High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein

doi:10.1016/0378-1119(84)90206-3

Gene

Volume 31, Issues 1–3, November 1984, Pages 155-164

https://doi.org/10.1016/0378-1119(84)90206-3 Get rights and content

Abstract

A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColEl rop gene under the control of the thermally inducible λ pl promoter. The rop gene product is a negative regulator ofColEl DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 °C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos.

Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.

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A self-inducing runaway-replication plasmid expression system utilizing the Rop protein
1989, Gene
A highly efficient prokaryotic expression system has been developed that produces proteins at levels exceeding 150 μg/ml of culture medium. The system consists of a temperature-sensitive-copy-number plasmid that carries the rop gene and promoter downstream from the trp promoter. Any sequence cloned into the PvuII site of the rop gene alters Rop protein activity and causes lethal runaway plasmid DNA replication. This plasmid replication can be suppressed in trans by complementation with a similar wild-type plasmid. Cells harboring both plasmids are quite stable, and induction of plasmid DNA synthesis occurs only after cells are grown for several generations under conditions that lead to the loss of the trans-acting repressor. Large amounts of Rop fusion proteins accumulate in the cell as the trp operon is gradually induced via repressor titration. All chimeric proteins accumulate as insoluble aggregates, and are therefore easily purified. They can be solubilized using relatively mild conditions, and the partially purified proteins are highly amenable to cleavage by chemical methods. Using this system we have made Rop fusions with the HIV Tat protein, the herpes simplex virus type-2 38K protein, and Chinese hamster metallothionein.
Spec2 genes of Strongylocentrotus purpuratus. Structure and differential expression in embryonic aboral ectoderm cells
1988, Journal of Molecular Biology
Members of the Spec gene family are expressed during embryonic development of the sea urchin, Strongylocentrotus purpuratus. The family encodes proteins related to the calmodulin/troponin C/myosin light chain group of calcium binding proteins and one gene, Specl, has been studied extensively in our laboratory. In this paper, we analyze other members of the family, collectively termed Spec2 genes. We make use of several hybridization probes derived from Spec1 and Spec2 cDNA clones, which recognize different members of the family. Genomic DNA gel blot and slot blot analyses show that there are approximately eight Spec genes in the S. purpuratus genome. The structures of three Spec2 genes, Spec2a, Spec2c and Spec2d, are described. A 60 kb (kb = 10³ bases or base-pairs) region of the genome contains the linked Spec1-Spec2c genes and two separate 20 kb regions contain the Spec2a and Spec2d genes. Six members of a repetitive sequence family are dispersed at various locations among the genes. The transcriptional initiation sites of the three Spec2 genes are mapped, and 400 to 500 base-pairs of 5′-flanking DNA sequenced. All three Spec2 genes initiate transcription approximately 120 base-pairs upstream from the 3′ end of the first exon. In contrast, the 5′ end of the Specl transcript begins about 107 base-pairs farther upstream, so it contains 5′ untranslated sequences that correspond to non-transcribed 5′-flanking sequences of the Spec2 genes. There is little similarity among the sequences upstream from the CAP site of the Spec2 genes except the TATA consensus sequence and a repeating trinucleotide, AAC. Measurements of Spec mRNA levels during embryogenesis show that Specl mRNA begins to accumulate at the early blastula stage and is the most abundant; Spec2a/Spec2c mRNAs begin accumulating several hours later at the late blastula-early gastrula stage and reach about 40 to 60% the levels of Spec1; and Spec2d mRNAs accumulate mostly during the gastrula and pluteus stages with levels reaching only 2% those of Spec1. In situ hybridization with probes that recognize either all Spec2 mRNAs or only Spec2d mRNAs show that, like Spec1, these mRNAs are restricted to aboral ectoderm cells and their precursors. The Spec gene family represents a group of related genes whose mRNAs all accumulate in the same cell type but at different times and to different levels during embryogenesis.
Suppression of ColE1 RNA-RNA mismatch mutations in vivo by the ColE1 rop protein
1987, Plasmid
In the bacterial plasmid ColE1 the control of initiation of DNA replication is mediated by the interaction of two complementary RNA molecules, the replication primer and RNA1. The rate of interaction between RNA1 and the primer RNA in vitro can be increased by the product of the ColE1 rop gene, a 63-amino-acid polypeptide. We have investigated the role of the Rop protein in suppressing the incompatibility defects of 13 RNA 1-mutant alleles. These RNA1 mutants are defective due to single nucleotide mismatches with their target, the primer RNA. The rop gene suppresses the defective behavior of most of the RNA1 point mismatch mutants in vivo. However, certain mutations that map in stems I and III of RNA1 are not suppressed by rop. The interaction of wild-type and mutant species of RNA1 with ColE1 replication primer transcripts was studied in vitro in the presence or absence of purified Rop protein. The Rop protein is known to increase the rate of wild-type RNA1-primer interaction about twofold in vitro. This enhancement was also observed for mutant RNA1 species having point alterations or a deletion of the 5′ terminus of RNA1, which is consistent with the in vivo suppression results. The implications of these results on the mechanism of Rop activity are considered.
Spec proteins: Calcium-Binding Proteins in the Embryonic Ectoderm Cells of Sea Urchins
1987, Methods in Enzymology
This chapter discusses spec proteins: the evolution and role of the troponin C superfamily of calcium-binding proteins. The sea urchin Strongylocentrotus purpuratus, during its embryonic and larval development, synthesizes and accumulates a set of calcium-binding proteins detectable exclusively in dorsal (aboral) ectoderm cells. These proteins, termed spec proteins, are related to the more extensively characterized calcium-binding proteins such as calmodulin, myosin light chains, troponin C, and parvalbumins. The in vivo function of the spec proteins is unknown, but it has been hypothesized that they play a role in changing the shape of the ectodermal cells. Supporting this hypothesis is the observation that two actin genes, cyIIIa and cyIIIb, are restrictively expressed in the same cells and at the same time in development as the spec genes. This suggests a structural and/or functional relationship between the spec proteins and the cyIII actin genes. Several eDNA libraries are available that have been constructed from RNA of S. purpuratus and Lytechinus pictus embryos. These include egg, blastula, gastrula, and plutcus stage libraries made from total or polysomal polyadenylated RNA. The recombinants are packaged in either phage or plasmid-harboring Escherichia coli. Spec sequences, as well as other cell- or stage-specific sequences, can be readily isolated from such libraries by routine differential-screening procedures. These libraries are available upon request.
The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes
1986, Cell
Fertilized clam embryos synthesize several new cell-cycle-related proteins. The cloned cDNA and derived amino acid sequences of one of these, cyclin A, are presented here. Immunoblots with an anti-cyclin A antibody reveal that cyclin A is undetectable in oocytes, appears within 15 min of fertilization, and is destroyed near the end of each meiosis and mitosis. We directly tested the ability of cyclin A to induce M phase by injecting SP6 cyclin A mRNA into Xenopus oocytes, which are arrested at the G2/M border of first meiosis. The injected mRNA was translated, with the result that the Xenopus oocytes entered meiosis. These findings indicate that the rise in cyclin A plays a direct and natural role in driving cells into M phase.
A large calcium-binding protein associated with the larval spicules of the sea urchin embryo
1986, Cell Differentiation
A tissue-specific, high molecular weight, calcium-binding protein from the sea urchin embryo is described. This protein, designated as CBP 180, has a molecular weight of 180000 under reducing conditions, and is extractable with 1% Triton X-100. It accumulates rapidly during development, starting roughly at the onset of spiculogenesis. When embryos are cultured in the presence of inhibitors of spicule formation, such as tunicamycin and zinc ions, accumulation of CBP180 is depressed or stopped. By immunofluorescence technique and by using an antibody specifically generated against this protein, CBP180 is mainly localized in primary mesenchyme cells and spicular syncytium of the pluteus larva. Little or none is detectable in ectoderm, endoderm or blastocoelar extracellular matrix. These results suggest that the protein is involved in calcium sequestration in the differentiation of larval spicules.

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^∗: Present address: Dept. of Molecular Biology Genentech, Inc., 460 Point San Bruno Blvd. South San Francisco, CA 94080 (U.S.A.).

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High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein

Abstract

Gene

Develop. Biol.

Develop. Biol.

Biochim. Biophys. Acta

Cell

J. Mol. Biol.

Gene

Cell

Gene

Gene

Gene