知花 博治

Background: In the budding yeasts, including Saccharomyces cerevisiae, in which individual chromosomes cannot be visualized by microscopy, the mitotic phases in the cell cycle have not been correlated with the chromosome behaviour. We used various repetitive sequences, namely, rDNA, telomeric sequences and RPSs, which are localized in limited regions in almost all chromosomes, as probes for fluorescence in situ hybridization (FISH) to analyse the cell cycle phases in a pathogenic yeast Candida albicans. The positioning of the FISH signals was analysed quantitatively in relation to the length of spindle microtubules in the nuclear domain. Results: RPSs were randomly distributed in the interphase nucleus, and they formed aggregates with the development of the spindle. DNA synthesis was complete before RPSs came closest to the spindle. As the spindle elongated, they were scattered along the spindle and then separated into two clusters at the spindle poles at the end of anaphase. rDNA was localized in the nucleolar domain, and telomere signals were randomly distributed throughout mitosis. Conclusion: By estimating quantitatively the proportions of mitotic cells with particular configurations of both microtubules and chromosomes in a population of rapidly proliferating cells, we were able to define various stages in the progression of mitosis. The S phase and pro-to-prometaphase were overlapping and the G2 phase was lacking. Unexpectedly, the pole-to-pole elongation of the spindle (anaphase B) was predominating and was followed by movement of chromosomes to the poles (anaphase A). © Blackwell Science Limited.

The location and organization of repetitive sequences, members of the RPS family, which are sequences specific to Candida albicans, were determined on each chromosome of C. albicans strain FC18. Using pulsed-field gel electrophoresis, we separated seven fractions from eight chromosomes. Each chromosome was cleaved by BamHI and XhoI to excise the RPSs, which were then detected by hybridization with an RPS probe. All chromosomes except chromosome 4 carried RPSs, and these RPSs were located within a limited region on each chromosome. From the digestion of each chromosome with SfiI and probing with the RPSs, we found that these recognition sites within the RPS region were conserved among all RPS-containing chromosomes. For further characterization of the RPSs, the locations and the boundary regions of the RPSs were examined on chromosome 6 of strain FC18 as a model chromosome. Using the restriction enzymes SfiI, SmaI, XhoI, BamHI, MluI and NruI, we constructed a semi-macro physical map of the RPSs and their boundary regions on this chromosome. We also determined which part of the RPS was adjacent to each boundary by using sub-fragments of RPS as probes. The physical configuration around the RPSs and their boundary regions are presented. The results obtained should be useful for future analysis of the function of these regions.

To study the mechanism of condensation of dispersed plastid (pt) nucleoids into a single pt nucleoid with aging of the cells of Chlamydomonas reinhardtii, two mutants, designated cond-1 and cond-2, were isolated. A plastid of a wild type cell, 6.5 μm in diameter, contained ten dispersed spherical pt nucleoids within one week of culture on an agar plate. At about one week of culture, the cell number was saturated and pt nucleoids began to associate with each other, condensing into a single pt nucleoid at three weeks of culture. In contrast, cond-1 and cond-2 cells, which had about 20 and 45 pt nucleoids and whose cell diameters were 7.8 and 9.5 μm at one week of culture respectively, still had about 10 and 20 pt nucleoids at even 7 weeks of culture. Doubling times of the three cell types were similar. From genetic analysis, each of the two mutants had one gene mutation. The two mutations are probably linked. The measurement of O2 evolution showed that the two mutations did not affect the photosynthetic system. Lipid contents of the two mutant cells were clearly higher than that of wild type cells. The role of a higher number of pt nucleoids is probably to increase the activity of lipid and/or membrane synthesis for lipid storage. © 1994 Springer-Verlag.

In a previous study, a repeated sequence, RPS1, was cloned from the genomic DNA of Candida albicans. It was 2.1 kb in length and was tandemly repeated in a limited region of almost all of the chromosomes. In this study, we examined and characterized the diversity of the repeating structure of the RPS units. RPS units were of 2.1, 2.3, 2.5, and 2.9 kbp in length after digestion of the genomic DNA with SmaI and 2.1 and 2.3 kbp after digestion with Pst1, with the differences being multiples of approximately 0.2 kbp. Moreover, one or two types of RPS unit were present specifically on each chromosome. We cloned 14 RPS units from the mixed DNA of chromosomes 1 and 2 and 59 RPS units from chromosome 6. These RPS units were classified into four types by their SfiI digestion profiles and chromosomal origins. Sequence comparisons revealed a tandem arrangement of internal, small repeating units of 172 bp. This unit of repetition was designated alt (C. albicans tandem repeating unit). The size of RPS units was variable, with sizes representing a series of increments of approximately 0.2 kbp that corresponded to the alt sequence. By contrast, the sequences other than the tandem repeats of alts were highly conserved, with homology of more than 98% among all cloned RPS units. These results suggested that RPS plays an important role in the organization and function of the chromosomes of C. albicans even though the actual function of RPS has not yet been clarified. Structural features of RPS that contains the repeated alt sequence are discussed in relation to human α-satellite DNA with its tandem repeats of about 170 bp that are similar in size to alt, the repetition of which is responsible for the variations in the size of the higher-order repeats.

The extracellular proteinase (EPR) of Candida albicans was induced in a medium containing bovine serum albumin as sole nitrogen source. There were two intracellular forms in cells induced to produce EPR, a 43 kDa protein (EPR) and a 45 kDa protein (cross-reacting material of EPR; CRM-EPR); these were detected by immunoblotting using anti-EPR antiserum. The 43 kDa protein (EPR) may be the same as the extracellular form judging by molecular mass, and the 45 kDa protein (CRM-EPR) may be a precursor form of EPR. Many dense granules were observed by electron microscopy near the plasma membrane of the mother cells in EPR-producing cells. Both the 43 and 45 kDa proteins were recovered in a membrane fraction and were solubilized by Triton X-100. When the membrane fraction was further fractionated by sucrose density gradient centrifugation, the 43 and 45 kDa proteins were differentially fractionated. This suggests that they were located in different membrane-bound structures and is consistent with an assumption that the 45 kDa protein is a precursor for EPR.

A repeated sequence, named RPS1, approximately 2 kb in size, is found mainly in chromosome 6, the second most variable chromosome among the eight chromosomes of Candida albicans. Most of the RPS1 units of chromosome 6 seem to be located within a single region of about 100 kb in strain FC18. In both strains FC18 and NUM812, a part of RPS1 is apparently tandemly repeated. A unit of RPS1 has been cloned and sequenced. It consists of 2114 bp and has a GC content of 40 mol%. The repeat unit contains smaller repeats of about 80-170 bp which are called REP1, REP2, REP3, REP4 and REP5; REP2 is duplicated. The small repeats are classified into two groups by their homology. One comprises REP1, REP2 and REPS, and the other REP3 and REP4. They are termed the REP1 and REP3 families, respectively. The two families both contain a common 29 bp sequence, called COM29. The dispersed repetitive sequence RPS1 may be involved in chromosomal rearrangements and may in part explain chromosome polymorphism in C. albicans. The origin of RPS1 was not determined.

Using Chlamydomonas reinhardtii cells of various ages, harvested after culture on agar plates for periods from 7 days to about one month and subsequently induced to form gametes for a period of 4 hours, we examined the formation of zygotes, the preferential digestion of chloroplast nucleoids (ct-nucleoids) of the mt- parent, nuclear fusion and maternal inheritance of chloroplast-specific traits.Previous studies have shown that, when young mt+ and mt- cells harvested from one-week-old cultures are mated, preferential digestion of ct-nucleoids occurs between 50 and 120 min and nuclear fusion between 70 and 180 min. We now report that, with later harvesting, although formation of zygotes and nuclear fusion are not affected, the occurrence of preferential digestion of ct-nucleoids can be delayed for up to about 5 h, depending on the age of the cultures of cells. This result suggests that the process of preferential digestion is independent of nuclear fusion.Mt+ cells dominated with respect to the occurrence of preferential digestion, when a comparison was made of the frequencies of preferential digestion in zygotes derived from the following two types of mating: young mt+ cells (harvested from one-week-old cultures) × aged mt- cells (harvested from about 3-week-old cultures); and aged mt+ cells × young mt- cells.Genetic analysis of chloroplast-specific genetic markers for resistance to antibiotics showed that delay in the occurrence of preferential digestion did not affect maternal chloroplast inheritance. © 1991. The Japanese Society of Plant Physiologists (JSPP).