Structure of an ectodermally expressed sea urchin metallothionein gene and characterization of its metal-responsive region.
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.
Harlow
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Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Watkins
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Thornton
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Nemer
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M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005091:Exons; D015870:Gene Expression; D015723:Gene Library; D005796:Genes; D008322:Mammals / Q000235:genetics; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012617:Sea Urchins / Q000196:embryology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D008670:Metals; D008668:Metallothionein