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Conjugation and autogamy
in ciliated protozoans
Paul R. Earl
[email protected]
Facultad de Ciencias Biológicas
Universidad Autónoma de Nuevo León
San Nicolás, NL 66450, Mexico
A little bit of history
Sexual processes in ciliates have been known since
they were discovered by the Dutch lens maker
Antoni van Leeuwenhoek over 300 years ago. The
Danish naturalist O. F. Müller (1786) was the second
observer of the sexual event conjugation. Others
like Balbiani (1858, 1861) followed, including
Hertwig (1889) and Maupas (1889). Giant steps in the
study of ciliate conjugation were taken by Gary N.
Calkins (1869-1943) with Paramecium caudatum as
in Calkins & Cull (1907). Herbert S. Jennings (18681947) in Paramecium had an analogous role to that
of Thomas H. Morgan (1866-1945) in Drosophila.
Autogamy was discovered around 1934 in P. aurelia
by William F. Diller (1902-1984), and Tracy M.
Sonneborn (1904-1981) discovered mating types in
1938 in Paramecium.
Ehrenberg (1838) suggested immortality for the ciliates,
and Maupas (1889) found that they had the periods of
youth, maturity and senescence. The rejuvenation of old
clones follows a sexual event. Without conjugation,
autogamy or cytogamy, the cell line will die out as found
by the remarkable French-Algerian protozoologist
Maupas (1842-1916).
Sexual processes
Autogamy is like conjugation, except that the entire
process occurs in single cells. Ralph Wichterman
(1904-1999) discovered cytogamy. See his 1986
book on Paramecium, and the book edited by Görtz
(1988). Cytogamy is like conjugation, except that
there is no nuclear exchange between the partners.
It is independent double autogamy.
Ciliates have several features of chromosome
segregation that are unique among eukaryotes,
including their maintenance of 2 nuclei: the
germline micronucleus (MI), which undergoes
conventional mitosis and meiosis, and a somatic
macronucleus (MA) with a great number of
minichromosomes that divide by a poorly
understood amitotic process. Ciliates can propogate
without the MI !
Each cell contains an MI with conventional
chromosomes and a transcriptionally much more
active polyploid MA that controls the cell. The MA
genome is com-posed exclusively of short linear
minichromosomes that typically contain single coding
regions. The MA DNA molecules are derived from a
copy of the MI genome during conjugation through an
extensive DNA reorganization process that includes
the fragmentation of the MI chromosomes and the
new addition of telomeres to the ends of the formed
MA minichromosomes via reverse transcriptase
subunit of telomerase or TERT.
P. aurelia does not really exist as a species any longer
since Sonneborn (1975) divided the aurela complex
into 14 syngens = species. A species, following Mayr
(1957), is a sexually isolated population. It cannot
cross with any others. How different species are
morphometrically, or in their DNA or RNA sequences
does not involve the species definition that is only
concerned with crossability.
The sequences of base pairs from several genes like
the histone centromere gene can be most helpful for
phylogenetic analyses. The polymerase chain
reaction, its wonderous results, blotting and other
molecular techniques are still in their protozoologic
infancy, although new insights are appearing at a fast
pace.
Know your Darlington ! NeoDarwinism combines
Charles Darwin's theory of evolution by natural selection
and Gregor Mendel's theory of genetics with random
mutation as the source of variation.
Cyril D. Darlington (1903-1981) discovered chromosomal
crossover and outlined the stages of meiosis in his 1937
textbook updated from his 1931 text.
A reduction division MUST OCCUR in meiosis. Reduction before
fertilization is gametic, whereas reduction after fertilization is zygotic
meiosis. Meiosis reduces the number of sets of chromosomes by
half, so that when gametic nuclei recombine the life cycle diploidy of
their parents will be reestablished.
In mitosis, 2 new chromosome sets segregate to 2 new cells. In
meiosis, homologous pairs of chromatids (bivalents) segregate to
become the gametes that can fuse to become the synkaryon
(fertilization nucleus).
Macronuclear regeneration. Reciprical fertilization in
the synkaryon produced a genetically new MA. Most
commonly the old MA is degenerated and resorped.
However when the new MA fails to develop, fragments of
the old MA are able to grow and resume control of the
exconjugant cell. Macronuclear regeneration was
discovered by Sonneborn in Paramecium in 1940.
The MA genome has only a small % of the MI genome (5–
10%); thus, the chromatin elimination involves as much
as 95% of the DNA. Two types of DNA processing form
the MA chromosomes:
1) deletion of DNA sequences that are ISEs and retention
of the MA genomic sequences--the MDSs; and
2) fragmentation of chromosomes 5' and 3' of genes
coupled with the new addition of telomeres.
As we move along, we find new directions leading still
farther from textbook cytology.
General conjugation with emphasis on Tetrahymena
In the crescent soon after pairing, the 10 diploid
chromosomes of Tetrahymena are reduced to 5 haploid
MIs in meiosis and are later exchanged between the
conjugants. In each cell of the pair, the migratory MI
fuses with the stationary MI to form a new zygote which
is the synkaryon.
The fertilization nucleus proceeds through 2 mitotic
divisions, usually, resulting in 4 daughter nuclei. Two of
the nuclei can serve as the germ line MI for the daughter
cells following the next round of cell division. One of the
nuclei will segregate to the new MA, and one to the new
MI. The old MA will be degraded unless needed to
salvage a malfunctioning new MA. One of 2 MI in the 1st
exconjugant is eliminated so that the 2nd excongugant
regains 1 MI + 1 MA.
Soon after mitosis, chromosomes within
the nucleus are destined to differentiate,
undergoing multiple rounds of replication
to form polytene chromosomes.
The chromosomes are radically fragmented
to minichromosomes, telomeres are added
to sequences of the MDSs, and the DNA of
the remaining non-MAs and the old MA
fragments are degraded.
The illustration above fits classic meiosis.
The stages of the crescent after Sugai &
Hiwatashi (1974)
First prize goes to the people of the Yao
laboratory
The best interpretation of conjugation is by
Marcella D. Cervantes, Xiaohui Xi, Danielle
Vermaak, Meng-Chao Yao and Harmit S. Malik
(J Biol Cell 17: 485-497, 2006), mainly because
they have used the most recent technology
which is that of immunofluorescence with
centromeric antibody comple-mented with
DAPI fluorescent dye specific to DNA as in
their 3 figures given here.
Remember that the somatic MA is highly polyploid and
consists of amplified, highly rearranged segments of the
MI genome. The MA is transcriptionally active and is
responsible for much of the gene expression in ciliates,
whereas the MI is active at special times like meiosis.
The completion of meiosis sets up a cascade of events
that leads up to the formation and diversification of the
germline MI & MA. One of 4 meiotic MIs is selected as a
gamete and undergoes 1 round of mitosis. The paired
cells exchange MIs, and then the 2 gametic nuclei, one
from each parent cell, fuse forming the diploid zygotic
nucleus--the synkaryon or zygote.
These MIs are the stationary and the migratory nuclei
and usually identical. Nevertheless, different
allopolyploids might well be encountered.
Tetrahymena thermophila has 10 diploid
chromosomes. They are represented by the
brilliant green staining of the appropriate
antigen, centromeric histone.
Stage III of the crescent and the distribution
of Histone 3. Credit to Cervantes et al, 2006
Destruction of
the old MA.
Credit to
Cervantes et al,
2006
Let's review conjugation again !
When conjugation is induced, the germ MI enters
meiosis and forms a synkaryon after reciprocal
exchange of gametic nuclei between the mates.
The synkaryon divides 3 times--usually--and
differentiates into new macro- and micronuclei.
The newly developed macronucleus (MA anlagen)
begins to function, the old MA degenerates and
the cells separate to be exconjugants. Eliminate
extras like 2 not 1 MIs in excongugants to fit the
condition. That's it !
FPD first prezygotic division, TPD third
prezygotic division, PZD post zygotic division.
Exconjugant of Paramecium with 4 MA anlagen. The Mis
are lost among MA fragments. Basic fuchsin, fast green.
An older exconjugant of Paramecium with 2 new MAs.