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• W2000786270 abstract "Human chromosome 17-specific genomic clones extending over 90 kilobases (kb) of DNA and coding for sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The presence of the D17S1828 genetic marker in the cosmid contig enabled us to map the SERCA3 gene (ATP2A3) 11 centimorgans from the top of the short arm p of chromosome 17, in the vicinity of the cystinosis gene locus. The SERCA3 gene contains 22 exons spread over 50 kb of genomic DNA. The exon/intron boundaries are well conserved between human SERCA3 and SERCA1 genes, except for the junction between exons 8 and 9 which is found in the SERCA1 gene but not in SERCA3 and SERCA2 genes. The transcription start site (+1) is located 152 nucleotides (nt) upstream of the AUG codon. The 5′-flanking region, including exon 1, is embedded in a 1.5-kb CpG island and is characterized by the absence of a TATA box and by the presence of 14 putative Sp1 sites, 11 CACCC boxes, 5 AP-2-binding motifs, 3 GGCTGGGG motifs, 3 CANNTG boxes, a GATA motif, as well as single sites for Ets-1, c-Myc, and TFIIIc. Functional promoter analysis indicated that the GC-rich region (87% G + C) from −135 to −31 is of critical importance in initiating SERCA3 gene transcription in Jurkat cells. Exon 21 (human, 101 base pairs; mouse, 86 base pairs) can be alternatively excluded, partially included, or totally included, thus generating, respectively, SERCA3a (human and mouse, 999 amino acids (aa)), SERCA3b (human, 1043 aa; mouse, 1038 aa), or SERCA3c (human, 1024 aa; mouse, 1021 aa) isoforms with different C termini. Expression of the mouse SERCA3 isoforms in COS-1 cells demonstrated their ability to function as active pumps, although with different apparent affinities for Ca2+. Human chromosome 17-specific genomic clones extending over 90 kilobases (kb) of DNA and coding for sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The presence of the D17S1828 genetic marker in the cosmid contig enabled us to map the SERCA3 gene (ATP2A3) 11 centimorgans from the top of the short arm p of chromosome 17, in the vicinity of the cystinosis gene locus. The SERCA3 gene contains 22 exons spread over 50 kb of genomic DNA. The exon/intron boundaries are well conserved between human SERCA3 and SERCA1 genes, except for the junction between exons 8 and 9 which is found in the SERCA1 gene but not in SERCA3 and SERCA2 genes. The transcription start site (+1) is located 152 nucleotides (nt) upstream of the AUG codon. The 5′-flanking region, including exon 1, is embedded in a 1.5-kb CpG island and is characterized by the absence of a TATA box and by the presence of 14 putative Sp1 sites, 11 CACCC boxes, 5 AP-2-binding motifs, 3 GGCTGGGG motifs, 3 CANNTG boxes, a GATA motif, as well as single sites for Ets-1, c-Myc, and TFIIIc. Functional promoter analysis indicated that the GC-rich region (87% G + C) from −135 to −31 is of critical importance in initiating SERCA3 gene transcription in Jurkat cells. Exon 21 (human, 101 base pairs; mouse, 86 base pairs) can be alternatively excluded, partially included, or totally included, thus generating, respectively, SERCA3a (human and mouse, 999 amino acids (aa)), SERCA3b (human, 1043 aa; mouse, 1038 aa), or SERCA3c (human, 1024 aa; mouse, 1021 aa) isoforms with different C termini. Expression of the mouse SERCA3 isoforms in COS-1 cells demonstrated their ability to function as active pumps, although with different apparent affinities for Ca2+. Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs), 1The abbreviations used are: SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; MOPS, 3-(N-morpholino)propanesulfonic acid; RT-PCR, reverse-transcribed-polymerase chain reaction; TFIIIc, transcription factor IIIc; aa, amino acids; bp, base pairs; cM, centimorgans; nt, nucleotides; kb, kilobase pairs; Inr, initiator.1The abbreviations used are: SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; MOPS, 3-(N-morpholino)propanesulfonic acid; RT-PCR, reverse-transcribed-polymerase chain reaction; TFIIIc, transcription factor IIIc; aa, amino acids; bp, base pairs; cM, centimorgans; nt, nucleotides; kb, kilobase pairs; Inr, initiator. mediating the uptake of Ca2+ into intracellular stores such as sarcoplasmic and endoplasmic reticulum, are encoded by three distinct genes in higher vertebrates (reviewed in Ref. 1Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar). SERCA1 is expressed only in the fast-twitch skeletal muscle as one of its developmentally spliced variants: the adult SERCA1a (994 aa) or the neonatal SERCA1b (1001 aa). Both isoforms present identical amino acid sequences up to amino acid 993. As a result of retention/excision of the penultimate exon (42 bp), respectively, in the SERCA1a/SERCA1b splice variants, the last amino acid (Gly) in SERCA1a is replaced in SERCA1b by a highly charged octapeptide sequence DPEDERRK (2Korczak B. Zarain-Herzberg A. Brandl C.J. Ingles C.J. Green N.M. MacLennan D.H. J. Biol. Chem. 1988; 263: 4813-4819Abstract Full Text PDF PubMed Google Scholar). COS cell expression studies showed no functional differences between SERCA1a and SERCA1b isoforms. The complete structures of the rabbit (2Korczak B. Zarain-Herzberg A. Brandl C.J. Ingles C.J. Green N.M. MacLennan D.H. J. Biol. Chem. 1988; 263: 4813-4819Abstract Full Text PDF PubMed Google Scholar) and human (3Zhang Y. Fujii J. Philips M.S. Chen H.-S. Karpati G. Yee W.-C. Schrank B. Cornblath D.R. Boylan K.V. MacLennan D.H. Genomics. 1995; 30: 415-424Crossref PubMed Scopus (64) Google Scholar) SERCA1 genes have been elucidated. The SERCA1 gene (ATP2A1) has been mapped to human chromosome 16p12.1 (4Callen D.F. Baker E. Lane S. Nancarrow J. Thompson A. Whitmore S.A. MacLennan D.H. Berger R. Cherif D. Jarvela I. Peltonen L. Sutherland G.R. Gardiner R.M. Am. J. Hum. Genet. 1991; 49: 1372-1377PubMed Google Scholar) and a deficiency in SERCA1 is responsible for at least one autosomal recessive form of Brody disease (5Odermatt A. Taschner P.E.M. Khanna V.K. Busch H.F.M. Karpati G. Jablecki C.K. Breuning M.H. MacLennan D.H. Nat. Genet. 1996; 14: 191-194Crossref PubMed Scopus (183) Google Scholar). Tissue-specific processing of the SERCA2 gene primary transcript generates up to four mRNA classes (6Wuytack F. Raeymaekers L. De Smedt H. Eggermont J.A. Missiaen L. Van Den Bosch L. De Jaegere S. Verboomen H. Plessers L. Casteels R. Ann. N. Y. Acad. Sci. 1992; 671: 82-91Crossref PubMed Scopus (48) Google Scholar), which code for two isoenzymes as follows: a cardiac/slow-twitch skeletal muscle protein (SERCA2a) and a ubiquitously expressed isoform (SERCA2b). As a result of alternative splicing, the SERCA2a-specific C terminus comprising the sequence AILE (aa 994–997) is replaced by a variant tail of 49 or 50 amino acids in SERCA2b (7Lytton J. MacLennan D.H. J. Biol. Chem. 1988; 263: 15024-15031Abstract Full Text PDF PubMed Google Scholar, 8Gunteski-Hamblin A.-M. Greeb J. Shull G.E. J. Biol. Chem. 1988; 263: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 9Eggermont J.A. Wuytack F. De Jaegere S. Nelles L. Casteels R. Biochem. J. 1989; 260: 757-761Crossref PubMed Scopus (48) Google Scholar). This extended tail contains a very hydrophobic stretch, which is suggested to represent a possible 11th transmembrane segment (7Lytton J. MacLennan D.H. J. Biol. Chem. 1988; 263: 15024-15031Abstract Full Text PDF PubMed Google Scholar, 8Gunteski-Hamblin A.-M. Greeb J. Shull G.E. J. Biol. Chem. 1988; 263: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 9Eggermont J.A. Wuytack F. De Jaegere S. Nelles L. Casteels R. Biochem. J. 1989; 260: 757-761Crossref PubMed Scopus (48) Google Scholar). The divergence in the C-terminal part is responsible for functional differences between SERCA2a and SERCA2b (10Lytton J. Westlin M. Burk S.E. Shull G.E. MacLennan D.H. J. Biol. Chem. 1992; 267: 14483-14489Abstract Full Text PDF PubMed Google Scholar,11Verboomen H. Wuytack F. De Smedt H. Himpens B. Casteels R. Biochem. J. 1992; 286: 591-596Crossref PubMed Scopus (125) Google Scholar); these differences were recently ascribed to the presence of the last 12 amino acids in SERCA2b (12Verboomen H. Wuytack F. Van Den Bosch L. Mertens L. Casteels R. Biochem. J. 1994; 303: 979-984Crossref PubMed Scopus (75) Google Scholar). Thus far, the complete structure of a SERCA2 gene is lacking, but partial characterization of the 5′- and/or 3′-ends of the gene has been reported for human (7Lytton J. MacLennan D.H. J. Biol. Chem. 1988; 263: 15024-15031Abstract Full Text PDF PubMed Google Scholar, 13Wankerl M. Boheler K.R. Fiszman M.Y. Schwartz K. J. Mol. Cell. Cardiol. 1996; 28: 2139-2150Abstract Full Text PDF PubMed Scopus (19) Google Scholar), rabbit (14Zarain-Herzberg A. MacLennan D.H. Periasamy M. J. Biol. Chem. 1990; 265: 4670-4677Abstract Full Text PDF PubMed Google Scholar), pig (15Eggermont J. Wuytack F. Casteels R. Biochem. J. 1990; 266: 901-907PubMed Google Scholar), and rat (16Rohrer D.K. Hartong R. Dillmann W.H. J. Biol. Chem. 1991; 266: 8638-8646Abstract Full Text PDF PubMed Google Scholar). The SERCA2 gene (ATP2A2) has been mapped to human chromosome 12q23-q24.1 (17Otsu K. Fujii J. Periasamy M. Difilippantonio M. Uppender M. Ward D.C. MacLennan D.H. Genomics. 1993; 17: 507-509Crossref PubMed Scopus (68) Google Scholar). Structural and functional analyses of the SERCA2 gene promoter in rabbit (18Fisher S.A. Buttrick P.M. Sukovich D. Periasamy M. Circ. Res. 1993; 73: 622-628Crossref PubMed Scopus (24) Google Scholar, 19Sukovich D.A. Shabbeer J. Periasamy M. Nucleic Acids Res. 1993; 21: 2723-2728Crossref PubMed Scopus (10) Google Scholar, 20Baker D.L. Dave V. Reed T. Periasamy M. J. Biol. Chem. 1996; 271: 5921-5928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), rat (16Rohrer D.K. Hartong R. Dillmann W.H. J. Biol. Chem. 1991; 266: 8638-8646Abstract Full Text PDF PubMed Google Scholar), and human (13Wankerl M. Boheler K.R. Fiszman M.Y. Schwartz K. J. Mol. Cell. Cardiol. 1996; 28: 2139-2150Abstract Full Text PDF PubMed Scopus (19) Google Scholar) identified the promoter regions required for transcriptional activity in NIH3T3 fibroblasts, primary cultured rat cardiomyocytes, C2C12 and Sol8 muscle cells. Several putativecis-acting elements have been described, among which Sp1 sites and thyroid-responsive elements have been proven to exert an important role in transcriptional regulation of the SERCA2 gene (20Baker D.L. Dave V. Reed T. Periasamy M. J. Biol. Chem. 1996; 271: 5921-5928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar,21Hartong R. Wang N. Kurokawa R. Lazar M.A. Glass C.K. Apriletti J.W. Dillmann W.H. J. Biol. Chem. 1994; 269: 13021-13029Abstract Full Text PDF PubMed Google Scholar). Unique SERCA genes have also been described in invertebrates, such as the crustacean Artemia franciscana (22Escalante R. Sastre L. J. Biol. Chem. 1994; 269: 13005-13012Abstract Full Text PDF PubMed Google Scholar) and the insectDrosophila melanogaster (23Magyar A. Bakos EÖ. VaÖradi A. Biochem. J. 1995; 310: 757-763Crossref PubMed Scopus (28) Google Scholar). The gene primary transcript is alternatively spliced in Artemia, and the expression of the two isoforms is regulated by tissue-specific alternative promoters (24Escalante R. Sastre L. DNA Cell Biol. 1995; 14: 893-900Crossref PubMed Scopus (7) Google Scholar). The first report describing the cloning of the SERCA3 cDNA from rat kidney (25Burk S.E. MacLennan D.H. Shull G.E. J. Biol. Chem. 1989; 264: 18561-18568Abstract Full Text PDF PubMed Google Scholar) indicated a broad expression pattern for its 4.8-kb transcript. Recent studies demonstrated that SERCA3 is always co-expressed along with the ubiquitous SERCA2b isoform (26Wuytack F. Dode L. Baba-Aissa F. Raeymaekers L. Biosci. Rep. 1995; 15: 299-306Crossref PubMed Scopus (72) Google Scholar), and high levels of SERCA3 mRNA have been documented in the hematopoietic cell lineage, arterial endothelial and secretory epithelial cells, as well as in cerebellar Purkinje neurons (27Wuytack F. Papp B. Verboomen H. Raeymaekers L. Dode L. Enouf J. Bokkala S. Authi K.S. Casteels R. J. Biol. Chem. 1994; 269: 1410-1416Abstract Full Text PDF PubMed Google Scholar, 28Wu K.-D. Lee W.-S. Wey J. Bungard D. Lytton J. Am J. Physiol. 1995; 269: C775-C784Crossref PubMed Google Scholar, 29Anger M. Samuel J.-L. Marotte F. Wuytack F. Rappaport L. Lompré A.-M. FEBS Lett. 1993; 334: 45-48Crossref PubMed Scopus (63) Google Scholar, 30Baba-Aissa F. Raeymaekers L. Wuytack F. Callewaert G. Dode L. Missiaen L. Casteels R. Mol. Brain Res. 1996; 41: 169-174Crossref PubMed Scopus (33) Google Scholar). Upon expression in COS-1 cells, SERCA3 presents a much lower apparent affinity for Ca2+, when compared with the other members of the SERCA family (10Lytton J. Westlin M. Burk S.E. Shull G.E. MacLennan D.H. J. Biol. Chem. 1992; 267: 14483-14489Abstract Full Text PDF PubMed Google Scholar). We have previously identified the 97-kDa SERCA3 (999 aa) in both human and rat platelets using a set of SERCA3-specific antisera (27Wuytack F. Papp B. Verboomen H. Raeymaekers L. Dode L. Enouf J. Bokkala S. Authi K.S. Casteels R. J. Biol. Chem. 1994; 269: 1410-1416Abstract Full Text PDF PubMed Google Scholar). Additionally, we cloned the human SERCA3 cDNA, isolated and partially characterized a genomic clone encoding all but the 5′-end of the gene, and localized the SERCA3 gene (ATP2A3) on human chromosome 17p13.3 (31Dode L. Wuytack F. Kools P.F.J. Baba-Aissa F. Raeymaekers L. Briké F. Van De Ven W.J.M. Casteels R. Biochem. J. 1996; 318: 689-699Crossref PubMed Scopus (49) Google Scholar). Until very recently, there were no indications that the SERCA3 pre-mRNA was subject to alternative splicing. Two mouse nucleotide sequences coding for SERCA3a and SERCA3b have been deposited in the EMBL/GenBankTM data bank. 2Deposited in the EMBL/GenBankTM data bank under the accession numbers U49394 and U49393, respectively, by Y. Tokuyama, X. Chen, M. W. Roe, and G. I. Bell.2Deposited in the EMBL/GenBankTM data bank under the accession numbers U49394 and U49393, respectively, by Y. Tokuyama, X. Chen, M. W. Roe, and G. I. Bell. So far, no indications regarding the alternative splicing mechanism were published. We now document the complete exon/intron organization of the human SERCA3 gene. The transcription initiation site and several upstream putative cis-regulatory elements were identified. The functional promoter analysis delineates the minimal promoter region responsible for efficient transcriptional activity and suggests the involvement of the Sp1 transcription factor. We also provide evidence that the human and mouse SERCA3 gene primary transcripts are alternatively spliced, thereby generating not two but three distinct isoforms with altered C termini as follows: SERCA3a, SERCA3b, and SERCA3c. Furthermore, the three mouse SERCA3 isoforms were overexpressed in COS cells and shown to be functionally active but with different apparent affinities for Ca2+. To isolate the entire gene, a human chromosome 17-specific library from Reference Library Data Base, ICRF (32Lehrach H. Drmanac R. Hoheisel J. Larin Z. Lennon G. Manaco A.P. Nizetic D. Zehetner G. Poustka A. Genome Analysis: Genetic and Physical Mapping. 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990: 39-81Google Scholar), was screened with the 1482-bpEcoRI insert of the human SERCA3 partial cDNA clone Z8 (31Dode L. Wuytack F. Kools P.F.J. Baba-Aissa F. Raeymaekers L. Briké F. Van De Ven W.J.M. Casteels R. Biochem. J. 1996; 318: 689-699Crossref PubMed Scopus (49) Google Scholar). The EcoRI fragment comprised 6 bp of the 5′-untranslated region and the first 1476 bp of the coding region. Six new positive clones (Fig. 1) were isolated and further characterized according to standard restriction mapping and sequencing protocols. Analysis of repetitive sequences was carried out using the CENSOR server. 3Available at the following on-line address: . The computer-assisted analysis of the putative transcription factor binding sites was performed using the Wisconsin Package Version 9.0 program from Genetics Computer Group (GCG), Madison, WI. Poly(A)+ RNA was isolated from human tonsils (31Dode L. Wuytack F. Kools P.F.J. Baba-Aissa F. Raeymaekers L. Briké F. Van De Ven W.J.M. Casteels R. Biochem. J. 1996; 318: 689-699Crossref PubMed Scopus (49) Google Scholar). Primer extension analysis was essentially performed as described (33Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1987) Current Protocols in Molecular Biology, pp. 4.8.1 4.8.5, John Wiley & Sons, Inc., New YorkGoogle Scholar). The extension primer used (5′-GAGGCCATGTCCGTGCTGGGAC-3′) corresponds to the inverse complement of nucleotides 25–46 (numbering relative to the determined transcription site; see Fig. 5 b). The 35S-labeled sequencing products (used as size markers) of a 5′ genomic fragment primed with the same extension primer and the extension products were separated on a 6% polyacrylamide, 7 m urea sequencing gel.Figure 5Structural and functional analysis of the 5′-flanking region of the human SERCA3 gene. a, CG (%) content analysis of an 11-kb nucleotide sequence surrounding the first exon. Exon 1 is shown schematically below the plot. The detected repetitive sequences are illustrated at the bottomof the panel: S, Alu type S; J, Alu types J and I, mammalian wide interspersed repeats. b shows the nucleotide sequence of the 5′-end of the gene. The nucleotide sequence of the first exon is in uppercase letters and the first intron and 5′-flanking sequences in lowercase letters. The amino acids encoded by exon 1 andView Large Image Figure ViewerDownload Hi-res image Download (PPT) A 6.6-kbBamHI-KasI genomic fragment containing the 5′-flanking region of the human SERCA3 gene (Fig. 1) was subcloned between the BglII and HindIII restriction sites of the luciferase expression vector pGL3 basic (Promega, Madison, WI). The resulting plasmid, p6.6BK, was used as a template for further generation of controlled deletions by making use of restriction sites present within the genomic insert and the luciferase cloning vector (Fig. 5 c). The 5′-end of each of the deletion constructs was confirmed by sequencing. The human SERCA3 promoter-luciferase constructs and the unmodified, promoterless pGL3 basic reporter vector were used for transient transfection of the human cell line Jurkat E6.1 cells by electroporation as described (34Berwaer M. Martial J.A. Davis J.R. Mol. Endocrinol. 1994; 8: 635-642Crossref PubMed Scopus (118) Google Scholar). To evaluate transfection efficiencies, the cells were co-transfected with 150 or 500 ng of a pEL1-βgal vector, containing the β-galactosidase reporter gene driven by the elongation factor 1 promoter (pEL1-βgal vector is a gift from Dr. F. Bulens). 4F. Bulens, I. Van Nerum, P. Merchiers, A. Belayew, and D. Collen, unpublished data.Reporter enzyme activities were assayed 40 h after electroporation according to the manufacturer's instructions. The measurements were performed with the MicroLumat LB 96P luminometer (EG & G, Berthold, Bad Wildbad, Germany) and corrected for protein concentration, as determined by the bicinchoninic acid method (Pierce), using bovine serum albumin as standard. Luciferase activities are expressed relative to the β-galactosidase activities and normalized to the value obtained with the promoterless pGL3 basic vector which is set at 1. The human RNA Master Blot™ (CLONTECH, Palo Alto, CA), to which high quality poly(A)+ RNAs from 50 different adult and fetal tissues have been immobilized along with several controls (Fig.6), was hybridized following the manufacturer's protocol. The synthesis of a 3′-end probe by PCR was described earlier (31Dode L. Wuytack F. Kools P.F.J. Baba-Aissa F. Raeymaekers L. Briké F. Van De Ven W.J.M. Casteels R. Biochem. J. 1996; 318: 689-699Crossref PubMed Scopus (49) Google Scholar). The probe corresponds to the nucleotides 3033–3405 (accession numberZ69881) found in the 3′-untranslated region of human SERCA3 cDNA. The blot was analyzed by means of a PhosphorImager model STORM 840 (Molecular Dynamics, Sunnyvale, CA). A common SERCA3b/SERCA3c probe (90-bp long) was PCR-synthesized using a 5′ primer N+ (5′-GCACGGCCTTCTCAGGACAGTCT-3′) and the 3′ primer P1 (5′-GGCTCATTTCTTCCGGTGTGGTCTGG-3′) and the GHS3 clone as template DNA; these primers (Fig. 8 a) span the exon/intron junctions involved in the alternative splicing. PCR amplification was carried out for 20 cycles, each cycle consisting of 30 s at 94 °C, 30 s at 65 °C, and 30 s at 72 °C.Figure 8Alternative splicing of the SERCA3 pre-mRNA in human and mouse. a, the genomic nucleotide sequence of the 3′-end of the human SERCA3 gene is compared with its mouse counterpart. The exon sequences are shown in uppercase letters and the introns in lowercase letters. For mouse, only the exons and the ends of the introns are aligned; thedashes in the mouse sequences denote the nucleotides that are identical with the human ones. To maintain the partial alignment, a single gap (depicted by a thin vertical arrow) was introduced in the mouse sequence downstream from a putative branch point sequence, 5′-ctctgac-3′ (shown in a rectangle withround corners). The 5′ donor sites D1 and D2 are indicatedabove their boxed sequences. If D1 is used, then the sequence of the exon 21 indicated in bold is joined to exon 22. When D2 is used, then the sequence in italics is included in exon 21 and then joined to exon 22. The human (hA) and mouse (mA) 3′ acceptor sites used are also indicated. The boxes Sa and Sb denote the overlapping human and mouse stop codons used in SERCA3a and SERCA3b, respectively. The human and mouse stop codons for SERCA3c (initalics) are shifted and shown in small boxes. The nucleotide numbering is shown relative to the human ATG codon inbold and italics for SERCA3b and SERCA3c, respectively. The determined or estimated (~) sizes of the human and mouse introns and of the last human exon are also indicated. P1 (thin arrow), P2 (dashed arrow), P3, N+, M − 1, and M + 1 (thick arrows) primers are depicted above or below their nucleotide sequence. The polyadenylation signal is underlined;n.d., sequence not determined. b, in the upper part the alternative splicing pattern is schematically illustrated. The exons (Exons 20, 21, and 22) are represented as boxes and introns as straight lines joining the exons. 3a, 3b, and 3cindicate the splicing pattern for SERCA3a, SERCA3b, and SERCA3c, respectively. The donor splice sites, D1 and D2, the stop codons for SERCA3a (Sa), SERCA3b (Sb), SERCA3c (Sc), and the polyadenylation signal are indicated.Below the diagram, the C-terminal parts of the human and mouse SERCA3 isoforms are compared. The sequence in bold, up to aa 993, is encoded by the last constitutively spliced exon (exon20). The common sequences of the SERCA3b and SERCA3c isoforms, encoded by exon 21 up to D1 site, are underlinedin both human isoforms and in their mouse counterparts. The size (in aa) of each isoform is indicated at the right of its corresponding sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Total RNA (0.5 μg) from mouse pancreatic islets (gift from D. L. Eizirik and D. Pipeleers, Department of Metabolism and Endocrinology, Vrije Universiteit, Brussels, Belgium) and 0.5 μg of poly(A)+RNA from human kidney (CLONTECH) were reverse-transcribed in an oligo(dT)-primed reaction as described (27Wuytack F. Papp B. Verboomen H. Raeymaekers L. Dode L. Enouf J. Bokkala S. Authi K.S. Casteels R. J. Biol. Chem. 1994; 269: 1410-1416Abstract Full Text PDF PubMed Google Scholar). The mouse SERCA3 primers used are as follows: a 5′ primer M + 1 (5′-GGGGTGGTGCTTCAGATGTCTCTGC-3′) corresponding to nucleotides 2948–2972 in mouse SERCA3a and SERCA3b nucleotide sequences (accession numbers U49394 and U49393, respectively) and a 3′ primer M − 1 (5′-GGACAAATGCCTGGATGCTCTCAGT-3′) corresponding to the inverse complement of nucleotide stretches 3086–3110 and 3159–3183 in mouse SERCA3a and SERCA3b cDNA nucleotide sequences, respectively. A specific 3′ primer for the mouse SERCA3c isoform P3 (5′-CTTCAGGTCCTTTTTTTCCAAGAAGCCAAC-3′) spans the splice boundary between the last exon and an optional exon. PCR amplifications were carried out for 35 cycles, each cycle consisting of 30 s at 94 °C, 30 s at 68 °C, and 30 s at 72 °C for both M + 1/M − 1, and M + 1/P3 pairs. The human SERCA3 primers used are as follows: a common 5′ primer 22+ (5′-CTGCACTTCCTCATCCTGCTCG-3′) corresponding to nucleotides 2833–2854 and a 3′ primer 1− (5′-ATGGGCACCATCAGTCTGAGG-3′) corresponding to the inverse complement of the nucleotide stretch 3040–3060; numbering according to the nucleotide sequence deposited under accession number Z69881. Two additional 3′ primers specific for the human SERCA3b and SERCA3c isoforms were designed as follows: the above mentioned primer P1 and the primer P2 (5′-GGCTCATTTCTTCAAAGAGGCCAAC-3′), respectively. The PCR conditions were the same for the 3 pairs of primers (22+/1−, 22+/P1, and 22+/P2): 35 cycles, each cycle consisting of 30 s at 94 °C, 30 s at 65 °C, and 30 s at 72 °C. All PCR amplifications were performed using a mixture of Pwo(proofreading activity) and Taq polymerases from Boehringer Mannheim, Brussels, Belgium. M + 1, M − 1, P2, and P3 primers are also represented in Fig. 8 a. PCR fragments were gel-purified and subcloned, and for each fragment several individual clones were sequenced. Amplification of the genetic marker D17S1828 (accession number, 602622) from human genomic DNA, ICRFc105-G1035 and -F10124 cosmid clones was performed using the 5′ primer L+ (5′-TGCACTCACAGATTTGCC-3′) and the 3′ primer L− (5′-TTAAGCCAAGTTCGGATTTG-3′) for 35 cycles, each cycle consisting of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C. Human SERCA2-specific primers were used to carry out PCR amplifications from both human genomic DNA and first-strand cDNA obtained from human kidney mRNA (CLONTECH) after reverse transcription. The 5′ primer SER2 + 8 (5′-TGACCCGGTTCATGGAGGG-3′) corresponds to nucleotides 840–858, and the 3′ primer SER2–9 (5′-TGCCATTCTGCGAGTTCCAAGA-3′) corresponds to the inverse complement of nucleotides 960–981 in the human SERCA2b cDNA nucleotide sequence (numbering relative to the translation initiation site in Ref. 7Lytton J. MacLennan D.H. J. Biol. Chem. 1988; 263: 15024-15031Abstract Full Text PDF PubMed Google Scholar). The cycling conditions were as follows: 35 cycles, each cycle consisting of 45 s at 94 °C, 45 s at 60 °C, and 45 s at 72 °C. The entire coding regions of the mouse SERCA3a, SERCA3b, and SERCA3c cDNAs were amplified by PCR from mouse pancreatic islets first-strand cDNA using a common 5′ primer MMLD (5′-AGAAGCGACCTGGACGTCGCGGAC-3′) corresponding to nucleotides 8–31 in mouse SERCA3a and SERCA3b cDNA sequences (numbering according to accession numbers U49394 and U49393, respectively) in combination with either the primer M − 1 (for SERCA3a and SERCA3b amplifications) or the SERCA3c-specific primer P3. PCR reactions were carried out for 35 cycles, each cycle consisting of 1 min at 94 °C and 4 min at 72 °C for both MMLD/M − 1 and MMLD/P3 primer pairs. PCR products were separated by 1% agarose gel electrophoresis, gel-purified, blunt-ended, phosphorylated, and transferred into theEcoRI-cut, dephosphorylated, and blunt-ended mammalian expression vector pMT2 (from R. J. Kaufman, Genetics Institute, Boston, MA). The cloning of the pig SERCA2b cDNA in pSV57 expression vector was described earlier (11Verboomen H. Wuytack F. De Smedt H. Himpens B. Casteels R. Biochem. J. 1992; 286: 591-596Crossref PubMed Scopus (125) Google Scholar). COS-1 cell culture and DEAE-dextran-mediated DNA transfections were performed as described (11Verboomen H. Wuytack F. De Smedt H. Himpens B. Casteels R. Biochem. J. 1992; 286: 591-596Crossref PubMed Scopus (125) Google Scholar). Microsomes were isolated from COS-1 cells expressing mouse SERCA3a, SERCA3b, SERCA3c, and pig SERCA2b according to Verboomen et al. (11Verboomen H. Wuytack F. De Smedt H. Himpens B. Casteels R. Biochem. J. 1992; 286: 591-596Crossref PubMed Scopus (125) Google Scholar). Preparation of the N89 anti-SERCA3 antibody, denaturing gel electrophoresis on 0.75-mm-thick 7.5% polyacrylamide slab gels, semi-dry blotting onto Immobilon-P membranes (Millipore, Brussels, Belgium), and immunostaining of the blots were done as reported earlier (27Wuytack F. Papp B. Verboomen H. Raeymaekers L. Dode L. Enouf J. Bokkala S. Authi K.S. Casteels R. J. Biol. Chem. 1994; 269: 1410-1416Abstract Full Text PDF PubMed Google Scholar). Oxalate-stimulated Ca2+ uptake was measured by a rapid filtration method in the absence or presence of 5 mm ATP at 27 °C as described (12Verboomen H. Wuytack F. Van Den Bosch L. Mertens L. Casteels R. Biochem. J. 1994; 303: 979-984Crossref PubMed Scopus (75) Google Scholar). We have previously described the isolation and partial characterization of the first genomic clone (GHS3, approximately 40 kb in length) specifying the 3′ region of the human SERCA3 gene and localized the gene by fluorescence in situ hybridization to human chromosome 17 (31Dode L. Wuytack F. Kools P.F.J. Baba-Aissa F. Raeymaekers L. Briké F. Van De Ven W.J.M. Casteels R. Biochem. J. 1996; 318: 689-699Crossref PubMed Scopus (49) Google Scholar). Subsequent screening of a human chromosome 17-specific cosmid library from the Reference Library Data Base, ICRF (32Lehrach H. Drmanac R. Hoheisel J. Larin Z. Lennon G. Manaco A.P. Nizetic D. Zehetner" @default.
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• W2000786270 title "Structure of the Human Sarco/Endoplasmic Reticulum Ca2+-ATPase 3 Gene" @default.
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