Human Molecular Genetics, 2003, Vol. 12, No. 7 759-769
DOI: 10.1093/hmg/ddg079
© 2003 Oxford University Press
Alternative 5' exons of the CFTR gene show developmental regulation
Paediatric Molecular Genetics, Weatherall Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford, OX3 9DS, UK
Received November 29, 2002; Accepted January 29, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a complex mechanism of tissue-specific and temporal regulation. Expression of the sheep and human CFTR genes shows a gradual decline during lung development, from the early mid-trimester through to term. Alternative upstream exons of CFTR have been identified in several species but their functional role remains obscure. We identified a novel 5' exon of the sheep CFTR gene (ov1a) that occurs in two splice forms (ov1aL and ov1aS), which are both mutually exclusive with exon 1. CFTR transcripts including ov1aL and ov1aS are present at low levels in many sheep tissues, however ov1aS shows temporal and spatial regulation during fetal lung development, being most abundant when CFTR expression levels start to decline. Alternative 5' exons -1a and 1a in the human CFTR gene also show changes in expression levels through lung development. Evaluation of ov1aL and ov1aS by Mfold reveals the potential to form extremely stable secondary structures which would cause ribosomal subunit detachment. Further, the loss of exon 1 from the CFTR transcript removes motifs that are crucial for normal trafficking of the CFTR protein. Recruitment of these alternative upstream exons may represent a novel mechanism of developmental regulation of CFTR expression.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Recruitment of alternative 5' exon(s) to a gene transcript to generate novel cell-type specific forms of a protein is a well-documented phenomenon. These additional 5' exons may be spliced directly to the conventional exon 1 of a gene or onto downstream exons so by-passing the first, or multiple exons. In some cases the resultant novel proteins show modulation of function (1), however, in other examples the biological role is not yet clear.
No clear functional model has yet emerged from the complexity of transcription initiation of the CFTR gene. Multiple start sites have been identified for the CFTR transcript and alternative exon usage at the 5' end of the CFTR gene has been described by several groups (2–5) (J. Rommens, personal communication), but the functional role of the alternative transcripts has not been elucidated. In human CFTR, the alternative 5' exons have been observed primarily in transcripts from intestinal carcinoma cell lines such as T84 so their role in normal CFTR expression in vivo remains to be confirmed. Two exons, termed -1a and 1a, were identified 5' of exon 1 of the human CFTR gene (3) both of which are spliced directly to exon 2, so excluding exon 1 from the transcript. Exon -1a may either be spliced to exon 1a or directly to exon 2. Exon 1a has termination codons in all three frames so inclusion of this exon instead of normal exon 1 was thought unlikely to result in a protein product. Exon -1a lacks an AUG but does contain a potential alternative initiation codon (CUG), alternatively, a downstream AUG in exon 4 might be used for translation initiation. Tissue-specific alternative 5' exon usage occurs in CFTR expressed in the mouse testis (6) and the alternative exon shows partial homology with human exon 1a. However, in contrast to human exons -1a and 1a, murine exon -1, is spliced directly to exon 1 as an alternative 5'-untranslated region instead of splicing to exon 2.
Alternative splicing of the 5' end of CFTR has not been investigated in other species. As part of a comparative genomic approach to elucidate the regulatory mechanisms of human CFTR expression we have evaluated the ovine CFTR gene (7). Ovine CFTR exhibits an equivalent developmental down-regulation in the lung to that seen in humans, with abundant CFTR mRNA during the second trimester of gestation throughout the airway epithelium followed by a gradual decrease until birth (8). The mechanism of down-regulation is unknown. While investigating alternative splicing of the ovine CFTR gene (Broackes-Carter et al., submitted) we noticed that transcripts in the ovine lung and trachea showed a substantial reduction in usage of exon 1 in comparison to other tissues. We now show that the ovine CFTR gene recruits two forms of a novel 5' exon, ov1a, located upstream of the native exon 1 which are spliced directly to exon 2, although neither form has obvious coding potential. Most importantly, the shorter form of ov1a appears transiently in lung tissue during gestation and its appearance and disappearance coincide with the start of developmental down-regulation of CFTR gene expression. Re-evaluation of the expression profile of human CFTR exons -1a and 1a in human lung through development showed that transcripts including exon -1a spliced to exon 2 or exon -1a/1a/2 show down-regulation from 14 weeks of gestation. The recruitment of these upstream exons to the CFTR transcript may play an important role in the developmental regulation of CFTR expression in the lung.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Tissue-specific inclusion of ovine CFTR exons 1 and 2
When characterizing alternative splicing in the ovine CFTR gene, we observed that the abundance of transcripts containing exon 1 was markedly reduced in lung tissue at 2 days post-term, in comparison to those containing exon 2. Figure 1 shows a 799 bp product from exons 1 to 6b of ovine CFTR generated with primers A2R and ov6abR and a 767 bp product from exons 2 to 6b generated from the same cDNA samples with primers A3R and ov6abR (individual cDNA synthesis reactions with primer ov6abR were divided into two aliquots for the PCR reactions with specific forward primers). Comparison of the abundance of the CFTR-specific fragment with that of subunit c of ovine ATP synthase (530 bp) amplified in the same reaction enables a semi-quantitative comparison between samples. In most tissues the amount of exon 1-containing cDNA is equivalent to exon 2-containing cDNA, however in lung and to a lesser extent the trachea and salivary gland, the relative abundance of exon 1-containing cDNA is reduced.
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Developmental aspects of exon 1 usage
We previously described developmental down-regulation of CFTR expression in ovine lung (8). To evaluate whether differential inclusion of exon 1 might contribute to the mechanism of this down-regulation, semi-quantitative RT–PCR was carried out on total lung RNA at 50, 74, 83, 90, 108, 129 and 143 days of gestation and 2 days post-term (Fig. 2). Transcripts containing exon 1 (amplified with primers A2R and ov6abR) were present at 50 days gestation and were of similar abundance until 90 days of gestation but then showed a rapid decline in levels by 108 days [Fig. 2B(ii), 799 bp]. In contrast, exon 2-containing transcripts (amplified with primers A3R and ov6abR) gradually declined in abundance after 83 days but remained detectable until at least 2 days post-term [Fig. 2B(iii), 767 bp]. (In Fig. 2B–D, cDNA generated with primer ov6abR was divided into three aliquots for the specific PCR reactions, hence the total amount of product was consistently lower than in Fig. 1, where reactions were divided into two aliquots.)
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Identification of an alternative 5' exon in ovine CFTR
The variation in exon 1 usage suggested that ovine CFTR might recruit alternative 5' exons as shown previously in human and murine transcripts. To test this hypothesis, 5' RACE was carried out on lung RNA at 83 days of gestation. cDNA was synthesized from nested primers OV3SL and 2S2L (Fig. 3 and Table 1), located in exons 3 and 2 respectively, as two rounds of PCR were necessary to amplify a 378 bp product. Sequence analysis of the 378bp product showed that it contained 284 bp of the ovine CFTR promoter region AF325415: 1154–1437 spliced directly onto exon 2. To identify the 5' end of this novel exon (ov1a) a further 5' RACE was performed on 83 day lung RNA generating cDNA with primer Ovex2R2 located at the 5' end of exon 2 (U20814: 208–225). After two rounds of semi-nested PCR using primer Ov1aR1 (AF32514: 1341–1359) or Ov1aR2 (AF32514: 1278–1296), located within the ov1a exon, 206 or 144 bp products, respectively, were obtained. The sequence of both these products commenced at the same base within the ovine CFTR promoter (AF325415: 1154). The 3' end of the ov1aL exon is located 931 bp 5' to the ATG and is spliced onto exon 2, so precluding ovine exon 1 from the transcript (Fig. 3).
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RT–PCR using 5' primer Ov1a (located in exon ov1a) and 3' primer ov6abR (located in exon 6b) of total RNA from ovine tissues generated two products of 917 (ov1aL) and 816 (ov1aS) bp. Direct sequencing of these forms revealed an alternative splice site located within exon ov1a so that the 816 bp (ov1aS, AF325415: 1154–1336) product only contained the 5' 183 bp of the ov1aL exon (AF325415: 1154–1437; Fig. 3).
Inclusion of exon ov1a in lung, pancreas and small intestine through development
Semi-quantitative RT–PCR of lung RNA using a 5' primer in exon ov1a (Ov1a) and a 3' primer in exon 6b (ov6abR) showed that the ov1aL exon was detectable at only very low levels that did not fluctuate through gestation [Fig. 2B(i)]. However, the ov1aS exon increased in abundance from 50 to 74 days gestation, with maximum levels seen at 90 days, before declining and becoming barely detectable after 129 days [Fig. 2B(i)]. This expression profile was in marked contrast to the exon 1-containing transcript [Fig. 2B(ii)], which was present at 50 days gestation but barely detectable after 90 days and exon 2-containing transcripts [Fig. 2B(iii)], which showed the gradual down-regulation through gestation that we reported previously for ovine CFTR, using a TaqMan quantitative RT–PCR assay (8).
Semi-quantitative RT–PCR of pancreas RNA during gestation used the same primer sets to detect ov1aL/S, exon 1- and exon 2-containing transcripts (Fig. 2C). In contrast to the transient expression of ov1aS-containing transcripts seen in lung RNA through gestation, all four forms showed similar expression profiles through gestation in the pancreas. Both ov1aL and ov1aS were present in low abundance as early as 50 days, and remained at constant levels, with more ov1aS than ov1aL until 90 days. By 108 days, the ov1a transcripts were barely detectable [Fig. 2C(i)]. Exon 1- and exon 2-containing transcripts in pancreas followed a similar pattern of expression, but with a relatively high abundance from 50 to 90 days gestation followed by a gradual decline until 2 days post-term [Fig. 2C(ii) and (iii)]. In RNA from small intestine the levels of all four forms remained constant throughout gestation, with minor individual fluctuations [Fig. 2D(i–iii)] though ov1aL and ov1aS were of a equal low abundance in comparison to exon 1- and exon 2-containing transcripts.
Quantitation of ov1a-containing transcripts by RNase protection
To confirm the relative abundance of ov1a-containing transcripts in vivo by a non RT–PCR-based method, RNase protection assays (RPA) were performed. Two riboprobes were used for RPA, the 
cDNA probe (9) (from base U20418:1–506) that spans the 5' UTR to 104 bp into exon 4 and the ov1a probe, the 339 bp RACE product [ov1a (AF325415: 1154–1437) joined to 55 bp at the 5' end of exon 2 (U20418: 183–238)]. Thirty micrograms of total RNA from pancreas, lung, small intestine and salivary glands at 83 days of gestation and 2 days post-term were evaluated with each probe (Fig. 4). Hybridization of the riboprobe to the RNA revealed two protected fragments of 506 and 323 bp (Fig. 4A and B). The 506 bp fragment arises from protection of mRNA containing the 5' UTR transcript and traditional exon 1 through to exon 4 (U20418: 129–506) and the 323 bp fragment is generated by the protection of a transcript containing exons 2–4 (U20418: 184–506) but lacking the 5' UTR and exon 1. In pancreas at 83 days of gestation the full-length product is more abundant than the form lacking exon 1, while at 2 days post-term both forms were of similar low abundance. In small intestine the two protected fragments were at equivalent levels at 83 days of gestation and 2 days post-term and remain in proportion to total CFTR expression at these two time points. However, at 83 days of gestation in lung and salivary gland the full-length 506 bp protected fragments containing the 5' UTR and exon 1 is of much lower abundance than the 323 bp form lacking these sequences. At 2 days post-term in lung only the 323 bp fragment is evident while neither form is detectable in salivary gland.
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Two protected fragments were also detected after hybridization of the 339 bp (ov1a) RACE product riboprobe to the RNA (Fig. 4C and D). The 339 bp fragment corresponds to a transcript containing exon ov1aL joined to 55 bp at the 5' end of exon 2 and the 238 bp fragment corresponds to ov1aS (183 bp, AF325415: 1154–1336) that lacks the 3' end of ov1aL but is also joined to exon 2. At 83 days gestation the ov1aS form is more abundant than ov1aL in lung and pancreas, but the two forms are at about the same level in salivary gland and were undetectable in small intestine. Both ov1aS and ov1aL were of very low abundance by 2 days post-term and were only detectable in pancreas and small intestine. These data are consistent with the observations made by RT–PCR (Fig. 2).
Developmental expression of human exon -1a and 1a in lung and small intestine
Alternative upstream exons have been identified for both the human and mouse CFTR/cftr genes (Fig. 5). The human data are derived largely from cell lines such as T84 (3,10). Using a reverse primer located in exon 6b (ov6abR) and forward primer HUM18632 in the region homologous to ov1a, Hu-1aK in exon -1a and Hu1aR in exon 1a, the presence of all three upstream exons in the human transcript was confirmed in RNA from T84 cells (data not shown). RNA from human fetal lung at 14, 17, 19 and 26 weeks gestation (equivalent to 74, 85, 93 and 120 days in the sheep) was then evaluated for the presence of the 5' exons by RT–PCR. In all cases expression of the CFTR transcript was compared to the human ‘housekeeping gene’ ß glucocerebrosidase (572 bp) (11). Both human exon -1a- and 1a-containing transcripts were detected at low levels but not transcripts with homology to ov1a. With the exception of exon -1a-containing transcripts at 14 weeks gestation, which were visible on ethidium-bromide-stained agarose gels, the products were only detected on Southern blots after probing with exon-specific probes. Figure 6 shows that although there is no dramatic change in the expression levels of human CFTR as detected by semi-quantitative RT–PCR of exons 2-6b between 14 and 26 weeks of gestation in the lung (Fig. 6C), the abundance of transcripts containing upstream exons -1a/1a/2 and -1a/2 (sequence confirmed) falls substantially during gestation. An equivalent profile was not observed for human 1a/2 in the lung (data not shown).
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Structural evaluation of upstream exon sequences
To evaluate the potential function of the ov1aL and ov1aS exons in sheep CFTR and human CFTR exon -1a and exon 1a these were subjected to structural analysis using the Mfold program (Fig. 7). Ovine ov1aL and ov1aS had
G values of -74.55 and -48.38, respectively, demonstrating significant stable secondary structure in these exons. The G values from human exon -1a and 1a individually were -29.7 and -97.01, respectively, and when joined in the -1a/1a/2 spliceform, -146.29.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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The molecular mechanism for developmental down-regulation of CFTR expression in the sheep and human lung remains to be elucidated. We have identified a novel alternative upstream exon of sheep CFTR that is spliced directly onto exon 2 of the gene. Recruitment of one form of this upstream exon shows developmental regulation, increasing in abundance as CFTR expression starts to decline in the sheep lung. An upstream exon of human CFTR was also detected at higher levels in the early mid-trimester of gestation and declined later during development. The transient appearance of these alternative 5' exons occurs at gestational ages corresponding to the end of the pseudoglandular and beginning of the canalicular phase of lung development. CFTR expression shows a major decline during the canalicular phase when lung epithelial differentiation takes place (8). Expression of alternative 5' exons during this developmental phase may be involved in the down-regulation. How might these alternative 5' exons function? One possibility is that ov1aS and human exon -1a exert a direct negative feedback on normal CFTR transcription for example by an RNA-interference-like mechanism. However, we have no evidence to support this. Although the abundance of transcripts containing the upstream exons is comparatively low, particularly in the human fetal lung, they might still be of physiological relevance. An alternative hypothesis is based on the structural evaluation of these exons by Mfold. The Gibbs free energy for ov1aS and human -1a/1a are -48.38 and -146.29, respectively. These
G values would be predicted to cause detachment of the 40s ribosomal subunit (12), which could provide a mechanism for reducing levels of CFTR protein at a time when other pathways were causing a gradual inhibition of CFTR mRNA expression during lung epithelial differentation.
Exon ov1a is not homologous to the alternative upstream exons previously documented in human CFTR (Fig. 3), but it has in common the lack of any obvious coding potential (3). All potential initiation codons in ov1a encounter downstream termination codons. The exclusion of exon 1 from a percentage of transcripts could be the most important consequence of the use of the upstream exons since this would be predicted to have several significant effects on the CFTR protein. The ov1aS 5' exon and human -1a splicing directly to exon 2 would force translation initiation to occur within downstream exons most likely utilizing an ATG in exon 4 of the CFTR transcript. A series of N-terminal truncations of the CFTR protein were generated to coincide with potential downstream initiator codons (13). When these mutants were injected into Xenopus oocytes, chloride channels with very similar properties to wild-type CFTR were observed if initiation occurred from M150 (exon 4) or M265 (exon 5), both of which conform strongly to the Kozak consensus. Ion channels with distinct N-terminal ends that confer novel properties on the protein have been described previously. For example, an N-terminal splice variant of the epithelial sodium channel ENaC, which deletes 49 amino acids of the mouse ENaC subunit resulted in altered sodium conductance properties when co-expressed with ß and 
subunits in Xenopus oocytes (14).
Loss of exon 1 would also be predicted to remove at least two motifs that are important in the intracellular localization and function of the CFTR protein: a diphenylalanine motif (F16/F17) that is conserved in human and sheep and has been shown to be important in normal trafficking of CFTR from the Golgi apparatus to the cell membrane (15). Further, a hydrophobic domain at the exon1/2 boundary is thought to play a role in the interaction of CFTR with syntaxin 1A, a component of the membrane vesicle fusion machinery. Syntaxin 1A, in complex with SNAP-23 inhibits trafficking of CFTR to the cell surface and so is a key negative regulator of CFTR chloride ion channel function (16–19). Hence the alternative upstream exons spliced directly to exon 2 would be predicted to alter CFTR trafficking and function.
Alternative upstream exons that remain to be identified may account for the observation by RNase protection that CFTR transcripts lacking exon 1 are also abundant in the sheep pancreas, even though there is very little ov1aS/L. Low levels of alternate transcripts are detected by RT–PCR throughout gestation in several sheep tissues and so might be considered of little significance. However the clear temporal regulation of the alternative CFTR transcripts in the mid-trimester sheep lung and probably also in human lung may represent a novel mechanism of regulation for this complex gene.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Isolation of total RNA from ovine and human tissues
Sheep breeds used were a cross between (ram) Poll Dorset and (ewes) cross-bred Suffolk and Mule, unless otherwise stated. Tissue was collected from fetuses and lambs at time points 50, 74, 83, 90, 108, 129 and 143 days of gestation, at birth and at 2 days after birth (post-term). Tissues were snap-frozen and stored in liquid nitrogen. Human fetal tissue was collected with ethical committee approval from social terminations at 14, 17 and 19 weeks and from a spontaneous abortion at 26 weeks. Tissue was homogenized in 4 M guanidinium isothiocyanate and RNA isolated from caesium chloride gradients and resuspended in 10 mM Tris (pH 7.4) 0.1 mM EDTA (20).
Reverse transcription and polymerase chain reaction for ovine and human RNA
CFTR cDNA synthesis and PCR was carried out as described previously (8,21), using primers shown in Table 1. RT–PCR products were extracted from TAE gels using Qiaspin columns (Qiagen) and direct sequenced using the ABI BIG Dye Terminator kit (Applied Biosystems). For semi-quantitative RT–PCR of ovine RNA, subunit c of the ovine ATP synthase was co-amplified as described previously (8,9) and for human RNA ß glucocerebrosidase was co-amplified (11). All reactions were evaluated in the exponential phase. For data shown in Figure 2 RT–PCR reactions were separated on agarose gels and the relative intensities of the CFTR-specific and ovine ATP-synthase products were measured by densitometry (ImageQuant, Amersham Biosciences). The amounts of ATP-synthase were normalized to one arbitrary reaction for each reaction set and samples loaded on gels to show variations in the CFTR-specific product in the presence of constant amounts of ATP synthase.
5' rapid amplification of cDNA ends (5' RACE)
5' RACE was carried out using the 5'/3' RACE Kit from Roche. cDNA was synthesized from 2 µg of RNA from 83 day lung using primers 2S2L, OV3SL or OVEX2R2 (Table 1). The dA-tailed cDNA was amplified at 52°C using the oligo-dT anchor primer and the specific primer used to synthesize the cDNA. Nested PCR was required to generate a visible product. One microlitre of the first round PCR reaction was amplified at 52°C using the anchor oligo dT and primer OV2S3L, OV1aR1 or OV1aR2 when the cDNA was synthesized from primer 2S2L or OV3SL/OVEX2R2, respectively. The PCR products were gel purified with the GFXTM kit (Amersham) and cloned into T-tailed pBluescript for sequence analysis.
Ribonuclease (RNase) protection
The 5' end of the ovine CFTR cDNA [fragment (9) U20418: 1–506] and the 339 bp RACE product containing the ov1aL exon and 55 bp of exon 2 (AF325415: 1154–1437 and U20418: 183–238) were sub-cloned into the vector pSP73. The constructs were linearized with BamHI or HindIII, respectively and radiolabelled transcripts were prepared by standard methods using RNA polymerase SP6 and 4 µl 32PdGTP (10 mCi/ml, Amersham). Thirty micrograms of RNA from ovine pancreas, lung, small intestine and salivary gland at 83 days of gestation and 2 days post-term were hybridized to radiolabelled transcripts (5x105 cpm) in 80% formamide buffer (80% formamide, 40 mM PIPES, 400 mM NaCl and 1 mM EDTA) overnight at 58°C. Annealed RNA duplexes were digested first with RNase T1 (2 µg/ml) and A (40 µg/ml) and then with proteinase K (1 mg/ml) sequentially for 30 min each at 37°C. After phenol–chloroform extraction and ethanol precipitation, the samples were run on 6% acrylamide gel. Autoradiographs were exposed for 3–14 days at -70°C.
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ACKNOWLEDGEMENTS |
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We thank Prof. Christopher Pugh and Kay Yeates for help with RNase protection assays; Dr Johanna Rommens for sharing unpublished data; also John Bassett, Cliff Hanson, Frances Knight and David Smith for assistance. This work was funded by Vaincre la Mucoviscidose and the Cystic Fibrosis Trust. F.C.B.-C. was in receipt of a Medical Research Council postgraduate studentship.
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FOOTNOTES |
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* The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
To whom correspondence should be addressed. Email: aharris{at}molbiol.ox.ac.uk
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 V. Chubanov, S. Waldegger, M. M. y Schnitzler, H. Vitzthum, M. C. Sassen, H. W. Seyberth, M. Konrad, and T. Gudermann Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia PNAS, March 2, 2004; 101(9): 2894 - 2899. [Abstract] [Full Text] [PDF]
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