Eduardo Orias

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.

Microarray analyses of gene expression during the Tetrahymena thermophila life cycle.

Background The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression. Methodology/Principal Findings A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organisms life cycle: growth, starvation and conjugation. Conclusions/Significance Of the ∼27,000 predicted open reading frames, transcripts homologous to only ∼5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5× corrected background and 95 genes are expressed at >250× corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

Chapter 13 Induction and Isolation of Mutants in Tetrabymena

Publisher Summary This chapter describes induction and isolation of mutants in Tetrahymena . Tetrahymena pyriformis is a unicellular animal well suited to studies of cell and molecular biology. It can be grown in pure culture to high concentrations, in practically unlimited volumes, with a remarkably short-vegetative doubling time (down to 2 hours under favorable conditions) and is thus extremely useful for the production and purification of cellular organelles and macromolecules. It is also well suited for genetic studies, having a diploid germ line with only five pairs of chromosomes, which are transmitted during conjugation according to the standard rules of the Mendelian genetics. The cells can be cloned efficiently, and conjugation is under precise experimental control. Clones can be readily frozen and indefinitely stored under liquid nitrogen. The chapter covers the methods used in the induction and isolation of Tetrahymena mutants, including (1) recent technical advances in handling the large numbers of cells required to isolate rare mutants and (2) recently developed strategies designed to increase the frequency of mutants. The chapter also describes elements of the Tetrahymena genetics, genetic analysis, and uses of the mutants. Design of conditions for isolating desired mutants is discussed in the chapter along with the limitations of the use of mutants.

Tetrahymena as a laboratory organism: useful strains, cell culture, and cell line maintenance.

Publisher Summary A key to the usefulness of Tetrahymena as a laboratory organism is its exceptionally fast growth rate under simple and inexpensive culture conditions. With a doubling time under 2 h, Tetrahymena is one of the fastest multiplying free-living eukaryotic cells. This chapter describes useful inbred and mutant strains, growth media, and some basic methods for laboratory storage and culture of Tetrahymena cells. Panels of meiotic segregants and terminal assortants derived from heterozygous progeny of inbred strains B × C3 are used to map genetic loci and DNA polymorphisms to the micronuclear and macronuclear genomes. Tetrahymena cells possess two efficient and sufficient routes of nutrient uptake: phagocytosis of particulate matter and active transport of nutrients in solution. Deionized and distilled H 2 O of high purity as well as dedicated glassware are used to make media, as Tetrahymena cells are sensitive to impurities in the water and soap residue on the glassware.

Control of rDNA replication in Tetrahymena involves a cis-acting upstream repeat of a promoter element

A novel genetic scheme was used to isolate mutants altered in the formation or maintenance of amplified rDNA in the Tetrahymena macronucleus. One such mutant had a cis-acting rDNA mutation that affected the ability of mutant rDNA molecules to replicate in macronuclei in the presence of a wild-type (B strain) rDNA. The mutant rDNA was lost from these heterozygous macronuclei during vegetative cell divisions, although it was maintained normally in the homozygous or hemizygous state. In contrast, wild-type macronuclear rDNA of the C3 strain used to obtain the mutant outreplicated B strain rDNA in B/C3 heterozygote macronuclei. Sequence differences were found between wild-type B and C3 and mutant C3 rDNAs in the replication origin region, changing an upstream repeat of a highly conserved rRNA promoter element. We propose that the various rDNA alleles differentially compete for limiting amounts of trans-acting factors that bind to these enhancer-like repeats and positively regulate rDNA replication.

Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure

BackgroundTetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymenas coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.ResultsWe addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.ConclusionWe report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.

Transcriptome Analysis of the Model Protozoan, Tetrahymena thermophila, Using Deep RNA Sequencing

Background The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation. Methodology/Principal Findings This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data. Conclusions/Significance RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.

Tetrahymena Metallothioneins Fall into Two Discrete Subfamilies

Background Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals. Methodology/Principal Findings We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported. Conclusion/Significance The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.

Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila.

Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (<500 bp) IES with precise elimination boundaries that can contribute the 3′ exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.

Tetrahymena thermophila, a unicellular eukaryote with separate germline and somatic genomes.

Tetrahymena thermophila is a ciliate--a unicellular eukaryote. Remarkably, every cell maintains differentiated germline and somatic genomes: one silent, the other expressed. Moreover, the two genomes undergo diverse processes, some as extreme as life and death, simultaneously in the same cytoplasm. Conserved eukaryotic mechanisms have been modified in ciliates to selectively deal with the two genomes. We describe research in several areas of Tetrahymena biology, including meiosis, amitosis, genetic assortment, selective nuclear pore transport, somatic RNAi-guided heterochromatin formation, DNA excision and programmed nuclear death by autophagy, which has enriched and broadened knowledge of those mechanisms.