Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in \u3cem\u3eTetrahymena Thermophila\u3c/em\u3e

Tlr elements are a novel family of ~30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleusretained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ~23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA

A Novel Family of Mobile Genetic Elements Is Limited to the Germline Genome in \u3cem\u3eTetrahymena Thermophila\u3c/em\u3e

In the ciliated protozoan Tetrahymena thermophila, extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germline micronucleus. This study describes the isolation and complete characterization of Tlr elements, a family of approximately 30 micronuclear DNA sequences that are efficiently eliminated from the developing macronucleus. The data indicate that Tlr elements are comprised of an ~22 kb internal region flanked by complex and variable termini. The Tlr internal region is highly conserved among family members and contains 15 open reading frames, some of which resemble genes encoded by transposons and viruses. The Tlr termini appear to be long inverted repeats consisting of (i) a variable region containing multiple direct repeats which differ in number and sequence from element to element and (ii) a conserved terminal 47 bp sequence. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the Tetrahymena germline genome. Possible mechanisms of developmentally programmed Tlr elimination are discussed

ProRepeat: an integrated repository for studying amino acid tandem repeats in proteins

ProRepeat (http://prorepeat.bioinformatics.nl/) is an integrated curated repository and analysis platform for in-depth research on the biological characteristics of amino acid tandem repeats. ProRepeat collects repeats from all proteins included in the UniProt knowledgebase, together with 85 completely sequenced eukaryotic proteomes contained within the RefSeq collection. It contains non-redundant perfect tandem repeats, approximate tandem repeats and simple, low-complexity sequences, covering the majority of the amino acid tandem repeat patterns found in proteins. The ProRepeat web interface allows querying the repeat database using repeat characteristics like repeat unit and length, number of repetitions of the repeat unit and position of the repeat in the protein. Users can also search for repeats by the characteristics of repeat containing proteins, such as entry ID, protein description, sequence length, gene name and taxon. ProRepeat offers powerful analysis tools for finding biological interesting properties of repeats, such as the strong position bias of leucine repeats in the N-terminus of eukaryotic protein sequences, the differences of repeat abundance among proteomes, the functional classification of repeat containing proteins and GC content constrains of repeats’ corresponding codons

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

Isolation and Characterization of the Tlr Family of Germ Line-Limited Mobile Genetic Elements in Tetrahymena Thermophila

In the ciliated protozoan Tetrahymena thermophila , extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germ line micronucleus. Tlr elements are a family of approximately 30 closely related DNA sequences in the micronuclear genome. All copies of these elements are deleted from the macronucleus during development. The primary goals of this research were to characterize the complete structure of Tlr elements and identify the cis -acting internal DNA signals that control their programmed elimination. The composite structure of a typical Tlr element was assembled from a series of overlapping DNA clones isolated by successively screening a plasmid library of micronuclear genomic DNA. Analysis of the resulting sequences revealed that Tlr elements consist of a ∼22 kb internal region flanked by long, complex terminal inverted repeats. The Tlr internal region is 90-97% conserved at the nucleotide level among family members and contains 15 major open reading frames. The conceptual translation products from several of the open reading frames resemble proteins encoded by transposable elements and viruses. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the germ line genome in Tetrahymena . In order to examine the mechanism by which Tlr elements are recognized for deletion from the macronuclear genome, an rDNA-based in vivo rearrangement assay was utilized to identify the internal sequences required in cis for faithful elimination of the most thoroughly characterized Tlr family member, Tlr1. These analyses revealed that multiple, non-overlapping regions of the Tlr1 element are independently sufficient to stimulate developmentally regulated DNA deletion within the context of normal flanking sequences. Since complete removal of Tlr1 DNA abolishes construct rearrangement activity, as does replacement of element sequences with macronuclear DNA, the data suggest that unique features of Tlr sequences provide cis -acting regulatory signals for programmed deletion from the differentiating macronuclear genome. Furthermore, replacement of Tlr1 DNA with the micronucleus-limited region of a non-Tlr internally eliminated sequence, the M element, resulted in accurate rearrangement activity. Thus, Tlr1 and M appear to contain functionally similar internal signals that promote programmed DNA elimination in Tetrahymen

Isolation and characterization of the Tlr family of germ line-limited mobile genetic elements in Tetrahymena thermophila

In the ciliated protozoan Tetrahymena thermophila , extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germ line micronucleus. Tlr elements are a family of approximately 30 closely related DNA sequences in the micronuclear genome. All copies of these elements are deleted from the macronucleus during development. The primary goals of this research were to characterize the complete structure of Tlr elements and identify the cis -acting internal DNA signals that control their programmed elimination. The composite structure of a typical Tlr element was assembled from a series of overlapping DNA clones isolated by successively screening a plasmid library of micronuclear genomic DNA. Analysis of the resulting sequences revealed that Tlr elements consist of a ∼22 kb internal region flanked by long, complex terminal inverted repeats. The Tlr internal region is 90-97% conserved at the nucleotide level among family members and contains 15 major open reading frames. The conceptual translation products from several of the open reading frames resemble proteins encoded by transposable elements and viruses. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the germ line genome in Tetrahymena . In order to examine the mechanism by which Tlr elements are recognized for deletion from the macronuclear genome, an rDNA-based in vivo rearrangement assay was utilized to identify the internal sequences required in cis for faithful elimination of the most thoroughly characterized Tlr family member, Tlr1. These analyses revealed that multiple, non-overlapping regions of the Tlr1 element are independently sufficient to stimulate developmentally regulated DNA deletion within the context of normal flanking sequences. Since complete removal of Tlr1 DNA abolishes construct rearrangement activity, as does replacement of element sequences with macronuclear DNA, the data suggest that unique features of Tlr sequences provide cis -acting regulatory signals for programmed deletion from the differentiating macronuclear genome. Furthermore, replacement of Tlr1 DNA with the micronucleus-limited region of a non-Tlr internally eliminated sequence, the M element, resulted in accurate rearrangement activity. Thus, Tlr1 and M appear to contain functionally similar internal signals that promote programmed DNA elimination in Tetrahymena

Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in Tetrahymena thermophila

Tlr elements are a novel family of ∼30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleus-retained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ∼23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA