Diverse sequences within Tlr elements target programmed DNA elimination in Tetrahymena thermophila

Abstract

Tlr elements are a novel family of approximately 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 approximately 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.

FIG. 1.

(A) rDNA-based in vivo rearrangement assay for T. thermophila. The pD5H8 processing vector consists of pUC18 plasmid DNA (thin black line) and Tetrahymena micronuclear rDNA (thick black line). The open box represents the 17S and 25S rRNA genes. The approximate positions of chromosome breakage sequences, the rDNA origin of replication (Ori), and a point mutation within the 17S rDNA (Pmr) conferring resistance to paromomycin are indicated. An IES (hatched box) and macronucleus-retained flanking sequences (gray boxes) are shown inserted into the unique NotI site in pD5H8. Upon transformation, pD5H8 and the cloned IES region undergo normal in vivo processing. (B) Tlr1 locus in the T. thermophila micronucleus (mic) and the predominant macronuclear (mac) product of programmed elimination. Hatched boxes depict the terminal regions of the Tlr1 element. Arrows represent the 825-bp terminal inverted repeat of Tlr1. Gray boxes depict Tlr1 flanking sequences. Positions of previously described elimination boundaries, including sites used in Tlr1 minor rearrangement variants, are indicated by arrowheads.

FIG. 2.

Either side of the Tlr1 terminal inverted repeat is sufficient to promote programmed DNA elimination within the context of normal Tlr1 flanking sequences. (A) Composite diagram of the micronuclear Tlr1 element indicating regions used to generate the Tflank.IRL and Tflank.IRR rearrangement constructs. The hatched boxes represent sequences from the terminal regions of the element, and the gray boxes represent Tlr1 flanking sequences. Thick arrows represent the Tlr1 terminal inverted repeat, and thin arrows indicate Tlr open reading frames. (B and C) (Left panels) Tflank.IRL (B) and Tflank.IRR (C) rearrangement constructs (filled arrowheads) and their in vivo processed macronuclear products (open arrowheads). Boxes represent the Tlr1 regions highlighted in panel A, and thin lines represent the vector polylinker included in the NotI fragments. Southern blotting probe Tlr1-L is depicted as a bar. Positions of primers used for PCR amplification of rearrangement junctions are indicated below the processed constructs. (Right panels) Southern blot analysis of Tflank.IRL (B) and Tflank.IRR (C) transformant DNAs probed with Tlr1-L. Lane P contains NotI-digested plasmid DNA, lanes 1 to 9 contain NotI-digested whole-cell DNA from independent Tetrahymena transformant lines, and lane wt contains NotI-digested whole-cell DNA from untransformed Tetrahymena. Hybridization to the chromosomal copy of Tlr1 was not detected at this level of exposure because the rDNA was amplified ∼200-fold.

FIG. 3.

Developmentally programmed DNA elimination requires IESs. (A) Composite diagram of the micronuclear Tlr1 element, the T. thermophila Cyd and α-tubulin (Tub) loci, and the Cat gene from E. coli transposon Tn9 indicating the DNA fragments used to generate control constructs. Thick arrows represent the Tlr1 terminal inverted repeat, and thin arrows indicate open reading frames. (B through E) (Left panels) Tflank, Tflank.Cyd, Tflank.Tub, and Tflank.Cat constructs (filled arrowheads) and their in vivo processed macronuclear products (open arrowheads). Boxes represent the DNA regions highlighted in panel A, and thin lines represent the vector polylinker included in the NotI fragments. Southern blotting probe Tlr1-L is depicted as a bar. Positions of primers used for PCR amplification of processed Tflank are indicated below the constructs. (Right panels) Southern blot analysis of Tflank (B), Tflank.Cyd (C), Tflank.Tub (D), and Tflank.Cat (E) transformant DNAs probed with Tlr1-L. Lane P contains NotI-digested plasmid DNA, lanes 1 to 9 contain NotI-digested whole-cell DNA from independent Tetrahymena transformant lines, lane wt contains NotI-digested whole-cell DNA from untransformed Tetrahymena, and lane ev contains NotI-digested whole-cell DNA from Tetrahymena transformed with pD5H8 containing no insert.

FIG. 4.

Internal fragments of Tlr elements promote programmed DNA elimination within the context of normal Tlr1 flanking DNA. (A) Composite diagram of the micronuclear Tlr1 element showing Tlr flanking sequences (gray boxes) and internal sequences (hatched boxes) used to generate Tflank.IN1 to Tflank.IN5 rearrangement constructs. Thick arrows represent the Tlr1 terminal inverted repeat, and thin arrows indicate Tlr open reading frames. (B through F) (Left panels) Tflank.IN1 to Tflank.IN5 constructs (filled arrowheads) and their in vivo processed macronuclear products (open arrowheads). Boxes represent the DNA regions highlighted in panel A, and thin lines represent the vector polylinker included in the NotI fragments. Southern blotting probe Tlr1-L is depicted as a bar. Positions of primers used for PCR amplification of processed Tflank.IN2 and Tflank.IN3 are indicated below the constructs. Deduced rearrangement activity of Tflank.IN1, Tflank.IN4, and Tflank.IN5 is represented by broken lines. (Right panels) Southern blot analysis of Tflank.IN1 to Tflank.IN5 transformant DNAs probed with Tlr1-L. Lane P contains NotI-digested plasmid DNA, lanes 1 to 8 (B and E) and 1 to 9 (C, D, and F) contain NotI-digested whole-cell DNA from independent Tetrahymena transformant lines, lane wt contains NotI-digested whole-cell DNA from untransformed Tetrahymena, and lane ev contains NotI-digested whole-cell DNA from Tetrahymena transformed with pD5H8 containing no insert.

FIG. 4.

Internal fragments of Tlr elements promote programmed DNA elimination within the context of normal Tlr1 flanking DNA. (A) Composite diagram of the micronuclear Tlr1 element showing Tlr flanking sequences (gray boxes) and internal sequences (hatched boxes) used to generate Tflank.IN1 to Tflank.IN5 rearrangement constructs. Thick arrows represent the Tlr1 terminal inverted repeat, and thin arrows indicate Tlr open reading frames. (B through F) (Left panels) Tflank.IN1 to Tflank.IN5 constructs (filled arrowheads) and their in vivo processed macronuclear products (open arrowheads). Boxes represent the DNA regions highlighted in panel A, and thin lines represent the vector polylinker included in the NotI fragments. Southern blotting probe Tlr1-L is depicted as a bar. Positions of primers used for PCR amplification of processed Tflank.IN2 and Tflank.IN3 are indicated below the constructs. Deduced rearrangement activity of Tflank.IN1, Tflank.IN4, and Tflank.IN5 is represented by broken lines. (Right panels) Southern blot analysis of Tflank.IN1 to Tflank.IN5 transformant DNAs probed with Tlr1-L. Lane P contains NotI-digested plasmid DNA, lanes 1 to 8 (B and E) and 1 to 9 (C, D, and F) contain NotI-digested whole-cell DNA from independent Tetrahymena transformant lines, lane wt contains NotI-digested whole-cell DNA from untransformed Tetrahymena, and lane ev contains NotI-digested whole-cell DNA from Tetrahymena transformed with pD5H8 containing no insert.

FIG. 5.

Tlr DNA is eliminated from within flanking sequences that are not normally associated with DNA rearrangement. (A) Diagram of the Tetrahymena Cyd1 locus and a composite of the Tlr1 element. Boxed areas indicate regions included in the Cflank.IRL, Cflank.IN2, and Cflank.IN5 constructs. Thick arrows represent the Tlr1 terminal inverted repeat, and thin arrows indicate open reading frames. (B through D) (Left panels) Cflank.IRL, Cflank.IN2, and Cflank.IN5 constructs (filled arrowheads) and their in vivo processed macronuclear products (open arrowheads). Boxes represent the regions highlighted in panel A, and thin lines represent the vector polylinker included in the NotI fragments. Southern blotting probe Cyd1.R is depicted as a bar. Positions of primers used for PCR amplification of processed Cflank.IRL and Cflank.IN2 are indicated below the constructs. Deduced rearrangement activity of Cflank.IN5 is represented by thick lines. (Right panels) Southern blot analysis of Cflank.IRL, Cflank.IN2, and Cflank.IN5 transformant DNAs. Lane P contains NotI-digested plasmid DNA, lanes 1 to 9 contain NotI-digested whole-cell DNA from independent Tetrahymena transformant lines, and lane wt contains NotI-digested whole-cell DNA from untransformed Tetrahymena.

FIG. 5.

Tlr DNA is eliminated from within flanking sequences that are not normally associated with DNA rearrangement. (A) Diagram of the Tetrahymena Cyd1 locus and a composite of the Tlr1 element. Boxed areas indicate regions included in the Cflank.IRL, Cflank.IN2, and Cflank.IN5 constructs. Thick arrows represent the Tlr1 terminal inverted repeat, and thin arrows indicate open reading frames. (B through D) (Left panels) Cflank.IRL, Cflank.IN2, and Cflank.IN5 constructs (filled arrowheads) and their in vivo processed macronuclear products (open arrowheads). Boxes represent the regions highlighted in panel A, and thin lines represent the vector polylinker included in the NotI fragments. Southern blotting probe Cyd1.R is depicted as a bar. Positions of primers used for PCR amplification of processed Cflank.IRL and Cflank.IN2 are indicated below the constructs. Deduced rearrangement activity of Cflank.IN5 is represented by thick lines. (Right panels) Southern blot analysis of Cflank.IRL, Cflank.IN2, and Cflank.IN5 transformant DNAs. Lane P contains NotI-digested plasmid DNA, lanes 1 to 9 contain NotI-digested whole-cell DNA from independent Tetrahymena transformant lines, and lane wt contains NotI-digested whole-cell DNA from untransformed Tetrahymena.