A family of developmentally excised DNA elements in Tetrahymena is under selective pressure to maintain an open reading frame encoding an integrase-like protein

Abstract

Tlr1 is a member of a family of approximately 20-30 DNA elements that undergo developmentally regulated excision during formation of the macronucleus in the ciliated protozoan TETRAHYMENA: Analysis of sequence internal to the right boundary of Tlr1 revealed the presence of a 2 kb open reading frame (ORF) encoding a deduced protein with similarity to retrotransposon integrases. The ORFs of five unique clones were sequenced. The ORFs have 98% sequence conservation and align without frameshifts, although one has an additional trinucleotide at codon 561. Nucleotide changes among the five clones are highly non-random with respect to the position in the codon and 93% of the nucleotide changes among the five clones encode identical or similar amino acids, suggesting that the ORF has evolved under selective pressure to preserve a functional protein. Nineteen T/C transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the TETRAHYMENA: genome, where TAA and TAG encode Gln. Similarities between the ORF and those encoding retrotransposon integrases suggest that the Tlr family of elements may encode a polynucleotide transferase. Possible roles for the protein in transposition of the elements within the micronuclear genome and/or their developmentally regulated excision from the macronucleus are discussed.

Figure 1

Restriction maps of the Tlr1 regions of micronuclear and macronuclear DNA. The cross-hatched line represents macronucleus-destined sequence. The thick solid line represents the micronuclear-limited sequence. The bold arrows represent the 825 bp Tlr1 inverted repeat. The symbol * marks the location of 19mer tandem repeats. The angled arrow depicts the location of the 2 kb open reading frame identified in this study. Arrowheads represent PCR primers. Restriction sites: B, BglII; C, ClaI; E, EcoRI; H, HindIII; S, Sau3A; X, XbaI. The HindIII restriction site indicated by the dashed line is not present in Tlr1, but is present in other family members. The boxes represent fragments used in hybridization for isolation of Tlr Int clones.

Figure 2

Alignment of Tlr clones according to shared sequence. The vertical line in clone Tlr1 Int indicates the location of the right Tlr1 boundary and the arrow the inverted repeat. Mic, micronuclear-limited sequence; mac, macronucleus-destined sequence; angled arrow, the 2 kb open reading frame; triangles, the primers used to PCR amplify clone Tlr1 Int from micronuclear DNA.

Figure 3

Alignment of the first 227 amino acid residues in the Tlr Int consensus sequence with proteins or putative proteins that have similarity according to NCBI BLAST. Amino acids that are identical in at least five sequences are shaded black. Amino acids that are similar in at least five sequences are shaded gray. The active site DDE residues are marked with an asterisk. The integrase HHCC domain residues are indicated by circles. E values and accession numbers of the sequences are: C.elegans cosmid Y57G11C, 1e^–22, Z99281; X.maculatus HASI putative protein, 3e^–22, U43331; Trichopulsia ni TED retrotransposon, 7e^–11, B36329; Drosophila melanogaster mdg3 retrotransposon, 1e^–7, X95908; Schizosaccharomyces pombe Tf1 retrotransposon 1e^–7, M38526; Zea mays Opie-2 retrotransposon, 2e^–7, AF105716; S.cerevisiae Ty1 retrotransposon, 4e^–5, Z47746.

Figure 4

An alignment of the residues surrounding the DDE amino acids in integrases and transposases with the corresponding Tlr1 Int amino acids. The amino acids common to all types of proteins are red. Blue letters correspond to the retroviral integrase consensus, green letters correspond to the retrotransposon integrase consensus and purple letters correspond to the class II element transposase consensus. Black letters show no consensus. Similar residues are included in the consensus and similarity for this analysis is defined as a score of 5 on the SG Matrix scoring system (38).

Figure 5

(A) Partial restriction map of restriction sites in the Tlr Int clones. Angled arrow, Int open reading frame; heavy arrow, Tlr1 inverted repeat; bars, the HindIII–XbaI and EcoRV–ClaI fragments used to probe the blots in (B) and (C), respectively. Restriction sites C, ClaI; H, HindIII; V, EcoRV; X, XbaI. (B) Southern blot of micronuclear (MI) and macronuclear (MA) DNA digested with EcoRI, XbaI, EcoRV and HindIII. (C) Southern blot of micronuclear (MI) and macronuclear (MA) DNA digested with ClaI + EcoRV, EcoRV, HindIII and RsaI.