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

Harlow, Patricia P.; Watkins, Ella; Thornton, Ruth D.; Nemer, Martin

doi:10.1128/mcb.9.12.5445

Cited by 45 publications

(29 citation statements)

References 63 publications

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“…The nucleotide composition of the region immediately upstream to the ATG codon agrees with Kozack's rules of ribosome-binding site consensus sequences. In contrast to the short length of the 5Ј-untranslated segment, the 3Ј-untranslated region, extending 595 bp before the polyadenylation site, is much longer than the corresponding regions of the mammalian and Drosophila MT genes and is only comparable with that of the sea urchin gene (36). The polyadenylation signal (AATAAA) follows the consensus pattern in eukaryotes exactly.…”

Section: Isolation and Characterization Of The Homarus Mtmentioning

confidence: 92%

A New Insight into Metallothionein (MT) Classification and Evolution

Valls¹,

Bofill²,

Gonzàlez‐Duarte³

et al. 2001

Journal of Biological Chemistry

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We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous Escherichia coli expression in zinc-and copper-supplemented medium was achieved for MTH, the two domains ␤␤MTH and ␤␣MTH and three site-directed mutants, ␤␤C9H, ␤␣C37H, and ␤␣E31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of M The metallothionein (MT) 1 superfamily comprises a wide range of low molecular weight polypeptides, sharing the ability to chelate heavy metal atoms (1). MT are present in all animal phyla and in most fungi and plants and have been related to metal homeostasis and detoxification, although their definite physiological function is still a matter of debate (2-5). To classify these extremely heterogeneous amino acid sequences, three different classes were proposed: Class I, comprising all the MT with clear sequence similarity to horse liver MT 1; Class II, including all the sequences with no clear similarity to horse liver MT 1; and Class III for the phytochelatins and vegetal ␥-glutamyl peptides of enzymatic origin (1). More recently, a new classification consisting of a singular set for each individual eukaryotic taxon has been proposed (www.unizh.ch/ ϳmtpage/classif.html). Both systematizations are based on primary structure data but do not include any functional or evolutionary information. Conventionally, terminology and standards corresponding to the canonical mammalian MT molecule have been extended to describe amino acid sequence and metal cluster features of newly described MT forms. Mammalian MT folds into a twodomain structure when coordinating divalent metal ions. The N-terminal segment (␤ domain), with 9 cysteines in Cys-XaaCys arrays, binds three M(II) ions, and the C-terminal segment (␣ domain), with 11 cysteines, some of which in Cys-Cys tandems binds four M(II) ions (6, 7). A higher binding affinity for monovalent (copper) than for divalent (zinc and cadmium) ions was described for the mammalian ␤ domain, with an opposite trend for the ␣ domain (8). Contemporarily, Saccharomyces cerevisiae MT CUP1 was characterized. The synthesis of this protein was induced by copper, and the MT recovered contained copper ions exclusively (9). Because the CUP1 cysteine distribution aligned well to the ␤ domain of mammalian MT, it was assumed that proteins with a primary structure similar to the mammalian ␤ domain would exhibit copper binding rather than zinc binding preferences. On the basis of their amino acid alignments, further application of the ␤/␣ domain terminology led to the consideration of crustacean peptides as ␤-␤ MT (10) and thus putative copper thioneins. This was corroborated by the three divalent ions contained in each domain of the crustacean Cd-MT structures solved by NMR (11, 12) and thus comparable with the Cd 3 -␤MT fragment in mammalian MT.A survey of the literature on naturally occurring and metalinduced crustacean MT ...

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Section: Isolation and Characterization Of The Homarus Mtmentioning

confidence: 92%

A New Insight into Metallothionein (MT) Classification and Evolution

Valls¹,

Bofill²,

Gonzàlez‐Duarte³

et al. 2001

Journal of Biological Chemistry

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“…In contrast to the short length of the 5′-untranslated segment (69·nt), the 3′-untranslated region extended 620·nt before the polyadenylation signal. This is much longer than the corresponding UTRs of the mammalian and Drosophila melanogaster MT sequences but is comparable to that of the sea urchin Strongylocentrotus purpuratus and lobster Homarus americanus (Harlow et al, 1989;Valls et al, 2001). A blastn homology search of GenBank using the L. littorea cDNA sequence as a query did not retrieve significant matches, so the translated ORF was used to classify the sequence.…”

Section: Cdna and Amino Acid Sequence Analysismentioning

confidence: 99%

Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastropodLittorina littorea

English

Storey

2003

Journal of Experimental Biology

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Differential screening of cDNA libraries constructed from the foot muscle of marine snails Littorina littorea revealed several cDNAs that are upregulated during anoxia or freezing exposures, environmental stresses that are naturally endured by this species. One full-length clone of 1196 nucleotides (GenBank accession number AY034179) hybridized with a 1200-nucleotide band on northern blots and encoded a 100-amino-acid protein that was identified as belonging to the metallothionein (MT) family. L. littorea MT shared 45% and 56% identity with the copper-and cadmium-binding MT isoforms, respectively, from another gastropod, Helix pomatia and 43-47% identity with marine bivalve MTs. The L. littorea sequence included the mollusc-specific C-terminal motif Cys-X-Cys-X(3)-Cys-Thr-Gly-X(3)-Cys-X-Cys-X(3)-Cys-X-Cys-Lys that identifies it as a family 2 (mollusc) MT.Northern blot analysis showed that L. littorea MT was upregulated in both foot muscle and hepatopancreas in response to both freezing and anoxia stresses; within 1·h of the beginning of the stress transcript levels rose 2.5-to sixfold of control levels, reaching maximal levels at 12 or 24·h. After 24·h recovery from either stress, transcript levels were reduced again in three cases but remained elevated in hepatopancreas from anoxia-treated snails. Upregulation of MT during environmental stress could serve one or more possible roles, including a function in antioxidant defense.

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“…Positive regulatory elements have been implicated in several genes expressed in the sea urchin embryo, and these together with negatively operative spatial-control elements have been identified in genes, such as those for cytoskeletal CyIIIa actin (48) and Spec 1 and Spec 2 troponins (16), which display the same aboral ectoderm specificity as SpMTA (34). Only one such element is indicated in the previously sequenced 0.6 kb of the 5' flank of the SpMTA gene (20). However, we have identified a cluster of elements in the first intron of this gene with homology to the above-mentioned positive and spatial-control elements.…”

mentioning

confidence: 87%

“…The SpMTA MT X206 clone from an S. purpuratus genomic DNA library (20) was the source of a previously restriction site-mapped subclone to be sequenced. A 1-kb region between AccI and PstI on May 9, 2018 by guest…”

Section: Methodsmentioning

confidence: 99%

“…The SpMTA gene of the sea urchin Strongylocentrotus purpuratus employs in its promoter a proximal pair of metal response elements (MREs), whose locations relative to each other and proximity to a TATA box are typical of metallothionein (MT) genes (20). Since MT genes generally have a multiplicity of MREs, which in the case of synthetic metalresponsive promoters can act synergistically (42), we have extended our analysis of the SpMTA gene here in order to identify any additional MREs, as well as other genic elements, that might regulate its metal-induced expression.…”

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confidence: 99%

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Combinatorial regulation by promoter and intron 1 regions of the metallothionein SpMTA gene in the sea urchin embryo.

Bai¹,

Stuebing²,

Parker³

et al. 1993

Mol. Cell. Biol.

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The SpMTA metallothionein gene of the sea urchin Strongylocentrotus purpuratus is regulated developmentally, histospecifically, and by heavy-metal induction. The mentally scheduled (35, 50) and histospecifically regulated (34). Since its expression is restricted to the aboral ectoderm in sea urchin gastrulae and pluteus larvae, cultured in either the absence or presence of added heavy metal ions, metal induction does not override the histospecificity but appears, indeed, to be linked to it (34). Positive regulatory elements have been implicated in several genes expressed in the sea urchin embryo, and these together with negatively operative spatial-control elements have been identified in genes, such as those for cytoskeletal CyIIIa actin (48) and Spec 1 and Spec 2 troponins (16), which display the same aboral ectoderm specificity as SpMTA (34). Only one such element is indicated in the previously sequenced 0.6 kb of the 5' flank of the SpMTA gene (20). However, we have identified a cluster of elements in the first intron of this gene with homology to the above-mentioned positive and spatial-control elements. Moreover, the absence of this intronic region from another MT gene, SpMTB1, which is not aboral ectoderm specific (34), suggests that it should be studied as a potentially specific regulator of SpMTA gene expression.To appreciate the full extent of promoter involvement in metal-induced expression, as well as the regulative participation of the first intron, we have obtained an additional 5'-flanking sequence and examined the activities in transgenic embryos of reporter gene constructs containing portions of 5'-flanking and intronic regions. Our observations indicate that the SpMTA gene is functionally bipartite, insofar as the 5' flank promotes metal-regulated transcription, while the first intron amplifies this activity. MATERIALS AND METHODSSequencing and preparation of plasmids. The SpMTA MT X206 clone from an S. purpuratus genomic DNA library (20) was the source of a previously restriction site-mapped subclone to be sequenced. A 1-kb region between AccI and PstI on May 9, 2018 by guest

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

Cited by 45 publications

References 63 publications

A New Insight into Metallothionein (MT) Classification and Evolution

A New Insight into Metallothionein (MT) Classification and Evolution

Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastropodLittorina littorea

Combinatorial regulation by promoter and intron 1 regions of the metallothionein SpMTA gene in the sea urchin embryo.

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