Cloning and expression of mouse-brain calmodulin as an activator of Bordetella pertussis adenylate cyclase in Escherichia coli

Danchin, Antoine; Sezer, Odile; Glaser, Philippe; Chalon, Pascale; Caput, Daniel

doi:10.1016/0378-1119(89)90259-x

Cited by 26 publications

(7 citation statements)

References 13 publications

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“…CaM is ubiqui-0 1993 Wiley-Liss, Inc. tously distributed over various tissues and abundantly in the brain and the testis (Kakiuchi et al, 1982;Zhou et al, 1985). The 148-amino acid sequence of CaM is highly conserved not only in mammals (Bender et al, 1988;Fischer et al, 1988;Danchin et al, 1989;Nojima, 1989) but also in fishes (Matsuo et al, 1992). In mammals, three nonallelic genes encode the single CaM protein by synonymous codon usage, although the sequence homology is low in the noncoding regions of these genes (Nojima, 1989).…”

Section: Introductionmentioning

confidence: 99%

Expression of three nonallelic genes coding calmodulin exhibits similar localization on the central nervous system of adult rats

Ikeshima

Yuasa

Matsuo

et al. 1993

J of Neuroscience Research

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By Northern blot analysis with the digoxigenin-labeled antisense RNA probes of the noncoding regions, the transcripts of three calmodulin (CaM) genes, CaMI, CaMII, and CaMIII, were separately detected in 12 different tissues of adult Wistar albino rats, without any cross-hybridization. The mRNAs of all three CaM genes were abundant in the central nervous system (CNS) as well as in the testis, although ubiquitous expression was detected at low levels in the other tissues. There were subtle but significant differences in the tissue-specific distribution of the three CaM gene RNAs. By in situ hybridization, strong hybridization of the three CaM gene probes was observed in common in large projection neurons of the CNS: the hippocampal pyramidal cells, the cerebellar Purkinje cells, and the large neurons of the cerebral neocortex, the pyriform cortex, the mesencephalon, the pons, and the spinal cord. The expression of the three CaM genes was at lower levels in small interneurons of the CNS. These profiles of expression were almost the same among the three CaM genes. Thus, all three CaM genes were coordinately expressed in neurons of the adult rat CNS. Certain regulatory mechanisms of the three CaM genes seemed to mediate similar tissue- and cell type-specific expression in the CNS.

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Section: Introductionmentioning

confidence: 99%

Expression of three nonallelic genes coding calmodulin exhibits similar localization on the central nervous system of adult rats

Ikeshima

Yuasa

Matsuo

et al. 1993

J of Neuroscience Research

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“…CaM purification. Mouse brain CaM was cloned and expressed in Escherichia coli as described [30]. The protein was recovered in the supernatant after bacterial disruption by sonication.…”

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confidence: 99%

Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy

Gallay

Vincent

Sierra

et al. 2004

European Journal of Biochemistry

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The interaction of the adenylate cyclase catalytic domain (AC) of the Bordetella pertussis major exotoxin with its activator calmodulin (CaM) was studied by time-resolved fluorescence spectroscopy using three fluorescent groups located in different regions of AC: tryptophan residues (W69 and W242), a nucleotide analogue (3¢-anthraniloyl-2¢-deoxyadenosine 5¢-triphosphate, Ant-dATP) and a cysteinespecific probe (acrylodan). CaM binding elicited large changes in the dynamics of W242, which dominates the fluorescence emission of both AC and AC-CaM, similar to that observed for isolated CaM-binding sequences of different lengths [Bouhss, A., Vincent, M., Munier, H., Gilles, A.M., Takahashi, M., Baˆrzu, O., Danchin, A. & Gallay, J. (1996) Eur. J. Biochem. 237, 619-628]. In contrast, AntdATP remains completely immobile and inaccessible to the solvent in both the AC and AC-CaM nucleotide-binding sites. As AC contains no cysteine residue, a single-Cys mutant at position 75 was constructed which allowed labeling of the catalytic domain with acrylodan. Its environment is strongly apolar and rigid, and only slightly affected by CaM. The protein's hydrodynamic properties were also studied by fluorescence anisotropy decay measurements. The average Brownian rotational correlation times of AC differed significantly according to the probe used (19 ns for W242, 25 ns for Ant-dATP, and 35 ns for acrylodan), suggesting an elongated protein shape (axial ratio of % 1.9). These values increased greatly with the addition of CaM (39 ns for W242, 60-70 ns for Ant-dATP and 56 ns for acrylodan). This suggests that (a) the orientation of the probes is altered with respect to the protein axes and (b) the protein becomes more elongated with an axial ratio of % 2.4. For comparison, the hydrodynamic properties of the anthrax AC exotoxin were computed by a mathematical approach (HYDROPRO), which uses the 3D structure [Drum, C.L., Yan, S. A change in axial ratio is also observed on CaM binding, but in the reverse direction from that for AC: from 1.7 to 1.3. The mechanisms of activation of the two proteins by CaM may therefore be different.

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“…Three non-allelic CaM genes encoding exactly the same 148 aa protein (17,000 daltons) were identified in the rat (Nojima, 1989 ), mouse (Bender et al, 1988;Danchin et al, 1989), and humans (Fischer et al, 1988); two were identified in Xenopus (Chien and Daxid, 1984), and four in the teleost fish medaka (Oryzias latipes) (Matsuo et al, 1992). Although the reason for the presence of multiple synonymous genes in animals is unclear, differences in the 5' and 3' non-coding region of the rat CaM genes suggest that each gene may be differentially regulated.…”

Section: Introductionmentioning

confidence: 99%

Three synonymous genes encode calmodulin in a reptile, the Japanese tortoise, Clemmys japonica

et al. 2002

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Three distinct calmodulin (CaM)-encoding cDNAs were isolated from a reptile, the Japanese tortoise (Clemmys japonica), based on degenerative primer PCR. Because of synonymous codon usages, the deduced amino acid (aa) sequences were exactly the same in all three genes and identical to the aa sequence of vertebrate CaM. The three cDNAs, referred to as CaM-A, -B, and -C, seemed to belong to the same type as CaMI, CaMII, and CaMIII, respectively, based on their sequence identity with those of the mammalian cDNAs and the glutamate codon biases. Northern blot analysis detected CaM-A and -B as bands corresponding to 1.8 kb, with the most abundant levels in the brain and testis, while CaM-C was detected most abundantly in the brain as bands of 1.4 and 2.0 kb. Our results indicate that, in the tortoise, CaM protein is encoded by at least three non-allelic genes, and that the 'multigene-one protein' principle of CaM synthesis is applicable to all classes of vertebrates, from fishes to mammals.

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Cloning and expression of mouse-brain calmodulin as an activator of Bordetella pertussis adenylate cyclase in Escherichia coli

Cited by 26 publications

References 13 publications

Expression of three nonallelic genes coding calmodulin exhibits similar localization on the central nervous system of adult rats

Expression of three nonallelic genes coding calmodulin exhibits similar localization on the central nervous system of adult rats

Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy

Three synonymous genes encode calmodulin in a reptile, the Japanese tortoise, Clemmys japonica

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