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Telomere function is mediated by a complex of proteins bound to double-stranded and single-stranded telomeric repeats. A key player in this complex is TRF1, which binds to duplex TTAGGG repeats and acts as a negative regulator of telomere length. This protein's domain structure, as defined by studies with mammalian orthologs, consists of an N-terminal acidic domain, a dimerization domain, and a C-terminal Myb DNA binding domain. TRF1 from Xenopus laevis was cloned and sequenced, and the encoded protein found to have a similar structure but with a very short acidic domain. This short acidic domain was confirmed in Xenopus tropicalis, a true diploid, by cloning of cDNA sequences by RACE and analysis of the genomic locus. The TRF1 transcript is expressed in developing and adult frogs. Compared to the mammalian orthologs, the Xenopus genes are the most distantly related vertebrate examples characterized to date. Since adult Xenopus ubiquitously express somatic telomerase activity, proteins that regulate telomerase access to the chromosome ends are important in regulating telomere length in normal somatic tissue. The structure of Xenopus TRF1 has implications for its regulation by tankyrase.
Fig. 1.
Alignment of vertebrate TRF1s. Human, mouse, chicken, and X. laevis proteins were aligned with ClustalW. Solid black backgrounds indicate identical residues, grey backgrounds mark similarities. Dimerization domain: human TRF1 residues 67â264 ( Smogorzewska and de Lange, 2004); Myb DNA binding domain: human TRF1 residues 379â429 (Marchler-Bauer and Bryant, 2004). An asterisk is shown above the position of the first intron in human, mouse, and Xenopus genes (see Fig. 3b).
Fig. 2.
Comparison of Xenopus TRF1s. (a) 5â² UTRs and start of open reading frames for X. laevis and X. tropicalis TRF1. Potential start codons underlined; predicted beginning of open reading frame (MEE) in bold. A variant of the X. laevis RACE product began with nucleotide 7 but was otherwise identical. The middle ATG for X. tropicalis and the first ATG for X. laevis are out of frame. (b) Comparison of predicted amino acid sequences for upstream regions of X. laevis and X. tropicalis TRF1s. X. tropicalis sequence from the upstream in-frame ATG, through the first 3 amino acids of the likely protein, is presented at the top of each pair; 3 possible reading frames of the entire 5â² UTR of the X. laevis transcript are presented below. (c) Conservation of the predicted amino acid sequences of laevis and tropicalis TRF1 proteins. X. tropicalis sequences cloned in this work constitute the first 154 amino acids; the remainder of the sequence was predicted from ESTs. Black and grey backgrounds indicate identical and similar residues, respectively.
Fig. 3.
Genomic TRF1 analyses. (a) Exonâintron structure of X. tropicalis genomic TRF1 locus. A cDNA assembled from RACE and EST products was compared by BLAST to identify exons and their position on genomic DNA. Black boxes represent the 12 exons and lengths in base pairs indicated above, and span 15,637 bp of genomic DNA. Intron lengths are roughly proportional to one another, but not to exons. (b) Comparison of the first exons of human, mouse, and X. tropicalis TRF1. BLAST searches of human and mouse genomic DNA with appropriate cDNA (NM_017489 and NM_009352) were used to identify the extent of the first exons in these species, and the encoded polypeptides then compared. Numbers refer to amino acid residues from the N-terminus. Grey boxes indicate dimerization domain as identified for human TRF1 in Smogorzewska and de Lange (2004).
Fig. 4.
Detection of TRF1 transcripts in tissues and embryos. RNA was isolated from various tissues and reverse-transcribed; 1 / 20th of the reaction was subjected to 30 cycles of PCR with primers to amplify either a portion of the TRF1 transcript (279 bp within the dimerization domain) or a housekeeping gene (G3PDH). Products were separated on 1.2% agarose gels and stained with ethidium bromide. The tissue source is indicated above each lane. An example no-reverse-transcription control (lane 1) is shown; all tissues were similarly tested. Stage 6 embryos are 32-celled and still dependent on maternally deposited transcripts (stage 8 marks the onset of zygotic transcription). Stage 10 is initial gastrula stage and stage 30 embryos are tail-bud.
