Ian Chambers, Jonathan Frampton, Peter Goldfarb',. Nabeel Affara2 ... leukotriene formation in other tissues (Samuelson, 1983; Bryant et al., 1982, 1983; Bryant ...
The EMBO Journal vol.5 no.6 pp.1221 -1227, 1986
The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the 'termination' codon, TGA
Ian Chambers, Jonathan Frampton, Peter Goldfarb', Nabeel Affara2, Wendy McBain and Paul R.Harrison The Beatson Insitute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 IBD, UK 'Present address: Department of Biochemistry, University of Surrey, Guildford, Surrey, UK 2Present address: Department of Medical Genetics, Yorkhill Hospital, Glasgow, UK Communicated by J.Paul
Glutathione peroxidase (GSHPx) is
an
iinporant selenium-
containing enzyme which protects cells from peroxide damage and also has a role in leukotriene formation. We report the identification of a genomic recombinant as encoding the entire mouse GSHPx gene. Surprisingly, the selenocysteine in the active site of the enzyme is encoded by TGA: this has been confirmed by primer extension/dideoxy sequencing experiments using reticulocyte mRNA. The same site of transcription initiation is used in three tissues in which the GSHPx mRNA is expressed at high levels (erythroblast, liver and kidney). Like some other regulated 'house-keeping' genes, the GSHPx gene has Spl binding site consensus sequences but no 'ATA' and 'CAAT' consensus sequences upstream of the transcription initiation site. Moreover, there is a cluster of two Spl binding site consensus sequences and two SV40 core enhancer sequences in the 3' region of the gene, close to the previously mapped position of a DNase I-hypersensitive site found only in tissues expressing the GSHPx mRNA at high levels. Key words: glutathione peroxidase/genes/selenocysteine/codons Introduction Glutathione peroxidase (GSHPx) is present in all cells, functioning to protect membranes (reviewed in Flohe, 1982; Chiu et al., 1982) and possibly DNA from damage by hydroperoxides (Christophersen, 1969). Also, by preventing peroxide-mediated inactivation of Cu/Zn superoxide dismutase (Hodgson and Fridovitch, 1975) it indirectly protects the cell from damage by the superoxide radical. Interestingly, superoxide dismutase protects GSHPx from inactivation by superoxide radicals (Blum and Fridovitch, 1985). Erythrocytes are at greater risk from peroxides than most cells due to their much higher concentration of oxygen: indeed it was within these cells that GSHPx activity was first detected (Mills, 1957). The enzyme has two main roles in circulating erythroid cells. First, by removing H202 produced during the spontaneous oxidation of haemoglobin to met-haemoglobin it prevents further oxidation of haemoglobin by H202 (Chiu et al., 1982). Secondly, by removing membrane hydroperoxides it maintains membrane flexibility and prevents the premature splenic clearance which would otherwise occur (Flohe, 1982). During reticulocyte maturation an erythroid-specific lipoxygenase attacks mitochondria and is responsible for their degradation (Rapoport et al., 1979). It seems likely that GSHPx reduces IRL Press Limited, Oxford, England
the resultant hydroperoxides following their removal from the mitochondrial membrane by phospholipase (Sevanian et al., 1983; Yasuda and Fujita, 1977): GSHPx has a similar role in reducing lipoxygenase-generated lipid hydroperoxides during leukotriene formation in other tissues (Samuelson, 1983; Bryant et al., 1982, 1983; Bryant and Bailey, 1982; Guidi et al., 1984). We report here the sequence and structure of the GSHPx gene, the nature of the control sequences regulating the gene and its promoter usage in different tissues. Results We have already reported the cloning of a mouse genomic DNA recombinant encoding a polypeptide of- 19 kd (epl9) whose mRNA is expressed most highly in erythroblasts, liver and kidney of all the tissues so far tested (Goldfarb et al., 1983; Affara et al., 1983, 1985). From the positions of hybridisation of two nonoverlapping cDNA recombinants derived from the epl9 mRNA, the main coding region of the epl9 gene was located in a 0.7-kb EcoRI fragment and the adjacent XbaI-EcoRI fragment (Figure 1). To attempt to identify the function of the epl9 polypeptide, the epl9 cDNA recombinants, pFA6 and pFC5,and appropriate regions of the epl9 gene were sequenced by the dideoxy method according to the strategy summarised in Figure 1. Searching the protein data base with translations of the ep 19 gene sequence so obtained revealed a very close homology to the complete amino acid sequence of bovine erythrocyte GSHPx (Gunzler et al., 1984) and with the sequence of a fragment of the rat liver enzyme (Condell and Tappel, 1982). This comparison (summarised in Figure 2) also reveals the presence of a single intron within the coding region of the GSHPx gene: this is confirmed by an interruption in one of the GSHPx cDNA sequences (in pFC5) when compared with the sequence of the gene. R
X
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r
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pFC5
xX
XRX X R R
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Fig. 1. Restriction map of the GSHPx gene and sequencing strategy. Top: map of lambda R68A, the genomic DNA recombinant containing the gene (Goldfarb et al., 1983). Middle: enlarged map of the gene showing restriction sites relevant to the sequencing strategy. Closed boxes represent protein coding regions and open boxes 5'- and 3' non-coding regions of the mRNA (as defined by the major transcription start site and the major polyadenylation site). Regions homologous to cloned cDNAs are shown by overlining. Bottom: sequencing strategy. Arrows indicate the extent and direction of sequencing of subclones. Abbreviations: R, EcoRI; X, Xbal; S, Sau3A; P, Pstl; M, MspI; H. HaeIlI; T, TaqI.
1221
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Fig. 2. Sequence of the mouse GSHPx gene. The nucleotide and deduced amino acid sequences of mouse GSHPx are aligned with those known for the bovine and rat enzymes. The position of the in-frame TGA codon is marked by an asterix and occurs at the same position selenocysteine (Sec) in the protein. Numbering begins at the transcription initiation site (marked START). Sites of polyadenylation are shown by pA(major) pA(minor) and preceded by presumptive polyadenylation signals which are underscored. Sequences with 8-9/10 homology to the Spl-binding site overlined. Sequences overlined with arrows ( _ =GGGGCGGPuPuPy) and those which also possess the hexanucleotide core (CCGCCC) have that with at least 7/8 homology witX the SV40 core enhancer (GTGGTTATG) sequence are boxed: the copy showing perfect homology is double boxed. as
or
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sequence
1222
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Structure of the mouse glutathione peroxidase gene 0
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To locate the limits of the GSHPx gene, the sites of initiation of transcription were mapped both by SI protection (Figure 4A) and primer extension techniques (Figure 4B). These experiments are all consistent with the transcription of the GSHPx gene beginning at a sequence 40 nucleotides upstream of the initiating methionine codon (see Figure 2). Interestingly, the same site of transcription initiation is used in each of three tissues expressing the GSHPx mRNA at high level (i.e. erythroblasts, liver and kidney, Figure 4). Examination of the sequence of the GSHPx gene immediately upstream of the site of initiation of transcription does not reveal either of the 'ATA' or 'CAAT' consensus sequences found upstream of the globin and other genes unless the CCAC and TTAAAA sequences at -55 and -31 are unusual variants of these consensus motifs. SI mapping of the 3' termini of the GSHPx mRNA transcripts (Figure 5) shows that these terminate in a region 17 nucleotides downstream of a AATAAA polyadenylation signal which in turn is 206 nucleotides downstream of the GSHPx stop codon (see Figure 2). There is also a minor termination site 280 nucleotides downstream of the termination codon, which is in turn 20 nucleotides away from a possible polyadenylation signal, GATAAG (Figure 2): each of these polyadenylation sites is used to the same relative extent in the three tissues expressing the GSHPx mRNA at a high level. We have also examined the GSHPx gene for potential regulatory elements. In fact there are five copies of the SpI-binding site hexanucleotide core sequence (GGGCGG): one upstream of the gene (at -188 nucleotides), one within the first exon (at +106 nucleotides), one within the intron (at + 326 nucleotides) and two towards the 3' end of the transcription unit (at +904 and +1157 nucleotides) (Figure 2). In addition to these, there are also eight sequences which, although they lack the hexanucleotide core sequence, show 8-9/10 nucleotide homology with the r cently exts8dd Spl-binding site consensus sequence (TGGGC GGAAT) (Kadonaga et al., 1986). Three of these are scattered throughout the protein coding region, the remainder being clustered upstream of the gene (Figure 2). There is also a sequence at +1009 nucleotides showing perfect homology with the SV40 core enhancer sequence (GTGGTATTG) (Khoury and Gruss, 1983) as well as other sequences snowing only a single base change from this consensus sequence (Figure 2). Perhaps significantly, the SV40 core enhancer consensus sequence and the related inverted sequence at +1027 nucleotides are centred within a 250-nucleotide sequence flanked by two inverted Sp I-binding site core sequences. -
is-
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31 4-
Primer
Fig. 3. Dideoxy sequencing of GSHPx mRNA around the selenocysteine codon. The reactions were carried out as described in Materials and methods. The primer is the 5' end-labelled 23-bp strand of the MspIlPstI fragment corresponding to nucleotides 193-215 of Figure 2. Lanes T,C,G,A and 0 are the products of reactions containing ddTTP, ddCTP, ddGTP, ddATP and no ddNTPs, respectively. The sequence shown at the side is that deduced for the mRNA. The position of the UGA codon is indicated.
A surprising point to emerge from this analysis was that the selenocysteine in the active site of the enzyme appeared to be encoded by one of the usual 'stop' codons, TGA. The accuracy of the sequence of the genomic recombinant in this region was first confirmed by sequencing both strands of the DNA. However, the possibility remained that a mutation had occurred during isolation of the genomic recombinant to produce the TGA codon. In this case selenocysteine would be encoded, not by TGA but by another codon, perhaps one of the normal cysteine codons (TGT, TGC). Unfortunately, this could not be excluded from the sequences of the two GSHPx cDNA sequences since neither is derived from this region of the mRNA. To clarify this point, the sequence of the mRNA in the relevant region was determined by reverse transcription using a short single-stranded 23-nucleotide primer (corresponding to a MspIlPstI fragment at nucleotides 193 -215 of the GSHPx gene sequence shown in Figure 2) under dideoxy sequencing conditions. The result (Figure 3) confirms that the GSHPx mRNA sequence encoding the selenocysteine residue is in fact UGA. There is no evidence for any further bands in the primer extension sequencing ladder other than very minor components present in the control lane. In contrast, the termination codon used in the GSHPx gene is TAA (Figure 2).
Discussion GSHPx is an enzyme of considerable importance for several reasons (see Introduction for references). First, it has an important protective role in removing lipid hydroperoxides and H202 formed during normal oxidative metabolism especially in the red cell but also in other tissues. Secondly, in certain tissues (e.g. platelets, leukocytes and lung) GSHPx reduces, and thereby cleaves, the hydroperoxy-fatty acids formed by the action of lipoxygenases on lipid fatty acids: this generates the immediate precursors of the leukotrienes which are involved in inflammatory responses, including asthma and rheumatoid arthritis. In this context, it is significant that certain drugs used in the treatment of arthritis have proven to be potent inhibitors of GSHPx (Chaudiere et al., 1984). In red cells, a red cell-specific lipoxygenase is responsible for the breakdown of mitochondira during reticulocyte maturation (Rapoport et al., 1979) and it is believed that GSHPx 1223
I.Chambers et al.
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Fig. 4. Analysis of GSHPx mRNA 5' ends. A and B. SI protection mapping: 5' end-labelled probes were derived as shown in the line diagram, panel B (abbreviations are as in Figure 1, except that the cross-hatched bars denote the two protein coding regions). In the experiment shown in (A) 50 /Ag of total RNA was hybridised to an excess of the probe indicated. Hybrids were treated with 5000 U of S1 nuclease (Boehringer) for 1 h at 37°C and the products resolved on a 6% denaturing acrylamide gel. Autoradiography was for 48 h at -70°C. The RNAs used were: tRNA, yeast tRNA: Fib, BALB/c-N cytoplasmic RNA; Ret, total BALB/c reticulocyte RNA; Fr+, total induced Friend cell RNA; Kid, total kidney RNA; and Liv, total liver RNA. Markers (M) were HaeIII fragments of pBR322. The gel origin (ORI) is also indicated. C and D. Primer extension mapping: 5' end-labelled SstII-Sau3AI single-stranded primer from the region of the GSHPx gene indicated in the line drawing (panel D see above for definitions) was used in the experiment shown in (C). 1 j4g of poly(A)+ RNA made up to 50 itg with yeast tRNA was hybridised to excess primer. Hybrids were extended with reverse transcriptase as described in Materials and methods section and the products separated on a 6% denaturing acrylamide gel. Autoradiography was for 48 h at -70°C. The RNAs used were: tRNA, yeast tRNA; Fr+, total poly(A)+RNA from induced Friend cells; and Fr-, total poly(A)+RNA from uninduced Friend cells. Markers (M) were as in (A). -
is involved in the metabolism of the lipid hydroperoxides thus formed. For these various reasons, it is important to elucidate how the GSHPx mRNA comes to be regulated to -50 times its constitutive level in a subset of tissues and how this compares with control of the expression of the different lipoxygenase mRNAS. An attempt to explore such questions should now be possible, at least in the red cell system, since we have now been successful in cloning the genes encoding both GSHPx and red cell lipoxygenase (this report, Harrison, 1984; Affara et al., 1985, and unpublished data). In each of the tissues tested in which the GSHPx mRNA is expressed at high levels, the GSHPx mRNAs have the same 5' and 3' termini and are derived by splicing out a single intron: this indicates that the same promoter and splicing mechanism is used in each tissue. It is interesting that the mouse GSHPx gene does not seem to utilise 'ATA' and 'CAAT' upstream control sequences as do and 3-globin and many other genes: in this a-
1224
respect it resembles certain regulated housekeeping genes, such
those encoding dihydrofolate reductase (Masters aand Attardi, 1985), adenosine deaminase (Valerio et al., 1985), hypoxanthine phosphoribosyltransferase (Melton et al., 1984), HMG CoA reductase (Reynolds et al., 1984; Osborne et al., 1985) and ,B-tubulin (Lee et al., 1983). It also resembles such genes in possessing potential Sp 1-binding sites in the upstream flanking region of the gene. Although only one of these possesses the exact hexanucleotide core sequence they may all nevertheless bind Spi: for instance, the acquired immune deficiency syndrome (AIDS) virus long terminal repeat promoter contains two SpI-binding sites which do not conform exactly to this consensus sequence (Kadonaga et al., 1986). Clearly, this can only be resolved by footprinting the binding of Spl to this region. Other potentially important regions of the GSHPx gene are the two inverted SV40 core enhancer sequences and Spl core sequence within the intron and the cluster of enhancer core sequences as
Structure of the mouse glutathione peroxidase gene
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3000 Ci/mmol) as described by Maniatis et al.' (1982). By careful choice of restriction enzymes used to generate the probe it was possible to label only the strand complementary to the GSHPx mRNA, this was separated from the unlabelled strand on a denaturing 6% polyacrylamide/7 M urea/TBE (Tris-borate-EDTA, pH 8.3) gel. SI nuclease protection. S1 mapping was carried out by the method of Berk and Sharp (1977) as modified by Weaver and Weissman (1979). Primer extension on RNA templates. cDNA was synthesized on RNA transcripts using AMV reverse transcriptase essentially as described by Maniatis et al. (1982). 5' End-labelled primer was hybridised to RNA as described in the Sl nuclease protection protocol. The reaction was then quenched in 100 Al of ice-cold 0.3 M sodium acetate, pH 5.5, and ethanol precipitated. The primer-template pellet was taken up in 50 A1 of reverse transcriptase 'mix' [100 mM Tris-HCI (pH 8.3 at 42°C)/10 mM MgC12/140 mM KCI/20 mM mercaptoethanol/l mM each dNTP/10 units reverse transcriptase (Boehringer Mannheim)] and incubated at 42°C for 2 h. RNA was then hydrolysed by the addition of 0.2 N NaOH at 420C for 60 min. Following neutralisation with HCI the cDNA was extracted with phenol and ether, ethanol precipitated and run on a 6% polyacrylamide/7 M urea/TBE gel (Sanger and Coulson, 1978). Sequencing of RNA by primer extension RNA was sequenced by a modification of the method of Hamlyn et al. (1978) using reverse transcription from a5' end-labelled primer in the presence of dideoxy nucleotides. 4 ng of primer was mixed with 80yg of reticulocyte poly(A)+ RNA in 7.5 II water and was heated to 70°C for 1 min followed by rapid cooling on ice. Potassium chloride was then added to 400 mM and the primer-RNA mix incubated at 200C for 2 h. The mixwas splitinto four equivalent aliquots. Each of these was made 60 iLM with respect to one of the four ddNTPs and the volume was adjusted to 50yl containing 100 mM Tris-HCI (pH 8.3 at 42°C)/10 mM
1226
MgCl2/140 mM KCI/20 mM ,3-mercaptoethanol/400 uM each dNTP/5 units reverse transcriptase (Boehringer Mannheim) and incubated at 42°C for 1 h. The cDNA was then processed as in the primer extension protocol and run on an 8% polyacrylamide/7 M urea/TBE gel.
Acknowledgements This work has been supported by the Cancer Research Campaign. I.C. is funded by an M.R.C. Research Studentship and a scholarship from I.C.I.
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Received on 26 Februarv 1986.
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