Download cDNA cloning, expression and chromosomal localization of the

Identification of a novel thioredoxin-1 pseudogene
on human chromosome 10
Antonio Miranda-Vizuete and Giannis Spyrou#
Department of Biosciences at Novum, Karolinska Institute, S-141 57
Huddinge, Sweden
# To whom correspondence should be addressed: Dept. of Biosciences, Center for
Biotechnology, Karolinska Institutet, Novum, S-141 57 Huddinge, Sweden.
Tel.: 46-8-6089162; Fax: 46-8-7745538; Email: [email protected]
The sequence data reported in this paper have been submitted to the GenBank Data
Libraries under the accession number AF146024
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Thioredoxin (Trx) is a small, ubiquitous protein of 12 kDa that acts as general
dithiol-disulfide oxidoreductase via its conserved redox active site Trp-Cys-GlyPro-Cys which is located in a protrusion of the protein (Holmgren 1985).
Mammalian cells contain two forms of thioredoxin: Trx1, a cytosolic enzyme able
to translocate to the nucleus under certain stimuli and be secreted from the cells
thus displaying cytokine-like properties and Trx2, a mitochondrial enzyme
(Tagaya et al. 1989; Spyrou et al 1997). While many functions have been described
for Trx1 for example antioxidant enzyme, modulator of transcription factors,
electron donor for enzymes like ribonucleotide reductase and PAPS reductase, etc.
(see introduction Spyrou et al. 1997), only an antioxidant function has been
assigned to Trx2. Trx2 acts as a reductant of a mitochondrial thioredoxindependent peroxidase which protects cells against hydrogen peroxide treatment
(Araki et al. 1999). Human Trx1 gene maps at chromosome 9q31 and several bands
hybridize with a Trx1 probe in Southern blots suggesting the existence of Trx1
derived sequences in the human genome (Heppell-Parton et al. 1995; Kaghad et al.
1994). As only one active Trx1 gene has been described, the other hybridizing
bands might correspond to different inactive pseudogenes (Kaghad et al. 1994), but
only one Trx1 pseudogene has been described so far (GenBank accession number:
AF146023 (Tonissen and Wells 1991)). We report here the sequence of a second
Trx1 retrotranscribed pseudogene and propose the nomenclature Trx1-1 and
Trx1-2.
We initiated our study identifying an EMBL entry (AC005874) corresponding
to a human genomic region of chromosome 10 that displayed high homology with
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Trx1 gene. We designed primers flanking the homology region (Forward 5´GGCTTGTGCTGGGATAGAGCTG-3´ and reverse 5´-CCCACACACACATACAC
ATCCCC-3´) and amplified by PCR a fragment from human genomic DNA
(Clontech). We cloned the fragment in pGEM-Teasy vector and sequenced it in
both directions confirming the sequence of the EMBL entry AC005874. The analysis
of the sequence cloned, strongly suggested that this genomic fragment might
correspond to a pseudogene rather than a functional copy of the gene. First, we
identified a unique intron-like sequence that showed homology with members of
the Alu sequence family while the human Trx1 gene is organized in 5 exons and 4
introns (Figure 1) (Kaghad et al. 1994; Tonissen and Wells 1991). Second, an
imperfect polyA tail is present exactly 3´ after the point at which homology with
Trx1 cDNA ceases. Third, the sequence Trx1-2 shows multiple nucleotide changes
when compared with Trx1 cDNA. The most striking change affects the conserved
active site which is mutated and the translation would then give a putative active
site WYGPC, where the Cys32 changing to tyrosine abolishes the enzymatic
activity (Tagaya et al. 1989). Furthermore, a one-base deletion would initiate a
frameshift resulting in a different C-terminus of the protein that has been found to
be necessary for protein-protein interaction (Eklund et al. 1991). Fourth, the Trx12 sequence is flanked by a 15 bp direct repeat (with only one mismatch) that is
believed to play a role in the insertion of the sequence into the genome (Vanin
1985). Fifth, the promoter regions described for human Trx1 (TATA box and SP1
binding site) have been replaced in Trx1-2 sequence, making it unlikely that this
gene would be expressed (Kaghad et al. 1994). Taken together, all these features
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correspond to a processed pseudogene that may have originated from a RNA
intermediate and integrated by retrotransposition in a different location in the
genome (Vanin 1985). The time of occurrence of a retrotransposition event can be
tentatively estimated based on the differences found in comparison with the coding
sequence of the functional gene. If a rate of ~2 x 10-9 per nucleotide per year is
taken as a model for spontaneous mutation in primates and assuming that the
nucleotide differences were accumulated by the pseudogene while the functional
gene remained unchanged since their divergence (Vanin, 1985), then this processed
pseudogene could have originated approximately 18 millions years ago. If a higher
mutation rate, 4.7 x 10-9 per nucleotide per year estimated for mammals (Li et al.
1987) is employed instead, the origin of the pseudogene would have been
approximately 7.8 millions years ago. In any case, the insertion of the Alu sequence
would have occurred after the retrotransposition event.
To determine whether Trx1-2 was transcribed, we performed RT-PCR on a
human multiple tissue cDNA panel (Clontech). When using specific primers for
Trx1-2 (forward 5´-GTGAAGCAAATTGAGAGCAAGGT-3´ and reverse 5´GTTGTCATCCAGATGGCAGTTG-3´) we were not able to amplify Trx1-2
although the same primers were used to amplify the corresponding fragment from
the
genomic
DNA.
Specific
primers
GTGAAGCAGATCGAGAGCAAGAC-3´
for
and
Trx1
(forward
reverse
5´5´-
ATTGTCACGCAGATGGCAACTG-3´) located at the same position as those used
for Trx1-2 were able to amplify Trx1 in all the tissues when RT-PCR was
performed under the same conditions as the Trx1-2 amplification (data not
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shown). Thus, we demonstrate that Trx1-2 is not expressed. Finally, we can map
Trx1-2 to position 10q25.2-10q25.3 as it has been assigned for the EMBL entry
AC005874, while Trx1 gene maps at chromosome 9q31 (Heppell-Parton et al. 1995).
This is in agreement with the fact that processed pseudogenes are usually not
syntenic with their respectives functional genes (Vanin 1985).
ACKNOWLEDGEMENTS
This work was supported by grants from the Swedish Medical Research
Council (Project 13X-10370) and the TMR Marie Curie Research Training Grants
(contract ERBFMBICT972824).
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LEGENDS TO THE FIGURES
Figure 1. Comparison of nucleotide sequence between homologous regions of
human Trx1 cDNA and Trx1-2 cDNA. The residues that differ between Trx1 and
Trx1-2 are boxed. Start and stop codons on Trx1 cDNA are indicated by asterisks
and the 15 bp direct repeats underlined. An arrow head indicates the critical
mutation (CY) at the active site of Trx1 and the one-base frameshift.
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