Introduction to the Genetics of Tetrahymena
Eduardo Orias - 2/12/97
Instructions
To go quickly to a particular major topic, click on the topic in
the Table of Contents. At the end of each major
topic section you may click to return to the Table of Contents or to
the Main Page.
Table of contents
- Tetrahymena thermophila
- The organism
- Nuclear dimorphism
- Life cycle
- Micronuclear (germline) genetics
- Mendelian genetics
- Inducible conjugation variants
- Mapping genetically the MIC genome
- Genetics of the macronucleus
- DNA rearrangements during macronuclear differentiation
- Phenotypic assortment
- Coassortment
- Mapping genetically and molecularly the MAC genome
- Special topics of MAC genetics
- Sexual maturity
- Mating type differentiation
- Genetics of the ribosomal RNA gene (rDNA)
- MAC DNA replication and copy number control
- Isolation and genetic characterization of mutants <to be
added>
- Other non-Mendelian inheritance
- Mitochondrial (Cytoplasmic) Genetics
- 10 Kb Plasmid
- Cortical Inheritance
- On the evolution of nuclear
dimorphism
- Genetic Protocols and strategies <To be added>
- Other Background
- Randomly Amplified Polymorphic DNA (RAPD)
- References
*************************
*************************
Tetrahymena thermophila
The organism
Tetrahymena thermophila (Fig. 1) belongs to the Ciliated
Protozoa-or Ciliates-a major, successful and diversified
evolutionary lineage of unicellular eukaryotes. It is a freshwater
organism that commonly inhabits streams, lakes
and ponds. The cells are large: 40-50 um along the anterior-posterior
axis. Like other ciliates, Tetrahymena cells
have a striking variety of highly complex and specialized cell
structures. Additional views of Tetrahymena cells
can be found in the published literature: transmission EM [ref. 1,
p. 5] and optical microscopy of stained cells
[ref. 2, p. 180]. The Tetrahymena genome size (roughly 220 Megabase
pairs) is of the same order of magnitude as
that of Drosophila-one order larger than yeast (Saccharomyces) and one
smaller than human.
Figure 1. Scanning electron micrograph of a Tetrahymena cell.
************************
Tetrahymena's versatile biology provides extremely useful
experimental tools. With a minimum doubling time below
2 hr, it is among the fastest growing eukaryotes. It readily grows to
high density over a wide range of scale (from
a few ul to fermentor scale) in a variety of media: totally defined
synthetic chemical medium, rich nutrient broth
or monobacterial culture. It grows over a wide temperature range: 12C
to 41C. It has powerful active transport
mechanisms and can be labeled with virtually any small molecule. The
sexual stage in its life cycle, conjugation,
can be induced at will, with high efficiency and synchrony. Cells can
maintain DNA constructs introduced by transformation.
Tetrahymena is also a promising system for biotechnological
applications. It is used for quick, reliable, sensitive
and inexpensive bioassays. For example, it is used for determining the
protein nutritional value of human foods,
the toxicity of compounds of chemical or biological origin, and for
monitoring water quality. It has potential
for industrial synthesis of pharmaceuticals. Its capacity for massive,
synchronous regulated secretion confers
the potential for industrial synthesis of useful proteins. Since
mosquitoes and snails are parasitized by other
Tetrahymena species and closely related genera, T. thermophila has the
potential for the development of tools for
the biological control of major world-wide human diseases for which
those organisms are intermediate vectors: schistosomiasis,
malaria, yellow fever. Tetrahymena-based biotech strategies to fight
fish hatchery and pet store epidemics of the
closely related ciliate Ichthyophthirius ("Ick") are
currently being explored.
This microbial eukaryote has great value as an experimental model
system due to the facility with which it can
be handled, its structural and functional differentiations, its
accessibility to genetic and molecular approaches,
its large evolutionary distance from other commonly used eukaryotic
genetic model systems, and its potential for
biotechnological applications coupled with its biosafety. It has
provided an excellent system for the discovery
and investigation of fundamental molecular and cellular mechanisms
(e.g. ribozymes and telomeres). The availability
of dozens of related species has proven extremely useful for
identifying evolutionarily conserved domains in macromolecules
and particularly for the dissection of important secondary structure
and functional domains in RNAs that have or
participate in catalytic activity. Its nuclear dimorphism provides
Tetrahymena with an additional and unique dimension
of biological versatility and genetic manipulability.
The species now called Tetrahymena thermophila was first considered
to be Tetrahymena pyriformis. Later, T.
pyriformis was found to comprise many sibling (cryptic) species,
sexually isolated from one another but morphologically
indistinguishable. As knowledge advanced, it was successively renamed
variety 1 and syngen 1 of Tetrahymena pyriformis,
before acquiring its current name [3]. Genetically, thermophila is the
Tetrahymena species by far most extensively
characterized.
Whenever possible I refer to useful reviews rather than primary
research articles. Nanney's [2] introduction
to the experimental biology of ciliates, with strong coverage of
Tetrahymena, is particularly useful. A broad review
of the biology of Tetrahymena was edited by Elliott [5]. A
comprehensive review of ciliate molecular biology was
edited by Gall [6]; it includes relevant chapters on conjugation,
genetics, and DNA organization, all with heavy
coverage of Tetrahymena thermophila. Prescott [4] comprehensive review
of ciliate molecular genetics includes much
about Tetrahymena. Bleyman [7] has recently reviewed the genetics of
ciliates and loci determining interesting
mutant phenotypes, including many in Tetrahymena. A shorter version of
this article will be published as a chapter
in ref. [16].
Nuclear dimorphism
As is typical of ciliates, the nuclear apparatus of Tetrahymena is
composed of two structurally and functionally
differentiated types of nuclei, a phenomenon known as nuclear
dimorphism. The micronucleus (MIC) is the germline,
i.e. the store of genetic information for the sexual progeny. It is
diploid and contains 5 pairs of chromosomes.
No known genes are expressed in the MIC. Amicronucleate Tetrahymena
cells (i.e., cells lacking a MIC) are frequently
collected in nature, but in laboratory strains of T. thermophila the
loss of the MIC leads to clone death. Only
one viable laboratory-obtained amicronucleate cell line has been
described. At cell division the MIC divides mitotically
with kinetochores and intranuclear mitotic spindle.
The macronucleus (MAC) is the somatic nucleus, i.e. the nucleus
actively expressed during vegetative multiplication.
No known MAC DNA is transmitted to the sexual progeny. The MAC
contains 200-300 autonomously replicating species
derived from the 5 MIC chromosomes by site-specific fragmentation
(further described below). The bulk of these
DNA species is present at the average level of 45 copies per
MAC. There are no visible structures expected for
mitotic distribution of MAC pieces , such as kinetochores or mitotic
spindle. The MAC is thus said to divide by
amitosis. Approximately (but seldom exactly) half of the MAC DNA is
distributed to each daughter MAC at cell division.
Alternative allele copies of a locus segregate at random during MAC
division (described later). Physical methods
are available to preparatively separate and purify MICs and MACs from
one another.
Life cycle
The life cycle consists of an alternation of haploid and diploid
stages (haplophase and diplophase, respectively)
with reference to the germline. Cell reproduction is exclusively by
binary fission; it is exclusively asexual and
occurs only in the diplophase. It is remarkable that such highly
differentiated cells can divide by binary fission
[e.g., see ref. 2, Plate II, p. 32]. Cell division is accompanied by a
variety of morphogenetic events that result
in the development of duplicate sets of cell structures, one for each
daughter.
Conjugation is the sexual stage of the Tetrahymena life cycle,
which includes no cell reproduction. During conjugation,
two cells pair, form a temporary junction, exchange gamete nuclei and
generate and differentiate the nuclear apparatus
of their sexual progeny. The nuclear events of conjugation (Fig. 2)
normally include meiosis, gamete nucleus formation,
fertilization and nuclear differentiation. It is remarkable that at
the time of exconjugant separation (stage 7
in Fig. 2) there are five nuclei experiencing four extremely diverse
fates, all within a common cytoplasm. Conjugation
includes the only-and very brief-haploid stage of the life cycle; it
follows meiosis and quickly ends at fertilization.
The haplophase is limited to a single nuclear division, without any
cell division.
Figure 2. Nuclear events in Tetrahymena conjugation.
0: Vegetative cells homozygous for alternative alleles at one
locus. The MIC (small circles) is shown nested
in-but physically separate from-the MAC (large circles) , the normal
arrangement in non-dividing vegetative cells.
1: Paired cells. 2: MICs undergo meiosis, and 4 haploid nuclei are
produced. Only the anterior meiotic product
remains functional; the other 3 disintegrate. This is the stage at
which meiotic crossing-over, used for making
genetic maps of the MIC genome, occurs. 3: Mitotic division of
functional meiotic product yields genetically identical
migratory (anterior) and stationary (posterior) gamete pronuclei. 4:
Migratory pronuclei are reciprocally exchanged,
and fuse with stationary pronuclei of the recipient cell, forming the
zygote nucleus. 5: The zygote nucleus undergoes
two mitotic divisions, giving rise to 4 genetically identical diploid
nuclei. 6: Anterior products differentiate
into macronuclei, while posterior products remain diploid
micronuclei. This is the stage at which site-specific
DNA rearrangements and mating type determination (discussed in the
text) occur in the MAC. 7: Exconjugants separate.
The old macronucleus, and one of the two new micronuclei, are
destroyed. 8: Exconjugants undergo the first postzygotic
cell division, forming the 4 karyonide cells. Each karyonide receives
an independently differentiated new macronucleus,
and a mitotic copy of the functional micronucleus. Karyonides then
begin vegetative multiplication by binary fission.
************************
In order to conjugate, Tetrahymena cells must satisfy the following
requirements: a) they must be starved for
at least one required nutrient; b) they must be of different mating
type; and c) they must have reached a sufficient
level of sexual maturity. Seven mating types (I-VII) have been known
since the earliest investigations of T. thermophila;
the number has not increased after testing thousands of additional
independent isolates from the wild. Sexual progeny
normally are unable to mate again immediately after finishing
conjugation. After 50-80 fissions they reach adolescence,
i.e., they can mate with mature but not adolescent cells. Cells reach
full sexual maturity 20-25 fissions later
[8]. In the wild, the frequencies of the seven mating types tend to be
equal and conjugation seems to occur frequently
[9].
It is virtually certain that T. thermophila is an outbreeder in
nature, i.e. with little or no mating among
close relatives. Nevertheless, inbred strains have been successfully
developed in the laboratory. Unlike Paramecium,
there is no programmed somatic (vegetative) senescence and death:
Tetrahymena cell lines are in practice immortal.
The MIC of a cell line tends to lose chromosomes at a variable and
unpredictable rate (germinal senescence). The
lack of MIC gene expression prevents direct selection against such
loss, just as in multicellular organisms possessing
differentiated germline and soma. A useful (and still up-to-date)
discussion of these topics is contained in Chapter.
9 of ref. [2].
Tetrahymena (and most Ciliates) are remarkably advanced among
unicellular eukaryotes: no cell reproduction in
the haplophase; differentiation of somatic and germ line nuclei;
binary fission in the face of highly specialized
compound cell structures; conjugation in diplophase; and internal
fertilization at conjugation. Given this enormous
biological versatility of ciliates, it is puzzling to this author why
this evolutionary group includes no known
multicellular forms.
Return to Table of
Contents <<<<<<000>>>>>>
Return to Main Page
Micronuclear (germline) genetics
Mendelian genetics
The nuclear events of conjugation (Fig. 2) have two noteworthy
genetic consequences: 1) they generate Mendelian
genetics, as the diploid MIC undergoes meiosis and the zygote nuclei
(from which progeny MIC and MAC are derived)
are formed by the fusion of two haploid gamete nuclei. 2) The MIC and
MAC of a karyonide start out genetically
identical to one another; normal progeny are thus said to be
homokaryons. 3) The differentiated MICs and MACs of
the entire set of four karyonides derived from a given conjugating
pair all have genetically identical diploid
progenitors, and thus the four karyonides are expected to have
identical phenotypes, no matter how different the
parental cells were. It follows that pairs-and not exconjugants or
karyonides-are the units to be counted in determining
phenotypic ratios among the progeny of a cross.
There are several useful and well characterized variants of the
normal conjugation pathway. Under some circumstances,
conjugation is aborted and the differentiation of new MAC does not
occur. The old MAC is retained instead. MAC
retention can be readily identified (and excluded from progeny
phenotypic ratio calculations) because the exconjugants
remain sexually mature and continue to express parental phenotypes,
including mating type. MAC retention is one
way to generate heterokaryons, i.e., cells with genetically different
MIC and MAC. MAC retention can be induced
in every conjugating pair in crosses to star strains (genomic
exclusion; see below). Heterokaryons are useful a)
for positively selecting true progeny of conjugation in mass culture
(e.g. parental cells with drug resistant MIC
and sensitive MAC), b) for perpetuating lethal genotypes in the MIC
(e.g., nullisomy, explained below) and c) for
sorting out the contributions of old and new MACs toward the execution
of early developmental events.
With low frequency, three cells can conjugate with one another in a
mixture of cells of two mating types. Triplet
conjugation usually generates either a set of three diploid
exconjugants or a haploid-diploid-triploid set, depending
on how well the three junctions are developed [see Chapter 7 in
ref. 2]. If all three junctions are well developed,
the triplet is symmetrical and each cell behaves equivalently: each
conjugant donates a migratory pronucleus to
the conjugant on its right, and receives one from the conjugant on its
left. Three diploid exconjugants result,
which generally are not genetically identical. If the junction between
the two cells of like mating type is not
well developed, then no pronucleus crosses this junction. The
conjugant with two good junctions donates a migratory
pronucleus to the conjugant on its right and receives a migratory
pronucleus from the two other conjugants, becoming
triploid. The other two exconjugants become diploid and haploid,
respectively. The first well characterized nullisomic
strains, i.e., strains lacking one or more pairs of chromosomes in the
MIC, were obtained after meiosis of a haploid
MIC within cells derived from a conjugating triplet.
Inducible conjugation variants
Other useful and well characterized conjugation variants that occur
spontaneously and can be induced at will
are: genomic exclusion, cytogamy (i.e. self-fertilization),
uniparental cytogamy and pronuclear fusion failure
[see refs. 10-12]. Some of these variant conjugation pathways generate
cells that are homozygous for their entire
genome, either in the MIC only (genomic exclusion) or in both MICs and
MACs (cytogamy and fusion failure). Cytogamy
generates homokaryons, while fusion failure generates both hetero- and
homokaryons. Cytogamy is used to efficiently
isolate laboratory-induced recessive mutants. Pronuclear fusion
failure is used for the generation of strains that
are homozygous and simultaneously heterokaryons for several loci.
Genomic exclusion is induced by conjugation with a star
strain. Such strains have a grossly defective MIC, with
only a few percent of the normal DNA amount. The name genomic
exclusion refers to the inability of the star strain
to contribute any genetic information to the sexual progeny. When a
normal cell conjugates with a star cell (i.e.
round I of genomic exclusion), the prezygotic nuclear events occur
normally in the normal conjugant. In the star
conjugant, meiosis is initiated but no meiotic products are
generated. The normal conjugant transfers its migratory
pronucleus to the star conjugant, but gets nothing in return. Each
conjugant thus ends up with a mitotic copy of
the single meiotic product saved in the normal conjugant. This
pronucleus diploidizes and becomes the MIC of the
exconjugants, while their old MAC is retained. The diploidization of
the MIC generates exconjugants that in one
step have become homozygotes for their entire MIC genome and,
depending on their initial genotype, often are heterokaryons.
The star-derived exconjugant (but not the other exconjugant) retains
the inability to maintain a normal MIC during
subsequent asexual multiplication and soon reverts to the star
state. The generation of homozygous heterokaryons
is the most common application of genomic exclusion often in
combination with phenotypic assortment (described
later). Genomic exclusion involving a haploid strain as the normal
conjugant was used to recover the first nullisomic
strains.
Since round I exconjugants in genomic exclusion retain their old
MACs, they retain their (different) mating
types and are sexually mature. If left alone and not re-fed, they
immediately undergo a second round of conjugation
(round II of genomic exclusion). This is now a normal round of
conjugation. If the two exconjugants from the same
round I pair undergo round II, homokaryons with both MIC and MAC
homozygous for the entire genome result. Ref.
[11] contains an excellent illustration of genomic exclusion.
Additional methods to obtain whole-genome homozygotes involve
induced blocks of fertilization. Cytogamy is a
self-fertilization that occurs when the exchange of gamete pronuclei
is blocked (e.g., by hyperosmotic shock or
microtubule inhibitors). The resulting diploid new MIC is homozygous
for the entire haploid genome of the functional
meiotic product saved in each conjugant. Nuclear differentiation
occurs normally and homokaryons are produced.
Generally, the two exconjugants are genetically different. Uniparental
cytogamy combines the induction of cytogamy
with a first round of genomic exclusion. The resulting progeny are
whole-genome homozygotes. In contrast to ordinary
genomic exclusion, the first round exconjugants differentiate new
macronuclei. The exconjugant clones mature sexually
very early, a fortuitous circumstance that has been used to isolate
mutants affecting the conjugation pathway [12].
Pronuclear fusion failure occurs when exchange of gamete pronuclei
occurs but their fusion is blocked (e.g.
with a pulse of microtubule inhibitors). Each conjugant at
fertilization ends up with two nuclei, each of which
is diploid and homozygous for an entire genome. Since each genome is
derived from a different conjugant, the two
nuclei are not necessarily genetically identical. Nuclear
differentiation proceeds normally. Of the four new nuclei
differentiated in each conjugant, one MIC and one MAC is derived from
each of the two unfused pronuclei. Since
one new MIC is destroyed, half of the karyonides are heterokaryons
[10].
Mapping genetically the MIC genome
Genetic differences in Tetrahymena are mapped to chromosome arms by
using nullisomic strains. These are strains
lacking both copies of a chromosome or chromosome arm in their
MIC. They survive because they are heterokaryons
with a normal MAC. When a diploid strain homozygous for a recessive
mutation is crossed to a nullisomic strain,
the resulting monosomic progeny express the recessive genotype if the
chromosome carrying the locus in question
is absent from the nullisomic parent [illustrated in ref. 13 for
mapping RAPD DNA polymorphisms]. By doing parallel
crosses of the mutant to an appropriate set of nullisomics, the locus
can be assigned to a chromosome arm. Bruns
[14] and this web site list the chromosome arm
assignments of many conventional loci,
cloned DNA sequences and DNA polymorphisms. Parenthetically, the newly
differentiated monosomic MAC starts out
in a state of major gene dosage unbalance. While in most eukaryotes
this condition would be lethal, Tetrahymena
cells monosomic even for several chromosomes survive to be useful and
reach normal growth rate because of mechanisms
that regulate the copy number of MAC pieces during asexual
multiplication.
As in other eukaryotes, genetic linkage maps of loci in individual
chromosomes are constructed by exploiting
the fact that meiotic crossing over generates recombinant genotypes
with a frequency which, in the linear portion
of the range, is roughly proportional to physical distance [see any
genetics textbook, e.g. Chapter 5, ref. 15].
Major progress has recently been made in constructing a solid
framework for a genetic map
of the Tetrahymena MIC genome, included in this web site. This
framework is currently based on the results of testing
for linkage nearly 400 "randomly amplified polymorphic DNA"
(RAPD) segments as well as some "classical"
Tetrahymena genetic loci.
Return to Table of
Contents <<<<<<000>>>>>>
Return to Main Page
Genetics of the macronucleus
DNA rearrangements during macronuclear differentiation
Several types of developmentally programmed DNA rearrangements
occur during MAC differentiation, diagrammed
in Fig. 3 and reviewed in [4]. One type is the site-specific
fragmentation of the 5 MIC chromosomes into 200-300
subchromosomal molecular DNA species, here called MAC ARPs
(autonomously replicating pieces). They have also been
called minichromosomes. The average size of these pieces is roughly
700 Kb, and they range in size from a few hundred
to a few thousand Kb; thus each ARP must contain many genes. The rDNA
ARP (described later) is exceptionally small
(21 Kb) and contains only the gene for the rRNA 45S precursor as an
inverted repeat.
Figure 3. Types of developmentally programmed DNA rearrangements
occurring during MAC differentiation.
Top line: Arbitrary segment of MIC DNA. Thick bar: MAC-destined DNA
segments. Open segments: internally deleted
(MIC limited) DNA sequences. Thin connector: DNA sequences (including
Cbs site-vertical arrow), lost during chromosome
fragmentation. Second line: MAC ARPs derived from MIC chromosome
segment. Wavy line: de novo added telomeric repeats.
Dashed lines: boundaries of MIC-limited sequences. Bottom lines:
Result of amplification in newly differentiated
MAC. MAC chromosomes (averaging roughly 700 kb) are not drawn at the
same scale as MIC limited sequences (generally
in the order of 1 kb) or telomeric repeats (hundreds of bp).
************************
The Cbs (Chromosome breakage sequence) is a unique DNA 15-mer
(5'AAAGAGGTTGGTTTA3' in one strand), necessary
and sufficient for chromosome fragmentation during MAC
differentiation. Telomeric repeats (GGGGTT at the 3' end)
are added by telomerase at the newly created ends of these pieces,
which are then amplified to the 45-ploid level;
the rDNA, with 10,000 copies per MAC, is the only known exception. The
Tetrahymena telomerase is a reverse transcriptase
that includes the RNA template for telomeric sequence synthesis as an
integral component of the enzyme. Telomeres
are hundreds of bp long, and the number of GGGGTT/CCCCAA repeats
varies from telomere to telomere. Tetrahymena
cells with certain mutant telomere sequences senesce and die. The very
high copy number of the rDNA telomere and
the intensive telomere synthesis occurring during MAC differentiation
made Tetrahymena a favorable system for the
discovery of the molecular basis of eukaryotic telomeres and of
telomerase, and for their continued investigation.
The regulation of telomere length has important implications for
carcinogenesis in mammalian cells [reviewed in
ref. 17].
A seemingly unrelated type of site-specific rearrangement involves
the deletion of internal DNA segments from
the MAC during its differentiation. These are known as MIC-limited
sequences or internally eliminated sequences
(IES) [reviewed in ref. 18]. There are roughly 6,000 different
deletion sites per haploid genome. At least a quarter
of them are estimated to have alternative deletion ends. The
alternative ends may be accurately specified or may
loosely fall within domains of the order of 100 bp. Some imprecision
may be tolerated in Tetrahymena because none
of the deletion systems so far characterized have ends within protein
coding sequences. The deletion size generally
ranges from a few hundred to a few thousand bp. In total, roughly 15%
of the MIC-derived DNA sequences are lost
during MAC differentiation. MIC-limited sequences are AT-rich DNA. So
far they have no known function and are proposed
to be remnants of ancient invasions and dispersions of
transposons.
Phenotypic assortment
When cells with a MAC initially heterozygous at a given locus
undergo asexual multiplication, subclones that
irreversibly express phenotypes associated with either homozygote are
generated. Because recessive phenotypes come
to expression in this way, the phenomenon was termed phenotypic
assortment. Starting with a heterozygous cell with
a mixed MAC, the steady state rate at which subclones pure for either
allele arise is 0.011/fission. Assortment
is attributed to the random distribution of allelic copies in a
compound MAC (Fig. 4; see also ref. 2). Mathematically,
the steady state rate of assortment of pure MACs from mixed MACs
approaches 1/2N-1 per fission for a large N, where
N is the number of copies just after MAC division (G1 stage). The
measured rate led to the first determination
of 45 as the average G1 ploidy of the MAC. This ploidy was
subsequently confirmed by molecular measurements of
MIC and MAC DNA amount and sequence complexity. When first described,
the assorting units were believed to be diploid
chromosome sets (subnuclei) showing allelic exclusion, i.e. with only
one allele of the pair being expressed in
each locus in each diploid set [see useful discussion in Chapter. 8 of
ref. 2]. With increased knowledge about
the molecular structure of the MAC, phenotypic assortment is now
attributed simply to the random distribution of
the acentromeric ARP copies at MAC division (Fig. 4). Phenotypic
assortment has some similarities to the segregation
of multicopy incompatible plasmids in bacterial clones.
Figure 4. Phenotypic assortment.
Only three of the 45 ARP copies are shown for simplicity. Circles:
G1 MACs; "peanut-shells": amitotically-dividing
MACs: each ARP copy has been replicated and its two copies have equal
probability of going to the same or to different
daughter MAC. Straight lines and solid loci symbols (diamonds): DNA
from one parental cell; wavy lines and open
symbols: DNA from the other parental cell. The diagram illustrates how
a cell with a mixed MAC generates, through
random distribution at successive MAC divisions, vegetative
descendants that are pure for one allele or the other
(phenotypic assortment). If one allele is dominant (e.g. solid
diamonds), the initial cell expresses the dominant
phenotype; later assortants pure for open diamonds, which express the
recessive phenotype, arise. Please note:
1) At the first MAC division, the two wavy copies could, with equal
probability, have been segregated to the two
daughter MACs. Thus the rate of assortment of pure MACs with 3 ARP
copies is 0.25 per fission. With 45 ARP copies,
the steady state rate assortment of pure MACs is merely slower --0.011 -->
--per fission. 2) While each ARP copy is shown
here to double exactly at each S period and the daughter MACs are
shown here to receive exactly the same number
of copies, such precision is probably rare in vivo in Tetrahymena MACs
with 45 copies at G1.
************************
Phenotypic assortment allows any recessive allele to come to full
expression. It also allows even a single mutant
allele, generated by mutation or by DNA-mediated transformation, to replace
completely
the 45 wild type alleles in the MAC. Independent assortment, in
combination with site-specific fragmentation, ensures
extensive hereditary phenotypic diversification of the members of a
vegetative clone, a situation commonly expected
for Tetrahymena in the wild given the multiple heterozygosis predicted
in an outbreeding species. Phenotypic assortment
thus provides a second, somatic, shorter-term level of natural
selection and adaptation for the species.
Coassortment
When doubly heterozygous cell lines are independently cultured
asexually for hundreds of fissions, they exhibit
virtually identical assortment pattern for certain pairwise
combinations of neighboring loci [19]. In other words,
starting with an AB/ab MAC, most of the vegetative descendants end up
pure for either the AB or the ab (parental)
combinations (Fig. 5). Less than 10% of the descendants become pure
for either of the recombinant types (Ab or
aB). This phenomenon is termed coassortment and allows the
identification of coassortment groups, i.e. groups of
loci such that its members all coassort with one another. A
coassortment group is the MAC analog of a MIC meiotic
linkage group. ARPs are the physical basis of coassortment groups;
i.e., loci carried on the same ARP coassort,
while loci carried on different ARPs assort
independently. Coassortment makes it possible to map loci to MAC ARPs
and determine ARP boundaries in the MAC purely by genetic
means. Finding the physical ARP that carries a conventional
locus can now be done indirectly by detecting its genetic coassortment
with a physically mapped DNA polymorphism.
These features may in the future facilitate the cloning of novel
mutant genes in Tetrahymena that can only be cloned
by complementation or chromosome walking.
Figure 5. Macronuclear coassortment of two loci in the course of
asexual multiplication.
A: Independent assortment of loci on two separate ARPs. B:
Coassortment of loci on same ARP. Circular figure:
MIC; "helmet"-shaped figure: MAC. Two different MAC ARPs
(long and short) and three different loci (circles,
triangles and diamonds) are shown. Only 8 of the 45 copies of each ARP
are shown. The short ARP is omitted in panel
B, as it is not needed for illustrating coassortment. Straight lines
and solid loci symbols: DNA from one parental
cell; wavy lines and open symbols: DNA from the other parental
cell. Both parental cells are double homozygotes.
P1 and P2: terminal assortants with parental genotypes; R1 and R2:
terminal assortants with recombinant genotypes.
Note that 1) Phenotypic assortment generated terminal assortants with
MACs that are pure at each locus. 2) Coassortment
between two loci is defined by a strong statistical excess of parental
over recombinant types, not seen for independently
assorting loci. 3) Loci on different MIC chromosomes (not shown here)
are expected always to assort independently.
4) There is no assortment in the mitotically-dividing MIC, which
remains doubly heterozygous.
************************
Mapping genetically and molecularly the MAC genome
Genetic mapping of the MAC on the basis of coassortment is now
underway. Currently identified coassortment
groups are listed in this web site. The most carefully mapped
coassortment groups so far represent contiguous
segments of MIC DNA, as expected from the simple model shown in
Fig. 3. A few cloned DNA segments have been physically
assigned to ARPs by hybridizing labeled probe to Southern blots of
whole cell DNA separated in agarose gels by
pulsed field electrophoresis [illustrated in ref. 19].
MIC-limited polymorphic DNA sequences are expected to show no
assortment during vegetative multiplication because
the signal originates in the MIC, which divides mitotically and
remains heterozygous. This lack of assortment has
been used to detect potential MIC-limited RAPD polymorphisms. Their
status has been confirmed by PCR or Southern
blot analysis: the signal disappears when whole cell DNA from a
nullisomic strain missing the chromosome that carries
the MIC-limited sequence is tested [unpublished observations, Orias
lab]. A list of MIC-limited
RAPDs and their MIC map location is given in this web site. Not
all apparent lack of assortment is due to MIC-limited
DNA. Exclusive assortment to a particular allele can result from
differential replication/maintenance of the ARP
copy carrying that allele [21]. For certain natural genetic
polymorphisms in surface antigen (serotype), extremely
biased initial amplification of one particular allele (in the absence
of differential vegetative replication) is
sufficient to explain the observed appearance of lack of assortment
[27]
Return to Table of
Contents <<<<<<000>>>>>>
Return to Main Page
Special topics of MAC
genetics
Sexual maturity
Sexual maturation is a stable, somatically inherited
differentiation of the MAC, remarkable in that it must
occur in response to some endogenous fission counting program, as
these are free-living unicells. The differentiation
of new MACs under conditions of physiological stress induces early
maturity.
Mating type differentiation
The MIC carries the potential for 5-7 mating types-depending on the
genotype at the mating type (mat) locus.
Nevertheless, a cell generally expresses only one mating type. This is
the result of a somatically inherited, irreversible,
stochastic event that occurs in the differentiating new
macronucleus. The frequencies with which various mating
types arise are not necessarily equal, and are affected by
environmental conditions prevailing at the time of MAC
differentiation, e.g. temperature and starvation [see ref. 20].
Roughly 50% of newly differentiated MACs normally are pure for
determinants for a single mating type. The rest
of the new MACs are mixed but, as a result of phenotypic assortment,
most MACs have become pure for a single mating
type determinant by the time sexual maturity is reached. Sexually
mature cells with MACs that remain mixed give
rise to clones within which pairing can occur; they are called
selfers. Continued asexual multiplication of selfers
generates subclones with pure MACs. The genetics of selfers was the
unusual context within which phenotypic assortment
was first described and analyzed in Tetrahymena. A model of mating
type determination based on an alternative deletion
system has been proposed for T. thermophila [20]. It explains
quantitatively the genetic phenomenology of mating
type determination and predicts the order of mating type genes within
the mat locus, but remains to be tested molecularly.
Genetics of the ribosomal RNA gene (rDNA)
Tetrahymena is an exceptional organism in having only a single copy
(per haploid genome) of the gene for the
45S ribosomal RNA precursor, which is processed posttranscriptionally
to the 18S and 28S rRNAs. During MAC differentiation,
this copy is excised by Cbs-directed fragmentation, turned into a 21
Kb inverted repeat (palindrome), supplied
with telomeres and amplified to the level of roughly 10,000 copies per
MAC [illustrated in ref. 21]. All but the
central 29 bp of the rDNA is palindromic. The two halves of the
palindrome are transcribed divergently. Each half
consists of a central transcribed region, flanked by the 5'- and the
3'-nontranscribed spacers (NTS). The 5'-NTS
contains the origin of rDNA replication. Because of its high copy
number, the rDNA telomere represents roughly
half of the telomeres in the MAC. Tetrahymena rDNA telomeres are built
into the vector used to clone large DNA
inserts in Saccharomyces as Yeast Artificial Chromosomes (YACs). The
28S rRNA coding sequence includes an intron;
understanding of its self-splicing nature led to the codiscovery of
ribozymes [22]. Paromomycin resistance is a
useful selectable trait, determined by a mutation located near the
3'-end of the 16S rRNA coding region. Most current
Tetrahymena transformation vectors incorporate the rDNA origin of
replication, which is required for their maintenance
in transformants.
Heterozygotes for Pmr assort to PM-S with kinetics that correspond
roughly to that of 45 randomly distributed
(G1) copies [28]. This number is much smaller than the total number of
rDNA copies (roughly 10,000) or the number
of nucleoli (roughly 500 in exponentially growing cells). Any strong,
systematic tendency for sister rDNA molecules
to go to the same daughter at MAC division would lower the apparent
number of assorting units. In addition, the
known differential growth of PM-S relative to PM-R cells would
spuriously lower the apparent number of assorting
units.
MAC DNA replication and copy number control
The rDNA is localized in nucleoli, of which there are about 500 in
exponentially growing cells [illustrated
in ref. 1, p. 5]. The origin of replication of the rDNA has been
mapped to the 5'-nontranscribed spacer. A natural
polymorphism and laboratory mutations that affect the relative
replicative maintenance of the rDNA during asexual
multiplication in heterozygotes have been described [reviewed in
ref. 21]. These studies reveal a coupling between
the initiation of rDNA replication and rRNA transcription. Inclusion
of the 5'-nontranscribed spacer of the rDNA
containing mutations that confer replicative advantage has proven to
be very useful for insuring the maintenance
of recombinant DNA constructs in high copy number in the MAC after
introduction by transformation. Fortunately,
the maintenance in high copy number of a plasmid carrying the rDNA
replication origin is compatible with the maintenance
of the endogenous rDNA also in high copy number.
Several observations imply the existence of mechanisms that
regulate ARP copy number in the bulk of the MAC
DNA. 1) The Tetrahymena MAC has no visible machinery to ensure the
regular distribution of daughter copies of DNA
molecules and the genetic evidence indicates that distribution of
allele copies is random. 2) The distribution
of total bulk DNA to daughter MACs is seldom equal. This imprecision
predicts descendants with lethal DNA sequence
deficiencies and imbalances in higher frequency than is actually
observed. 3) Cells that start out with haploid,
triploid or extensively aneuploid MACs generate descendants with the
normal amount of MAC DNA. Two levels of regulation
must be envisioned: a global mechanism that maintains every ARP (other
than the rDNA) at an average of 45 copies,
and a specific mechanism that corrects copy number deviations of each
individual ARP independently of the deviations
occurring at other ARPs. Unusual cell cycles (with reference to the
MAC) are triggered in cells with a total G1
amount of MAC DNA outside a predetermined range. When the upper
threshold is exceeded, the cell divides without
MAC DNA replication; when the G1 amount is below the lower threshold,
two rounds of MAC DNA replication occur before
cell division. Virtually everything remains to be learned about the
mechanisms that control replication and copy
number in the Tetrahymena MAC.
Return to Table of
Contents <<<<<<000>>>>>>
Return to Main Page
Other non-Mendelian
inheritance
Mitochondrial (Cytoplasmic) Genetics
Tetrahymena cells contain 600-800 mitochondria [see ref. 1,
p. 75]. Their DNA is a linear DNA molecule, an uncommon
occurrence among eukaryotic mitochondria. Its telomeres consist of a
variable number of repeats of a 53-bp unique
sequence. Chloramphenicol resistance, determined by a mutation
virtually certain to reside in the mitochondria,
shows cytoplasmic inheritance [see ref. 7]: i.e., the exconjugant
clone derived from a resistant conjugant remains
resistant, while the exconjugant clone derived from the sensitive
parent remains sensitive. The genetic evidence
suggests that in Tetrahymena the exchange of mitochondria across the
conjugal junction occurs rarely, if ever.
10 Kb Plasmid
A linear 10 Kb plasmid (pTtL10), present in roughly 20,000 copies
per cell, has been described in certain wild
isolates of T. thermophila [23]. Its ends are terminal inverted
repeats of a 47 bp unique sequence. This organization-but
not the repeat sequence itself-is reminiscent of the mitochondrial DNA
telomeres. Its 5' (but not 3') ends are
resistant to exonuclease digestion. This plasmid is suspected of
having protein-primed (rather than RNA-primed)
DNA replication, such as found in adenovirus, for example. When
plasmid-free cells conjugate with plasmid-containing
cells, the plasmid is transferred to the plasmid-free conjugant in
some but not all the pairs. No plasmid transfer
has been detected in mixed vegetative culture. The cellular location
of the plasmid DNA may well be mitochondrial.
It has been proposed that in the course of evolution the Tetrahymena
mitochondrial DNA became linear as a consequence
of recombination with a pTtL10-type plasmid.
Cortical Inheritance
The somatic ciliature in Tetrahymena is distributed throughout the
cell and organized in a regular array of
rows (or meridians) of ciliary units in the cell cortex [illustrated
in ref. 2, p.180]. Each ciliary unit carries
one cilium and associated cytoskeletal structures, including several
microtubule arrays. The ciliary unit has anterior-posterior
and left-right asymmetry. Variant cells having ciliary rows with
reversed polarity can be generated in the absence
of genic mutations; the reversed polarity is hereditarily transmitted
at cell division. The (nongenic) basis of
this form of inheritance (cortical inheritance or cytotaxis) is the
highly structured addition of new ciliary units
that compensates for their dilution at cell division. Overall
orientation of a ciliary row is perpetuated through
cell division because a) a new ciliary unit always differentiates
anteriorly (i.e. along the meridian) and in tandem
orientation with regard to the local organization of a preexisting
ciliary unit and b) fission occurs in the equatorial
plane [see useful in Chapter. 11 of ref. 2].
Patterns of inheritance with uncertain basis
An unusual pattern of inheritance was observed when analyzing
certain mutagenized progeny selected for the absence
of expression of the immobilization (cell surface) antigen specified
by the serH locus[29]. By analogy to well
established genetic observations in Paramecium [30], it has been
proposed that in these variants the altered cytoplasm
in which a new MAC differentiates can hereditarily determine the state
of expression of the serH locus in the asexual
clone that carries descendants of that MAC. It has further been
speculated that this phenomenon is mediated at
the level of DNA rearrangements in the differentiating MAC. These
experiments need to be repeated under conditions
where independent genetic markers (now available) provide a rigorous
check against unexpected cytogenetic accidents
that could distort interpretations.
A remarkable phenomenon has been recently reported in Tetrahymena
[31] which may provide a basis for the above
observations. Cell lines can be obtained in which a certain
MIC-limited sequence fails to be removed from the MAC.
When a new MAC differentiates in the presence of such an old MAC, this
condition is perpetuated, i.e. the normally
MIC limited sequence is retained in the newly differentiated MAC. The
basis for this observation remains to be
elucidated, but the phenomenon appears at least superficially related
to analogous observations made in species
of the Paramecium aurelia group of ciliates [32].
On the evolution of nuclear
dimorphism
The nuclear dimorphism of the ciliates is a remarkable
phenomenon. It staggers the imagination to consider how
amitosis of an acentromeric fragmented somatic genome could have
evolved from mitosis through a series of functional
intermediates. The following steps have been proposed for the
evolution of the MAC. The first major step would
have occurred in distant eukaryotic ancestors having an alternation of
haploid and diploid phases in their life
cycle. A differentiated, non-dividing somatic MAC could then have
evolved in the context of a diplophase consisting
exclusively of a non-dividing, unicellular trophic (feeding) stage
[24]. This would have previously required the
evolution of mechanisms that inhibit diplophase cell division and
could well have been accompanied by the loss
of mitotic mechanisms and components in the diplophase, in the absence
of selection against such unnecessary mechanisms.
Such a life cycle and type of nuclear dimorphism is found today among
certain living Foraminifera [25], a group
of shelled, marine unicellular eukaryotes.
The proposed second major step is the reacquisition of cell
division in the diplophase-through events akin to
cancer-causing mutations in multicellular organisms-and the gradual
shortening of the haplophase, giving rise to
Karyorelict ciliate type of nuclear dimorphism [24]. These ciliates
have MACs that do not divide. The loss of the
MAC during cell multiplication is averted by the fact that at every
vegetative cell cycle the MIC divides twice;
half of the products differentiate into MACs and are segregated to the
two daughter cells (much like in Tetrahymena's
first cell cycle after zygote formation; step 7-8 in Fig. 2). The
maintenance of a non-dividing type of nucleus
in a dividing cell is an unprecedented feature of Karyorelict
ciliates, readily rationalized by this proposed evolutionary
history. Absent nuclear division, chromosome fragmentation could have
evolved at this or the previous stage without
any risk of chromosomal imbalance caused by the lack of centromeres on
the fragments. Ploidy increase may have
happened at this stage to support the evolutionary increase in size of
the cells.
The last major step would be the reacquisition of division in MACs
with relatively high ploidy as a superior
alternative to MAC differentiation at every cell cycle [26]. Absent
mitotic mechanisms and functional centromeres
in the MAC, the early evolutionary intermediates would have found
irregular, serendipitous ways to distribute daughter
DNA copies at MAC division. The possession of high ploidy in the MAC
would have lessened the risks of chromosomal
loss or serious imbalance associated with unequal distributions. The
validity of these evolutionary speculations
remains to be determined by future research.
Return to Table of
Contents <<<<<<000>>>>>>
Return to Main Page
Other background
Randomly Amplified Polymorphic DNA (RAPD)
The RAPD method (randomly-amplified polymorphic DNA) is an
efficient method for detecting DNA polymorphisms
[33]. This method uses 10-mers of arbitrary sequence as primers for
PCR amplification. Up to roughly a dozen discrete
ethidium bromide- stained bands are revealed with each primer. The
pattern is specific for each primer, and is
sensitive to single base changes in the primer; thus each band checks
20 base pairs for single (or more) base polymorphisms.
By screening two different wild type or lab inbred strains the method
uncovers in effect a practically inexhaustible
source of "instant mutations".
The RAPD method appears to provide an efficient, general,
relatively inexpensive and safe method to obtain genetic
maps of complex genomes. RAPD polymorphisms have the additional
advantage that the polymorphic DNA is molecularly
cloned by the very first act of polymorphism detection. Minute amounts
of template DNA are required (25 ng per
reaction). Anyone can reproduce the polymorphism and generate a sample
of the polymorphic DNA segment simply by
using the corresponding 10-mers, which are commercially available at
an inexpensive price. A list of the RAPD
PCR primers used in mapping the Tetrahymena genome is available.
Special features of Tetrahymena genetics, uniquely related to their
nuclear dimorphism, have further enhanced
the power of RAPD mapping [13,34].
1) Genomic exclusion allows the isolation of meiotic segregants as
"instant" whole-genome homozygotes
from independent, single meiotic products of a multiple-heterozygous
F1. This represents a far greater degree of
homozygosity than can be accomplished by ordinary selfing of an F1
individual. The direct generation of whole-genome
homozygotes is extremely valuable in view of the "dominance"
of the band+ RAPD phenotype, i.e. the inability
of the RAPD method to distinguish between band+/band- heterozygotes
and band+ homozygotes.
2) The availability of nullisomic strains has allowed us to assign
linkage groups to particular MIC chromosome
arms, to target RAPD screens to particular chromosome segments and to
identify MIC-limited RAPDs.
3) The ability to map RAPDs to MAC ARPs by coassortment provide an
additional, experimentally independent check
on the maps generated by meiotic linkage.
Return to Table of
Contents <<<<<<000>>>>>>
Return to Main Page
References
1. Hill,DL (1972): The Biochemistry and Physiology of
Tetrahymena. Academic Press, New York & London. 230
pages.
2. Nanney,DL (1980): Experimental Ciliatology: An introduction to
genetic and developmental analysis in Ciliates.
John Wiley & Sons, New York, NY.
3. Nanney,DL; McCoy,JW (1976): Characterization of the species of
the Tetrahymena pyriformis complex. Trans.
Amer. Microsc. Soc. 95, 664-682.
4. Prescott,DM (1994): The DNA of ciliated
protozoa. Microbiol. Rev. 58, 233-267.
5. Elliott,AM (1973): Biology of Tetrahymena. Dowden, Hutchinson
and Ross, Stroudsburg, PA. 508 pages.
6. Gall,JG (1986): Molecular Biology of the Ciliated
Protozoa. Academic Press, New York, NY.
7. Bleyman,LK (1996): Ciliate genetics. In: Ciliates. Cells as
Organisms. (Eds: Hausmann,K; Bradbury,PC) Gustav
Fisher Verlag, Stuttgart - Jena - New York, 291-324.
8. Rogers,MB; Karrer,KM (1985): Adolescence in
T. thermophila. Proc. Nat. Acad. Sci. USA 82, 436-439.
9. Doerder,FP; Gates,MA; Eberhardt,FP; Arslanyolu,M (1995): High
frequency of sex and equal frequencies of mating
types in natural populations of the ciliate Tetrahymena
thermophila. Proc. Natl. Acad. Sci. U. S. A. 92, 8715-8718.
10. Orias,E (1986): Ciliate Conjugation. In: Molecular Biology of
the Ciliated Protozoa. (Ed: Gall,JG) Academic
Press, New York, NY, 45-84.
11. Bruns,PJ (1986): Genetic organization of Tetrahymena. In: The
Molecular Biology of Ciliated Protozoa. (Ed:
Gall,JG) Academic Press, New York, NY, 27-44.
12. Cole,ES; Bruns,PJ (1992): Uniparental cytogamy: a novel method
for bringing micronuclear mutations of Tetrahymena
into homozygous macronuclear expression with precocious sexual
maturity. Genetics 132, 1017-1031.
13. Brickner,JH; Lynch,TJ; Zeilinger,D; Orias,E (1996):
Identification, mapping and linkage analysis of randomly
amplified DNA polymorphisms in Tetrahymena thermophila. Genetics 141,
1315-1325.
14. Bruns,PJ; Cassidy-Hanley,D (1993): Tetrahymena thermophila. In:
Genetic Maps: Locus Maps of Complex Genomes,
6th ed. (Ed: O'Brien,SJ) Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, 2175-2179.
15. Griffiths,AJF; Miller,JH; Suzuki,DT; Lewontin,RC; Gelbart,WM
(1996): An Introduction to Genetic Analysis.
6th ed. W.H.Freeman and Company, New York. 915 pages.
16. Reeve,ECR (1998): Encyclopedia of Genetics. Fitzroy Dearborn
Publishers, London. 000 pages.
17. Greider,CW; Blackburn,EH (1996): Telomeres, telomerase and
cancer. Sci. Am. 274, 92-97.
18. Yao,M-C (1996): Programmed DNA deletions in Tetrahymena:
Mechanisms and implications. Trends in Genetics
12, 26-30.
19. Longcor,MA; Wickert,SA; Chau,M-F; Orias,E (1996): Coassortment
of genetic loci during macronuclear division
in Tetrahymena thermophila. Eur. J. Protistol. 32, 85-89.
20. Orias,E (1981): Probable somatic DNA rearrangements in mating
type determination in Tetrahymena thermophila:
A review and a model. Devel. Genet. 2, 185-202.
21. Kapler,GM (1993): Developmentally regulated processing and
replication of the Tetrahymena rDNA minichromosome.
Curr. Opin. Genet. Dev. 3, 730-735.
22. Kruger,K; Grabowski,PJ; Zaug,AJ; Sands,J; Gottschling,DE;
Cech,TR (1982): Self-splicing RNA: Autoexcision
and autocyclization of the ribosomal RNA intervening sequence in
Tetrahymena. Cell 31, 147-157.
23. Ortiz,JM (1993): Characterization of an Extrachromosomal Linear
DNA molecule discovered in Tetrahymena.
Ph.D. Dissertation, University of California at Santa Barbara, Santa
Barbara, CA. 101 p.
24. Orias,E (1991): On the evolution of the karyorelict ciliate
life cycle: heterophasic ciliates and the origin
of ciliate binary fission. BioSystems 25, 67-73.
25. Grell,KG (1979): Cytogenetic systems and evolution in
Foraminifera. J. Foraminif. Res. 9, 1-13.
26. Orias,E (1991): Evolution of amitosis of the ciliate
macronucleus: Gain of the capacity to divide. J. Protozool.
38, 217-221.
27. Nanney,DL; Preparata,RM (1979): Genetic evidence concerning the
structure of the Tetrahymena thermophila
macronucleus. J. Protozool. 26, 2-9.
28. Merriam,EV; Bruns,PJ (1988): Phenotypic assortment in
Tetrahymena thermophila: assortment kinetics of antibiotic-resistance
markers, tsA, death, and the highly amplified rDNA locus. Genetics
120, 389-395.
29. Doerder,FP; Berkowitz,MS (1987): Nucleo-cytoplasmic interaction
during macronuclear differentiation in ciliate
protists: genetic basis for cytoplasmic control of SerH expression
during macronuclear development in Tetrahymena
thermophila. Genetics 117, 13-23.
30. Sonneborn,TM (1977): Genetics of cellular differentiation:
stable nuclear differentiation in eucaryotic
unicells. Ann. Rev. Genet. 11, 349-367.
31. Chalker,DL; Yao,MC (1996): Non-Mendelian, heritable blocks to
DNA rearrangement are induced by loading the
somatic nucleus of Tetrahymena thermophila with germ line-limited
DNA. Mol. Cell. Biol. 16, 3658-3667.
32. Meyer,E; Duharcourt,S (1996): Epigenetic regulation of
programmed genomic rearrangements in Paramecium aurelia.
J. Eukaryot. Microbiol. 43, 453-461.
33. Williams,JGK; Kubelik,AR; Livak,KJ; Rafalski,JA; Tingey,SV
(1990): DNA polymorphisms amplified by arbitrary
primers are useful genetic markers. Nucl. Acids Res. 18,
6531-6535.
34. Lynch,TJ; Brickner,JH; Nakano,KJ; Orias,E (1995): Genetic map
of randomly amplified DNA polymorphisms closely
linked to the mating type locus of {ITetrahymena
thermophila}. Genetics 141, 1315-1325.
Return to Table of
Contents <<<<<<000>>>>>>
Return to Main Page
