Biomagnification of cadmium selenide quantum dots in a simple experimental microbial food chain.

Previous studies have shown that engineered nanomaterials can be transferred from prey to predator, but the ecological impacts of this are mostly unknown. In particular, it is not known if these materials can be biomagnified-a process in which higher concentrations of materials accumulate in organisms higher up in the food chain. Here, we show that bare CdSe quantum dots that have accumulated in Pseudomonas aeruginosa bacteria can be transferred to and biomagnified in the Tetrahymena thermophila protozoa that prey on the bacteria. Cadmium concentrations in the protozoa predator were approximately five times higher than their bacterial prey. Quantum-dot-treated bacteria were differentially toxic to the protozoa, in that they inhibited their own digestion in the protozoan food vacuoles. Because the protozoa did not lyse, largely intact quantum dots remain available to higher trophic levels. The observed biomagnification from bacterial prey is significant because bacteria are at the base of environmental food webs. Our findings illustrate the potential for biomagnification as an ecological impact of nanomaterials.

Autonomously replicating macronuclear DNA pieces are the physical basis of genetic coassortment groups in Tetrahymena thermophila.

The macronucleus of the ciliate Tetrahymena thermophila contains a fragmented somatic genome consisting of several hundred identifiable chromosome pieces. These pieces are generated by site-specific fragmentation of the germline chromosomes and most of them are represented at an average of 45 copies per macronucleus. In the course of successive divisions of an initially heterozygous macronucleus, the random distribution of alleles of loci carried on these copies eventually generates macronuclei that are pure for one allele or the other. This phenomenon is called phenotypic assortment. We have previously reported the existence of loci that assort together (coassort) and hypothesized that these loci reside on the same macronuclear piece. The work reported here provides new, rigorous genetic support for the hypothesis that macronuclear autonomously replicating chromosome pieces are the physical basis of coassortment groups. Thus, coassortment allows the mapping of the somatic genome by purely genetic means. The data also strongly suggest that the random distribution of alleles in the Tetrahymena macronucleus is due to the random distribution of the MAC chromosome pieces that carry them.

Tetrahymena micronuclear genome mapping. a high-resolution meiotic map of chromosome 1l.

The ciliate Tetrahymena thermophila is a useful model organism that combines diverse experimental advantages with powerful capabilities for genetic manipulation. The genetics of Tetrahymena are especially rich among eukaryotic cells, because it possesses two distinct but related nuclear genomes within one cytoplasm, contained separately in the micronucleus (MIC) and the macronucleus (MAC). In an effort to advance fulfillment of Tetrahymena's potential as a genetic system, we are mapping both genomes and investigating the correspondence between them. With the latter goal especially in mind, we report here a high-resolution meiotic linkage map of the left arm of chromosome 1, one of Tetrahymena's five chromosomes. The map consists of 40 markers, with an average spacing of 2.3 cM in the Haldane function and a total length of 88.6 cM. This study represents the first mapping of any large region of the Tetrahymena genome that has been done at this level of detail. Results of a parallel mapping effort in the macronucleus, and the correspondence between the two genomes, can be found in this issue as a companion to this article.

Tetrahymena macronuclear genome mapping: colinearity Of macronuclear coassortment groups and the micronuclear map on chromosome 1l.

The genetics of the ciliate Tetrahymena thermophila are richer than for most other eukaryotic cells, because Tetrahymena possesses two genomes: a germline (micronuclear) genome that follows a Mendelian model of genetic transmission and a somatic (macronuclear) genome, derived from the micronuclear genome by fragmentation, which follows a different genetic transmission model called phenotypic assortment. While genetic markers in the micronucleus fall into classical linkage groups under meiotic recombination and segregation, the same markers in the macronucleus fall into coassortment groups (CAGs) under phenotypic assortment by the random distribution of MAC chromosome pieces. We set out to determine whether genomic mapping in the macronucleus by genetic means is feasible. To investigate the relationship between the micronuclear map and coassortment groups, we systematically placed into CAGs all of the markers lying on chromosome 1L that are also found in the macronucleus. Sixteen CAGs were identified, 7 of which contain at least two loci. We have concluded that CAGs represent a fundamental genetic feature of the MAC. The MIC and MAC maps on 1L are colinear; that is, CAGs consist exclusively of markers that map to a continuous segment in a given region of the micronuclear map, with no intervening markers from other CAGs. These findings provide a solid foundation for exploiting the MAC chromosome pieces to build a physical map of the Tetrahymena genome.

Toward sequencing the Tetrahymena genome: exploiting the gift of nuclear dimorphism.

Important scientific discoveries have utilized the unique advantages of Tetrahymena thermophila as a research organism. Recently developed molecular genetic manipulations allow full exploitation of the many scientific dividends that would result from having its genome sequenced. As a typical ciliated protozoan, Tetrahymena exhibits "nuclear dimorphism". It possesses two differentiated forms of its nuclear genome: a globally repressed, diploid germline or micronuclear genome, and a polyploid, site-specifically fragmented somatic or macronuclear genome. The macronuclear genome is, in effect, a natural, large-insert library of the micronuclear genome. This presentation describes how the gifts of nuclear dimorphism are being exploited in the experimental analysis of molecular and cell biology. Mechanisms present in humans that are either absent in other eukaryotic microbial model systems, or not as readily accessible in them as in Tetrahymena, are especially relevant. This presentation also reviews unique tools generated by nuclear dimorphism that are being used for genetically and physically mapping the Tetrahymena genome.

Mapping the germ-line and somatic genomes of a ciliated protozoan, Tetrahymena thermophila.

Ciliates are among the very few eukaryotes in which the powers of molecular biology, conventional genetics, and microbial methodology can be synergistically combined. Because ciliates also are distant relatives of vertebrates, fungi, and plants, the sequencing of a ciliate genome will be of import to our understanding of eukaryotic biology. Tetrahymena thermophila is the only ciliate in which a systematic genetic mapping of DNA polymorphisms has begun. Tetrahymena has many biological features that make it a specially or uniquely useful experimental system for fundamental research in cell and molecular biology and for biotechnological applications. A key factor in the usefulness of Tetrahymena is the speed, facility, and versatility with which it can be cultivated under a wide range of nutrient conditions, temperature, and scale. This article describes the progress made in genetically and physically mapping the genomes of T. thermophila: the micronuclear (germ-line) genome, which is not transcriptionally expressed, and the macronuclear (somatic) fragmented genome, which is actively expressed and determines the cell's phenotype.

Mapping three classical isozyme loci in tetrahymena: meiotic linkage of EstA to the ChxA linkage group.

We demonstrate a reliable method for mapping conventional loci and obtaining meiotic linkage data for the ciliated protozoan Tetrahymena thermophila. By coupling nullisomic deletion mapping with meiotic linkage mapping, loci known to be located on a particular chromosome or chromosome arm can be tested for recombination. This approach has been used to map three isozyme loci, EstA (Esterase A), EstB (Esterase B), and AcpA (Acid Phosphatase A), with respect to the ChxA locus (cycloheximide resistance) and 11 RAPDs (randomly amplified polymorphic DNAs). To assign isozyme loci to chromosomes, clones of inbred strains C3 or C2 were crossed to inbred strain B nullisomics. EstA, EstB and AcpA were mapped to chromosomes 1R, 3L and 3R, respectively. To test EstA and AcpA for linkage to known RAPD loci on their respective chromosomes, a panel of Round II (genomic exclusion) segregants from a B/C3 heterozygote was used. Using the MAPMAKER program, EstA was assigned to the ChxA linkage group on chromosome 1R, and a detailed map was constructed that includes 10 RAPDs. AcpA (on 3R), while unlinked to all the RAPDs assigned to chromosome 3 by nullisomic mapping, does show linkage to a RAPD not yet assignable to chromosomes by nullisomic mapping.

Identification, mapping and linkage analysis of randomly amplified DNA polymorphisms in Tetrahymena thermophila.

Using the random amplified polymorphic DNA (RAPD) technique and exploiting the unique genetics of Tetrahymena thermophila, we have identified and characterized 40 DNA polymorphisms occurring between two inbred strains (B and C3) of this ciliated protozoan. These RAPD markers permit the PCR amplification of a DNA species using template DNA from SB1969 (B strain) but fail to do so using DNA from C3-368-5 (C3 strain). Polymorphisms were mapped to chromosomes using a panel of monosomic strains constructed by crossing B strain-derived nullisomic strains to inbred strain C3. They map to all five chromosomes and appear to be evenly distributed throughout the genome. Chromosomal groups were then analyzed for linkage using meiotic segregants; four linkage groups were identified in chromosomes 1R 2L, 3 and 5. The RAPD method appears useful for the construction of a genetic map of the Tetrahymena genome based on DNA polymorphisms.

An improved method to obtain high molecular weight DNA from purified micro- and macronuclei of Tetrahymena thermophila.

An improved method to obtain high molecular weight DNA from purified macro- and micronuclei of Tetrahymena thermophila is described. Micro- and macronuclear DNA obtained using previously described protocols was degraded and not suitable for the cloning of large (>100 kb) DNA fragments. Based on the data reported here, we propose that DNA degradation is mainly due to nuclease activity; some micronuclear DNA degradation is due to mechanical shearing as a result of extended periods of blending. We have made modifications to reduce nuclease degradation by minimizing cell lysis, by the early addition of EDTA and by increasing the EDTA concentration (23 mM). To reduce mechanical shearing, cell and nuclear suspensions were blended for shorter periods. High molecular weight micro- and macronuclear DNA was obtained using the new protocol.

Developmentally programmed DNA rearrangement in Tetrahymena thermophila: isolation and sequence characterization of three new alternative deletion systems.

Extensive developmentally programmed DNA rearrangements, including thousands of internal deletions, occur in the differentiating somatic macronucleus in Tetrahymena thermophila. Some deletion systems involve the use of multiple alternative deletion sites. We report here the cloning and the sequences of three new alternative deletion systems (RR, RP and B) obtained using genomic subtraction. The RP and RR deletion systems are 2 kb apart on chromosome 1R, and both involve the removal of < 2 kb of micronuclear sequences. The B deletion system is on chromosome 5 and involves a deletion of > 5 kb. All three deleted regions are very AT rich (approximately 80%) and do not appear to encode any protein. Sequences of the regions flanking the deletion junctions of all three systems revealed no sequence similarity among them nor with any previously reported deletion systems, suggesting that different cis-acting elements are involved for rearrangement. Unlike other deletion systems in ciliates, the B deletion system lacks short terminal direct repeats. Our results suggest an average of at least one alternative deletion system per 134 kb of micronuclear DNA and lead to an estimate that at least 25% of all deletion systems in Tetrahymena utilize alternative ends. The genomic subtraction method employed in this study could prove useful for the isolation of alternatively deleted DNA in special-purpose cases in Tetrahymena and other ciliates. The hybridization parameters for genomic subtraction worked out here for highly AT-rich DNA may have wider usefulness.

Genetic map of randomly amplified DNA polymorphisms closely linked to the mating type locus of Tetrahymena thermophila.

We have used the PCR-based randomly amplified polymorphic DNA (RAPD) method to efficiently identify and map DNA polymorphisms in the ciliated protozoan Tetrahymena thermophila. The polymorphisms segregate as Mendelian genetic markers. A targeted screen, using DNA from pooled meiotic segregants, yielded the polymorphisms most closely linked to the mat locus. A total of 10 polymorphisms linked to the mat-Pmr segment of the left arm of micronuclear chromosome 2 have been identified. This constitutes the largest linkage group described in T. thermophila. We also provide here the first crude estimate of the frequency of meiotic recombination in the mat region, 20 kb/cM. This frequency is much higher than that observed in most other eukaryotes. Special features of Tetrahymena genetics enhanced the power of the RAPD method: the ability to obtain in a single step meiotic segregants that are whole-genome homozygotes and the availability of nullisomic strains permitting quick deletion mapping of polymorphisms to micronuclear chromosomes or chromosome segments. The RAPD method appears to provide a practical and relatively inexpensive approach to the construction of a high-resolution map of the Tetrahymena genome.

Tetrahymena thermophila mutants defective in the developmentally programmed maturation and maintenance of the rDNA minichromosome.

The abundant rDNA minichromosome of Tetrahymena thermophila is generated by a series of developmentally controlled processing steps, termed rDNA maturation, during the formation of the new macronucleus in conjugating cells. rDNA maturation involves excision of a region encoding the single copy rRNA gene (rDNA) from its germline location, rearrangement of the rDNA into a palindromic minichromosome, de novo telomere addition, and amplification to approximately 10(4) copies. The rDNA is maintained at this high level in vegetatively growing cells. Using a previously developed genetic scheme for studying rDNA maturation and maintenance, we report the isolation of a new class of mutants defective for rDNA maturation. Several new rDNA maintenance mutants were also obtained. The maturation mutant, rmm10, is severely defective for the production of both monomeric and palindromic rDNA in the developing macronucleus. The mm10 mutation is recessive-lethal and cis-acting. None of the previously identified DNA sequence elements that control rDNA maturation or maintenance is mutated in rmm10. Therefore, additional cis-acting sequence elements must be required for rDNA maturation. Based on our current understanding of rDNA maturation processes, we suggest that the rmm10 mutation affects rDNA excision rather than subsequent rDNA amplification/replication.

Genetic characterization of Tetrahymena thermophila mutants unable to secrete capsules.

Under appropriate conditions, Alcian Blue-induced exocytosis of Tetrahymena mucocysts leads to formation of a capsule that surrounds the cell. This phenomenon is an example of regulated secretion, a mechanism of fundamental significance in eukaryotic cells. In order to dissect genetically the mechanism of mucocyst biogenesis and regulated exocytosis, mutants unable to form capsules (Caps-) were isolated. In this paper we report a genetic characterization of Caps- mutants in this collection. The mutations in mutants SB255 and SB281 behave as single recessive Mendelian mutations. The mutation in SB251 is restricted to the macronucleus, and could not be further characterized by the genetic methods we used. Complementation tests suggest the existence of at least 2 genes, named exoA and exoB; additional mutant loci are likely to be included in the mutant collection. Deletion mapping using nullisomic strains showed that exoA and exoB are located on the left arm of chromosome 4. The exo-3 mutation, which behaves as recessive and complements with exoA1 in SB255 and exoB2 in SB281, maps to chromosome 3. These Caps- mutants may be useful for the elucidation of the developmental pathway of mucocyst biogenesis and the control of regulated secretion in eukaryotic cells.

Mapping the mating type locus of Tetrahymena thermophila: meiotic linkage of mat to the ribosomal RNA gene.

Tetrahymena thermophila has a multiple mating type system. While a sexually mature cell usually expresses only one mating type, its germline (micronucleus) carries the genetic potential for 5 to 7 mating types. The set of allowed mating types is specified by the mat locus. The choice of which particular mating type is expressed by a cell reflects a somatically inherited, developmentally programmed differentiation of the somatic nucleus (macronucleus). In this work we report that the mat locus maps to the left arm of chromosome 2, as determined by nullisomic deletion mapping. We also report a distance of 29 cM between the mat locus and the ribosomal RNA gene, previously mapped to chromosome 2L. This represents another (rare) case of meiotic linkage in Tetrahymena.