Progress in Mapping the Tetrahymena Micro- and Macronuclear Genomes
Eduardo Orias and Eileen P. Hamilton
Department of Molecular, Cellular and Developmental Biology, University of California
at Santa Barbara, Santa Barbara, CA 93106, USA
Tetrahymena thermophila is a unicellular eukaryote that belongs to the Ciliated Protozoa. Tetrahymena is a very successful genus; almost any freshwater sample in nature contains cells of some species of this genus. Tetrahymena has a number of useful features (reviewed by Orias, 1996) that make it very useful as a model system for research in cellular and molecular biology, as a toxicity test system and potentially for biotech applications. Tetrahymena is one of the most distantly related systems where one can combine the advantages of conventional genetics and molecular biology.
Recently we initiated a project to map the genome of Tetrahymena thermophila. Mapping the genome is useful for a variety of purposes: to investigate a variety of genetic phenomena, to verify the genetic composition of genetic constructs, to sort out a mutant collection, to check for authenticity when cloning suppressor genes, to check for correct insertion when making gene replacements or knockouts, and to help positional cloning of genes with novel functions identified mutationally. I will present here a brief summary of our progress. I will also discuss the phenomenon of macronuclear coassortment and present molecular genetic evidence that allows us to conclude that macronuclear autonomously replicating DNA pieces are the physical basis for coassortment groups.
Nuclear dimorphism and conjugation.
As a typical ciliate, Tetrahymena possess nuclear dimorphism, a rare condition in which the genetic functions of the nucleus have been separately assigned to two types of nucleus: the micronucleus (MIC) and the macronucleus (MAC). The micronucleus is the germline, i.e., the reservoir of genetic information for the sexual progeny. It is diploid and contains 5 pairs of chromosomes, numbered 1 through 5. No gene is known to be expressed in the MIC. The MIC divides mitotically at every binary fission. In contrast, the MAC is the somatic nucleus, i.e. the nucleus where active gene expression occurs. Thus, the MAC determines the phenotype of the cell. The bulk of the MAC DNA is 45-ploid. During MAC differentiation, its DNA undergoes a site specific fragmentation that generates 200-300 autonomously-replicating molecular species of subchromosomal size. The MAC divides "amitotically", i.e., it lacks mitotic spindle apparatus and kinetochores. These unusual features of the MAC, which we have exploited for mapping the MAC, will be discussed in more detail later.
To understand the genetic and developmental relationship between the MIC and MAC of a Tetrahymena cell it is important to review the nuclear events associated with conjugation, the sexual stage of the life cycle. These events are summarized briefly here; they are described in more detail by Orias (1986,1996). In order to pair (conjugate) cells must be starved for at least one required nutrient, must have different mating type and must be sexually mature. When the cells pair, the MIC in each cell first undergoes meiosis, generating 4 haploid products. It is at this stage that the meiotic recombination, exploited for making the linkage maps described below, occurs. Only one of the meiotic products remains functional while the other three are destroyed. The surviving meiotic product in each conjugant undergoes one mitotic division, generating a migratory and a stationary pronucleus, genetically identical to one another. The conjugants reciprocally exchange migratory pronuclei, which fuse to the resident stationary pronuclei and generate a diploid zygote nucleus in each cell. The rest of the conjugation events involve the differentiation of new MICs and MACs, and the destruction of the old MAC. The diploid zygote nucleus of each conjugant divides mitotically twice and generates four identical products, two at each end of the cell. The anterior pair differentiates into MACs while the posterior pair remains diploid and become MICs. MAC differentiation is accompanied by several types of DNA rearrangements that will be further described later. After the first cell division of each exconjugant, the normal complement of one new MIC and one new MAC is achieved. During subsequent asexual multiplication, MAC and MIC divide at each binary fission.
Mapping the germline (micronucleus).
We are mapping the genome using naturally-occurring DNA polymorphisms between inbred strains B and C3, detected by the Randomly Amplified Polymorphic DNA (RAPD) method (Williams et al., 1990). This method involves PCR amplification using short (10-mer) DNA primers of arbitrary sequence that bind at random places on the genome. When primers bind to genomic DNA in opposite orientation and within a few Kb of one another they generate DNA bands that can be separated by gel electrophoresis. By running amplification products derived from B and C3 DNA in parallel gel lanes, polymorphisms are detected when a particular band present in one lane is missing in the other. The use of this method for detecting RAPD loci in Tetrahymena is described in (Lynch et al., 1995; Brickner et al., 1996). RAPDs are next assigned to chromosome arms by using a panel of monosomic strains; these strains were obtained by crossing B nullisomics with a C3 diploid. They have a complete C3 genome but lack a particular combination of B chromosome or chromosome arms (see Brickner et al., 1996, for a more detailed description). To determine linkage and map linkage groups, we have used a panel of meiotic segregants. The members of this panel are whole-genome homozygotes derived from independent haploid meiotic products of a B/C3 heterozygote (Lynch et al., 1996). Linkage between pair of loci is inferred when the frequency of recombinants is statistically significantly lower than the frequency of parental types. Genetic distance between linked loci in cM is given by the frequency of recombinants, corrected for expected but unobserved multiple crossovers (Brickner et al., 1996). We have used the MAPMAKER program (Lander et al., 1987) to test new RAPDs for linkage to all previously analyzed loci, and to determine statistical significance of linkage and of map orders by means of log odds (LOD) scores.
The current status of the MIC mapping is summarized by Orias (1996). Nearly 400 RAPDs have been mapped to 25 linkage groups distributed among all the chromosomes and chromosome arms that our nullisomics allow us to distinguish (Table 1). The linkage maps includes a number of classical Tetrahymena loci as well. One potential centromere region has been identified in chromosome 4 by the criterion that a locus assigned to chromosome 4R by monosomic mapping shows linkage to loci assigned to 4L by monosomic mapping.. The probability that a new RAPD is linked to a previously mapped RAPD is now 95%. 4,300 cM have been linked so far. When this is compared to the roughly 220 Mb genome size, one obtains a maximum recombination rate of 51 Kb/cM, a very high rate of recombination among eukaryotes. This means that in comparison to most other eukaryotes, two loci have to be physically much closer in Tetrahymena to show linkage. This high rate of recombination explains why efforts to detect linkage using a limited number of conventional loci yielded very few examples of linkage in Tetrahymena.
DNA rearrangements during MAC differentiation.
Before describing our progress in mapping the macronuclear genome, it is important to understand the molecular phenomena associated with MAC differentiation. Several types of developmentally programmed, site-specific DNA rearrangements of the MIC form of the DNA occur during MAC differentiation, reviewed by Yao (1989). 1) The chromosomes are cut at the Cbs sequence, a 15 bp double stranded DNA segment that is necessary and sufficient for chromosome fragmentation during MAC differentiation. This process generates 200-300 autonomously replicating pieces (ARPs), also referred to as minichromosomes. The size of these pieces ranges from 21 Kb to a few Mb, with an average of roughly 700 Kb (Fig. 1). 2) Telomeres, consisting of GGGGTT repeats at the 3'end, are added de novo to each of these pieces. 3) Internal deletions lead to the loss of DNA segments, generally a few hundred to a few thousand base pairs. An estimated 6,000 deletion events occur per haploid genome in the differentiating MAC, leading to the loss of about 15% of the genome derived from the germline. 4) the bulk of the DNA pieces is amplified to an average level of 45 copies per MAC. The only known exception is the 21 Kb piece that carries an inverted repeat of the genes for the 18S and 28S rRNA, amplified to the level of roughly 10,000 copies per MAC.
Phenotypic assortment.
The MAC has no detectable machinery that would distribute daughter DNA copies regularly at division. Instead, daughter DNA copies are distributed randomly at MAC division. In a heterozygous MAC this random distribution produces a drift in the frequency of the two alleles in the MAC of the descendants. Occasionally, by chance alone, a descendant MAC is generated that has only a single type of allele. This phenomenon is called phenotypic assortment because it allows recessive or codominant alleles to be expressed exclusively. A cell with a pure MAC "breeds true" in the asexual descendants. The end result is that as a function of the number of fissions in a clone of a heterozygous cell, the fraction of cells with a MAC pure for either allele constantly increases, while that of cells with mixed MACs constantly decreases. By the time a heterozygous cell has undergone 500 fissions, the probability that a descendant will have a pure MAC is greater than 99%. We call such cells terminal assortants. For a more detailed treatment of assortment, see Orias (1996).
We expected that the RAPDs whose MIC genetics we had characterized should show assortment in the MAC. To this end, we obtained a panel of 36 terminal assortants derived from B/C3 heterozygotes and tested each member of the panel for assortment with a variety of RAPDs. We found that indeed RAPDs assort as expected (Fig. 2). This is the first demonstration of assortment of a DNA polymorphism and shows that phenotypic assortment is due to the physical loss of one allele from the MAC and its replacement by the alternative allele (Longcor et al., 1996).
Coassortment groups.
It has long been speculated that in double heterozygotes one might detect the linked assortment (coassortment) of parental alleles. With knowledge of the chromosome fragmentation in the MAC, we postulated that if two loci were carried on the same MAC piece they might show coassortment. That is, staring with a AB/ab heterozygotes (where A and B are loci carried on the same MAC piece), terminal assortants that have assorted for the A allele might more often than not, if not always, have also assorted for the B allele. If so, recombinant MACs, pure for Ab or aB, would in be the minority (see Fig. 2). We indeed found the first examples of coassortment in Tetrahymena (Longcor et al., 1996). Two loci that coassort with one another define a coassortment group. We have found additional examples of coassortment groups in all five chromosomes, confirming the expected generality of the phenomenon (see Fig. 3). The coassortment groups best investigated so far contain loci that map to continuous MIC chromosome segments. Less than 10% recombinants have been observed among the panel of 36 terminal assortants among the coassortment groups we have identified. This is true even for a coassortment group in which the outer loci are 40 cM apart in the MIC. This low frequency of MAC recombinants provides a useful research tool because it complements the high frequency of meiotic recombination in the MIC.
We have shown by molecular methods that two coassorting loci, 1PM8 and 1KF2, closely linked in the MIC, were carried on the same DNA MAC piece, while a third closely linked locus, 1KN3, which assorts independently of both, is carried on a separate MAC DNA piece (Longcor et al., 1996). This evidence is consistent with the hypothesis that the DNA pieces are the physical basis of coassortment groups. In order to obtain more rigorous evidence for this hypothesis, we have exploited a size polymorphism of the 1PM8 ARP in inbred strains B and C3 (Wong, Shapiro, Orias and Hamilton, unpublished results). We detected assortment of the ARP size polymorphism in our standard panel of 36 terminal assortants, indicating that the polymorphism behaves as an allelic pair in the MAC. Furthermore, the ARP size polymorphism coassorts with the 1PM8 and 1KF2 RAPDs. This finding provides additional molecular genetic evidence that ARPs are the physical basis of coassortment groups, and provides a basis for mapping MAC ARPs purely by genetic means. Such mapping is now underway in our laboratory. The objective is to systematically delineate coassortment groups and thereby determine which MIC loci are carried in each ARP.
A coassortment map of the MAC may be a very useful tool for studying Tetrahymena genes detected by mutational approaches whose functions are so novel that no molecular probes are yet available for cloning them. The low frequency of MAC recombinants allows the detection of coassortment between a novel mutant gene and a DNA polymorphism. By probing with the polymorphic DNA one can then indirectly identify the size of the physical ARP that carries the mutant gene. This in turn should lower the number of Tetrahymena transformants needed to clone the gene by complementation. Alternatively, identification of the ARP should shorten the distance of a chromosome walk to a mutant gene of interest by making it a "within-ARP" walk. Coassortment may well be useful also for checking correct gene replacements or knockouts in the MAC.
Acknowledgments
E.O. thanks the Japanese Society of Protozoology and Prof. Hiroshi Hosoya for inviting him to present this work and for their gracious hospitality. We thank the following collaborators who contributed to our mapping effort in the past year: Virginia Merriam, Loyola Marymount University, Los Angeles; Sally L. Allen, University of Michigan; Mihoko Takahashi, University of Tsukuba, Japan; Judy Orias, Steve Wickert, Bruce Bauer, Fernando Bautista, Ceri Van Slike, Gerlinde Chan, Robert Elliott, Yvonne Hughes, Meade Johnson, Tim Joiner, Megan Longcor, Erin McCaskill, Gina Mecagni, Christie Miller, Leslie Nangle, Shauna Paxson, Francisco Rosas, Lori Siu, Lana Shapiro, Diana Thorpe, Jorge Torres, and Laura Wong at UC Santa Barbara. We thank the USA National Institutes of Health for support of this project (grant RR09231 to E.O.).
REFERENCES
Brickner, J. H., Lynch, T. J., Zeilinger, D. and Orias, E. (1996). Identification, mapping and linkage analysis of randomly amplified DNA polymorphisms in Tetrahymena thermophila. Genetics 141, 1315-1325.
Lander, E. S., Green, P., Abrahamson, J., Barlow, A., Daly, M. J., Lincoln, S. E. and Newburg, L. (1987). MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1, 174-181.
Longcor, M. A., Wickert, S. A., Chau, M.-F. and Orias, E. (1996). Coassortment of genetic loci during macronuclear division in Tetrahymena thermophila. Eur. J. Protistol. 32, Suppl. 1, 85-89.
Lynch, T. J., Brickner, J. H., Nakano, K. J. and Orias, E. (1995). Genetic map of randomly amplified DNA polymorphisms closely linked to the mating type locus of Tetrahymena thermophila. Genetics 141, 1315-1325.
Orias, E. (1986). Ciliate Conjugation. In: Molecular Biology of the Ciliated Protozoa. (Ed: Gall, J. G.) Academic Press, New York, NY, 45-84.
Orias, E. (1996). Tetrahymena Genome Project Web Site. http://lifesci.ucsb.edu/~genome/Tetrahymena, Santa Barbara, CA.
Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V. (1990). DNA polymorphisms amplified by arbitrary primers are useful genetic markers. Nucl. Acids Res. 18, 6531-6535.
Yao, M.-C. (1989). Site-specific chromosome breakage and DNA deletion in ciliates. In: Mobile
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Table 1. Distribution of linkage groups among chromosome arms
1L CEN 2R 2L CEN 1R --------------0-------------- --------------0-------------- 1L-MJ10 2R-KP02R 2L-BB01R 1R-AT01 1L-SP13* 2R-JO66 2L-mat 1R-ChxA
3L CEN 3R 4L CEN 4R --------------0-------------- --------------0-------------- 3L-JB36 3R-AcpA 4L-AS01 4L-BR19 4R-LC01 3L-JO25 3R-JO06 4L-JB06 3L-MJ18R 3R-LC08 4L-QD02 3L-XS43 4L-QD02R
5 X
----------------------------- ---------
5-JB04 X-KP04R
5-JP02
5-LC16a
L and R: Left and right chromosome arms. CEN: centromere.
Linkage groups cannot be assigned to chromosome 5 arms because the required nullisomic is not
available. Linkage group X has not yet been assigned a chromosome for technical reasons. The
1L.2R and 2L.1R chromosome arm associations shown here reflect the hypothesis of Bruns,
Hanley-Cassidy & Merriam (personal communication) regarding wild type T. thermophila strains.
* Linkage group 1L-SP13 was originally assigned to chromosome 1L but may be in 2R (currently
under investigation in our laboratory).
FIGURE LEGENDS AND FIGURES
Fig 1. Pulse field blot of whole-cell DNA of Tetrahymena thermophila, inbred strain B. The outside lanes are Saccharomyces cerevisiae chromosomes used as size markers. The lowest band is the 21 Kb rDNA piece. The brightest band at the bottom is the mitochondrial DNA. The rest of the bands are bulk MAC DNA ARPs present at 45 copies per MAC. Brighter bands most likely represent coincidences of unrelated ARPs of the same size. MIC DNA stays in the wells under these conditions.
Fig. 2. Expectations for independent assortment and coassortment of two loci in the Tetrahymena macronucleus. 1. MAC genotype in a young double heterozygote; 2 and 3: terminal assortants of parental MAC genotype; 4 and 5: terminal assortants of recombinant MAC genotype. Only five out of the 45 copies of each ARP are shown for simplicity. A. Independent assortment of two loci carried on different MAC ARPs. No statistical excess of parental assortants is expected. B. Coassortment of two loci carried on the same MAC ARP. A statistical excess of parental assortants is expected.
Fig. 3. Phenotypic assortment and coassortment demonstrated at the DNA level. Lane M: 1Kb
ladder (BRL) size markers. Visible markers: 1.6 and 1.0 Kb. Lanes 1-16: Amplification products
using whole-cell DNA from the following strains: B homozygotes, C3 homozygotes, young B/C3
heterozygotes, 13 terminal assortants from B/C3 heterozygotes, respectively. The letters between
the panels represent the allele, B or C3 (represented by C), present in each terminal assortant at
each locus. A. Assortment of RAPD 1BR2, located in linkage group 5-JB4. This RAPD behaves
as a codominant length polymorphism: the DNA segment bracketed by the primer binding sites is
shorter in inbred strain B (~0.9 Kb) than in C3 (~1.1 Kb). Note that the young B/C3
heterozygotes show both bands but every terminal assortant shows only one. (The faint ~1.1 Kb
band seen in B homozygotes and some terminal assortants is not polymorphic.) B. Assortment of
two unrelated RAPDs, LS15 (bright band at ~1.1 Kb, located in linkage group 2L-mat on
chromosome 2L) and LS15R (faint band at ~1.3Kb, located in linkage group 5-JB4 on
chromosome 5). Both of these are typical dominant RAPDs. Note that B/C3 heterozygotes show
the polymorphic bands, but some of the terminal assortants have lost it. Note also that 1LS15R and
1BR2 (which are 3 cM apart in the MIC) coassort with one another: only one recombinant was
found among the total of 32 terminal assortants tested (lane 13, B allele at 1BR2 and C3 allele at
1LS15R). The two RAPDs are thus in the same coassortment group. RAPD 1LS15 assorts
independently of 1LS15R and 1BR2, as expected given its location on a different MIC
chromosome: twelve recombinants were found among 32 terminal assortants tested.
FIGURE 1
FIGURE 2
FIGURE 3
