Nuclear Division Cycle

The cell cycle is divided into four phases: G1 phase (between mitosis and the beginning of DNA synthesis), S phase (the period of DNA synthesis), G2 phase (the interval following the S phase and the beginning of mitosis) and M phase (separation of daughter nuclei). The G1, S and G2 phases are typically a longer part of the cell cycle than the M phase (Figure 2.2). Whereas in plant and animal cells the nuclear membrane breaks down during cell division, fungi—including yeasts—have a closed mitosis: spindle microtubules are inside the nucleus and the spindle pole bodies are formed within an intact nuclear membrane. For a transmission electron micrograph of mitosis in fungi, see Alexopoulos et al. (1996). Since nuclear division in fungi is generally not followed by cytokinesis, some prefer to use the term nuclear division cycle in place of cell cycle for the sequence of events by which the nucleus duplicates. However, because of the common use of the term cell cycle, it will be used here, often interchangeably.

Aspergillus nidulans is an attractive fungus for cell cycle studies because its conidia are uninucleate. Therefore, the rounds of division that a nucleus has undergone at different times can be determined by counting the number of stained nuclei. The first three divisions of nuclei are synchronous and result in the formation of eight nuclei in conidium. In this fungus, three nuclear divisions in conidium are essential for the emergence of a germ tube. The genes that are required for nuclear division as well as nuclear movement into the germ tube are being identified by isolating temperature-sensitive (conditional) mutants in A. nidulans.

Figure 2.2 Aspergillus nidulans cell cycle. The duration of different phases is shown as percentage of total cell cycle.

2.3.1 Temperature-Sensitive Mutants

Nuclear division, nuclear movement and septation are indispensable for hyphal growth. Therefore, to identify the genes that control these events, the mutants in which growth fails only at a higher temperature must be isolated. A. nidulans grows normally between 15 to 44°C, whereas a temperature-sensitive mutant does not grow at a higher temperature (44°C) as the mutated gene-encoded protein takes a non-functional or denatured structure. Bergen and Morris (1983) spread mutagenized conidia on agar plates and searched for the rare microscopic colonies in which growth was arrested either before or soon after germination at 44°C but which grew normally if shifted to 32°C (a permissive temperature). When such cells were picked and grown at the permissive temperature, four major classes of mutants were identified: (i) nuclei in the nim (never in mitosis) mutants did not divide; (ii) those in the bim mutants were blocked in mitosis; (iii) the nud mutants were defective in nuclear distribution; and (iv) the sep mutants were unable to form a septum following the third nuclear division (Figure 2.3). The mutant approach showed there is no obligatory coupling between nuclear division and cytokinesis. However, nuclear division and septation are required for hypha development as the nud and the sep mutants fail to grow at 44°C — a temperature at which the wild type can grow.

Figure 2.3 Temperature-sensitive mutants of Aspergillus nidulans defective in nuclear division and nuclear movement. Redrawn from Plamann (1996).

2.3.2 Kinetics of Nuclear Division Cycle

Using the nim mutant of A. nidulans blocked in the G2 phase, the nuclear division cycle was deduced from the number of nuclei in the cell (Bergen and Morris, 1983). The first step was to synchronize the multinuclear germlings at the beginning of the S phase. Dormant conidia were germinated for six hours in presence of hydroxyurea (a DNA synthesis inhibitor) at the restrictive temperature. The conidia underwent one nuclear division, indicated by the appearance of binucleate germlings, and this synchronized the germlings at the beginning of the second cycle S phase. The binucleate germlings were shifted to a hydroxyurea-free medium at the permissive temperature and samples removed at intervals and retreated with hydroxyurea before they became sensitive to it, i.e., complete S phase (become tetranucleate). The time required to pass the hydroxyurea-sensitive phase (S phase) was around 40 min. The rate of nuclear doubling was determined from a plot of nuclear division per cell vs. time using the equation:

division per cell = log

x log 2

where N is the number of nuclei and C is the number of cells analyzed. The nuclear doubling time (generation time or Gt), calculated from the slope of the line was 95 min. The length of M phase, calculated from mitotic index (percentage mitotic figures in hyphal tips) of culture was 5 min. The G2 phase, found by subtracting the aggregate length of M and S from the generation time, was 40 min. The G1 phase, determined as Gt - (S + G2 + M), was 10 min.

In N. crassa, morphological differences in the nuclear shape of different phases were used to determine the nuclear division cycle (Martegani et al., 1980). The G1 nuclei are compact and globular; the S and G2 nuclei are ring-shaped and the M phase nuclei are double ring or horseshoe-shaped. Treatment with picolinic acid blocked nuclei in G1. Release from picolinic acid inhibition was followed by a wave of synchronous DNA replication. From the frequencies of phase-specific nuclear shapes, the duration of the G1 phase and S + G2 phase can be estimated. In conidia germinated in sucrose medium, the duplication (generation) time was 100 min (G1 = 20 min, S = 30 min, G2 = 40 min and M = 10 min). The extended G2, relative to G1, is characteristic of fungi. These data on A. nidulans and N. crassa show that the cell cycle in fungi is completed faster than in animals or plants. For example, a human cell growing in culture has G1 = 9 hours, S = 10 hours, G2 = 4.5 hours and M = 30 to 45 min; the complete cycle takes about 24 hours.

0 0

Post a comment