The MRS is an intriguing feature of the C. albicans genome, in part because it has no known homologues in the eukaryotic world. In this study, we have found that the MRS plays a role (whose mechanism is presently unknown) in the proper maintenance of chromosomes. This effect on chromosome stability is dependent on the size of an MRS allele; the larger the MRS allele, the higher the likelihood of loss of that homologue. The analysis of naturally occurring disparities in the size of the MRS on chromosome homologues showed a preferential appearance of the smaller MRS in the monosomic progeny selected on sorbose. Although the MRS on chromosome 5 in CAI-4 is approximately 52 kb, we were still able to delete it with our standard methods.
This is the first report of deletion of a sequence of that size by oligonucleotide-mediated gene disruption. When one MRS was eliminated entirely by gene replacement, the segregation ratio continued to be in favor of the appearance of the homologue bearing the smaller (or absent) MRS. The ratio of monosomic strains in all the experiments did not appear to be a function of the size difference between the two MRSs, but it did seem to be associated with the absolute size of the remaining MRS (Tables and ). Thus, the 52-kb MRS of the deletants was lost at about the same frequency (60 to 61%) as the 50-kb MRS of 3153A (64%). In the first case, the homologue preferentially appearing had an MRS of 0 kb, while in the second it was 16 kb. Similarly, the 66-kb MRS of FC18 was lost 74% of the time, while the 92-kb MRS of NUM46 was lost 80% of the time.
There are several possible explanations for the association of MRS size with chromosome loss. The MRS could be a preferred site for cohesin binding, making sister chromatids with larger sequences late in separating, leading to nondisjunction. Cohesin appears not to have a sequence preference for binding, but cohesin sites appear to be incompatible with transcription (12
). This would predict that the areas of the chromosomes including the MRS would lag during normal mitosis. In the only study done looking at the MRS in mitosis, Chibana and Tanaka (5
), using fluorescence in situ hybridization to the repeated RPS subunit, found that the MRS appeared to separate in an asynchronous manner throughout anaphase. Thus, while the separation of MRSs occurs both early and late, it is possible that the larger MRSs may be the late-separating ones, accounting for the variability seen in the times of separation in the fluorescence in situ hybridization experiment.
The general (but not absolute) sequence conservation of the MRS (4
) and its high frequency of appearance within the genome speak to the importance of the MRS, yet its absence from chromosome 3 and the results of the deletion experiments in this paper clearly demonstrate that it is not an essential part of C. albicans
chromosomes. The concept of genetic drift implies that random base pair changes should become fixed in DNA that is not performing a function and should result in a loss of conservation of that genomic locus over time. C. albicans
is thought to exist mainly as a clonal population (13
), in which unselected random changes should accumulate rapidly, and divergence of nonfunctional DNA should be seen in different lineages of the species. However, such divergence does not seem to be occurring in the MRS, as it is conserved not only within the genome of a single strain but also among the different strains of C. albicans
that we have analyzed.
What important function, then, could the MRS be fulfilling for C. albicans
? The nonessential nature of the lone RB2 subunit on chromosome 3 and the complete MRS on chromosome 5 seems to negate the possibility that the MRS has a centromeric role, and indeed, the centromeres have been shown to be localized in unique regions of the chromosomes (32
). However, here we show evidence for a possible role of the MRS in adaptation of C. albicans
to its many growth niches. Chromosome nondisjunction leading to aneuploidy can affect cell phenotype, as shown by selection for l
-sorbose utilization, d
-arabinose utilization, or fluconazole resistance (17
), and we have shown that a large MRS increases the frequency of loss of a chromosome homologue after growth on sorbose. Perhaps strains that have large MRS alleles on homologous chromosomes are more readily able to undergo beneficial mitotic nondisjunction events and adapt more rapidly to a new environment. Strains that have smaller MRS alleles or nonfunctional MRS alleles may then be at a disadvantage, as they would not be able to adapt as quickly as the large-MRS strain and may be at a competitive disadvantage in that niche.
Previous research has shown that the MRS is a preferred site for translocations between heterologous chromosomes (3
) and that translocations can alter the phenotype of the resultant cell (30
). The control of the translocation process is not understood, and most translocations seem to occur either during growth in the host or when induced by chromosome damage in vitro (15
). If the MRS is the location for translocations between heterologous chromosomes, as the data would suggest, it may also play a role in translocations between homologous chromosomes via mitotic recombination. Although heterologous translocations are difficult to detect in a high-throughput manner, it is possible to study the mitotic recombination rate across the MRS under a variety of conditions. Research on the effect of the MRS on mitotic recombination is ongoing.
If the translocations occur exactly within the MRS, then there is no clear explanation as to how this can affect the resultant phenotype of the cell unless the MRS exerts transcriptional control on genes located near it. A role in silencing of proximal genes would not be unusual for a genetic structure such as the MRS. Large repeat regions in eukaryotes are often heterochromatic in nature, and heterochromatin frequently exerts an inhibitory transcriptional effect on adjacent genes (9
) Interestingly, while there is no obvious homologue of the MRS in eukaryotes, limited structural but not sequence homology to the MRS is found in the human D4Z4 repeat system, which is known to have an inhibitory effect on transcription (11
). The D4Z4 repeat consists of 3.3-kb subunits with 11 to 150 repeats per structure, compared to ≈2-kb RPS subunits with 1 to 50 repeats per MRS. The base subunit of the D4Z4 repeat also contains a highly conserved 27-bp sequence that is a binding site for a transcriptional repression complex, while similarly, each RPS subunit of an MRS contains four to five highly conserved 29-bp sequences that have no known function. Thus, the function of the D4Z4 repeat is the inhibition of transcription of genes that are up to 3 Mb away from the repeat, with the extent of the inhibition being dependent on the number of repeats. The MRS may have a similar effect on nearby genes, and this effect may be dependent on MRS size (RPS number) as well. Experiments are under way to study the expression of genes located up to 50 kb proximal to the MRS in strains CAI4 and in the MRS deletion strains created in this study.
The MRS remains a cryptic structure within the genome of C. albicans; it has tantalizing connections to essential functions and chromosome dynamics, yet is clearly not essential to the chromosome. We have shown in this paper that while neither the MRS nor the lone RB2 is necessary for chromosome maintenance and function in vitro, the MRS does have an effect on chromosome mitotic nondisjunction. Further work will be required to determine how this effect is mediated and whether the MRS has any other phenotypic effects.