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

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

Analysis of Genomic G + C Content, Codon Usage, Initiator Codon Context and Translation Termination Sites in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

In recent years, the amount of molecular sequencing data from Tetrahymena thermophila has dramatically increased. We analyzed G + C content, codon usage, initiator codon context and stop codon sites in the extremely A + T rich genome of this ciliate. Average G + C content was 38% for protein coding regions. 21% for 5′ non-coding sequences, 19% for 3′ non-coding sequences, 15% for introns, 19% for micronuclear limited sequences and 17% for macronuclear retained sequences flanking micronuclear specific regions. the 75 available T. thermophila protein coding sequences favored codons ending in T and, where possible, avoided those with G in the third position. Highly expressed genes were relatively G + C-rich and exhibited an extremely biased pattern of codon usage while developmentally regulated genes were more A + T-rich and showed less codon usage bias. Regions immediately preceding Tetrahymena translation initiator codons were generally A-rich. For the 60 stop codons examined, the frequency of G in the end + 1 site was much higher than expected whereas C never occupied this position

Homing Endonucleases Encoded by Germ Line-Limited Genes in Tetrahymena thermophila Have APETELA2 DNA Binding Domains

Three insertion elements were previously found in a family of germ line-limited mobile elements, the Tlr elements, in the ciliate Tetrahymena. Each of the insertions contains an open reading frame (ORF). Sequence analysis of the deduced proteins encoded by the elements suggests that they are homing endonucleases. The genes are designated TIE1-1, TIE2-1, and TIE3-1 for Tetrahymena insertion-homing endonuclease. The endonuclease motif occupies the amino terminal half of each TIE protein. The C-terminal regions of the proteins are similar to the APETELA2 DNA binding domain of plant transcription factors. The TIE1 and TIE3 elements belong to families of repeated sequences in the germ line micronuclear genome. Comparison of the genes and the deduced proteins they encode suggests that there are at least two distinct families of homing endonuclease genes, each of which appears to be preferentially associated with a specific region of the Tlr elements. The TIE1 and TIE3 elements and their cognates undergo programmed elimination from the developing somatic macronucleus of Tetrahymena. The possible role of homing endonuclease-like genes in the DNA breakage step in developmentally programmed DNA elimination in Tetrahymena is discussed