Download Mamm_Genome yTrx1-2 + refs

☰





Search
Explore
Log in
Create new account
Upload
×
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
1
Thioredoxin (Trx) is a small, ubiquitous protein of 12 kDa that acts as
general
dithiol-disulfide oxidoreductase via its conserved redox active site Trp-CysGlyPro-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
2
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
3
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
4
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).
5
REFERENCES
Araki, M., Nanri, H., Ejima, K., Murasato, Y., Fujiwara, T., Nakashima, Y.
and
Ikeda, M. (1999). Antioxidant function of the mitochondrial protein SP-22 in
the
cardiovascular system. J. Biol. Chem. 274, 2271-2278.
Eklund, H., Gleason, F. K. and Holmgren, A. (1991). Structural and functional
relations among thioredoxins of different species. Proteins: Structure,
function
and genetics. 11, 13-28.
Heppell-Parton, A., Cahn, A., Bench, A., Lowe, N., Lehrach, H., Zehetner, G.
and
Rabbitts, P. (1995). Thioredoxin, a mediator of growth inhibition, maps to
9q31.
Genomics. 26, 379-381.
Holmgren, A. (1985). Thioredoxin. Annu. Rev. Biochem. 54, 237-271.
Kaghad, M., Dessarps, F., Jacquemin-Sablon, H., Caput, D., Fradizeli, D. and
Wollman, E. E. (1994). Genomic cloning of human thioredoxin-encoding gene:
mapping of the transcription start point and analysis of the promoter. Gene.
140, 273-278.
Li, W.-H., Tanimura, M. and Sahrp, P. (1987). An evaluation of the molecular
clock
hypothesis using mammalian DNA sequences. J. Mol. Evol. 25, 333-342.
Spyrou, G., Enmark, E., Miranda-Vizuete, A. and Gustafsson, J.-Å. (1997).
Cloning
and expression of a novel mammalian thioredoxin. J. Biol. Chem. 272,
29362941.
Tagaya, Y., Maeda, Y., Mitsui, A., Kondo, N., Matsui, H., Hamuro, J., Brown,
N.,
Arai, K., Yokota, T., Wakasugi, H. and Yodoi, J. (1989). ATL derived factor
6
(ADF), an IL-2 receptor/Tac inducer homologous to thioredoxin; possible
involvement of dithiol-reduction in the IL-2 receptor induction. EMBO J. 8,
757764.
Tonissen, K. F. and Wells, J. R. E. (1991). Isolation and characterization of
human
thioredoxin-encoding genes. Gene. 102, 221-228.
Vanin, E. (1985). Processed pseudogenes: characteristics and evolution. Annu.
Rev.
Genet. 19, 253-272.
7
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.
8
Download
1.
2.
3.
4.
Science
Biology
Biochemistry
Genetics
Mamm_Genome yTrx1-2 + refs.doc
Interactions in genes and ecosystems
Genetics crossword
Modeling Gene Drive Systems
Variation Heredtiy & Genes SA
COSI-178A Computational Biology Instructor: Pengyu Hong Description:
Gene Drive
Exam-20121020_tfke38_englishversion
Computational Approaches to Gene Regulation
PAPERS
NJG1686MHH SP10T ANTENNA SWITCH GaAs MMIC
studylib © 2017
Report