Homing endonucleases encoded by germ line-limited genes in Tetrahymena thermophila have APETELA2 DNA binding domains

Abstract

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.

FIG. 1.

Insertion elements in the Tlr clones. A physical map of the Tlr elements, drawn as a composite of sequences from nine genomic clones. Clones containing the TIE element insertions and homologous clones lacking the TIE elements are shown below the map of the Tlr family. The Tlr Int clones and the pMBR clones were described previously (13, 29).

FIG. 2.

Alignment of the partial sequence of the deduced proteins encoded by TIE1-1, TIE2-1, and TIE3-1 with the amino acid sequences of seven viral homing endonucleases, two proteins containing the AP2 domains, and the AP2 consensus sequence. Red, identical residues in the first eight proteins; blue, conserved residues; green, the AP2 DNA binding domain consensus sequence and identical sequences in the last five proteins. A consensus sequence for the N-terminal region is above the sequence alignment in red. Uppercase letters designate amino acids that are invariant among these proteins. The HNH domain as designated by the Pfam program is underlined. Motifs I, II, and III are additional motifs that are conserved among the TIE elements and the bacteriophage endonucleases. In motif II amino acids that may be either positively or negatively charged are indicated (±). The sequences of the termini of several of the proteins, including the N termini of Tie1-1p and Tie2-1p were omitted from the figure where they do not align with consensus domains. Accession numbers: Bacillus subtilis SPO1, P34081; Lactococcus lactis phage r1t, AAB18716; B. subtilis, CAB13895; phage SP82, AAA56884; lactococcal bacteriophage bIL170, AAC27218; Bacteroides thetaiotaomicron VPI-5482, NP_810939; bacteriophage phi-E, U04813; TIE1-1, AF451862; TIE2-1 and TIE3-1, AF451865; Atriplex hortensis AP2 domain protein, AAF76898; A. thaliana AP2 domain protein, NP_177931.

FIG. 3.

TIE elements are repeated in the micronuclear genome and are germ line limited. Southern blots of genomic DNA probed with the TIE elements TIE1-1 (A) and TIE3-1 (B). Open circles indicate the expected positions of bands if the TIE3 probe had cross-hybridized to TIE2-1 sequences. The sizes of the hybridizing fragments were determined relative to Ho-Lo DNA marker (Minnesota Molecular). Mi, micronuclear DNA; Ma, macronuclear DNA; p8E1, plasmid pMBR8E1 DNA; p8, plasmid pMBR8 DNA; H, HindIII; E, EcoRV; X, XbaI.

FIG. 4.

Probes for screening duplicate colony lifts. Thin line, micronuclear DNA in plasmids pMBR8E1 (the plasmid with the TIE1-1 insertion) and pMBR4C1 (a plasmid covering the same region of the Tlr elements but lacking the TIE1-1 insertion); heavy line, TIE1-1 insertion; angled arrow, TIE1-1 ORF; open box, TIE1-1 probe; gray boxes, Tlr probe obtained by PCR amplification of pMBR4C1 and the corresponding regions on the pMBR8E1 plasmid.

FIG. 5.

Alignment of the TIE sequence reads from the Tetrahymena genome project. (A) Alignment of plasmid pMBR4C1 with plasmid pMBR8E1 (29), containing the TIE1-1 insertion and four reads from clones sequenced as part of the Tetrahymena genome project. Box, TIE1-1 insertion element; angled arrows, TIE1 ORFs, with the initiation codon represented by a vertical bar and the stop codon indicated by the arrowhead; black boxes above the clone, regions of homology with pMBR8E1; gray boxes below the clone, homology with pMBR4C1. (B) Alignment of plasmid Tlr Int C (13) with the homologous region of plasmid pMBR8 (29), containing the TIE3-1 insertion and with four reads from clones in the Tetrahymena genome project. For the TIE3-1 insertion element and TIE3 ORFs, the format corresponds to that of panel A. Black boxes, homology to pMBR8; gray boxes, homology to Tlr Int C. For both panels, letters to the right are the plasmid name or the identification number for the read in the Tetrahymena genome project database.

FIG. 6.

The TIE3 ORFs extend into the Tlr elements. The sequences of four clones containing the 3′ end of the TIE3 elements were aligned with the corresponding sequence from Tlr Int B and three additional empty site clones from the TIGR database. The single letter consensus code for the Tie3p deduced proteins is above the alignment, and the last two amino acids of the deduced protein Tlr4Rp are below. Stop codons are in bold letters.