BMC Genomics
BioMed Central
Open Access
Research article
Molecular cloning, genomic characterization and over-expression of
a novel gene, XRRA1, identified from human colorectal cancer cell
HCT116Clone2_XRR and macaque testis
Felix M Mesak1,2, Naoki Osada3,4, Katsuyuki Hashimoto3, Qing Y Liu5 and
Cheng E Ng*1,2
Address: 1Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, 503 Smyth Rd., Ottawa, ON, K1H 1C4, Canada, 2Faculty of Medicine,
University of Ottawa, 451 Smyth Rd., Ottawa, ON, K1H 8M5, Canada, 3Division of Genetic Resources, National Institute of Infectious Disease,
Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan, 4Current address: Department of Ecology and Evolution, University of Chicago, 1101 E57th
Str., Chicago, IL 60637, USA and 5Institute for Biological Sciences, National Research Council of Canada, 1200 Montreal Rd, M-54, Ottawa, ON,
K1A 0R6, Canada
Email: Felix M Mesak - Felix.Mesak@orcc.on.ca; Naoki Osada - nosada@uchicago.edu; Katsuyuki Hashimoto - khashi@nih.go.jp;
Qing Y Liu - Qing_Yan.Liu@nrc.ca; Cheng E Ng* - Cheng.Ng@orcc.on.ca
* Corresponding author
Published: 09 August 2003
BMC Genomics 2003, 4:32
Received: 10 April 2003
Accepted: 09 August 2003
This article is available from: http://www.biomedcentral.com/1471-2164/4/32
© 2003 Mesak et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
Abstract
Background: As part of our investigation into the genetic basis of tumor cell radioresponse, we
have isolated several clones with a wide range of responses to X-radiation (XR) from an
unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we
recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone,
HCT116Clone2_XRR. We have named this gene as X-ray radiation resistance associated 1 (XRRA1)
(GenBank BK000541). Here, we present the first report on the molecular cloning, genomic
characterization and over-expression of the XRRA1 gene.
Results: We found that XRRA1 was expressed predominantly in testis of both human and
macaque. cDNA microarray analysis showed three-fold higher expression of XRRA1 in macaque
testis relative to other tissues. We further cloned the macaque XRRA1 cDNA (GenBank
AB072776) and a human XRRA1 splice variant from HCT116Clone2_XRR (GenBank AY163836). In
silico analysis revealed the full-length human XRRA1, mouse, rat and bovine Xrra1 cDNAs. The
XRRA1 gene comprises 11 exons and spans 64 kb on chromosome 11q13.3. Human and macaque
cDNAs share 96% homology. Human XRRA1 cDNA is 1987 nt long and encodes a protein of 559
aa. XRRA1 protein is highly conserved in human, macaque, mouse, rat, pig, and bovine. GFPXRRA1 fusion protein was detected in both the nucleus and cytoplasm of HCT116 clones and
COS-7 cells. Interestingly, we found evidence that COS-7 cells which over-expressed XRRA1
lacked Ku86 (Ku80, XRCC5), a non-homologous end joining (NHEJ) DNA repair molecule, in the
nucleus. RT-PCR analysis showed differential expression of XRRA1 after XR in HCT116 clones
manifesting significantly different XR responses. Further, we found that XRRA1 was expressed in
most tumor cell types. Surprisingly, mouse Xrra1 was detected in mouse embryonic stem cells R1.
Conclusions: Both XRRA1 cDNA and protein are highly conserved among mammals, suggesting
that XRRA1 may have similar functions. Our results also suggest that the genetic modulation of
XRRA1 may affect the XR responses of HCT116 clones and that XRRA1 may have a role in the
response of human tumor and normal cells to XR. XRRA1 might be correlated with cancer
development and might also be an early expressed gene.
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Background
Treatment with X-radiation (XR) remains a major modality for eradicating cancer and it has been estimated that
half of all cancer patients undergo radiotherapy at some
point during their treatment [1]. At least partly because of
the relevance of radiotherapy to cancer treatment, the
genetic control of the expression of resistance, or conversely, sensitivity to XR has been investigated for several
decades. It is now generally accepted that there are several
components to the cellular response to XR stress: sensing
of the DNA damage, cell signaling pathways, and repair of
the DNA damage [2]. However, the molecular mechanisms of how cells detect, communicate, and cope with
XR-induced DNA damage are not well characterized.
There is strong evidence that ataxia telangiectasia mutated
(ATM) protein act as damage sensors and that their activation initiates mobilization and/or activation of repair
complexes [3]. In addition, the ATM kinase activity mediates the prompt induction of various signaling pathways
with some of these events leading to either cellular apoptosis or cell cycle arrest [4]. There is also evidence that
non-homologous end joining involving Ku86/Ku70 is
likely the major DNA double strand breaks rejoining
pathway following XR with a smaller role for homologous
recombination in post replicated DNA [5].
Many of the studies on the genetic basis of cellular
response to XR have benefited either from mutants that
have increased ionizing radiation (IR) sensitivity compared to the parental cell lines or from the study of human
genetic syndromes that are associated with increased IR
sensitivity (i.e. Fanconi's anemia, Bloom's, and Werner's
syndromes) [6,7]. By contrast, mutants or human genetic
syndromes associated with increased IR resistance (as
exemplified by Li Fraumeni) are rare [8]. Furthermore,
Bennett et al [9] recently identified 107 new loci that are
associated with response to γ-radiation in yeast. While
over 50% of these newly identified putative yeast genes
shared homology with human genes, it remains to be
established that the human counterparts of these genes
are actually associated with similar responses to γ-radiation.
In order to further investigate the genetic basis of tumor
cell radioresponse, we have isolated several clones with a
wide range of responses to X-radiation from an unirradiated human colorectal tumor cell line, HCT116. Interestingly, we found a cell clone, HCT116Clone2_XRR that was
resistant to both fractionated and single dose X-radiation
when compared to the parental cells. In addition, we also
isolated two other clones: (a) HCT116CloneK_XRS that was
more sensitive than the parental cells, and (b)
HCT116Clone10 that possessed similar X-radiation
responses as the parental cells, to both of these types of Xradiation (Qutob, S.S., Ng, C.E., to be published else-
http://www.biomedcentral.com/1471-2164/4/32
where).
Global
gene
analysis
between
cells
HCT116Clone2_XRR and HCT116Clone10 using cDNA microarray identified an unknown GenBank expression
sequence tag (EST) accession no. R40588, as one of the
spots on the 19,200 human cDNA microarray (University
Health Network, Ontario Cancer Institute, Toronto), that
was down regulated by approximately two-fold in
HCT116Clone2_XRR in comparison with HCT116Clone10 cells
(Qutob, S.S., Ng, C.E., to be published elsewhere).
Here we describe the in silico analysis of R40588 that
resulted in the identification of a novel gene, XRRA1. We
cloned the XRRA1 full-length cDNA from macaque testis
and an XRRA1 splice variant from HCT116Clone2_XRR cells.
We then extended our analysis to identify XRRA1 in several mammals (i.e. mouse, rat, pig and bovine). This
paper also reports the experiments to evaluate XRRA1
expression in human and macaque normal tissue/organs
and in various human cancer and normal cell lines, as
well as over-expression of GFP-XRRA1 fusion protein in
HCT116 clones and COS-7 cells. We did immunostaining
of Ku86 in the presence of over-expressed GFP-XRRA1 24
hours after XR treatment at 4 and 10 Gy of COS-7 cells.
This is the first report on the molecular cloning, genomic
characterization and over-expression of the novel XRRA1
gene.
Results
Genomic approaches revealed a novel XRRA1 gene
Using the EST sequence of R40588 as template, we performed ESTs "walking" either towards the 5' or 3' direction on both the sense and anti-sense strands that we refer
to as ESTs-based ORF assembling (EBOA). By EBOA, we
acquired a cDNA candidate that putatively contained
start-, stop-codon and poly-A signal. ESTs that overlapped
each other sequentially were as follows: GenBank accession nos R40588, BQ648278, BU187847, BE782795,
BI819448, BQ925455, and BM563944. Out of this ORF
candidate, we found 31 ESTs as primary sequences that
could be constructed into one potential novel gene in silico (Table 1, Fig. 1). We have named this gene as X-ray
radiation resistance associated 1 (XRRA1) (GenBank
accession no. BK000541). BLAST [10] analysis demonstrated that it was a novel gene.
We used human genomic clones RP11-147I3 and CMB98M21 (GenBank accession nos AP001992 and
AP000560) of chromosome 11q13.3 as template for
GrailEXP [11] analysis. The analysis predicted 11 exons
including a putative promoter region for the human
(Homo sapiens, Hs) XRRA1 gene (Fig. 2, Fig. 3A). These 11
exons spliced perfectly to create the mRNA for Hs XRRA1.
The exon-intron junctions, comprising the 5' splice donor
and 3' splice acceptor, follow Kozak consensus (Table 3)
[12]. Cloning of the 5' region of XRRA1 gene that
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Table 1: ESTs that were used to build Hs XRRA1 and Mm Xrra1.
Organism
EST
Nucleotides
Exons
Genomic Clone
Exons
Homo sapiens XRRA1 (1987 nt)
1. BM563944.1
2. BE780143.1
3. AW962535.1
4. BM921116.1
5. BQ925455.1
6. AI651142.1 (+/-)
7. AW448937.1 (+/-)
8. AI651143.1 (+/-)
9. AW197451.1 (+/-)
10. AW895676.1 (+/-)
11. BE836017.1 (+/-)
12. BF810439.1
13. AI652186.1 (+/-)
14. BE313013.1
15. BF891539.1 (+/-)
16. BI819118.1
17. BQ319609.1
18. BU620818.1 (+/-)
19. BU620757.1 (+/-)
20. AI057634.1
21. BQ051594.1
22. BE782795.1
23. AW189295.1
24. AI655334.1
25. AA854636.1
26. AI218588.1
27. AI457916.1
28. AW513519.1 (+/-)
29. AA961245.1 (+/-)
30. BU187847.1
31. AA953084.1 (+/-)
1. BI990168.1
2. AV046823.1
3. BE650319.1
4. BU610007.1
5. AA144723.1
6. AI647383.1
7. BE956083.1 (+/-)
8. BQ840215.1
9. BE631695.1
10. BQ175761.1 (+/-)
11. BE956870.1 (+/-)
12. AI449753.1 (+/-)
13. AW125246.1 (+/-)
1 – 822
1 – 233
1 – 175
1 – 107
1 – 348, 513 – 1066
225 – 348, 513 – 726
234 – 348, 513 – 726
245 – 348, 513 – 726
270 – 348, 513 – 726
513–609
513 – 609
513 – 589
526 – 726
583 – 1190
607 – 694
897 – 1755
1271 – 1388
1275 – 1976
1312 – 1911
1474 – 1981
1492 – 1979
1500 – 1898, 1939–1987
1502 – 1979
1546 – 1976
1556 – 1981
1593 – 1979
1594 – 1981
1614 – 1758
1680 – 1977
1796 – 1987
1845 – 1977
277 – 870
715 – 978
857 – 1380
1134 – 1459
1144 – 1396
1184 – 1372
1381 – 1903
1398 – 1649
1458 – 1604
1458 – 1903
1471 – 1903
1600 – 1901
1704 – 1903
1–7
1–2
1–2
1
1 – 3, 4 – 7
2 – 3, 4 – 6
2 – 3, 4 – 6
2 – 3, 4 – 6
2 – 3, 4 – 6
4–5
4–5
4–5
5–6
5–8
5–6
7 – 11
8 – 10
9 – 11
9 – 11
10 – 11
10 – 11
11
11
11
11
11
11
11
11
11
11
2–7
6–7
7–9
8 – 10
8 – 10
8–9
9 – 11
10 – 11
10 – 11
10 – 11
10 – 11
11
11
AP001992.4
AP000560.4
AP001324.4
1–5
3 – 11
5 – 11
NW_000328
1 – 11
Mus musculus Xrra1 (1903 nt)
comprised the first four exons from HCT116Clone2_XRR and
HCT116Clone10 revealed that Hs XRRA1 had those exons.
However, in addition to the one from HCT116Clone2_XRR,
we obtained one splice variant that lacked exon three and
ended on exon four (GenBank accession no. AY163836)
(Fig. 3A).
To test whether these exon-intron borders were actually
unique to XRRA1, we searched for possible XRRA1 homologues from other organisms. Thus far, we could only
retrieve Xrra1 containing genomic clones from mouse
(Mus musculus, Mm) (GenBank accession no.
NW_000328) and rat (Rattus norvegicus, Rn) (GenBank
accession no. AC127923). Mm Xrra1 gene is located on
chromosome 7. GrailEXP analyses showed that Mm Xrra1
gene also contained 11 exons. Alignment of mouse and
rat genomic sequences resulted in information for rat
exons, which also consist of 11 exons. Interestingly, all the
exon-intron borders of Hs XRRA1, Mm and Rn Xrra1
genes are well conserved (Table 3). We again employed
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Figure XRRA1
Human
1
cDNA sequences and its translation
Human XRRA1 cDNA sequences and its translation. The gene comprises 11 exons, and produces 559 aa protein. XRRA1 protein consists several motifs i.e. leucine rich repeats, PEST sequence, nuclear localization signal, and two sites of tyrosine phosphorylation (*). Poly A-signal, AATAA is underlined.
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Promoter
Figure 2 region of human XRRA1 gene as predicted by GrailExp [11]
Promoter region of human XRRA1 gene as predicted by GrailExp [11]. Two putative sites of TATA box were found at -506 and
-400 from the start codon. Predicted transcription factor binding sites revealed several members of bZIP family such as C/
EBPa, c-Jun, c-Fos, AP-1 and CREB.
EBOA to look for all possible ESTs of mouse and other
organisms (Table 2). We used the longest human EST containing XRRA1 cDNA sequence as template for the analysis. From the mouse dbEST, we obtained 13 ESTs as
primary sequences, which assembled Mm Xrra1 (GenBank accession no. BK000542). However, unlike the
GrailEXP analysis, Mm Xrra1 lacks exon one and part of
exon two. Nevertheless, these two approaches verified the
cDNA sequence for Mm Xrra1. We could not find any
publicly available ESTs for rat. Other ESTs found to be
homologues to XRRA1 were from pig (Sus scrofa, Sc),
horse (Equus caballus, Eq) and bovine (Bos taurus, Bt). We
assembled Bovine (Bt) Xrra1 (GenBank accession no.
BK000644) from 5 ESTs. The Bt Xrra1 gene is likely to
have 11 exons with truncated exons 4 and 8 (Table 2, Fig.
3A).
We used cDNA libraries from macaque (Macaca fascicularis, Mf) brain and testis to obtain Mf XRRA1. Since we
have created and sequenced these libraries, we were able
to look for the Mf XRRA1. We found only three testis
clones (QtsA-12093, 10089 and 20433) from 10,400 testis and 53,000 brain cDNAs that were homologous to Hs
XRRA1. One of the clones, QtsA-20433 (GenBank
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Figure 3
Representation
of the XRRA1 genomic structure
Representation of the XRRA1 genomic structure. (A) The XRRA1 gene spans 64 kb and comprises 11 exons. HCT116Clone2_XRR
cells contain 2 splice variants, one of which lacks exon 3 and is truncated. Other cDNA clones such as BC032794 lack exon 4,
AK074152 lacks exon 3–4 and possible partial deletion of exon 5, and AK056364 lacks exon 2–4 and 10. The gene is located
between genomic markers D11S916-D11S911 (80.1–84.2 cM) or at position 625.9 cR of NCBI RH Map. (B) The assembled Hs
XRRA1 comprises a putative promoter region, start/stop codons and poly-A signal, and predicted a protein with motifs such as
LRR, PEST, and NLS. Scale bar is multiplied by 100.
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Table 2: ESTs of Mm, Rn, Bt, Sc, and Eq Xrra1 with corresponding nucleotides and exons to Hs XRRA1
Organism
EST
Corresponding Nucleotides
of Hs XRRA1
Corresponding
Exons of Hs XRRA1
Genomic Clone
Exons
Mus musculus Xrra1 (1903 nt)
BI990168.1
AV046823.1
BE650319.1
BU610007.1
AA144723.1
AI647383.1
BE956870.1 (+/-)
312 – 776
660 – 1011
1017 – 1093, 1215–1414
1215 – 1383, 1400 – 1491
1215 – 1414
1215 – 1383, 1400 – 1491
1437 – 1491, 1529 – 1599, 1668 –
1737, 1933–1966
1436 – 1599
1529 – 1599
1529 – 1599, 1668 – 1737, 1933 –
1966
1529 – 1599, 1668 – 1737, 1933 –
1966
1668 – 1737, 1933 – 1966
1933 – 1966
2–7
6–7
7 – 8, 8 – 10
8 – 9, 10
8 – 10
8 – 9, 10
10, 11
NW_000328
1 – 11
AC127923.2
1 – 11
BQ840215.1
BE631695.1
BQ175761.1 (+/-)
BE956083.1 (+/-)
AI449753.1 (+/-)
AW125246.1 (+/-)
Rattus norvegicus Xrra1
(1909 nt)
Bos taurus Xrra1
(1759 nt)
Sus scrofa Xrra1 (970 nt)
Equus caballus Xrra1 (621 nt)
10 – 11
11
11
11
11
11
BM106446.1
1 – 422
1–4
BI540915.1
BE722755.1
BM254248.1
BE751905.1
BF442510.1
BF442490.1
BF444411.1
BI341438.1
542 – 703
797 – 1248
1308 – 1760
1366 – 1761
407 – 708
407 – 703
407 – 514, 1208 – 1269
581 – 810, 877 – 978, 1006 –
1079
1336 – 1575, 1658 – 1761, 1902 –
1970
5–6
7–8
9 – 11
9 – 11
4–6
4–6
4, 8 – 9
5 – 7, 7, 7 – 8
BM735121.1
accession no. AB072776) had the full-coding sequence
and showed strong identity to Hs XRRA1, sharing 96 and
95% identities in cDNA and protein sequences, respectively. SIM4 [13] and BLAST analysis showed that Mf
XRRA1 sequence dispersed properly into 11 exons on
human chromosomal band 11q13 (Fig. 3A).
XRRA1 is conserved among human, macaque, mouse, rat,
pig and bovine
Multiple alignments of both XRRA1 cDNA and protein
sequence from human, macaque, mouse, rat, pig, and
bovine Xrra1s showed a conservative molecule. Percent
identities at both the nucleotide and protein level among
these mammals resulting from two by two alignments are
listed in Table 4. As expected, phylogenetically, human
XRRA1 is closer to macaque XRRA1 and both of them are
closer to bovine and pig Xrra1s than murine and rat
Xrra1s. We failed to find possible XRRA1 homologues in
other organisms ranging from microorganisms, plants,
and animals. The latter included organisms whose
9 – 11, 11, 11
genomes have been sequenced completely such as yeast,
plasmodium, fruit fly, zebra fish and fugu.
The Hs XRRA1 gene produces a novel 559 aa protein (Fig.
3B). The XRRA1 protein contains two possible sites of leucine-rich repeats (aa 20–41 and aa 138–164), a PEST
sequence at aa 247–264, two tyrosine phosphorylation
sites at aa 81 and aa 142, and bipartite nuclear targeting
sequences are found at aa 357–373 and aa 485–501.
Besides human, Mf XRRA1, Mm, Rn, and Bt Xrra1s retain
the above-mentioned motifs.
XRRA1 responded differentially to XR in clones of varying
radiation responses
The RT-PCR results confirmed earlier observation from
our cDNA microarray analysis that unirradiated
HCT116Clone2_XRR cells had lower basal expression of
XRRA1 than unirradiated HCT116Clone10 cells (Fig. 4,
Table
5).
However,
XRRA1
expression
in
HCT116Clone2_XRR cells increased almost immediately
(~10 minutes) after XR and decreased thereafter. By
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Table 3: Exon-intron junctions of human (Hs) XRRA1, murine (Mm) Xrra1 and rat (Rn) Xrra1
Exon
No.
1
2
3
4
5
6
7
8
9
10
11
Exon Size
(bp)
5' Splice Donor
128
133
133
219
206
206
43
44
44
127
126
126
96
94
94
123
116
116
351
324
324
202
196
199
117
116
116
108
106
106
484
445
445
GGCTCAGGAGgtaaagccataa
GGCTCAGGAGgtattgtcacag
GGCTCAGGAGgtattatcacag
GACCGGACAGgtgagtgacacc
GACCGCACAGgtgagctatggc
GACCGCACAGgtgagcccgccc
AACCTCAGAGgtaggaactgac
AACCTCAGAGgtaggaggacct
AACCTCAGAGgtaggaggaacc
CTACAACAAGgtgactttctgc
CTACAACAAGgtaactgctttg
CTACAACAAGgtaaactgcttt
CATACACGAGgtatagtgcccg
CACACACGAGgtatggtggcca
CACACACGAGgtatggtggcca
ATCATGGAAGgtagggcttcca
GTCACGGAAGgtaagccttcca
ATCGCGGAAGgtaagccttcca
CCTGACCCAGgtacctgtatcc
CCTGACCCAGgtgacagccccc
CCTGACCCAGgtgacagccccc
GAGCCAAAGGgtatgtgagggc
GAGCCAAAGGgtatgggattaa
GAGCCAAAGGgtacgggattaa
AGAGAAACGGgtaaacatccag
GGAGAAGAGGgtaagcacttca
AGAGAAGAGGgtaagcacttca
GCTCCACTAGgtacggctctgc
GCTCCACTGGgtatggtgccag
GCTCCACTGGgtatggtgccag
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Hs XRRA1
Mm Xrra1
Rn Xrra1
Intron
No.
1
2
3
4
5
6
7
8
9
10
Intron Size
(bp)a
3' Splice Acceptor
ttgcactcacagGGAGGCATCT
ctgtgcccacagGGAGGCATCC
ctgtgcccacagGGAGGCATCT
ccttaattgaagACTGAAGAAG
ccttaattgaagACTGAAGAAG
tcttaattgaagACTGAAGAAG
cttctgtttcagAGGTTGTGTT
tttctgtttcagAGGTTGTGTT
tttatgtttcagAGGTTGTGTT
cttctttctaagACAACCAAGA
tttcctctgaagACAGCCAAGG
ttccctcctaagACAACCAAGG
cattcattagagATCGCAAAAG
cattcattagagATCGCAAAAG
tgcactttccagATCGCAAAGG
tcatggcaccagGGGTCCCTCC
ccttcataccagGGATCCACAC
ccctcacaccagGGATCCCACC
tctccatgacagGTGAAAAGCG
tctctcatccagGTGAAGACCT
tctctcatacagGTGAAAACCT
tcctgggcacagGTGAGTGAAC
ttcctggcacagGTGAATGAGC
ttcctggcacagGTGAATGAGC
ttcttgccctagGGAATCCAGA
tgcatcctctagGCATCCAGAA
tgcatcctccagGCATCCAGAA
accttctgccagGCCCAGAGAA
tgcatcctctagGCCGGGAGGA
cgtttgtgccagGTTGCGAGGA
cttttcctgcagGTGCTGTCCT
tcattcctgtagGCACTGTGTT
tcatccctgtagGCGCTGTGTG
687
685
43215
4118
3696
3706
7073
4393
779
736
2622
2256
2839
1421
788
594
207
169
397
294
-
aThe intron sizes of Rn Xrra1 gene could not be determined since the sequencing of rat genomic clone CH230-188L13 (GenBank AC127923)
containing the gene was in progress. Nevertheless, their splicing sites can be identified, thus the exon sizes are also known.
Table 4: Percent identities of XRRA1 cDNA and protein among several mammals
Percent Identity (cDNA)*
Percent Identity
(Protein)*
ORGANISM
Hs XRRA1
Mf XRRA1
Mm Xrra1
Rn Xrra1
Sc Xrra1
Bt Xrra1
HsXRRA1
Mf XRRA1
MmXrra1
Rn Xrra1
Sc Xrra1
Bt Xrra1
95.0
71.8
69.6
77.7
68.3
96.0
74.1
71.7
79.4
69.5
75.9
76.2
90.2
67.7
62.0
75.9
76.3
93.6
62.6
61.6
83.4
83.6
74.6
74.7
70.1
76.8
77.2
70.9
70.1
86.4
-
*Hs, Homo sapiens; Mf, Macaque fascicularis; Mm, Mus musculus; Rn, Rattus norvegicus; Sc, Sus scrofa; Bt, Bos taurus.
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BMC Genomics 2003, 4
contrast, HCT116Clone10 cells down regulated XRRA1
expression approximately ten minutes following XR.
Interestingly, XRRA1 expression in HCT116CloneK_XRS cells
was initially similar to HCT116Clone10 cells but regained
the basal levels by 24 hours following XR.
XRRA1 was expressed predominantly in the testis in
human and macaque
We carried out DNA microarray where PCR-amplified
QtsA-20433 (Mf XRRA1) was included. Because we found
that Mf XRRA1 was more commonly present in testis than
in brain cDNA libraries, we further investigated whether
Mf XRRA1 was predominantly expressed in the testis. The
ratio of Mf XRRA1 expression in testis to that in mixed tissues was 3.02. We also compared the expression of XRRA1
between human and macaque testis. The ratio of XRRA1
expression in human testis to macaque testis was 1.04. In
addition, we examined Hs XRRA1 expression from various normal human tissues/organs. We found that Hs
XRRA1 was expressed predominantly in testis followed by
prostate and ovary (Fig. 4, Table 5). Other tissues/organs
such as peripheral blood leukocyte, spleen, thymus, small
intestine and colon demonstrated low expression of Hs
XRRA1.
XRRA1 was expressed ubiquitously in various types of
cancer cells
We evaluated the expression of Hs XRRA1 in various cancer cell lines (neuroblastoma, glioma, breast, lung, leukemia, renal, ovarian, prostate, another colorectal). XRRA1
was present in all of those cells, although the expression
level was variable (Fig. 4, Table 5). We also detected
XRRA1 expression in immortalized normal fibroblast
AG1522 and COS-7 cells, as well as in a dermal microvascular endothelial cell line (HDMEC). Surprisingly, besides
mouse fibrosarcoma cells and tumor biopsy from a
mouse model of fibrosarcoma, we were also able to detect
Mm Xrra1 expression in pluripotent cells such as murine
embryonic stem cells R1 (Fig. 4, Table 5).
Over-expression of GFP-XRRA1 fusion protein was only
achieved transiently and showed nucleo-cytoplasmic
protein distribution
To understand further the possible function of the XRRA1
gene, we fused the XRRA1 cDNA immediately downstream of a green fluorescent protein (GFP) cDNA. We
over-expressed the GFP-XRRA1 in HCT116 clones, but
after 48 hours post-transfection the fluorescence clearly
decreased both in the number of cells and intensity of fluorescence. We obtained similar results when we overexpressed GFP-XRRA1 in COS-7 cells. After three weeks
selection in 500 µg/ml G418, very few COS-7 cells
remained fluorescent (Fig. 5). Only transient expression
of the GFP-XRRA1 was observed in both types of cells. The
fluorescence of the GFP-XRRA1 did not show an exclusive
http://www.biomedcentral.com/1471-2164/4/32
nuclear localization (although the predicted XRRA1
protein contains NLS motif). Instead, the fluorescence
appeared in most cells to be nucleo-cytoplasmic (Fig. 5).
Over-expressed COS-7 cells with GFP-XRRA1 might
modulate Ku86 expression
Ku86 was exclusively localized in nuclei with or without
XR (Fig 6). The over-expression of GFP-XRRA1 abolished
nuclear immunostaining of Ku86 in COS-7 (Fig 6A).
Immunostaining of α-Tubulin, as additional control for
the experiment, did not change in the presence of GFPXRRA1 (Fig 6B). The secondary antibody conjugated with
Cyanine-3 (Cy3) was used for both Ku86 and α-Tubulin
immunostaining.
Discussion
EBOA and in silico analysis helped us to identify a novel
gene XRRA1 from a previously unknown EST R40588 not
only in human, but also in mouse, bovine, and rat.
Recently, several clones that contained Hs XRRA1 spliced
variants have been deposited to GenBank database by
others. Hs XRRA1 clone from testis (GenBank accession
no. BC037294) lacked exon 4; the one from spleen (GenBank accession no. AK074152) lacked exons 3, 4 and a
possible partial deletion of exon 5, and the one from NT2
cells (GenBank accession no. AK056364) lacked exons 2,
3, 4, and 10 (Fig. 3A). The testis clone with GenBank no.
BC037294 did not appear to have a proper open reading
frame. EBOA and GrailExp methods failed to produce a
start site further upstream of Hs XRRA1 (GenBank no.
BK000541).
Surprisingly, when we searched from approximately
63,400 macaque brain and testis cDNAs, we found only
three testis clones that were homologous to Hs XRRA1,
suggesting that the Mf XRRA1 gene is expressed only at a
very low level in brain. The Mf XRRA1 is highly
homologous with Hs XRRA1. Because the Hs XRRA1 gene
consists of 11 exons, it is likely that the Mf XRRA1 gene
also contains 11 exons (see Fig. 3A). In general, the XRRA1
gene seems to be highly conserved among mammals (i.e.
human, macaque, mouse, rat, pig, and bovine), suggesting that it has similar function(s). Moreover, we were unable to find any convincing evidence of XRRA1 gene from
fully sequenced genomic DNA of fish, fly, worm, yeast
and microbes. This suggests that XRRA1 might be specific
for, or evolutionary-enriched in, mammals. A specific
role(s) for XRRA1 in mammals was further supported by
the observation that the predicted protein sequences for
motifs such as LRR, PEST, and NLS, as well as Tyr
phosphorylation sites, are preserved among the abovementioned mammals. Additionally, these motifs suggested that the XRRA1 protein may interact dynamically
with other proteins, or might be subjected to rapid degra-
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cells,
Figure
RT-PCR
and4ofnormal
XRRA1tissues/organs
and a house-keeping gene G3PDH from HCT116 clones treated with X-radiation, various cancer and normal
RT-PCR of XRRA1 and a house-keeping gene G3PDH from HCT116 clones treated with X-radiation, various cancer and normal
cells, and normal tissues/organs. The same set of primers was used to amplify corresponding mRNA from mouse embryonic
stem cells and fibrosarcoma cells/tumor.
dation via the proteasomal pathway, or might suggest a
possible role for XRRA1 in cellular signaling.
Although the protein contains a NLS motif, it appears that
XRRA1 is not exclusively located in the nucleus, as shown
by the over-expression of the GFP-XRRA1 fusion protein.
HCT116 clones and COS-7 cells containing the fusion
protein (either with full length or truncated XRRA1 protein) had lost the auto-fluorescence. This suggested that
the protein concentration above the basal expression levels of XRRA1 might be lethal to the cells.
It is noteworthy that the expression of XRRA1 in HCT116
clones was variably affected by 4 Gy XR. The results sug-
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Table 5: Differential expression of human XRRA1 in XR-treated HCT116 clones, in normal tissues/organs and cells, as well as in various
cancer cell types.
Cells/Tissues/Organs
Relative Expression Level (Normalized
against G3PDH)*
HCT116Clone2_XRR
+++
HCT116Clone10
++++
HCT116CloneK_XRS
++++++
HCT116Clone2_XRR
HCT116Clone2_XRR M10
HCT116Clone2_XRR H6
HCT116Clone2_XRR H24
HCT116Clone10
HCT116Clone10 M10
HCT116Clone10 H6
HCT116Clone10 H24
HCT116CloneK_XRS
HCT116CloneK_XRS M10
HCT116CloneK_XRS H6
HCT116CloneK_XRS H24
Testis
Prostate
Ovary
Leukocyte
Spleen
Thymus
Small Intestine
Colon
AG1522
COS-7
HDMEC
MCF7
MDA MB435
U251MG
SKMEL-3
HT144
HTB56
A594
Daoy
SKNA
SHSY
A2780CP
OCCL
OV2008
C13
433
SKOV3
HEY
429
PC-3
DU145
SN12C
HT29
K-562
Mm TM28
Mm FS-TM28
Mm ES-R1
+++
++++
++
+
++++
+
+
+
++++++
+
+
++++
++++++++
+++++
+++++
++
+
++
++
+
++++
+++
++++
+
+++++
+
+
+
+++
++++
++++
+++
++++
+
++++
++++
+++
+
++
++++
+++
+
+++++
+
+
+++++
+++
+
++
Cell/Tissue/Organ type; Cancer Type
Colorectal, more resistant to X-radiation than
HCT116
Colorectal, similar radiation response to
HCT116
Colorectal, more sensitive to X-radiation than
HCT116
Without X-radiation
10 Minutes after X-radiation
6 Hours after X-radiation
24 Hours after X-radiation
Without X-radiation
10 Minutes after X-radiation
6 Hours after X-radiation
24 Hours after X-radiation
Without X-radiation
10 Minutes after X-radiation
6 Hours after X-radiation
24 Hours after X-radiation
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal fibroblast (immortalized)
Normal fibroblast (immortalized)
Normal microvascular endothelial (primary)
Breast cancer
Breast cancer
Glioma
Melanoma
Melanoma
Lung cancer
Lung cancer
Neuroblastoma
Neuroblastoma
Neuroblastoma
Ovarian cancer, resistant to cisplatin
Ovarian cancer
Ovarian cancer
Ovarian cancer
Ovarian cancer
Ovarian cancer
Ovarian cancer
Ovarian cancer
Prostate cancer
Prostate cancer
Renal cancer
Colorectal cancer
Leukemia
(Murine) Fibrosarcoma
(Murine) Fibrosarcoma biopsy
(Murine) Embryonic Stem Cell
*+, very low; ++, low; +++ moderate; >++++, high.
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Figure 5
GFP-XRRA1
fusion protein in COS-7 and HCT116 clones
GFP-XRRA1 fusion protein in COS-7 and HCT116 clones. A, B, and C are transfected COS-7 cells. D, E, and F are transfected
HCT116Clone2_XRR cells. Control with GFP cassette alone is on panel A and D. Expression of GFP-truncated XRRA1 is shown in
panels B and E. Expression of GFP-full length XRRA1 is shown in panels C and F. Transfected COS-7 cells were examined by
fluorescence microscopy after 3 weeks of G418-selection. Expression of GFP-XRRA1 fusion protein was detected only transiently in HCT116 clones. The leftmost panel is a phase-contrast picture of the cells. The middle panel is green fluorescence
shown with FITC filter. The rightmost panel is an overlay of the two previous panels. Magnification used was 400×.
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Figure 6
Immunocytochemistry
of COS-7 cells over-expressing GFP-XRRA1 with Ku86 (A) and α-Tubulin (B)
Immunocytochemistry of COS-7 cells over-expressing GFP-XRRA1 with Ku86 (A) and α-Tubulin (B). (A) Ku86 (red fluorescence) was localized in nuclei. Nuclear Ku86 immunostaining was eliminated (asterisk) in the presence of GFP-XRRA1 (green
fluorescence), but not with the GFP alone. (B) Neither GFP nor GFP-XRRA1 changed the immunostaining of α-Tubulin (red
fluorescence). DAPI-stained nuclei are shown in blue fluorescence. Magnification used was 1000×.
gested that the XRRA1 gene might be involved in the
immediate response following XR and might further be
linked to XR resistance and/or sensitivity. Thus the return
of XRRA1 to almost pre-irradiated basal levels by 24 hours
post-irradiation in radiosensitive HCT116CloneK_XRS cells
(but not in the other two clones) might be necessary for
the subsequent manifestation of enhanced sensitivity to
XR by this clone. One possibility is that the XRRA1 gene
may play a role either in the expression of cell death and/
or signaling of DNA damage following XR stress. Therefore, genetic modulation of the expression of XRRA1 in
these clones may, specifically lead to either enhanced or
decreased XR-induced lethality, and, generally, may be
relevant for determining cellular response to XR in both
tumor and normal cells.
In mammalian cells, DNA double strand breaks caused by
XR are preferentially repaired by non-homologous end
joining (NHEJ) mechanism. The major molecules
involved in the NHEJ pathway are heterodimeric DNA
end-binding complexes Ku86/Ku70 [14]. Therefore, we
used Ku86 as the first candidate to test whether XRRA1 is
involved in the modulation of the NHEJ pathway. The
immunocytochemistry study on both HCT116Clone2_XRR
and COS-7 cells showed that there was no significant
modulation of Ku86 with XR. However, COS-7 cells overexpressing GFP-XRRA1 were devoid of nuclear Ku86. This
phenomenon suggested that over-expression of XRRA1
might have resulted in the down-regulation of Ku-86. Further studies are required to determine whether there is a
negative correlation between XRRA1 and Ku86 expressions during cellular response to XR.
The varying expression of XRRA1 following XR described
above could be related to the role of transcription factors.
In fact, we were able to detect a possible region between
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BMC Genomics 2003, 4
nt -506 and -400 from the start codon for transcription
factor binding sites that regulate the expression of XRRA1.
These factors were C/EBPα, HSF-1, c-Jun, c-Fos, AP-1, and
CREB. Interestingly, most of the previously mentioned
transcription factors are members of the bZIP superfamily. The latter is associated with various functions,
including mediating G1 arrests [15,16], regulating
responses against environmental stresses including IR and
UV [17], acting as an immediate-early response factor
[18], controlling wide range plasticity processes [19], and
serving as a promoter element that mediates transcriptional activation in response to increased levels of intracellular essential secondary messenger cAMP [20]. Thus,
putative binding sites for these transcription factors further strengthen the suggestion that the expression of
XRRA1 was rapidly affected after XR treatment.
XRRA1 was expressed predominantly in the testis in both
human and macaque. It may be relevant to note that the
testis is generally regarded as one of the most sensitive
organs to XR [21]. Thus upregulation of XRRA1 in an XRsensitive organ would appear to be generally consistent
with our observation that it is downregulated in the XRresistant HCT116Clone2_XRR cells. Because there was comparable expression level of XRRA1 in both human and
macaque, it suggests that there is a functional necessity for
XRRA1 in mammalian testis (i.e. XRRA1 might serve as a
testis-specific molecule). By contrast, the low expression
of XRRA1 in healthy colon and relatively much stronger
expression of XRRA1 in HCT116 cells, may suggest an
upregulation of this gene in cancer development. The
ubiquitous expression of XRRA1 in numerous cancer cells
and in immortalized normal cells might be indicative of a
role of this gene in tumor development.
Interestingly, from the SAGEmap, we identified three tags
that specifically represent XRRA1 anchored with restriction enzyme NlaIII. The tags were TATTCAGGGG, ACCTGGTGCC, and GAATCAAGTG. SAGE libraries where
XRRA1 was less than 20 tags per million were excluded.
We found that the XRRA1 was present in mammary gland
epithelium ductal in situ carcinoma, normal cerebellum,
normal gastric body epithelium, well differentiated oligodendroglioma, pancreatic epithelium ductal adenocarcinoma, metastasis mammary gland carcinoma, ovarian
clear cell poorly differentiated carcinoma, brain glioblastoma multiform cell line, ovarian serous adenocarcinoma,
mammary gland ductal in situ high grade carcinoma, prostate carcinoma, brain juvenile ependymoma, and medulloblastoma cerebellum. The foregoing data further
suggested that XRRA1 might be correlated with carcinomas in breast, brain, prostate, pancreas and ovary. Finally,
the expression of Mm Xrra1 expression in mouse embryonic stem (ES) cells (R1) as well as in mouse fibrosarcoma
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cells and in fibrosarcoma biopsy suggest that XRRA1
might function as an early expressed gene.
Conclusions
XRRA1 is a novel molecule that is expressed selectively in
normal healthy tissues/organs. It is expressed at relatively
higher levels in primary sex organs such as testis (of both
human and macaque), prostate and ovary. The expression
of XRRA1 in normal proliferative cells, embryonic stem
cells, and various tumor cells is also generally high and
comparable to testicular expression of XRRA1. These findings suggest that XRRA1 expression may be important for
cell proliferation, development, and differentiation as
well as carcinogenesis. In regard to XR, we found that
XRRA1 expression was rapidly modulated after treatment,
suggesting the potential involvement of this gene/protein
in the manifestation/expression of radioresistance/sensitivity by tumor cells such as human colorectal cancer cells.
Over-expression of GFP-XRRA1 fusion protein was
located in both nucleus and cytoplasm of HCT116 clones
or COS-7 cells. A possible correlation with NHEJ pathway
for repairing DNA double strand break after XR is shown
by the lack of Ku86 immunostaining in COS-7 cells that
over-expressed the GFP-XRRA1. Further experiments to
determine the function of XRRA1 in DNA damage sensor,
repair and apoptosis following radio- and or chemotherapy are ongoing. Also the function of XRRA1 in
carcinogenesis as well as embryogenesis and reproduction
biology needs to be elucidated.
Methods
Normal/Cancer cell cultures
Clones of colorectal cancer cells (HCT116Clone2_XRR,
HCT116Clone10, and HCT116CloneK_XRS), normal fibroblast
cells (COS-7 and AG1522), breast cancer cells (MCF-7
and MDA MB435), glioma cells (U251 MG), melanoma
cells (SKMEL-3 and HT144), lung cancer cells (HTB56
and A549), ovarian cancer cells (A2780CP and A2780S),
prostate cancer cells (PC-3 and DU145), leukemia cells
(K-562), colorectal cancer cells (HT29), renal cancer cells
(SN12C), and mouse fibrosarcoma cells (TM28) were
grown in DMEM/Ham' s F12 1:1 mix (Wisent Inc., St.
Bruno, QC, Canada) supplemented with 10% fetal bovine
serum (FBS) (Wisent Inc.) and 15 mM HEPES in a humidified atmosphere of 95% air, 5% CO2 at 37°C. Various
individuals from the ORCC, Ottawa, Canada, kindly provided us with the cells. They were: J.C. Bell (COS-7, PC-3,
DU145, K-562, HT29, and SN12C cells), C.L. Addison
(MCF-7 and MDA MB435 cells, neuroblastoma cells
(Daoy, SKNA, and SHSY), normal human dermal microvascular cells (HDMEC)), HC Birnboim (TM28 cells), B.
Vanderhyden (ovarian cancer cells OCCL, OV2008, C13,
SKOV3, 433, HEY, and 429), and M. McBurney (mouse
embryonic stem cells R1). H.C. Birnboim also donated a
C57BL/6 mouse with a solid fibrosarcoma (TM28)
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BMC Genomics 2003, 4
growing in the flank. A. Gatignon (Lady Davis Insitute,
Montreal, Canada) kindly provided the U251 MG cells.
Clones of HCT116, AG1522, SKMEL-3, HT144, A2780CP,
and A2780S cells were from our own laboratory collection
(C.E. Ng, ORCC, Ottawa, Canada).
Cloning and sequencing of Hs XRRA1 splice variant
A specific primer pair was created to amplify the 5' region
of the XRRA1 cDNA. They were 5'-GCGCTGGAGACACTGATGCTGG ATGACAAC-3' and 5'-GGCCAGGCTAAGGTATCTCAGCTCTGGG-3'. These primers generated a
product of 452-bp that comprised the first 4 exons. Total
RNA from HCT116Clone2_XRR and HCT116Clone10 cells were
used as template for RT-PCR. PCR fragments were gel
purified using Gel Extraction Kit (Qiagen Inc., Mississauga, ON, Canada) and cloned into pTZ57R plasmid
using InsT/Aclone PCR Product Cloning Kit (MBI Fermentas, Burlington, ON, Canada). Sequencing of the clones
was performed by DNA Sequencer model 4000L
according to the manufacturer' s protocol (LI-COR Biosciences, Lincoln, NE, USA) using M13 primer.
Construction of macaque cDNA libraries and DNA
sequencing of their cDNA inserts
Oligo-capped cDNA libraries were made from macaque
brain and testis. cDNAs were isolated according to the
method described previously [22,23]. The 5'-end
sequences of the clones were sequenced using ABI 3700
sequencer (Applied Biosystems, Tokyo, Japan) and categorized using DYNACLUST (Dynacom Co., Chiba, Japan)
based on a BLAST search against the GenBank database.
The entire sequences of clones were determined by the
primer walking method with an ABI PRISM BigDye Terminator Sequencing kit (Applied Biosystems) according to
the manufacturer' s instructions.
Construction of plasmid expressing Mf XRRA1 fused to
GFP
Macaque cDNA clone of QtsA-20344 was found from the
cDNA libraries to contain Mf XRRA1 transcript. A 1709-bp
XhoI fragment of Mf XRRA1 was removed from pME18SFL3 and sub-cloned into XhoI sites of pEGFP-C1 (BD Biosciences, Mississauga, ON, Canada), immediately downstream of the GFP sequence (pFM1709). To create a
truncated Mf GFP-XRRA1 (pFM584), 1.1-kb EcoRI fragment was removed from pFM1709 leaving a 584-bp of the
5' XRRA1 cDNA fused with GFP.
Transfection of GFP-XRRA1 into HCT116Clone2_XRR,
HCT116Clone10, HCT116CloneK_XRS, and COS-7 cells
HCT116 clones and COS-7 cell were transfected with a
mixture of pFM584, pFM1709 or pEGFP-C1 and lipid
complexes FuGENE 6 (Roche Diagnostics, Laval, QC,
Canada) transiently or stably. Briefly, HCT116 clones and
COS-7 were grown on cover slips in a 6-well plate and
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reached 60–70% confluences before transfection. The
ratio of FuGENE 6 and pure plasmid was 6:1 in serum free
medium. G418 (Sigma-Aldrich, Oakville Ltd., ON,
Canada) at a concentration of 400 µg/ml was used for
selection. Cells were fixed with 3.7% formaldehyde in PBS
for 10 min, and then washed three times with 1x PBS.
Coverslips were mounted upside down onto slides with
Dako Fluorescent Mounting Medium (DAKO, Carpinteria, CA, USA). Visualization was performed using a fluorescence microscope Axioskop 2 MOT (Carl Zeiss
MicroImaging Inc., Thornwood, NY, USA). Images of cells
were captured with a CCD camera fitted with a FITC filter.
Immunocytochemistry
Transfected COS-7 and HCT116Clone2_XRR cells with
pFM1709 or pEGFP-C1 were grown on cover slips until
they reached 70–80% confluency before being treated
with 4 or 10 Gy XR. Cells were fixed in 2% formaldehyde
in 1x PBS for 15 min 24 h after XR treatment. Cells were
then permeabilized with 0.2% Triton X-100 for 10 min.
Ku86 antibody was obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. The secondary antibody
was conjugated with Cy3 (Amersham Biosciences Corp,
Baie d' Urfe, QC, Canada). Controls for the immunostaining were the secondary antibody alone, and control for
the experiment was α-Tubulin antibody that was obtained
from Calbiochem, San Diego, CA, USA.
RT-PCR
Total RNAs of normal/cancer cells were extracted using
RNAeasy kit (Qiagen Inc.) according to the manufacturer'
s instructions. TRIZOL Reagent (Invitrogen Inc., Burlington, ON, Canada) was used to isolate RNA of mouse fibrosarcoma tumor biopsy. RNAs of human peripheral
blood leukocyte, spleen, thymus, small intestine, colon,
ovary, testis and prostate were purchased from BD Biosciences. First Strand cDNA Synthesis kit (MBI Fermentas)
was used to create first strand cDNA from RNA samples. A
specific primer pair that amplified ~1 kb house keeping
gene
glyceraldehyde-3-phosphate
dehydrogenase
(G3PDH) was used as the control. The primers were 5'TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CATGTAGGCCA TGAGGTCCACCAC-3'. Conditions of PCR
were as follows: 94°C for 30 s, 61°C for 15 s, and 72°C
for 40–60 s for 35 cycles. The PCR products were then separated on 1.5% agarose gels, stained with ethidium bromide, and visualized with the gel documentation imager
Epi Chemi II (UVP Inc., Upland, CA, USA). Densitometry
of bands were analyzed using Labworks™ software (UVP).
Relative expression of XRRA1 was calculated as percentages of the mean raw densities of grayscale level of XRRA1
to G3PDH.
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X-radiation of HCT116Clone2_XRR, HCT116Clone10, and
HCT116CloneK_XRS cells
Clones of HCT116 cells were cultured at 1 × 105 cells/ml.
Cultures reached 70–80% confluence before being treated
with XR. Each clone was treated with a single dose of 4 or
10 Gy using a 250 kVp X-ray unit (Pantak, CT, USA) at a
dose rate of 150 cGy/min. Total RNAs were collected 5
minutes, 6 hours and 24 hours following the XR. Specific
primers for Hs XRRA1 as mentioned above were used.
G3PDH was used as control. Relative expression of XRRA1
to G3PDH was evaluated
cDNA microarray of Mf XRRA1 from testis versus mixed
tissues or human testis
512 fully sequenced testis cDNA clones were amplified
using 5'-CTTCTGCTCTAAAAGCTGCG-'3 as a forward
primer and 5'-CGACCTGCAGCTCGAGCACA-'3 as a
reverse primer. Successful amplification was confirmed by
agarose gel electrophoresis. Approximately 300 µg /ml
DNA in 2 × Solution-T reagent (Takara Bio Inc., Shiga,
Japan) were printed on duplicate glass-slides with a GMS
417 arrayer (Genetic MicroSystems, Woburn, MA, USA).
RNA was isolated with TRIZOL Reagent (Invitrogen K.K.,
Tokyo, Japan) and purified with Oligo-Tex (Takara Bio
Inc.). Macaque testis RNA was labeled with Cy3-dUTP
(Pharmacia K.K., Tokyo, Japan). A mixture of macaque
RNA was also obtained from 10 tissues: brain, heart, skin,
liver, spleen, renal, pancreas, stomach, small intestine,
large intestine, and labeled with Cy5-dUTP (Pharmacia).
We mixed equal amount of RNA of these ten tissues. In a
separate experiment, RNA from human testis (BD Biosciences) was labeled with Cy5-dUTP. Both labeled RNAs
were co-hybridized to DNA spots. After the hybridization
and washing procedure, slides were scanned with ScanArray (PerkinElmer Life Sciences Co., Tokyo, Japan). Beta
actin and/or total signal intensity was used as the control
for hybridization. The methods and results of DNA microarray have been deposited in the Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo with the accession
numbers GPL206, GSM2387, and GSM2388.
ESTs-based ORF assembling (EBOA) and other in silico
analysis
In order to elucidate what gene R40588 might represent,
we employed EBOA to obtain the full-length cDNA containing start-, stop-codon, and poly-A signal or a complete
open reading frame (ORF). The sequence of R40588 was
used as a template to do BLAST search for all possible
overlapping ESTs from human dbEST http://
www.ncbi.nlm.nih.gov/dbEST. These retrieved ESTs,
either sense-antisense or 5'- and 3'-direction, were
selected and were again used as template for searching
other overlapping ESTs. To confirm the ORF candidates,
(i) we carried out a BLAST search against full-length
cDNAs that were available on public databases, and (ii)
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genomic sequences comprising the ORF candidates were
analyzed with GrailEXP version 3.3 http://comp
bio.ornl.gov/grailexp[11]. Multiple alignments were done
according to ClustalW method [24]. Transcription factors
and their binding sites were predicted by employing the
Alibaba2 version 2.1 program http://www.gene-regula
tion.com/pub/programs.html that used the TRANSFAC
database http://transfac.gbf.de/TRANSFAC[25]. Possible
tissues/organs expression of XRRA1 was explored from the
SAGEmap http://www.ncbi.nlm.nih.gov/SAGE[26]. Protein motifs were searched using software available on
these web sites: http://www.expasy.ch/prosite[27], http://
www.at.embnet.org/embnet/tools/bio/PESTfind[28], and
http://www.cbs.dtu.dk/services/NetPhos[29].
Data deposition
The sequences reported in this paper have been deposited
in the DDBJ/EMBL/GenBank databases under the accession numbers BK000541, AY163836, AB072776,
BK000542, and BK000644, respectively. HUGO Gene
Nomenclature Committee and MGD Nomenclature
Committee have approved the names and symbols of the
XRRA1 and Xrra1 genes.
List of abbreviations
Bt, Bos taurus (bovine); EBOA, ESTs-based ORF assembling; EST, expressed sequence tag; Hs, Homo sapiens
(human), Gy, Gray; Macaca fascicularis (macaque); Mm,
Mus musculus (mouse); ORF, open reading frame; Rn, Rattus norvegicus (rat); SAGE, serial analysis gene expression;
Sc, Sus scrofa (pig); XRRA1, X-ray radiation resistance associated 1, XR, X-radiation.
Authors' contributions
FMM carried out the in silico analysis including the multiple sequence alignments, cloning of the splice variant, XR
assay, cells cultures, RT-PCR of XR treated, various
human/mouse cancer and normal cells/tissues/organs,
construction and expression of the fusion protein, immunocytochemistry study, and drafted the manuscript. NO
carried out the construction and sequencing of macaque
testis and brain cDNA libraries, and cloning and cDNA
microarray of Mf XRRA1. KH was funded to carry out the
construction of cDNA libraries and cDNA microarray of
macaque brain and testis, conceived of the Mf XRRA1
study and the in silico analysis. QYL contributed technical
expertise to the cDNA microarray analysis of HCT116
clones. CEN is the Principal Investigator and was funded
to carry out the cDNA microarray of HCT116 clones,
supervised this work, provided the technical radiobiological expertise and contributed to the writing of this manuscript. All authors read and approved the final
manuscript.
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Acknowledgments
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