Structure, Function, and Regulation of a Subfamily of Mouse Zinc Transporter Genes
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m304163200
ISSN1083-351X
AutoresJodi Dufner‐Beattie, S. Joshua Langmade, Fudi Wang, David Eide, Glen K. Andrews,
Tópico(s)Heavy Metal Exposure and Toxicity
ResumoZinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eight-transmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of zinc in transfected cultured cells, similar to the previously demonstrated functions of human ZIP1 and ZIP2 (Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560–5564; Gaither, L. A., and Eide, D. J. (2001) J. Biol. Chem. 276, 22258–22264). No ZIP3 orthologs have been previously studied. Furthermore, this first systematic comparative study of the in vivo expression and dietary zinc regulation of this subfamily of zip genes revealed that 1) zip1 mRNA is abundant in many mouse tissues, whereas zip2 and zip3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted in their accumulation; and 2) unlike mouse metallothionein I and zip4 mRNAs (Dufner-Beattie, J., Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D., and Andrews, G. K. (2003) J. Biol. Chem. 278, 33474–33481), the abundance of zip1, zip2, and zip3 mRNAs is not regulated by dietary zinc in the intestine and visceral endoderm, tissues involved in nutrient absorption. These studies suggest that all three of these ZIP proteins may play cell-specific roles in zinc homeostasis rather than primary roles in the acquisition of dietary zinc. Zinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eight-transmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of zinc in transfected cultured cells, similar to the previously demonstrated functions of human ZIP1 and ZIP2 (Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560–5564; Gaither, L. A., and Eide, D. J. (2001) J. Biol. Chem. 276, 22258–22264). No ZIP3 orthologs have been previously studied. Furthermore, this first systematic comparative study of the in vivo expression and dietary zinc regulation of this subfamily of zip genes revealed that 1) zip1 mRNA is abundant in many mouse tissues, whereas zip2 and zip3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted in their accumulation; and 2) unlike mouse metallothionein I and zip4 mRNAs (Dufner-Beattie, J., Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D., and Andrews, G. K. (2003) J. Biol. Chem. 278, 33474–33481), the abundance of zip1, zip2, and zip3 mRNAs is not regulated by dietary zinc in the intestine and visceral endoderm, tissues involved in nutrient absorption. These studies suggest that all three of these ZIP proteins may play cell-specific roles in zinc homeostasis rather than primary roles in the acquisition of dietary zinc. Zinc is an essential trace element that is required for the catalytic activity of numerous metalloenzymes (4.Vallee B.L. Auld D.S. Biochemistry. 1990; 29: 5647-5659Crossref PubMed Scopus (1536) Google Scholar, 5.Berg J.M. Shi Y.G. Science. 1996; 271: 1081-1085Crossref PubMed Scopus (1687) Google Scholar) and can also serve a purely structural role by stabilizing the conformation of certain zinc-dependent protein domains, such as zinc fingers, zinc clusters, and RING fingers, that are commonly found in transcriptional regulatory proteins (5.Berg J.M. Shi Y.G. Science. 1996; 271: 1081-1085Crossref PubMed Scopus (1687) Google Scholar, 6.Krishna S.S. Majumdar I. Grishin N.V. Nucleic Acids Res. 2003; 31: 532-550Crossref PubMed Scopus (659) Google Scholar). Deficiency of this essential metal can have devastating effects. In mammals, inadequate levels of zinc in the diet lead to dermatologic lesions, growth retardation, mental disorders, and compromised function of the immune and reproductive systems (7.Fraker P.J. King L.E. Laakko T. Vollmer T.L. J. 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In mammals, the zinc transporter (ZnT) 1The abbreviations used are: ZnTzinc transporterMTmetallothioneinZIPZrt/Irt-like proteinIrtiron-regulated transporterZrtzinc-regulated transporterESTexpressed sequence tagRTreverse transcriptionHAhemagglutininBACbacterial artificial chromosome. family of proteins function in a tissue-, cell-, and organelle-specific manner to regulate intracellular zinc homeostasis. These proteins contain six predicted transmembrane domains and are thought to function as multimers. Seven members of the Znt family have been identified to date in mammals (Znt1–7), and genetic studies have confirmed the importance of many of these genes in mammalian zinc metabolism (10.Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (643) Google Scholar, 11.Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (397) Google Scholar, 12.Palmiter R.D. Cole T.B. Quaife C.J. Findley S.D. Proc. Natl. Acad. Sci. U. S. 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Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar), and dietary zinc modulates Znt1 and Znt2 mRNA levels in the kidney, intestine, and liver (19.Liuzzi J.P. Blanchard R.K. Cousins R.J. J. Nutr. 2001; 131: 46-52Crossref PubMed Scopus (203) Google Scholar). Zinc can also regulate the intracellular localization of the ZnT4 and ZnT6 proteins (16.Huang L.P. Kirschke C.P. Gitschier J. J. Biol. Chem. 2002; 277: 26389-26395Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). zinc transporter metallothionein Zrt/Irt-like protein iron-regulated transporter zinc-regulated transporter expressed sequence tag reverse transcription hemagglutinin bacterial artificial chromosome. Intracellular zinc is bound by small cysteine-rich proteins called metallothioneins (MTs) (20.Coyle P. Philcox J.C. Carey L.C. Rofe A.M. Cell. Mol. Life Sci. 2002; 59: 627-647Crossref PubMed Scopus (1084) Google Scholar). In the mouse, the MT family consists of four members. Exposure to high levels of zinc causes MT accumulation through increased gene expression, whereas dietary zinc deficiency leads to decreased MT abundance through decreased gene expression and protein destabilization (21.Andrews G.K. Prog. Food Nutr. Sci. 1990; 14: 193-258PubMed Google Scholar). These proteins are thought to sequester zinc when present at high levels, protecting against heavy metal toxicity, and to provide a labile pool of zinc under limiting conditions that can be released for use by other proteins. In eukaryotes, uptake of several essential metals is mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters (22.Eide D. Curr. Opin. Cell Biol. 1997; 9: 573-577Crossref PubMed Scopus (83) Google Scholar, 23.Guerinot M.L. Biochim. Biophys. Acta. 2000; 1465: 190-198Crossref PubMed Scopus (891) Google Scholar). The first member of this family to be identified was iron-regulated transporter-1 (Irt1) from Arabidopsis. Irt1 was found to preferentially import iron, but can also transport manganese, cadmium, and zinc (24.Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1083) Google Scholar, 25.Eng B.H. Guerinot M.L. Eide D. Saier Jr., M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (209) Google Scholar, 26.Korshunova Y.O. Eide D. Clark W.G. Guerinot M.L. Pakrasi H.B. Plant Mol. Biol. 1999; 40: 37-44Crossref PubMed Scopus (619) Google Scholar, 27.Rogers E.E. Eide D.J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12356-12360Crossref PubMed Scopus (375) Google Scholar). Subsequently, two other members of this superfamily (zinc-regulated transporter-1 (Zrt1) and Zrt2) were identified that function in zinc uptake in yeast (28.Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar, 29.Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The acronym for the superfamily (i.e. ZIP) reflects Zrt- and Irt-like proteins. ZIP proteins contain eight putative transmembrane domains with conserved histidine, serine, and glycine residues within the fourth transmembrane domain, which may play a role in metal binding during transport. These proteins also often contain a histidine-rich loop between the third and fourth transmembrane domains. Computer searches of the human and mouse genome sequences reveal multiple members of the ZIP superfamily (12 in human) (30.Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (433) Google Scholar). However, little is known about the regulation and function of most of these mammalian ZIP genes. A conserved subfamily of three zip genes (zip1, zip2, and zip3) has been identified in mice and humans. Human ZIP1 (SLC39A1) and ZIP2 (SLC39A2) have been demonstrated to function as zinc transporters, and ZIP1 is the major zinc uptake protein in K562 cells (1.Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 2.Gaither L.A. Eide D.J. J. Biol. Chem. 2001; 276: 22258-22264Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). No studies on the function of ZIP3 have been reported. The recent findings that the human ZIP4 gene (SLC39A4) is mutated in the genetic zinc metabolism disorder acrodermatitis enteropathica (31.Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar) and that this conserved gene encodes a zinc-regulated zinc transporter in mice (3.Dufner-Beattie J. Wang F. Kuo Y.-M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) suggest that mammalian ZIP genes are integral components of the zinc homeostatic mechanism. Although several recent studies have examined the expression and regulation of members of this conserved subfamily of three zip genes, there have been no systematic comparative studies of their expression or regulation in vivo. zip1 mRNA has been detected in most adult rat and many embryonic mouse tissues (32.Lioumi M. Ferguson C.A. Sharpe P.T. Freeman T. Marenholz I. Mischke D. Heizmann C. Ragoussis J. Genomics. 1999; 62: 272-280Crossref PubMed Scopus (48) Google Scholar), and ZIP1 mRNA is also found in most human tissues (2.Gaither L.A. Eide D.J. J. Biol. Chem. 2001; 276: 22258-22264Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) and cultured human cell lines. In contrast, ZIP2 mRNA was not detectable by Northern blot analysis of human tissue RNAs (1.Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Expression of ZIP3 has not been examined in any system, and expression of zip1 and zip2 has not been examined in adult mouse tissues. A modest hormonal regulation of ZIP1 mRNA in cultured prostate cell lines has been reported (33.Costello L.C. Liu Y. Zou J. Franklin R.B. J. Biol. Chem. 1999; 274: 17499-17504Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar); and in human monocytes and THP-1 cells treated with a zinc chelator, ZIP2 mRNA abundance increased significantly, whereas ZIP1, ZIP3, and ZIP4 mRNA abundance did not change (34.Cousins R.J. Blanchard R.K. Popp M.P. Liu L. Cao J. Moore J.B. Green C.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6952-6957Crossref PubMed Scopus (153) Google Scholar, 35.Cao J. Bobo J.A. Liuzzi J.P. Cousins R.J. J. Leukocyte Biol. 2001; 70: 559-566PubMed Google Scholar). The objective of the studies presented herein was to characterize in detail the structure and function the mouse zip1, zip2, and zip3 genes and to compare and contrast their regulation in vivo in nutrient-absorptive tissues (intestine and embryonic visceral endoderm) during periods of dietary zinc deficiency. Computer Analyses of Sequence Data—Multiple sequence alignments were performed using the AlignX application within the Vector NTI Suite program (Informax, Bethesda, MD). Plasmid Construction—The expressed sequence tags (ESTs) for zip1 (GenBank™/EBI accession number BE572790) and zip3 (accession number BF100710) were obtained from Incyte Genomics (St. Louis, MO). Sequencing of these ESTs revealed that they contain the expected open reading frames. The mouse zip2 cDNA was obtained by reverse transcription (RT)-PCR from poly(A)+ RNA isolated from the placentas of day 12 zinc-deficient pregnant mice (described below). Reverse transcription was carried out using Improm-II reverse transcriptase (Promega, Madison, WI), and PCR of the open reading frame was carried out using Pfu DNA Polymerase (Stratagene, La Jolla, CA). For zinc uptake studies, selectable mammalian pcDNA3.1Puro(+) expression plasmids (36.Thomas L.R. Stillman D.J. Thorburn A. J. Biol. Chem. 2002; 277: 34343-34348Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) encoding each of the three mouse ZIP proteins with C-terminal hemagglutinin (HA) tags were constructed through a multistep cloning strategy. The resulting plasmids contained the open reading frame, including at least 6 bp preceding the initiator methionine for optimal translation fused in-frame at the 3′-end with an oligomer encoding an HA tag. In the case of zip3, the potential upstream initiator methionine that does not adhere to the Kozak consensus sequence (37.Kozak M. Nucleic Acids Res. 1981; 9: 5233-5262Crossref PubMed Scopus (833) Google Scholar) (see “Results”) was included in the plasmid in case it is the actual translation start point. Each plasmid was sequenced in its entirety to confirm the absence of mutations introduced during the subcloning process. Animal Care and Use—All experiments involving mice were performed in accordance with National Institutes of Health guidelines for the care and use of animals and were approved by the Institutional Animal Care Committee. CD-1 mice (48–60 days old) were purchased from Charles River Breeding Laboratories (Raleigh, NC). Mouse diets were purchased from Harlan Teklad (Madison, WI) and have been described in detail previously (38.Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (107) Google Scholar). Zinc levels in the diets were as follows: zinc-deficient, 1 ppm zinc; and zinc-adequate, 50 ppm zinc. These diets are otherwise identical. To examine tissue-specific expression, female or male CD-1 mice (six per group) maintained on zinc-adequate feed were killed, and the indicated tissues were harvested and snap-frozen in liquid nitrogen for subsequent extraction of RNA for Northern analysis. Pancreatic RNA was extracted immediately from fresh tissue. To examine the effect of dietary zinc deficiency during pregnancy on mouse zip mRNA levels, pregnant mice were subjected to moderate dietary zinc deficiency as described previously (39.Andrews G.K. Geiser J. J. Nutr. 1999; 129: 1643-1648Crossref PubMed Scopus (62) Google Scholar, 40.Andrews G.K. Lee D.K. Ravindra R. Lichtlen P. Sirito M. Sawadogo M. Schaffner W. EMBO J. 2001; 20: 1114-1122Crossref PubMed Scopus (86) Google Scholar, 41.Lee D.K. Geiser J. Dufner-Beattie J. Andrews G.K. J. Nutr. 2003; 133: 45-50Crossref PubMed Scopus (20) Google Scholar). CD-1 females were mated with CD-1 males; the day a vaginal plug was found was considered day 1 of pregnancy. On day 1, mice were placed in pairs in cages and provided free access to zinc-adequate feed and deionized distilled water. On day 8, mice were placed in pairs in cages with stainless steel false bottoms to reduce the recycling of zinc (42.Cook Mills J.M. Fraker P.J. Br. J. Nutr. 1993; 69: 835-848Crossref PubMed Scopus (53) Google Scholar). Water bottles were washed with 4 m HCl and rinsed with deionized water to remove zinc. The diet was changed to the zinc-deficient diet, or where indicated, the zinc-adequate diet was maintained. The visceral yolk sacs, placentas, and maternal small intestines were harvested on days 11–15 (six mice per group) and snap-frozen in liquid nitrogen for RNA extraction and subsequent Northern analysis or RT-PCR. RNA Extraction and Northern Analysis—Total RNA was isolated either using TRIzol reagent (Invitrogen) according to the manufacturer's instructions or as described previously (18.Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 40.Andrews G.K. Lee D.K. Ravindra R. Lichtlen P. Sirito M. Sawadogo M. Schaffner W. EMBO J. 2001; 20: 1114-1122Crossref PubMed Scopus (86) Google Scholar). Polyadenylated RNA was purified from total RNA using the Oligotex mRNA isolation kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. Total RNA (3 μg/lane) was fractionated on formaldehyde-containing 1% agarose gels, transferred to nylon membranes, and immobilized via UV cross-linking (43.Dalton T.P. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (251) Google Scholar). Duplicate gels were stained with acridine orange to monitor RNA integrity and to normalize for loading. DNA fragments containing full-length zip1, zip2, or zip3 cDNA were excised from plasmids and used as probes for Northern blot hybridization. Probes were labeled with [32P]dCTP using the Random Primers DNA labeling system (Invitrogen) according to the manufacturer's instructions. The mouse MT-I probe was as described previously (38.Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (107) Google Scholar). Membranes were hybridized and washed under stringent conditions as described previously (18.Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 38.Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (107) Google Scholar, 43.Dalton T.P. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (251) Google Scholar). Hybrids were detected by autoradiography with intensifying screens at –70 °C. Cell Culture and Transient Transfections—HEK293 cells were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine at 37 °C in a humidified 5% CO2 incubator. For transient transfections, cells were seeded onto poly-l-lysine-treated 24-well plates at a density of 2 × 105 cells/well and were transfected the following day. Transfections were carried out using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's instructions. Zinc uptake assays were performed 36–48 h later. Zinc Uptake Assays—Zinc uptake assays were performed as described previously (1.Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 2.Gaither L.A. Eide D.J. J. Biol. Chem. 2001; 276: 22258-22264Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). After transfection, cells were washed once with cold uptake buffer (15 mm HEPES, 100 mm glucose, and 150 mm KCl, pH 7.0), followed by a 10-min incubation with prewarmed uptake buffer. Cells were then incubated in prewarmed uptake buffer containing the specified concentration of 65ZnCl2 (PerkinElmer Life Sciences) in a 37 °C shaking incubator for 15 min unless otherwise indicated. Assays were stopped by addition of an equal volume of cold uptake buffer supplemented with 1 mm EDTA (stop buffer). Cells were collected by filtration on glass fiber filters (Type A/E, Gelman Sciences) and washed three times with stop buffer (total of 10 ml). Cell-associated radioactivity was measured with a Packard Auto-Gamma 5650 γ-counter. Metal salts were obtained from Sigma. Stock solutions of the chloride salts of various metals (CuCl2, CdCl2, MnCl2, CoCl2, MgCl2, and NiCl2) and AgNO3 were prepared at 100 mm in distilled water. A ZnCl2 stock was prepared at 100 mm in 0.02 n HCl, and an FeCl3 stock was prepared at 50 mm in 0.1 n HCl. Sodium ascorbate (1 mm) was used to reduce Fe3+ to Fe2+. Ascorbate treatment alone did not alter zinc uptake activity (data not shown). Cells grown in parallel to those used for uptake experiments were washed three times with ice-cold uptake buffer, resuspended in phosphate-buffered saline containing 0.1% SDS and 1% Triton X-100 for cell lysis, and then assayed for protein content using a Bradford assay kit. Zinc accumulation and uptake rates were normalized to protein concentrations of these cell lysates. Michaelis-Menten constants were determined by nonlinear interpolation of the data using Prism (Version 3.0a for Macintosh, GraphPAD Software, San Diego, CA). RT-PCR Detection of Mouse zip2 mRNA—Tissue-specific expression of zip2 was examined by RT-PCR. Total RNA from various mouse tissues (1 μg/reaction) was DNase I-treated according to the manufacturer's instructions (Invitrogen). DNase I was inactivated by addition of EDTA to 2.5 mm, followed by a 10-min incubation at 65 °C. Reverse transcription was subsequently carried out using Improm-II reverse transcriptase. Samples were then amplified using Platinum Taq DNA polymerase (Stratagene) for 30 cycles. Mouse zip2 was amplified using primers 2TXTS (5′-CTTCTTGGGAGCAGGGTTGATGC-3′) and 210073AS (5′-CGCCACTGTGGCCTGTAGTCC-3′) to give a 406-bp product. Mouse zip1 was used as a control for RT reactions because it is widely expressed and was amplified using primers mZIP1KOZS (5′-CGGGATCCACAGCCACCATGGGGCCCT-3′) and mZIP1GFPAS (5′-CGGAATTCTTAGATTTGGACAAAGAGAAGGCCAGTGAGC-3′) to give a 1000-bp product. RT-PCR was used to examine the effect of dietary zinc deficiency during pregnancy on mouse zip2 mRNA abundance. Total RNA (1 μg; isolated from embryonic visceral yolk sacs from the zinc-deficient pregnant mice described above) was DNase I-treated and reverse-transcribed as described above. Samples were then amplified using Platinum Taq DNA polymerase for increasing increments of three cycles, starting at 21 cycles and proceeding to 33 cycles. Mouse zip2 was amplified using primers 2TXTS and 210073AS. Mouse zip1 was used as a negative control for regulation by dietary zinc and was amplified using primers mZIP1KOZS and mZIP1GFPAS. Mouse MT-I was used as a positive control for regulation by dietary zinc and was amplified using primers MT1S3 (5′-CACCACGACTTCAACGTCCTG-3′) and MT1AS604 (5′-TCTTGCAGGCGCAGGAGCTG-3′) to give a 138-bp product. Accession Numbers—The GenBank™/EBI accession numbers are as follows: zip1 bacterial artificial chromosome (BAC), AC096622 and AC096623; zip1 pseudogene BAC, AC021434; zip2 BAC, AC079536; zip3 BAC, AC073816; zip1 EST, BE572790; and zip3 EST, BF100710. Characterization of the Mouse zip1, zip2, and zip3 Genes and cDNAs—To identify mouse ZIP subfamily members, the conserved 12-amino acid signature sequence (HSVFEGLAVGLQ) from the human ZIP1, ZIP2, and ZIP3 proteins (Fig. 1) was used to search the mouse translated high throughput genome sequences data base on the NCBI Protein Database server. This search identified three different BAC entries that were derived from the C57/B6 strain of mice (see “Experimental Procedures” for accession numbers). Alignment of the cDNA sequences of the human ZIP genes with the nucleotide sequences flanking the signature sequences within each of these three mouse BACs (Fig. 1) provided the identity of the mouse zip1, zip2, and zip3 genes. The cDNA corresponding to each gene was identified by alignment of ESTs from the mouse EST data base to generate overlapping contiguous consensus sequences. This approach worked well to identify the cDNAs for zip1 and zip3, which are ∼2300 and 3750 bp in length, respectively, because there were several ESTs for each of these genes in the data base. Unfortunately, it did not work for zip2 because there were only three zip2 EST entries in the data base. Therefore, the full-length human ZIP2 cDNA sequence was aligned with the genomic sequence in the zip2 BAC. The high degree of homology shared between the human ZIP2 cDNA sequence and the mouse zip2 genomic sequence helped reveal the zip2 cDNA sequence, which was found to be ∼1350 bp in length. PCR primers were designed using this information to amplify the cDNA containing the coding sequence. Rapid amplification of cDNA ends was carried out for each of these three mouse zip genes to identify the 5′-end of the cDNA. In each case, the 5′-end was extended either by only a few base pairs or not at all compared with the known cDNA and EST sequences in the data bases. The 3′-end of each cDNA is demarcated by a poly(A) tail. In the case of zip3, comparison of ESTs revealed two different messages that are identical in coding sequence, but one ends prematurely due to the use of a non-canonical polyadenylation signal (Fig. 2C). The number of ESTs corresponding to this shorter transcript suggests that this message is significantly less abundant than the longer message. As this alternative poly(A) tail occurs within the 3′-untranslated region, the same protein is predicted to be encoded by both messages. Both zip1 and zip3 cDNAs contain a thymidine-rich region within their long 3′-untranslated regions, but the significance of this is not yet known. Comparison of the zip1 cDNA and gene sequences initially revealed a zip1 pseudogene within this BAC in the data base. The pseudogene has no introns, terminates in a poly(A) tract, and displays mismatches and codon deletions compared with the EST sequences. To identify a BAC containing the actual expressed zip1 gene, primers were designed to differentiate between the functional gene and the pseudogene based on the sequence mismatches. These primers were sent to Incyte Genomics for PCR screening of a mouse genomic BAC library. Two positive clones were identified, and partial sequence analysis revealed that they were devoid of the nucleotide mismatches seen in the pseudogene. These BACs were then sequenced in their entirety at the Mouse Genomic Sequencing Group of the University of Oklahoma. The exon-intron structure for each gene was determined by aligning the gene with the cDNA and by identifying splice donor and acceptor consensus sequences (Fig. 2). All three genes are relatively small, spanning <10 kilobase pairs of DNA, and contain four exons. Using accession numbers for the BACs containing each zip gene, the chromosomal localization was assigned using the mouse genome server. 2Available at www.ensembl.org. Localization of zip1 to chromosome 3 is consistent with a previous report (32.Lioumi M. Ferguson C.A. Sharpe P.T. Freeman T. Marenholz I. Mischke D. Heizmann C. Ragoussis J. Genomics. 1999; 62: 272-280Crossref PubMed Scopus (48) Google Scholar). The zip2 gene was localized to chromosome 14, the zip3 gene to chromosome 10, and the zip1 pseudogene to chromosome 2. Analysis of the cDNA sequence revealed that the predicted mouse ZIP1, ZIP2, and ZIP3 proteins are 324, 309, and 317 amino acids in length, respectively. Alignment of the predicted human and mouse ZIP proteins revealed significant sequence conservation along the entire length of each of the three proteins (Fig. 3). The mouse and human ZIP1, ZIP2, and ZIP3 proteins are 93, 78, and 83% identical, and 96, 85, and 90% similar, respectively. Computer analysis of the zip3 cDNA identified an open reading frame for mouse ZIP3 that contains an additional 31 amino acids N-terminal to the initiator methionine shown in Fig. 3C. This extension is due to the presence of an in-frame methionine codon located 93 nucleotides farther upstream. This potential upstream start codon does
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