Biology 332 - Protistology Term 2 - 2002-2003

Ciliates - Life Cycle and Life History - Macronuclear Development

Life cycle and life history

Conjugation. The basic sexual process in ciliates is conjugation. Conjugation involves the

temporary union of two cells of complementary mating type followed by
micronuclear meiosis (first and second pregamic divisions) and
a third pregamic division that produces two gamete nuclei from a single meiotic product. All other meiotic products are destroyed.  The old macronucleus begins to disintegrate at this stage.
Gamete nuclei are exchanged. In each cell one gamete nucleus (the micratory pronucleus) is passed to the other cell, and the other (the stationary pronucleus) stays behind.
The zygote nucleus is then formed by fusion of the migratory and stationary pronuclei (See below).
The zygote nucleus then undergoes one or more postzygotic divisions.
The nuclei produced by the postzygotic divisions differentiate into new macronuclei and micronuclei.

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Life cycle. The life cycle consists of all events between one fertilization event and the next (one sexual generation). The main issue in thinking about the ciliate life cycle is to understand that the cell is controlled by the macronucleus which is a somatic nucleus (that is, it is not involved in transmission of information across sexual generations). It is formed at the beginning of a sexual generation and it is destroyed during conjugation prior to the start of a new sexual generation. The micronuclei are the germ line and provide continuity across sexual generations.

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At each fertilization a zygote nucleus is formed that gives rise to a new   macronucleus and a new micronucleus. This new cell gives rise to a clone of cells that are all progeny of the initial zygote. When fertilization occurs again a new zygote gives rise to a new clone.

The parallel with animal development is direct. A new zygote gives rise to a clone of cells that form an embryo. All of the cell in the embryo are the progeny of the initial zygote. Each clone of ciliates can be thought of as an individual. The individual comes into being at fertilization and then undergoes the subsequent proceses of maturation and development, leading eventually to senescence and death.

Nature of the macronucleus.

In the observations avout life span you noticed that there is something special about conjugation that sets back the ageing clock. That something is the production of a new macronucleus. After fertilization the zygot nucleus divides  one or more times and some of the daughter nuclei differentiate into macronuclei.

There is a special localized region in the cytoplasm that contains a factor (presumably a protein) that is responsible for causing differentiation of the diploid nuclei into macronuclei. If the nuclei do not get into this special region, they become micronuclei. In Tetrahymena the special cytoplasmic region is at the front of the cell and in Paramecium it is at the back. Differentiation as micronuclei is the default. On the other hand if nuclei do get into this special region at the right time they are exposed to the 'macronucleus inducing factor' they differentiate into macronuclei.   This involves a number of drastic changes.  athe chromosomal structure of the nucleus is broken down. The genome consists of several hundred to several thousand different linear DNA molecules.  Each of these DNA molecules is a linear DNA plasmid with telomeres at each end, but with no centromeric sequence. Once macronuclei are formed chromosomes are never seen again.  As macronuclei develop mahy DNA sequences are lost. The percentage of sequence loss varies from a low of about 15 % to a high of 95%.  The nuclei undergo a series of rounds of DNA replication so that the final macronucleus will contain many copies of the macronuclear genome. This ranges from about 45 copies in Tetrahymena, to 960 copies in Paramecium to many thousands in large ciliates like Spirostomum.

In oligohymenophoran ciliates like Tetrahymena or Paramecium the macronuclear  DNA  are very large  and contain many gene loci, while in hypotrich ciliates, like Euplotes, the molecules are small and contain only one or a very few genes.

During the development of the macronucleus DNA is lost. These losses may be either between macronucleus destined sequences or they may occur within a sequence destined for the macronucleus. These sequences that are cut out from within a macronucelar molecule are called ies (internal eliminated sequences). In hypotrich ciliates there are further complications that reach tryly amazing. Not only is a slngle gene sized molecule in the macronucleus derived from several non-contiguous micronucelar Sequences, but the order of these macronucleus destined sequences is changed during the process of macronuclear development.

In the figure below MDS = macronucleus destines sequence. IES = internal eliminated sequence. During macronuclear development the IESs are removed and the order of MDSs is changed. Finally telomeres are added at the ends of the macronuclear sequence. The location of the open reading frame for the gene is indicated in the lower figure.

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This is the most extreme case of sequence alteration during macronucelar development. However, in all ciliates examined so far, there are losses of sequence and destruction of chromosomes.

After the macronuclear genome has been established through loss of centromeres and other eliminated DNA sequences, the copy number is increased. The precise copy number varies with the size of the cell and the particular group of ciliates in question. In general larger cells have higher copy numbers. Here are some examples:

Genus Copies in G1 macs
Tetrahymena 45 copies
Paramecium 960 copies
Spirostomum 16,500 copies

Regulation of macronuclear DNA content.

One of the consequences of having macronuclei that consist of plasmids rather than chromosomes is that there is no regular process for distribution of exactly equal DNA complements to daughter cells. For example, in Tetrahymena the mean difference in DNA content between daughter cells is 12%. This means that the G1 DNA content of macronuclei is also variable (coefficient of variation = 20%).  During the S period the DNA content of the macronucleus doubles. This means that if no regulatory process occurred that the variance in macronucelar DNA contetn would increase indefinitely.   This obviously does not happen, hence DNA content must be regulated.

Regulation of macronuclear DNA content in Tetrahymena is accomplished by a simple threshold-based system. If the DNA content is higher than a certain level cells proceed to division without replicatioin. If it is lower than the threshold level, replication occurs.  If it is lower than a lower threshold, two rounds of replication occur within a single cell cycle.

Condition Consequence
G1 DNA content high (above 1.4 times mean) No DNA replicaton during cell cycle
G1 DNA content intermediate (between 0.7 and 1.4) One round of DNA replication
G1 DAN content low (below 0.7 times mean) Two rounds of DNA replication in one cell cycle

Thus with a large nucleus (e.g. 150% of mean)  there is no replication and daughter cells have an average of 75% of the mean DNA content. With an average sized nucleus (100%) there is one round of replication producing 200% of G1 level in G2 and daughter macronuclei have an average of 100% of the mean level. If the initial macronuclear DNA content is low (60% of G1 mean) then two rounds of replication occur produginf a final DNA content of 240% of the mean level and daughter macronculei will have an average DNA content of 120 of the mean level. The waythat this really works is that there is probably one test, whether macs are big enough to enable cell division. If it is the cell divides. In not, it replicates and then tests again. I

In Paramecium there is a more sophisticated scheme: All macronuclei synthesize the same amount of DNA (100% of the mean G1 DNA content) regardless of their initial DNA content.

Life history. The life history of a ciliate typically looks like this: An initial period of sexual immaturiny during which cells can not mate is followed by a period of sexual maturity during which mating occurs readily. As cells age they start to become senescent and mating offurs less readily and survival or progeny is poorer. Finally the clone dies out. This means that all cells descended from an initial zygote die. Ciliates, unlike most protists, show somatic ageing.

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Organisms (species) differ in their typical life span. There are short lives species (e.g. 120 fissions) and long lived species (>600 fissions). The life span and the details of the life history of the organism are part of the organism's ecogenetic strategy. For example, short life span organisms typically have short or negligible periods of sexual immaturity. They also tend to be much more strongly inbreeding than long life span organisms. Long life span organisms tend to have long immature periods and to be much more strongly outbreeding. Life history is an adaptive complex of traits that relates strongly to the ecology of the organisms. If you are interested in eclogy you might wish to explore this set of ideas more extensibvely in an essay. There is a lot of material on ciliate life histories and breeding patterns.

One of the interesting things about  conjugation is that it resets the ageing clock. When a cell makes a new macronucleus it is young again. Thus progeny will usuall outlive parents even though both might die at the same clonal age.

This rejuevenation effect is not complete. Progeny derived from an old parent have shorter life spans than do progeny derived from the same parent clone in its youth. Compare Clone 2 and Clone 3 lifespans in the diagram below.

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Examination of Clones 2 and 4 in the figure above also shows a further point: Progenty of long-lived parents have longer life spans than do progeny of short-lived parents, even when they originate at the same parental age.

How to make sense of this?

Observation.  Cells treated with caffeine (inhibits DNA repair processes) have a drastically reduced life span. What do you might be the basis of this?

If DNA repair (photoreactivation) is induced in cells that have first been treated with UV light they live much longer that the UV-treated non-photoreactivated cells, but also longer than the non-UV-treated controls?  This effect can be repeated over the course of the life span as shown in this figure (data from J. Smith-Sonneborn).

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What can you conclude from this experiment? What assumptions do you make?