occur, but at frequencies too low to be observed in our experiments.
High-frequency recombinational events were observed only in RPS-containing regions with one or more full-length RPS units. Reorganizational events resulted in molecular mass changes consistent in each case with the addition or deletion of one or more full-length RPS units. In one case (strain RP5.1), a 7·5 kb band, estimated to contain one RPS unit, increased in size to 9·7 kb after 800 generations, then decreased back to 7·5 kb after 1400 generations. This appeared to represent first the addition of a complete RPS unit, then the deletion of a complete RPS unit. There was no case in which an RPS-containing fragment decreased in size below that which accommodated one complete RPS unit. Therefore, duplication appears to require a minimum of one complete RPS unit, while deletion appears to require at least two RPS units.
Our results are consistent with two models of intrachromosomal recombination: unequal sister-chromatid exchange and slipped misalignment at the replication fork. Unequal sister-chromatid exchange occurs during or soon after DNA replication (Kuzminov, 1996 ), and involves the association and homologous recombination of out-of-phase tandem repeat arrays. A model of unequal sister-chromatid exchange has been adapted to a cluster of two full-length RPS units in Fig. 7
. In an unequal cross-over between RPS clusters, deletion and insertion occur concomitantly. After the unequal cross-over event and subsequent cell division, one daughter cell would receive the chromatid with deleted RPS and the other cell the chromatid with inserted RPS. Since the protocol we used to follow changes over 3000 generations in the four test strains involved cloning every 200 generations, the fingerprint obtained each 200 generations could include only one of the two genotypes resulting from sister chromatid exchange. Consistent with this model was the observation that the number of duplications was equal to the number of deletions in the combined 12000 generations of the four strains analysed.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 March 1999;
revised 14 June 1999;
accepted 17 June 1999.