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BMC Genomics :10 Research2 2001, article Genomic organisation and alternative splicing of mouse and human 1 Simone A Osborne and Kathryn F Tonissen*

Address: School of Biomolecular and Biomedical Science, Griffith University, Nathan, Queensland 4111, Australia E-mail: Simone A Osborne - [email protected]; Kathryn F Tonissen* - [email protected] *Corresponding author

Published: 22 November 2001 Received: 28 August 2001 Accepted: 22 November 2001 BMC Genomics 2001, 2:10 This article is available from: http://www.biomedcentral.com/1471-2164/2/10 © 2001 Osborne and Tonissen; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any non-commercial purpose, provided this notice is preserved along with the article's original URL. For commercial use, contact [email protected]

Abstract Background: Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining. Results: The human TXNRD1 spans 100 kb of genomic DNA organised into 16 exons and the mouse Txnrd1 gene has a similar exon/intron arrangement. We also analysed the alternative splicing patterns displayed by the mouse and human thioredoxin reductase 1 genes and mapped the different mRNA isoforms with respect to genomic organisation. These isoforms differ at the 5' end and encode putative proteins of different molecular mass. Genomic DNA sequences upstream of mouse exon 1 were compared to the human promoter to identify conserved elements. Conclusions: The human and mouse thioredoxin reductase 1 gene organisation is highly conserved and both genes exhibit alternative splicing at the 5' end. The mouse and human promoters share some conserved sequences.

Background an thioredoxin reductase: TR1, TR3 and TGR, that share The thioredoxin system is comprised of thioredoxin a similar domain organisation and all contain seleno- (Trx) and thioredoxin reductase (TR) and plays an im- cysteine [8–14]. TR1 was the first thioredoxin reductase portant role in maintaining the redox state of the cell [1]. to be characterised and is known as the cytosolic form This system is involved in many cellular functions in- [8]. TR3 is the mitochondrial form and is also known as cluding synthesis of deoxyribonucleotides [2], redox TrxR2 [9–11]. TGR, previously known as the novel form control of transcription factors [3], protection against of thioredoxin reductase (or TR2) functions as a Trx and oxidative stress [4], cell growth and cancer [5]. The re- GSSG reductase [11,12]. In this study, we focus upon the duced form of Trx is maintained by TR, an enzyme that cytosolic form of mouse thioredoxin reductase (mTR1) contains a redox active disulphide group and a FAD mol- and human thioredoxin reductase (hTR1). The mouse ecule that uses the reducing power of NADPH [6,7]. Txnrd1 gene is located on 10 and yields a Presently, there are three recognised forms of mammali- cDNA sequence approximately 3200 bp long. This se- BMC Genomics 2001, 2:10 http://www.biomedcentral.com/1471-2164/2/10

quence encodes a protein with 499 amino acids and a (respectively) although intron lengths could not be de- molecular mass of 54.5 kDa [15]. The human TXNRD1 termined. Table 1 shows the length of each exon ranging gene is located on [16] and the first from the shortest, exon 5 (73 bp), to the longest, exon 3b cDNA sequence reported was 3826 bp and encoded a (338 bp). The principal ATG start codon is located in protein of 495 amino acids with a molecular mass of 54.1 exon 4 and the TAA stop codon in exon 16. Another ATG kDa [8]. Recently it was reported that the mouse Txnrd1 start codon that is utilised by one mRNA isoform gener- gene is alternatively spliced, producing three isoforms of ated by alternative gene splicing is found in exon 3b. Ta- mouse Txnrd1 mRNA that vary in their 5' sequences but ble 1 also shows the intron lengths in the region spanning have common downstream sequences [17]. Also recently, exons 5 to 15. These sizes range from the shortest, intron the human TXNRD1 gene has been reported to exhibit 13 (94 bp), to the longest, intron 15 (3.13 kb). All introns possible alternative splicing around its first exon [18]. display splice signals consistent with the GT/AG rule [19], except intron 4, in which the 5' splice donor is GC Here we report the genomic organisation of the human instead of GT. This discrepancy in the 5' splice donor in and mouse thioredoxin reductase 1 genes, subsequently intron 4 is consistent with that also found in intron 4 of detailing the alternative splicing pattern of the first exon the human TXNRD1 gene (Table 1). in both species. We also present a potential mouse Txnrd1 promoter region and compare this genomic DNA Human TXNRD1 genomic organisation sequence with the recently studied TXNRD1 gene pro- To determine the genomic structure of the human moter [18] to identify conserved sequences. TXNRD1 gene, the data from the NCBI database [www.ncbi.nlm.nih.gov] was searched using Results and Discussion the TXNRD1 cDNA [8] sequence. Alignments were used Mouse Txnrd1 genomic organisation to establish intron/exon junctions, and these results con- To determine the genomic structure of the mouse Txnrd1 firmed through the adherence of predicted splice signals gene we initially screened a mouse genomic DNA library to the AG/GT rule [19] and by comparisons made with using a mouse Txnrd1 cDNA fragment as a probe. Suc- the mouse intron/exon junctions described above. The cessful screening, restriction mapping and Southern blot cDNA sequences for mouse and human thioredoxin re- analysis revealed two overlapping Txnrd1 genomic ductase are approximately 80% homologous, and the hu- clones approximately 15 kb in length. One clone was ex- man [20] and mouse [21] thioredoxin genes have an tensively mapped and sequenced and aligned with the identical genomic organisation. Therefore we predicted Txnrd1 cDNA sequence [15] to reveal intron/exon junc- that the human and mouse thioredoxin reductase genes tions. The genomic sequences contained exons 5, 6, 9, 10, would also have a similar organisation. After searching 11, 12, 13, 14 and 15 (accession numbers AF394929, approximately 100 kb of human TXNRD1 genomic DNA AF394930, AF394931, AF394932, AF394933, sequence, all exons were mapped. AF394934, AF394935, AF394936 and AF394937 re- spectively). The location of exons 7 and 8 was deter- The mapping of the human TXNRD1 gene is detailed in mined by restriction enzyme mapping, however the Table 1. Exon lengths range from the shortest, exon 1a precise intron/exon junctions were established using (42 bp), to the longest, exon 3b (341 bp). The start codon mouse genomic sequences from the National Centre for is located in exon 4, however another ATG is found in Biotechnology Information (NCBI) Trace Archive exon 1 and may be utilised in at least one mRNA isoform [www.ncbi.nlm.nih.gov] . Two sequences numbered [18]. The stop codon is positioned in exon 16, as is the 18222987 and 20784325 in the archive were used to con- 3'UT region. Intron sizes vary greatly from the shortest, firm exons 7 and 8 respectively. These alignments ena- intron 10 (917 bp), to the longest mapped intron, intron bled the intron/exon boundaries to be fully mapped 12 (26.97 kb). Table 1 also lists the intron/exon splice sig- between exons 5 and 15 (Table 1). Exons 5 to 15 span ap- nals which all conform to the AG/GT rule [19], except in- proximately 14 kb which is consistent with the size of the tron 4. Like intron 4 in the mouse Txnrd1 gene, the mouse genomic Txnrd1 clone obtained from the library human 5' splice donor contains a GC instead of GT. The screen. The intron/exon junctions for exons 1 to 4 and 16 occurrence of this splice signal deviation in both mouse were determined by database mining using genomic and human thioredoxin reductase intron 4 sequences is Txnrd1 DNA sequences deposited in the Trace Archive of interesting since the 5' region of both genes are involved the NCBI. Database searches using cDNA sequences cor- in alternative splicing and this could represent a poten- responding to exons 1 to 4 and exon 16 [15] produced a tial regulatory site. number of 1 kb sequences. The following mouse Txnrd1 genomic sequences 19029770, 19679815, 18378992, Alternative splicing of mouse and human TXNRD1 genes 22011035, 17424013 and 18272097 were used to deduce Recent reports of alternative splicing in both the mouse intron/exon boundaries for exons 1, 2, 3a, 3b, 4 and 16 [17,22] and human [18] thioredoxin reductase genes lead BMC Genomics 2001, 2:10 http://www.biomedcentral.com/1471-2164/2/10

Table 1: Intron/Exon boundaries of human TXNRD1 and mouse Txnrd1 genes

Exon Intron Human TXNRD1 Gene Mouse Txnrd1 Gene Number Numbera TXNRD1/ TXNRD1/ Txnrd1 Txnrd 1

Exon Intron Sequence at intron/EXON/intron Exon Intron Sequence at intron/EXON/intron junctionb size size junctionb size size (bp) (kb) (bp) (kb)

1a 1 -- 42-ctgcagTCCTGA--ATTCTTgtaagc 112 - ccggagCCACTA--GTTCAT gtaagc 1---279-ctgcagTCCTGA--CATGTGgtaagt - - 2 2 1a 1 110 1.86 acacagAGGACG---TTGAAGgtagga 107 ND ccacagAGGGGG------CTGAAGgtagga 1- -1.62 - 3a - 2a - - 115 ND gctaagGTTCAT------ACTAAGgtaagg 3b 3b 2 2b 341 25.56 ccacagTGCTTT-----GGGCTGgtaagt 338 ND ccacag TGCCCT-----GAGCTGgtaagt 431231.19ctttagGCTTAT------GCTAAGgcaagg 123 ND ctgcagCCGAAC------GCTAAGgcaaga 5 4 73 1.83 ttccagGAGGCA-----GATGGGgtaagc 73 ND tcccagGAGGCA------GATGGGgtgagt 651202.46ttgtagGTCTTG-----AGACAGgtatga 120 1.62 ttgcagGTCTCG------ACACAGgtgtgg 761531.76atacagTTAAGC-----ATTAAGgtaatt 143 1.4 acgcagTGAAGC------ATTGTGgtaatt 8 7 116 0.414 tgttagGCAACA------CAGCAGgtaaag 116 0.411 ttgcagGCGACA------CAGCAGgtgggt 9 8 226 1.51 ccccagTGATGA----ATTAAAgtaagt 226 1.59 ctccagTGATGA------ACGAAAgtaagt 10 9 93 9.29 ttacagGTTGAA-----AATACGgtaagg 93 0.559 ttgtagATTGAA------AACACAgtaagg 11 10 77 0.917 tttcagGTGATG------TGAAAAgtaaga 77 0.416 ttttagGTGTTG------CGAAAAgtaagg 12 11 157 6.19 ttgcagGACTGG-----GTCAAGgtgagt 158 0.950 ttgcagAACCGG------GTCAAAgtgaga 13 12 108 26.97 aaacagTGTGAC----ATTGAGgtaagt 109 0.094 acacagTGTGAC------ATTGAAgtaagt 14 13 96 9.67 tcttagGTTTAC------GACAATgtaagt 96 2.03 ttccagGTTTAC------GACGATgtaagt 15 14 135 1.58 ccttagGAACGT-----GCAGAGgtgggt 135 3.126 tcttagGAACGT------GCAGAGgtgagt 16 15 69 9.08 ctgcagGTATTC-----GGTTAA 3'UT 138 ND ctgcagATATTC------GGTGAAgtgcNg

a Introns are numbered at 3' splice acceptor ie. intron 3 precedes exon 4. b Nucleotide sequences indicated at the intron (lowercase letters) and exon (uppercase letters) junctions.

us to investigate the link between splicing events and ge- • Isoform II nomic organisation. This investigation was carried out through comparisons of mouse and human thioredoxin With respect to isoform II, we noted that ESTs (for exam- reductase expressed sequence tags (EST) with the ge- ple, accession number AI956288) containing exon 2 al- nomic organisation as described in this report. ways displayed exon 1 immediately upstream, or did not extend far enough to be informative. Thus isoform II ap- Alternative splicing of mouse Txnrd1 gene pears to contain exons 1, 2, 4, 5, 6, 7 etc (Figure 1). The The Gladyshev group [17] proposed the first three exons only ATG start codon in the correct reading frame is lo- in the mouse Txnrd1 gene are alternatively spliced, pro- cated in exon 4, hence translation of this isoform would ducing three isoforms of mouse Txnrd1 mRNA (Figure also yield the 54.5 kDa mouse TR1 protein [15]. 1). These three mRNA isoforms were reported to contain exons 1 and 4, 2 and 4, and 3 and 4 to generate isoforms • Isoform III I, II and III respectively. Transcripts consistent with some of these isoforms were described in another study With respect to form III, ESTs containing exon 3 (acces- [22], although they were labelled differently. sion numbers AA168412, AI607108) were aligned with mouse Txnrd1 genomic DNA sequences in order to es- • Isoform I tablish the intron/exon junctions. Alignment of a mouse Txnrd1 EST containing exon 3 (accession number As reported by Sun et al [17], isoform I contains the ATG AI607108) with mouse genomic Txnrd1 sequence (Trace start codon positioned within exon 4 and translation of Archive 22011035) showed exon 3 is actually comprised the resulting sequence produces the first reported mouse of two exons, designated as exons 3a and 3b in this study. TR1 protein of 54.5 kDa [15]. The splicing signals around exon 3b are consistent with BMC Genomics 2001, 2:10 http://www.biomedcentral.com/1471-2164/2/10

Alternative splicing of human TXNRD1 gene Alternative splicing of the human TXNRD1 gene was in- vestigated based on that already determined for the Mouse Txnrd1 mouse Txnrd1 gene and also on recent reports of possi- ble alternative splicing in the human TXNRD1 gene

EXONS 1 2 3a 3b 4 [17,18]. The Arner group [18] reported three isoforms of human TXNRD1 mRNA – I, II and III, however these

mRNA Isoform I isoforms do not align with the three mouse mRNA iso- ∆ mRNA Isoform II forms previously discussed. In this report the human ∆ TXNRD1 isoforms are numbered according to structural mRNA Isoform III ∆∆ and sequence similarity displayed with the mouse Txnrd1 isoforms where possible. Subsequently the iso-

Human TXNRD1 forms I, II and III reported by the Arner group are denot- ed here as isoforms V, II and I (respectively). In addition to the isoforms reported by the Arner group we identified

A two further isoforms (isoforms IV and VI) and proposed another (isoform III), that would align with the mouse EXONS 1A 1 2 3b 4 5 isoform III.

mRNA Isoform I ∆ ∆ • Isoform I mRNA Isoform II ∆ mRNA Isoform IV ∆ Isoform I contains exons 1, 2, 4, 5, 6, 7 etc (accession

mRNA Isoform V number BF182740) [8] and represents the human ∆ TXNRD1 cDNA sequence first reported [8]. There are mRNA Isoform VI ? ∆ two ATG start codons in the correct reading frame in ex- ons 1 and 4. The ATG in exon 4 directs translation of the orginal 54.1 kDa hTR1 protein [8]. Translation of this Figure 1 mRNA isoform from the ATG in exon 1 yields a protein Exon 1 alternative splicing pattern in mouse and human with a predicted molecular mass of approximately 60 thioredoxin reductase 1 genes. The shaded area (A) shown kDa [18]. This protein coincides with a 60 kDa hTR1 pro- in TXNRD1 exon 1 is 42 bp in length and represents an alter- tein previously reported [22]. native splicing site within the exon. The mRNA isoforms of Txnrd1 and TXNRD1 are represented by: a green line for Iso- • Isoform II form I; a red line for Isoform II; a blue line for Isoform III; a purple line for Isoform IV; and a pink line for Isoform V. The Isoform II is comprised of exons 1, 4, 5, 6, 7 etc (acces- resulting mRNA isoforms are also shown with predicted sion number AU077310). The ATG start codon is in exon start codons indicated by the ∆ symbol. 4 and directs translation of the original 54.1 kDa hTR1 protein. the AG/GT rule [19]. Exon 3a was aligned with the mouse • Isoform III genomic Txnrd1 DNA sequence (Trace Archive 18378992) to confirm a consensus splice sequence at the Isoform III is predicted to contain exons 3b, 4, 5, 6, 7 etc, 5' splice donor site to form the intron between exon 3a however no human thioredoxin reductase EST was and 3b. The 5' end of exon 3a could not be accurately de- found to contain exon 3b. Therefore isoform III is not de- fined since the 5'end of isoform III has not been accu- noted in Figure 1. This isoform prediction was based on rately mapped. All ESTs containing exon 3 sequences sequence comparisons made between the mouse exon 3a always contain both exon 3a and 3b and therefore iso- and 3b sequences with human genomic TXNRD1 se- form III is predicted to contain exons 3a, 3b, 4, 5, 6, 7 etc quences. A human TXNRD1 genomic region with homol- (Figure 1). Two ATG start codons in the same reading ogy to mouse exon 3b was detected (Figure 2). This frame are present in exons 3b and 4. Translation from alignment was flanked by canonical GT/AG splice sig- the start codon encoded in exon 4 would produce the nals at equivalent positions to that observed for mouse original 54.5 kDa protein, however use of the start codon exon 3b, indicating the possibility of the human TXNRD1 in exon 3b yields a protein with a predicted molecular mRNA isoform III. Exon 3a was not identified in the hu- mass of 67 kDa. This 67 kDa protein coincides with the man genome sequences possibly due to low homology 67 kDa mTR1 protein previously reported [17]. between the mouse and human 5' UT region. An align- BMC Genomics 2001, 2:10 http://www.biomedcentral.com/1471-2164/2/10

Isoform IV was discovered following an EST database TXNRD1 1 CCACAGT G C TTT G T A T T G A A G G A G G A A C C T 30 search. This isoform contains exon 1A, 4, 5, 6, 7 etc (ac- Txnrd1 1 ------T G C CCT G C A T T G A A G G A G A A G C C C 24 cession number AU132293). Exon 1A is a product of an TXNRD1 31 T G G C C A TAA T G C C A G T T G A T G A C T A C T G G C 60 Txnrd1 25 T G G T C A CCA T G C C A G T T G A T G A C T G C T G G C 54 internal splice site located within exon 1. This splice site

TXNRD1 61 T G T GTT T A C C A G C C T C T T G T G C T A G A C C T T 90 generates a 42 bp fragment that corresponds to the 5' Txnrd1 55 T G T ACT T C C C A G C T T C T C G A G G T A G A A C C T 84 end of exon 1. There is one ATG start codon in the correct

TXNRD1 91 T T G T G C A G A C T G T C A G A G T G G TGC AGT C T T 120 reading frame in exon 4 and translation from this ATG Txnrd1 85 T T G T G C A G A C T G T C T G G G T G G CAC CCAC T T 114 yields the original 54.1 kDa hTR1 protein [8]. TXNRD1 121 G C C C C C A C T G T T G C T G G T T T C C A G G T G T T C 150 Txnrd1 115 G C C C C A A C T G T T G C T G G T T T C C A G G T T T T C 144 • Isoforms V TXNRD1 151 T C C C T T C A G T C C C TGAG C C A C T A C G T A T G C 180 Txnrd1 145 T C C C T C C A G T C C C CCGG C C A C C A C A T G T G C 174

TXNRD1 181 C C GCCAT G C T G C CAA CAG G TAGCC AC AG T G 210 This isoform contains exons 1A, 2, 4, 5, 6, 7 etc (accession Txnrd1 175 C C CGTGT G C T G C TGA GGG G CCCTC GTGG G G 204 number BG771986). The ATG start codon is in exon 4 TXNRD1 211 C T G T G C T T C C T C C T T C A C A T TGC T C C A CCG 240 and directs translation to produce the original 54.1 kDa Txnrd1 205 C T G T G C T T C C T G C T T C A C G T CCC T C C A AGA 234 human TR1 protein [8]. TXNRD1 241 C A C C C C C T T C C A C A T C C C A A G A ACC TTC T T 270 Txnrd1 235 C A C T C C C C T C C T C A T C C C A - G A --C GC C C T 261 • Isoforms VI TXNRD1 271 C T T C CGC T G A C C C C AAGCT C T G C C T T T C A C 300 Txnrd1 262 G T C C TAC T G A C C C C TGTAT C T G C C C T C C A C 291

TXNRD1 301 C C C C T A C A T C T G A T A G T A G G C A A G A G A G A A 330 Isoform VI was discovered via an EST database search. Txnrd1 292 C C T C C A C A C C T G A T A G T A G G C A G G A A A A A A 321 This isoform contains exons 2, 4, 5, 6, 7 etc (accession TXNRD1 331 A T GTG C A G T T T G G G C T G GTAAGT 353 number BG772375) and the ATG codon is found in exon Txnrd1 A T ACG C A A T C T G A G C T G 322 338 4 where it directs translation to potentially produce the original 54.1 kDa TR1 protein [8]. The EST that revealed Figure 2 this isoform extends a further 300 bp immediately up- Alignment of mouse thioredoxin reductase 1 (Txnrd1) exon stream of the 5' end of exon 2. The nature of this 300 bp 3b with partial human thioredoxin reductase 1 (TXNRD1) region is unknown as it did not display homology with genomic DNA sequence. Splice junctions (AG/GT) are out- human TXNRD1 cDNA sequences or any sequence in lined in TXNRD1. The ATG codon that yields the 67 kDa GenBank. Subsequently the 5' end of this isoform may protein form is conserved in both genes and is boxed. not terminate with exon 2.

Potential thioredoxin reductase regulatory elements ment between the deduced amino acid sequences de- The alternative splicing pattern utilised by both the rived from the mouse and human exon 3b (Figure 3) TXNRD1 and Txnrd1 genes generate mRNA isoforms reveals a 50% identity between the two species (as out- that are heterogenous at the 5' end. This alternative lined in Figure 3). If conservative amino acid substitu- splicing presents consequences for possible gene regula- tions are taken into consideration the homology tion and location of control elements. Indeed Rundlof percentage increases to approximately 70%. The high de- and co-workers [22] recently reported that the alterna- gree of sequence conservation further substantiates the likelihood of exon 3b being utilised in an as yet unde- scribed human mRNA variant. hTR1-3b 1 M P V D D Y W L C L P A S CAR P F V Q T V R V 24 mTR1-3b 1 M P V D D C W L Y F P A S RGR T F V Q T V W V 24

An analysis of the reading frame in exon 3b revealed hTR1-3b 25 VQS C P H C C W F P G V L P S V P E P LRMP 48 three in frame ATG codons that could potentially initiate mTR1-3b 25 APT C P N C C W F P G F L P P V P R P PHVP 48 translation. Translation of this mRNA isoform from the hTR1-3b 49 AML PTG S H S A V L P P S HCS TAPP S T 72 most 5' ATG start codon in exon 3b would produce a pro- mTR1-3b 49 RVL LRG P R G A V L P A S RPS KTLP S S 72 tein with a predicted molecular mass of approximately hTR1-3b 73 S Q E P S S S A D P K L C L S P P T S D S R Q E 96 67 kDa. A human TR1 protein with a mass of 67 kDa has mTR1-3b 73 S Q T P C P T - D P C I C P P P S T P D S R Q E 95 been detected [17] substantiating the possibility that hTR1-3b 97 R N V Q FGL 103 exon 3b is present in some mRNA transcripts. A search mTR1-3b 96 K N T Q SEL 102 of the EST and nucleotide databases failed to identify any expressed sequences that contain exon 3b. Figure 3 Alignment of human thioredoxin reductase (hTR1) exon 3b • Isoform IV deduced amino acid sequence with mouse thioredoxin reductase (mTR1) exon 3b deduced amino acid sequence. BMC Genomics 2001, 2:10 http://www.biomedcentral.com/1471-2164/2/10

of mouse DNA (NCBI Trace Archive 19029770) that ex- TXNRD1 1 A C C T TAAG G C A G T G GGTC C C A A ACT T T G C T G CC 33 Txnrd1 1 A - C T CG - G A C A C T G AAAC T C C A --T G T G G T G NG 29 tends 450 bp upstream from exon 1 has been examined

TXNRD1 34 A A - C T CAATC A CCT T T GGAGT T T C T T T T T T T T T 65 for possible control elements. This sequence, when Txnrd1 30 A A T C T TTGGC A TTT T T T---T T T T T T T T T T T T T 59 aligned with the human TXNRD1 core promoter region TXNRD1 66 TTA A A CGC A GAGGCC - - -C A G G - T C A C A CTC CA 94 [18] produced a nucleotide match of approximately 70% Txnrd1 60 AAA A A AAC A ACTATTTGGC A G G G T C A C A GCC AG 92 (Figure 4). This potential mouse promoter region, like TXNRD1 95 CAC C A ATG A C A T C A G A ATG T C T AGG GGT G G G A G 127 Txnrd1 93 TGC C A GGG G C A T C A G A TCG T C T --G TAT G G G T G 123 the corresponding human region, lacks a classical TATA

TXNRD1 128 C C A G G C ACC A GGA T T C T A A A G A C-C C T C A C G T G 159 box. Unlike the human region, it also lacks consensus Txnrd1 124 C C G G G C GTC A AT A T T T T T A A A A TTC C T C A T G T G 156 Sp1, Sp3 and Oct 1 binding sites at equivalent positions. TXNRD1 160 A - T G A C AACA G C T A G CAAA G TTCTGT A G C TAC T 191 Txnrd1 157 A C T A A C GCAA G C T T G TGTA G ----AT A G C AGC - 184 However it does contain potential AP-1 binding sites, one

TXNRD1 192 G C C T T A G G G C AT A G T C TAATT T CTT C A G T AAAA 224 of which is conserved in the human promoter. There is Txnrd1 185 G C C T T A G G G C TGA G C C CGGGT T ACT C A C T CC - - 215 also a potential CAAT box present at position 325 in both TXNRD1 225 A C A C A C T T A T T C CAA A T T T G G T T C C AGA - A T T G 256 the mouse and human sequences (Figure 4). Promoter Txnrd1 216 A C A C T C T T A C T C -GA T T C T G G T T C C CAA C A T T - 246 studies with the human promoter suggest this CAAT box TXNRD1 257 C C T T A A A T T G TTTTTG C TCTG TTCT T A G G T T G G 289 Txnrd1 247 C T T T G A A T A G CAGCCG C --GG CCT T T G G G T C G G 277 is not required as part of the core promoter [18].

TXNRD1 290 G G G - C G G C T ATG A G C A G G C A G A G G A T G T G G T GT 321 Txnrd1 278 G G G G C G G T T CCG A G C A G - C - G A G G A T G T T G T AC 308 The mouse Txnrd1 mRNA isoform III does not contain TXNRD1 322 CACC C A A T T A G G A G C T C T C A GC T T A C TAG G - C A 353 exon 1, instead exon 3a is the most 5' exon. This infers Txnrd1 309 GGT C C A A T T A G G A G C T C T C A CTT T G C GGG G A C A 341 that a potential promoter region may be present just up- TXNRD1 354 A TTAG C A T A G G T T G C C A G G G C T G C A C G A G G A G T 386 Txnrd1 342 A CCGG G A C A G G T T G C C G G G G C T G C G C G G G G A C T 374 stream from exon 3a. This isoform encodes a 67 kDa pro-

TXNRD1 387 G G A T T T C T G C T T T G T C A T T C T G A C T C T G G C A G T 419 tein that is expressed at lower levels [17] than the 54.5 Txnrd1 375 G G A T T T T T G C T T T G T C A T T C T G A C A C G G G C A G T 407 AP-1 kDa mouse TR1 protein first identified [15]. The differ- TXNRD1 420 T A G C C C G C C CGC T CGG C G C A G G G C G T G G C T T C T 452 Txnrd1 408 T C G C C C G C C TAC T AAG A G C A G G G C G T G G C T T C C 440 ence in expression levels of these two forms of mouse

TXNRD1 453 C G T A G C C A T T A G G A A A 468 Txnrd1 may be a reflection of the use of alternate pro- Txnrd1 441 C G G A G C C A C T A G G A A C Txnrd1 exon 1 456 moter regions. In the human gene exons 1 and 4 span ap- proximately 28 kb and similarly an alternative promoter may exist in this region to direct transcription of mRNA Figure 4 Alignment of human thioredoxin reductase (TXNRD1) pro- isoforms that lack exon 1. moter region with predicted mouse thioredoxin reductase (Txnrd1) promoter region. Two potential AP-1 consensus In addition to the transcriptional regulation of the binding sites are outlined on Txnrd1 at positions 383 and 393. thioredoxin reductase 1 genes previously discussed, it is The 5' end of Txnrd1 exon 1 is outlined at position 448. also important to consider post-transcriptional control. Recent studies [23,24] have investigated AU rich insta- bility elements present in the 3' UT region of the human tively spliced mouse transcripts are expressed in a tissue TXNRD1 gene that facilitate mRNA degradation, leading specific manner. Currently the only genome information to rapid mRNA turnover. There are four AU elements regarding transcriptional control of thioredoxin reduct- present in the human 3'UT region and three of these ele- ase 1 genes is available in the recent identification and ments are also present in the 3' UT region of the mouse characterisation of the core promoter for the human Txnrd1 gene. Also, a non-AU rich instability element TXNRD1 gene [18]. This core promoter of approximately present in the human TXNRD1 3'UT region [24] is 83% 180 bp contains Oct 1, Sp1 and Sp3 binding sites and has conserved in the mouse Txnrd1 3'UT region [15] (Figure an increased GC content, suggesting the human 5). Thus, control of thioredoxin reductase 1 mRNA levels TXNRD1 gene is a house keeping gene. However this from both the human and mouse genomes appears to be does not explain the response of TXNRD1 to cellular sig- directed from elements present in both the 5' and 3' UT nalling since thioredoxin reductase protein and mRNA regions. levels are known to increase quickly and significantly in human cells in response to exogenous agents. It is possi- Conclusions ble that the core promoter for the human TXNRD1 gene In conclusion we report the genomic organisation of is accompanied by other promoter elements that have mouse and human thioredoxin reductase 1 genes. These not yet been identified. Comparisons between mouse genes display a conserved genomic organisation as the and human promoter regions may reveal these potential coding regions of both genes have an almost identical regulatory elements. exon/intron structure. Comparison of mouse and human 5' sequences allows possible regulatory elements to be A promoter region for the mouse Txnrd1 gene has yet to identified and also in this study has enabled alternative be elucidated. However in this study a genomic sequence splicing events at the 5' end of each gene to be reviewed BMC Genomics 2001, 2:10 http://www.biomedcentral.com/1471-2164/2/10

• Mapping of mouse Txnrd1 TXNRD1-3'UT1 A G G T T A A G C C C C A G T G T G G A T G C T Txnrd1-3'UT 1 A G G T T A A G C C C C A G T G T G G A T G C T One of the clones obtained from the mouse genomic li- TXNRD1-3'UT25 G T T G C C A A G A C T G C A A A C C A C T G G brary screen was extensively mapped using restriction Txnrd1-3'UT 25 G T T G C C A A G A C T A C A G A C C A T T G C enzyme digests and Southern blot analysis. The resulting TXNRD1-3'UT49 C T C G T T T C C G T G C C C A AATC C A A G sequences were aligned with the mouse Txnrd1 cDNA se- Txnrd1-3'UT 49 C T T G C T T C C T T G C C C A CGCC C A - G quence [15] using MacVector™ software (Oxford Molec- TXNRD1-3'UT73 G C G A A G T T TT ular Group) to reveal the intron/exon junctions for exons Txnrd1-3'UT 72 G T G A A G T T CA 5, 6, 9, 10, 11, 12, 13, 14 and 15.

Figure 5 To complete the intron/exon map of the mouse Txnrd1 Alignment of the non-AU rich instability element present in gene, the mouse Txnrd1 cDNA sequence was used to the human TXNRD1 3'UT region with a sequence element BLAST-search [25] the NCBI Trace Archive for mouse present in the mouse Txnrd1 3'UT region. genomic DNA fragments containing exons 1, 2, 3, 4, 7, 8 and 16. Numerous 1 kb genomic sequences were ob- tained and aligned with the relevant exon sequences via and further defined. Further work will be required to link MacVector™ software to reveal the intron/exon junc- the different transcripts with specific promoter regulato- tions. ry elements. Genomic organisation of the human TXNRD1 gene Materials and methods The human TXNRD1 cDNA sequence [8] was used to All chemicals were purchased from Sigma (Castle Hill, BLAST-search the NCBI human genome database for the TXNRD1 NSW, Australia) unless otherwise indicated. genomic DNA sequence. The resulting genomic sequence was aligned with the human TXNRD1 cDNA Genomic organisation of mouse Txnrd1 gene sequence using MacVector™ software to reveal intron/ • Cloning of mouse genomic Txnrd1 DNA exon junctions.

To obtain mouse genomic DNA clones for the determina- Alternative splicing patterns tion of the genomic structure of the mouse Txnrd1 gene, Mouse we initially screened a mouse genomic DNA library in λ BLAST-searches [25] of the NCBI EST and Trace Archive phage (Stratagene, La Jolla, CA, USA) with an αP32- databases were used to analyse the alternative splicing Txnrd1 dATP labelled mouse Txnrd1 cDNA fragment as a probe. patterns in the mouse gene. Approximately 5 × 105 plaques were prehybridised in 10% w/v dextran sulphate (Astral, Gymea, NSA, Austral- Human ia), 2% w/v sodium dodecyl sulphate, 0.04% w/v bovine BLAST-searches of the NCBI EST and human genome serum albumin, 0.04% w/v polyvinyl pyrollidone (MW databases were used to analyse the alternative splicing TXNRD1 40000), 0.04% w/v Ficoll and 1 M sodium chloride at 61 patterns in the human gene. C for 4 hours. The probe was hybridised to the filters at 61 C for 12 hours. The filters were washed three times at Promoter Elements TXNRD1 40 C in 2XSSC (0.3 M sodium chloride, 0.33 M sodium The human promoter sequence was used to citrate) and 0.1% w/v sodium dodecyl sulphate. Success- BLAST-search the NCBI Trace Archive for the potential Txnrd1 ful screening of the mouse genomic DNA library pro- mouse promoter region. duced two overlapping mouse Txnrd1 genomic clones approximately 15 kb in length. Abbreviations BLAST – Basic Local Alignment Search Tool The Txnrd1 cDNA fragment used in the probe was gener- ated using reverse transcriptase PCR that utilised mRNA EST – Expressed Sequence Tags isolated from mouse liver tissue (Oligotex Direct mRNA Mini Kit, Qiagen, Victoria, Australia) as the template GSSG – Oxidised Glutathione with oligonucleotides designed from the mouse Txnrd1 cDNA sequence [15]. These two oligonucleotides (for- NCBI – National Centre of Biotechnology Information ward primer 5' ACATCTACGCCATTGGTGAC and re- verse primer 5' TGGGGCTTAACCTCAGCAGC PCR – Polymerase Chain Reaction (Geneworks, Adelaide, Australia)) amplified a region of mouse Txnrd1 cDNA approximately 520 bp in length. Trx – Thioredoxin BMC Genomics 2001, 2:10 http://www.biomedcentral.com/1471-2164/2/10

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Rundlof AK, Carlsten M, Arner ES: The core promoter of human thioredoxin reductase 1: cloning, transcriptional activity, yours - you keep the copyright and oct-1, sp1, and sp3 binding reveal a housekeeping-type BioMedcentral.com promoter for the au-rich element-regulated gene. J Biol Chem Submit your manuscript here: 2001, 276:30542-30551 http://www.biomedcentral.com/manuscript/ [email protected]