Thermodynamic parameters for an expanded nearest-neighbor model for the formation of RNA duplexes with single nucleotide bulges

Thirty-four RNA duplexes containing single nucleotide bulges were optically melted, and the thermodynamic parameters deltaH degrees, deltaS degrees, deltaG degrees (37), and T(M) for each sequence were determined. Data from this study were combined with data from previous thermodynamic data [Longfellow, C. E., Kierzek, R., and Turner, D. H. (1990) Biochemistry 29, 278-85] to develop a model that will more accurately predict the free energy of an RNA duplex containing a single nucleotide bulge. Differences between purine and pyrimidine bulges as well as differences between Group I duplexes, those in which the bulge is not identical to either neighboring nucleotide, and Group II duplexes, those in which the bulge is identical to at least one neighboring nucleotide, were considered. The length of the duplex, non-nearest-neighbor effects, and bulge location were also examined. A model was developed which divides sequences into two groups: those with pyrimidine bulges and those with purine bulges. The proposed model for pyrimidine bulges predicts deltaG degrees (37,bulge) = 3.9 kcal/mol + 0.10deltaG degrees (37,nn) + beta, while the model for purine bulges predicts deltaG degrees (37,bulge) = 3.3 kcal/mol - 0.30deltaG degrees (37,nn) + beta, where beta has a value of 0.0 and -0.8 kcal/mol for Group I and Group II sequences, respectively, and deltaG degrees (37,nn) is the nearest-neighbor free energy of the base pairs surrounding the bulge. The conformation of bulge loops present in rRNA was examined. Three distinct families of structures were identified. The bulge loop was either extrahelical, intercalated, or in a "side-step" conformation.     

The effect of base sequence on the stability of RNA and DNA single base bulges

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.     

Investigation of potential RNA bulge stabilizing elements

As a part of our interest in recognition and cleavage of RNA we carried out thermal melting studies with the aim of screening a number of simple oligonucleotide modifications for their potential as modifying elements for RNA bulge stabilizing oligonucleotides. A specific model system from our studies on oligonucleotide-based artificial nuclease (OBAN) systems was chosen and the bulge size was varied from one to five nucleotides. Introduction of single 2'-modified nucleoside moieties (2'-O-methyl, 2'-deoxy and 2'-deoxy-2'-amino) with different conformational preferences adjacent to the bulge revealed that a higher preference for the north conformers gave more stable bulges across the whole range of bulge sizes. Changing a bulge closing a G-U wobble base pair to a G-C pair resulted in the interesting observation that, although the fully complementary complex and small bulges were highly stabilized, there was little difference in the stability of the larger bulges. The wobble base pair even gave a slight stabilization of the 5 nt bulge system. Introduction of a uridine C-5 linker with a single ammonium group was clearly bulge stabilizing (DeltaT(m) + 4.6 to + 5.4 degrees C for the three most stabilized bulges), although with limited selectivity for different bulge sizes since the fully complementary duplex was also stabilized. Introduction of a naphthoyl group on a 2'-aminolinker mostly gave a destabilizing effect, while introduction of a 5-aminoneocuproine moiety on the same linker resulted in stabilization of all bulges, in particular those with two or four unpaired nucleotides (DeltaT(m) + 3.6 and + 2.9 degrees C respectively). The aromatic groups destabilize the fully complementary duplex, resulting in higher selectivity towards stabilization of bulges. A combination of the studied partial element should be suitable for future designs of modified oligonucleotides that, apart from standard base pairing, can also provide additional non-Watson-Crick recognition of RNA.     

Conformational transitions in RNA single uridine and adenosine bulge structures: a molecular dynamics free energy simulation study

Extra unmatched nucleotides (single base bulges) are common structural motifs in folded RNA molecules and can participate in RNA-ligand binding and RNA tertiary structure formation. Often these processes are associated with conformational transitions in the bulge region such as flipping out of the bulge base from an intrahelical stacked toward a looped out state. Knowledge of the flexibility of bulge structures and energetics of conformational transitions is an important prerequisite to better understand the function of this RNA motif. Molecular dynamics simulations were performed on single uridine and adenosine bulge nucleotides at the center of eight basepair RNA molecules and indicated larger flexibility of the bulge bases compared to basepaired regions. The umbrella sampling method was applied to study the bulge base looping out process and accompanying conformational and free energy changes. Looping out toward the major groove resulted in partial disruption of adjacent basepairs and was found to be less favorable compared to looping out toward the minor groove. For both uridine and adenosine bulges, a positive free energy change for full looping out was obtained which was approximately 1.5 kcal mol-1 higher in the case of the adenosine compared to the uridine bulge system. The simulations also indicated stable partially looped out states with the bulge bases located in the RNA minor groove and forming base triples with 5'-neighboring basepairs. In the case of the uridine bulge this state was more stable than the intrahelical stacked bulge structure. Induced looping out toward the minor groove involved crossing of an energy barrier of approximately 3.5 kcal mol-1 before reaching the base triple state. A continuum solvent analysis of intermediate bulge states indicated that electrostatic interactions stabilize looped out and base triple states, whereas van der Waals interactions and nonpolar contributions favor the stacked bulge conformation.     

Solution structure of dAATAA and dAAUAA DNA bulges

The NMR structure analysis is described for two DNA molecules of identical stem sequences with a five base loop containing a pyrimidine, thymin or uracil, in between purines. These five unpaired nucleotides are bulged out and are known to induce a kink in the duplex structure. The dAATAA bulge DNA is kinked between the third and the fourth nucleotide. This contrasts with the previously studied dAAAAA bulge DNA where we found a kink between the fourth and fifth nucleotide. The total kinking angle is ~104° for the dAATAA bulge. The findings were supported by electrophoretic data and fluorescence resonance energy transfer measurements of a similar DNA molecule end-labeled by suitable fluorescent dyes. For the dAAUAA bulge the NMR data result in a similar structure as reported for the dAATAA bulge with a kinking angle of ~87°. The results are discussed in comparison with a rAAUAA RNA bulge found in a group I intron. Generally, the sequence-dependent structure of bulges is important to understand the role of DNA bulges in protein recognition.     

Bulge loops used to measure the helical twist of RNA in solution.

Bulge loops are commonly found in helical segments of cellular RNAs. When incorporated into long double-stranded RNAs, they may introduce points of flexibility or permanent bend that can be detected by the altered electrophoretic gel mobility of the RNA. We find that a single An or Un bulge loop near the middle of a long RNA helix significantly retards the RNA during polyacrylamide gel electrophoresis if n greater than or equal to 2. The mobility of an RNA containing two A2 bulges various periodically with the number of base pairs between the bulges. We interpret this to mean that A2 bulges varies periodically with the number of base pairs between the bulges. We interpret this to mean that Z2 bulges form torsionally stiff bends in the helix; the gel mobility reaches a minimum when the total helical twist between the bulges rotates the arms of the molecule into a cis conformation. The gel mobilities are proportional to the predicted end-to-end distance of the RNA if the average RNA helical repeat is 11.8 +/- 0.2 bp/turn and there is no helical twist (3 +/- 9 degrees) associated with the bulge (data obtained in 0.15 M Na+). Other sizes and sequences of bulges have very different effects on RNA helix conformation and flexibility. U2 bulges bend the helix to a much smaller degree than A2 bulges, while longer A or U bulge sequences probably allow bends of 90 degrees or more; all of these may be fairly flexible joints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

RNA bulge entropies in the unbound state correlate with peptide binding strengths for HIV-1 and BIV TAR RNA because of improved conformational access.

For the binding of peptides to wild-type HIV-1 and BIV TAR RNA and to mutants with bulges of various sizes, changes in the DeltaDelta G values of binding were determined from experimental K d values. The corresponding entropies of these bulges are estimated by enumerating all possible RNA bulge conformations on a lattice and then applying the Boltzmann relationship. Independent calculations of entropies from fluctuations are also carried out using the Gaussian network model (GNM) recently introduced for analyzing folded structures. Strong correlations are seen between the changes in free energy determined for binding and the two different unbound entropy calculations. The fact that the calculated entropy increase with larger bulge size is correlated with the enhanced experimental binding free energy is unusual. This system exhibits a dependence on the entropy of the unbound form that is opposite to usual binding models. Instead of a large initial entropy being unfavorable since it would be reduced upon binding, here the larger entropies actually favor binding. Several interpretations are possible: (i) the higher conformational freedom implies a higher competence for binding with a minimal strain, by suitable selection amongst the set of already accessible conformations; (ii) larger bulge entropies enhance the probability of the specific favorable conformation of the bound state; (iii) the increased freedom of the larger bulges contri-butes more to the bound state than to the unbound state; (iv) indirectly the large entropy of the bound state might have an unfavorable effect on the solvent structure. Nonetheless, this unusual effect is interesting.     

Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29.

A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging. This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops. Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging. The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated. Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101. A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints. Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly. Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities. However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly. Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery. Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other packaging components.     

Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP

DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A5 bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5'-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed.     

Microsatellite Evolution: Testing the Ascertainment Bias Hypothesis

Previous studies suggest the median allele length of microsatellites is longest in the species from which the markers were derived, suggesting that an ascertainment bias was operating. We have examined whether the size distribution of microsatellite alleles between sheep and cattle is source dependent using a set of 472 microsatellites that can be amplified in both species. For those markers that were polymorphic in both species we report a significantly greater number of markers (P < 0.001) with longer median allele sizes in sheep, regardless of microsatellite origin. This finding suggests that any ascertainment bias operating during microsatellite selection is only a minor contributor to the variation observed. Received: 6 January 1997 / Accepted: 19 May 1997     

Thyroid hormone signaling controls hair follicle stem cell function

Observations in thyroid patients and experimental animals show that the skin is an important target for the thyroid hormones. We previously showed that deletion in mice of the thyroid hormone nuclear receptors TRα1 and TRβ (the main thyroid hormone–binding isoforms) results in impaired epidermal proliferation, hair growth, and wound healing. Stem cells located at the bulges of the hair follicles are responsible for hair cycling and contribute to the regeneration of the new epidermis after wounding. Therefore a reduction in the number or function of the bulge stem cells could be responsible for this phenotype. Bulge cells show increased levels of epigenetic repressive marks, can retain bromodeoxyuridine labeling for a long time, and have colony-forming efficiency (CFE) in vitro. Here we demonstrate that mice lacking TRs do not have a decrease of the bulge stem cell population. Instead, they show an increase of label-retaining cells (LRCs) in the bulges and enhanced CFE in vitro. Reduced activation of stem cells leading to their accumulation in the bulges is indicated by a strongly reduced response to mobilization by 12-O-tetradecanolyphorbol-13-acetate. Altered function of the bulge stem cells is associated with aberrant activation of Smad signaling, leading to reduced nuclear accumulation of β-catenin, which is crucial for stem cell proliferation and mobilization. LRCs of TR-deficient mice also show increased levels of epigenetic repressive marks. We conclude that thyroid hormone signaling is an important determinant of the mobilization of stem cells out of their niche in the hair bulge. These findings correlate with skin defects observed in mice and alterations found in human thyroid disorders.     

Population-Specific Links Between Heterozygosity and the Rate Human Microsatellite Evolution

Microsatellites form an abundant class of DNA sequences used widely as genetic markers. Surprisingly, the length of human microsatellites varies highly predictably with distance from Africa, apparently following the linear decline in variability that arose as we colonised the world. Such patterns have been used to argue that heterozygosity modulates the rate of microsatellite evolution. Here I test the ensuing prediction that variation in demographic history will cause individual populations predictably either to lead or to lag any given trend in length. I find that they do: larger populations with locally higher heterozygosity have microsatellites that are longer when a locus is expanding and shorter when a locus is contracting. These patterns remain even after controlling for the stepwise way in which heterozygosity and allele lengths decline across the world. This analysis provides support for a strongly discontinuous model for how human genetic variability is distributed and shows how individual populations differ in the average rate their microsatellites are evolving. Such patterns have the potential to provide a new window onto historical demography.     

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.     

Isolation and characterization of microsatellite loci in the Caribbean gorgonian Pseudopterogorgia elisabethae

We report on the isolation and characterization of five polymorphic microsatellites in the gorgonian Pseudopterogorgia elisabethae from genomic DNA‐enriched libraries. Forty‐four microsatellites were screened from the libraries with the oligonucleotide probe (CA)₁₂. Five of the screened microsatellites were polymorphic. The levels of polymorphism found in 50 genotyped individuals from a single population suggest that microsatellites are useful tools for the study of genetic variation in gorgonians. These are the first microsatellite loci reported from any gorgonian species.     

Thermodynamic and spectroscopic study of bulge loops in oligoribonucleotides

Thermodynamic parameters for bulge loops of one to three nucleotides in oligoribonucleotide duplexes have been measured by optical melting. The results indicate bulges Bn of An and Un have similar stabilities in the duplexes, GCGBmGCG + CGCCGC. The stability increment for a bulge depends on more than its adjacent base pairs. For example, the stability increment for a bulge is affected more than 1 kcal/mol by changing two nonadjacent base pairs or by adding terminal unpaired nucleotides (dangling ends) three base pairs away. Thus a nearest-neighbor approximation for helixes with bulges is oversimplified. Many of the non-self-complementary strands used in this study were observed to form homoduplexes. Such duplexes with GA mismatches were particularly stable.     

Microsatellite DNA markers for rice chromosomes

We found 369 complete microsatellites, of which (CGG/GCC)_n was the most frequent, in 11 798 rice sequences in the database. Of these microsatellites, 35 out of 45 could be successfully converted into microsatellite DNA markers using sequence information in their flanking regions. Thus, the time and labor used to develop new microsatellite DNA markers could be saved by using these published sequences. Twenty eight polymorphic markers between Asominori (japonica) and IR24 (indica) have been correctly mapped on the rice genome and microsatellites appear to be randomly distributed in the rice chromosomes. Integration of these markers with the published microsatellite DNA markers showed that about 35% of the rice chromosomes were covered by the 56 microsatellite DNA markers. These microsatellites were hypervariable and were easily to assay by PCR; they were distributed to all chromosomes and therefore, one can easily select plants carrying desired chromosome regions using these microsatellite DNA markers. Thus, microsatellite maps should aid the development of new breeds of rice saving time, labor, and money.     

Non-nearest-neighbor dependence of the stability for RNA group II single-nucleotide bulge loops

Thirty-one RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each sequence were determined. The bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The data were used to develop a model to predict the free energy of an RNA duplex containing a single-nucleotide bulge. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned farther from the hairpin loop.     

Ability of polymerase eta and T7 DNA polymerase to bypass bulge structures

DNA misalignment occurs in homopolymer tracts during replication and can lead to frameshift mutations. Polymerase (pol) recognition of primer-templates containing bulge structures and the transmission of a bulge through a polymerase binding site or replication complex are important components of frameshift mutagenesis. In this report, we describe the interaction of the catalytic core of pol eta with primer-templates containing bulge structures by single round primer extension. We found that pol eta could stabilize a frayed primer terminus, which enhances its ability to extend primer-templates containing bulges. Based on methylphosphonate-DNA mapping, pol eta interacts with the single strand template but not appreciably with the template strand of the DNA stem greater than two nucleotides from the primer terminus. These latter characteristics, combined with the ability to stabilize a frayed primer terminus, may explain why primer-templates containing template bulges are extended so efficiently by pol eta. Although pol eta could accommodate large bulges and continue synthesis without obstruction, bulge structures in the template, but not in the primer, caused termination of the T7 DNA replication complex. Terminations occurred when the template bulge neared the helix-loop-helix domain of the polymerase thumb. Terminations were not observed, however, when bulge structures approached the site of interaction of the DNA with the extended thumb and thioredoxin. At low temperature, however, terminations did occur at this site.     

Use of polymorphic short and clustered coding-region microsatellites to distinguish strains of Candida albicans

Abstract We describe the identification of polymorphic microsatellite loci in the pathogenic yeast, Candida albicans . A search for all coding-region microsatellites with more than four repeats that can be found in Candida sequences in GenBank was conducted. Nine such microsatellite sequences consisting of trinucleotide motifs were found. Three of these were perfect microsatellites while the remaining six sequences were found in one imperfect microsatellite and two compound microsatellites. Because of the close proximity of some of these repeats, all could be assayed with six PCR primer pairs. All of these microsatellite sequences were found in five nuclear genes, ZNF1, CCN1, CPH1, EFG1 , and MNT2 . Except for a single (CTT)₅ serine tract, all coded for polyglutamine tracts. Another locus with seven alleles, a region of the ERK1 protein kinase gene, was also examined, and may be a representative of a new class of highly polymorphic ‘clustered’ microsatellites. Such loci, in which several non-contiguous but closely linked microsatellites are clustered together, may be a useful source of DNA polymorphisms in microorganisms in which long microsatellite sequences are unavailable. All seven regions amplified were polymorphic, having between two and seven variable length alleles in the 11 strains of Candida albicans examined. The results of this and similar searches will facilitate epidemiological and evolutionary studies of Candida and other microorganisms.