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Mol Cell Biol. Dec 1989; 9(12): 5445–5455.
PMCID: PMC363713

Structure of an ectodermally expressed sea urchin metallothionein gene and characterization of its metal-responsive region.

Abstract

The metallothionein-A gene in the metallothionein gene family of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) was sequenced and found to contain three coding exons plus a 3' entirely noncoding exon. Putative alpha and beta MT domains were encoded, by its exons 2 and 3, respectively, in reverse of the order in vertebrate metallothionein genes. The SpMTA promoter was characterized through the expression of recombinant constructs containing various portions of the proximal 678-base-pair (bp) 5'-flanking region of the SpMTA gene. Zygotes injected with constructs were cultured to the blastula stage in the presence of a heavy-metal chelator and then incubated in the presence or absence of cadmium. The longest constructs were expressed only when heavy-metal ion was present. Two putative metal-responsive elements (MREs a and b) within 240 bp of the transcription start site resembled mammalian MREs in their critical 8-bp cores (TGCRCNCS) and in their locations relative to each other and to the TATA box. Elimination of activity by site-specific mutations in MREs a and b, separately or in both, identified them as metal regulatory elements. Thus, MRE recognition in this invertebrate resembles that in vertebrates. Upstream sites with single-mismatched MREs neither acted as MREs nor amplified the activity of MREs a and b. The SpMTA, Spec1, and CyIIIa actin genes, which have the same ectodermal specificity, have common DNA elements at relatively similar locations in their promoter regions; however, these elements are insufficient in themselves to promote gene expression.

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Selected References

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  • Akhurst RJ, Calzone FJ, Lee JJ, Britten RJ, Davidson EH. Structure and organization of the CyIII actin gene subfamily of the sea urchin, Strongylocentrotus purpuratus. J Mol Biol. 1987 Mar 20;194(2):193–203. [PubMed]
  • Andersen RD, Birren BW, Taplitz SJ, Herschman HR. Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences. Mol Cell Biol. 1986 Jan;6(1):302–314. [PMC free article] [PubMed]
  • Angerer LM, Kawczynski G, Wilkinson DG, Nemer M, Angerer RC. Spatial patterns of metallothionein mRNA expression in the sea urchin embryo. Dev Biol. 1986 Aug;116(2):543–547. [PubMed]
  • Benson S, Sucov H, Stephens L, Davidson E, Wilt F. A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. I. Authentication of the cloned gene and its developmental expression. Dev Biol. 1987 Apr;120(2):499–506. [PubMed]
  • Bond-Matthews B, Davidson N. Transcription from each of the Drosophila act5C leader exons is driven by a separate functional promoter. Gene. 1988;62(2):289–300. [PubMed]
  • Breathnach R, Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. [PubMed]
  • Britten RJ, Cetta A, Davidson EH. The single-copy DNA sequence polymorphism of the sea urchin Strongylocentrotus purpuratus. Cell. 1978 Dec;15(4):1175–1186. [PubMed]
  • Brunk CF, Jones KC, James TW. Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem. 1979 Jan 15;92(2):497–500. [PubMed]
  • Calzone FJ, Thézé N, Thiebaud P, Hill RL, Britten RJ, Davidson EH. Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo. Genes Dev. 1988 Sep;2(9):1074–1088. [PubMed]
  • Conlon RA, Tufaro F, Brandhorst BP. Post-transcriptional restriction of gene expression in sea urchin interspecies hybrid embryos. Genes Dev. 1987 Jun;1(4):337–346. [PubMed]
  • Cox KH, Angerer LM, Lee JJ, Davidson EH, Angerer RC. Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis. J Mol Biol. 1986 Mar 20;188(2):159–172. [PubMed]
  • Culotta VC, Hamer DH. Fine mapping of a mouse metallothionein gene metal response element. Mol Cell Biol. 1989 Mar;9(3):1376–1380. [PMC free article] [PubMed]
  • Davidson EH. Lineage-specific gene expression and the regulative capacities of the sea urchin embryo: a proposed mechanism. Development. 1989 Mar;105(3):421–445. [PubMed]
  • DePamphilis ML, Herman SA, Martínez-Salas E, Chalifour LE, Wirak DO, Cupo DY, Miranda M. Microinjecting DNA into mouse ova to study DNA replication and gene expression and to produce transgenic animals. Biotechniques. 1988 Jul-Aug;6(7):662–680. [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Flytzanis CN, Britten RJ, Davidson EH. Ontogenic activation of a fusion gene introduced into sea urchin eggs. Proc Natl Acad Sci U S A. 1987 Jan;84(1):151–155. [PMC free article] [PubMed]
  • Foss K, McClain WH. Rapid site-specific mutagenesis in plasmids. Gene. 1987;59(2-3):285–290. [PubMed]
  • Franks RR, Hough-Evans BR, Britten RJ, Davidson EH. Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA. Development. 1988 Feb;102(2):287–299. [PubMed]
  • Furey WF, Robbins AH, Clancy LL, Winge DR, Wang BC, Stout CD. Crystal structure of Cd,Zn metallothionein. Science. 1986 Feb 14;231(4739):704–710. [PubMed]
  • Glanville N, Durnam DM, Palmiter RD. Structure of mouse metallothionein-I gene and its mRNA. Nature. 1981 Jul 16;292(5820):267–269. [PubMed]
  • Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. [PMC free article] [PubMed]
  • Hamer DH. Metallothionein. Annu Rev Biochem. 1986;55:913–951. [PubMed]
  • Hardin SH, Carpenter CD, Hardin PE, Bruskin AM, Klein WH. Structure of the Spec1 gene encoding a major calcium-binding protein in the embryonic ectoderm of the sea urchin, Strongylocentrotus purpuratus. J Mol Biol. 1985 Nov 20;186(2):243–255. [PubMed]
  • Harlow P, Nemer M. Developmental and tissue-specific regulation of beta-tubulin gene expression in the embryo of the sea urchin Strongylocentrotus purpuratus. Genes Dev. 1987 Apr;1(2):147–160. [PubMed]
  • Haslinger A, Karin M. Upstream promoter element of the human metallothionein-IIA gene can act like an enhancer element. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8572–8576. [PMC free article] [PubMed]
  • Heguy A, West A, Richards RI, Karin M. Structure and tissue-specific expression of the human metallothionein IB gene. Mol Cell Biol. 1986 Jun;6(6):2149–2157. [PMC free article] [PubMed]
  • Hinegardner R. Cellular DNA content of the echinodermata. Comp Biochem Physiol B. 1974 Oct 15;49(2):219–226. [PubMed]
  • Hough-Evans BR, Britten RJ, Davidson EH. Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo. Dev Biol. 1988 Sep;129(1):198–208. [PubMed]
  • Hunt CT, Boulanger Y, Fesik SW, Armitage IM. NMR analysis of the structure and metal sequestering properties of metallothioneins. Environ Health Perspect. 1984 Mar;54:135–145. [PMC free article] [PubMed]
  • Jones NC, Rigby PW, Ziff EB. Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 1988 Mar;2(3):267–281. [PubMed]
  • Karin M, Haslinger A, Heguy A, Dietlin T, Cooke T. Metal-responsive elements act as positive modulators of human metallothionein-IIA enhancer activity. Mol Cell Biol. 1987 Feb;7(2):606–613. [PMC free article] [PubMed]
  • Karin M, Richards RI. Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene. Nature. 1982 Oct 28;299(5886):797–802. [PubMed]
  • Katula KS, Hough-Evans BR, Britten RJ, Davidson EH. Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs. Development. 1987 Nov;101(3):437–447. [PubMed]
  • Kadesch T, Berg P. Effects of the position of the simian virus 40 enhancer on expression of multiple transcription units in a single plasmid. Mol Cell Biol. 1986 Jul;6(7):2593–2601. [PMC free article] [PubMed]
  • Lee JJ, Shott RJ, Rose SJ, 3rd, Thomas TL, Britten RJ, Davidson EH. Sea urchin actin gene subtypes. Gene number, linkage and evolution. J Mol Biol. 1984 Jan 15;172(2):149–176. [PubMed]
  • Lynn DA, Angerer LM, Bruskin AM, Klein WH, Angerer RC. Localization of a family of MRNAS in a single cell type and its precursors in sea urchin embryos. Proc Natl Acad Sci U S A. 1983 May;80(9):2656–2660. [PMC free article] [PubMed]
  • Maroni G, Otto E, Lastowski-Perry D. Molecular and cytogenetic characterization of a metallothionein gene of Drosophila. Genetics. 1986 Mar;112(3):493–504. [PMC free article] [PubMed]
  • McClay DR, Chambers AF. Identification of four classes of cell surface antigens appearing at gastrulation in sea urchin embryos. Dev Biol. 1978 Mar;63(1):179–186. [PubMed]
  • McMahon AP, Flytzanis CN, Hough-Evans BR, Katula KS, Britten RJ, Davidson EH. Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev Biol. 1985 Apr;108(2):420–430. [PubMed]
  • Mueller PR, Salser SJ, Wold B. Constitutive and metal-inducible protein:DNA interactions at the mouse metallothionein I promoter examined by in vivo and in vitro footprinting. Genes Dev. 1988 Apr;2(4):412–427. [PubMed]
  • Nemer M, Travaglini EC, Rondinelli E, D'Alonzo J. Developmental regulation, induction, and embryonic tissue specificity of sea urchin metallothionein gene expression. Dev Biol. 1984 Apr;102(2):471–482. [PubMed]
  • Nemer M, Wilkinson DG, Travaglini EC, Sternberg EJ, Butt TR. Sea urchin metallothionein sequence: key to an evolutionary diversity. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4992–4994. [PMC free article] [PubMed]
  • Nielson KB, Winge DR. Independence of the domains of metallothionein in metal binding. J Biol Chem. 1985 Jul 25;260(15):8698–8701. [PubMed]
  • Otto E, Allen JM, Young JE, Palmiter RD, Maroni G. A DNA segment controlling metal-regulated expression of the Drosophila melanogaster metallothionein gene Mtn. Mol Cell Biol. 1987 May;7(5):1710–1715. [PMC free article] [PubMed]
  • Palmiter RD. Molecular biology of metallothionein gene expression. Experientia Suppl. 1987;52:63–80. [PubMed]
  • Peterson MG, Mercer JF. Structure and regulation of the sheep metallothionein-Ia gene. Eur J Biochem. 1986 Nov 3;160(3):579–585. [PubMed]
  • Richards RI, Heguy A, Karin M. Structural and functional analysis of the human metallothionein-IA gene: differential induction by metal ions and glucocorticoids. Cell. 1984 May;37(1):263–272. [PubMed]
  • Searle PF, Davison BL, Stuart GW, Wilkie TM, Norstedt G, Palmiter RD. Regulation, linkage, and sequence of mouse metallothionein I and II genes. Mol Cell Biol. 1984 Jul;4(7):1221–1230. [PMC free article] [PubMed]
  • Searle PF, Stuart GW, Palmiter RD. Building a metal-responsive promoter with synthetic regulatory elements. Mol Cell Biol. 1985 Jun;5(6):1480–1489. [PMC free article] [PubMed]
  • Searle PF, Stuart GW, Palmiter RD. Metal regulatory elements of the mouse metallothionein-I gene. Experientia Suppl. 1987;52:407–414. [PubMed]
  • Sharp PA, Berk AJ, Berget SM. Transcription maps of adenovirus. Methods Enzymol. 1980;65(1):750–768. [PubMed]
  • Stuart GW, Searle PF, Chen HY, Brinster RL, Palmiter RD. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7318–7322. [PMC free article] [PubMed]
  • Stuart GW, Searle PF, Palmiter RD. Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. Nature. 317(6040):828–831. [PubMed]
  • Sucov HM, Benson S, Robinson JJ, Britten RJ, Wilt F, Davidson EH. A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. II. Structure of the gene and derived sequence of the protein. Dev Biol. 1987 Apr;120(2):507–519. [PubMed]
  • Sucov HM, Hough-Evans BR, Franks RR, Britten RJ, Davidson EH. A regulatory domain that directs lineage-specific expression of a skeletal matrix protein gene in the sea urchin embryo. Genes Dev. 1988 Oct;2(10):1238–1250. [PubMed]
  • Thiele DJ, Hamer DH. Tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene. Mol Cell Biol. 1986 Apr;6(4):1158–1163. [PMC free article] [PubMed]
  • Varshney U, Jahroudi N, Foster R, Gedamu L. Structure, organization, and regulation of human metallothionein IF gene: differential and cell-type-specific expression in response to heavy metals and glucocorticoids. Mol Cell Biol. 1986 Jan;6(1):26–37. [PMC free article] [PubMed]
  • Wilkinson DG, Nemer M. Metallothionein genes MTa and MTb expressed under distinct quantitative and tissue-specific regulation in sea urchin embryos. Mol Cell Biol. 1987 Jan;7(1):48–58. [PMC free article] [PubMed]
  • Zafarullah M, Bonham K, Gedamu L. Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region. Mol Cell Biol. 1988 Oct;8(10):4469–4476. [PMC free article] [PubMed]

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