title: Cloning, expression pattern and essentiality of the high-affinity copper transporter 1 (ctr1) gene in zebrafish.
abstract: The high-affinity copper transporter 1 (Ctr1) is a highly conserved transmembrane protein that mediates the internalization of copper ions from the extracellular medium. In this study, we have isolated the zebrafish ctr1 gene. The zebrafish ctr1 cDNA encodes a protein with 69% identity to the human orthologue and shows conservation of specific amino acid residues involved in copper transport. We find only a single ctr1 gene in the zebrafish genome which maps to linkage group 5. The genomic structure of the zebrafish gene shows that it consists of five exons and that exon-intron boundaries are absolutely conserved with the mammalian ctr1 genes. Expression in embryos was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Zebrafish ctr1 is maternally loaded, and transcripts can be detected throughout development and in adult fish. Distribution of ctr1 message appears ubiquitous during early stages becoming restricted to the brain and ventral tissues by 24 h post fertilization (hpf). Beginning at 3 days post fertilization (dpf), expression is found mainly in the developing intestine. Specific knockdown of ctr1 by antisense morpholino oligonucleotides (MOs) causes early larval lethality. Defects include cell death in tissues where ctr1 is most heavily expressed, a finding similar to that described for a mouse knockout of mCtr1. Despite the existence of at least one other copper transport mechanism in the fish, our studies show that zebrafish ctr1 is an essential gene for development.
authors.affiliation: Millennium Nucleus in Developmental Biology, Departamento de Biología, Facultad de Ciencias Universidad de Chile, Casilla 653 Santiago, Chile.
authors.forename: Natalia C
authors.initials: NC
authors.lastname: Mackenzie
authors.forename: Mónica
authors.initials: M
authors.lastname: Brito
authors.forename: Ariel E
authors.initials: AE
authors.lastname: Reyes
authors.forename: Miguel L
authors.initials: ML
authors.lastname: Allende
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D000079923:Copper Transporter 1; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism; D047108:Embryonic Development; D005091:Exons; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D005796:Genes / Q000235:genetics; D020043:Genes, Essential / Q000235:genetics; D017403:In Situ Hybridization; D007438:Introns; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics*; D008845:Microinjections; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D018076:DNA, Complementary; D026901:Membrane Transport Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C490079:SLC31A1 protein, human; C490077:Slc31a1 protein, mouse; C486454:Slc31a1 protein, zebrafish; D029961:Zebrafish Proteins