Calmodulin Promoter Research Articles

Neuronal calmodulin levels are controlled by CAMTA transcription factors.

The ubiquitous Ca2+ sensor calmodulin (CaM) binds and regulates many proteins, including ion channels, CaM kinases, and calcineurin, according to Ca2+-CaM levels. What regulates neuronal CaM levels, is, however, unclear. CaM-binding transcription activators (CAMTAs) are ancient proteins expressed broadly in nervous systems and whose loss confers pleiotropic behavioral defects in flies, mice, and humans. Using Caenorhabditis elegans and Drosophila, we show that CAMTAs control neuronal CaM levels. The behavioral and neuronal Ca2+ signaling defects in mutants lacking camt-1, the sole C. elegans CAMTA, can be rescued by supplementing neuronal CaM. CAMT-1 binds multiple sites in the CaM promoter and deleting these sites phenocopies camt-1. Our data suggest CAMTAs mediate a conserved and general mechanism that controls neuronal CaM levels, thereby regulating Ca2+ signaling, physiology, and behavior.

Nuclear Repositioning Precedes Promoter Accessibility and Is Linked to the Switching Frequency of a Plasmodium falciparum Invasion Gene

Variation of surface adhesins, such as the Plasmodium falciparum erythrocyte invasion ligand PfRh4, is critical for virulence and immune evasion in many microbes. While phenotypic switching is linked to transcriptional changes and chromatin function, the determinants of switching frequency remain poorly defined. By expressing a prokaryotic DNA methylase in P.falciparum, we directly assayed accessibility of transcriptionally active and silent chromatin at the PfRh4 locus. Parasites selected for invivo PfRh4 activation show a reversible increase in promoter accessibility and exhibit perinuclear repositioning of the locus from a silent to a conserved activation domain. Forced activation of a proximal gene results in a similar repositioning of the PfRh4 locus, witha concomitant increase in PfRh4 activation ina subpopulation of parasites and promoter accessibility correlating with actively transcribed loci. Thus, nuclear repositioning is associated with increased P.falciparum switching frequency, while promoter accessibility is tightly linked to clonally active PfRh4 promoters.

Investigations into the role of the Plasmodium falciparum SERCA (PfATP6) L263E mutation in artemisinin action and resistance.

Artemisinin-based combination therapies (ACTs) are highly effective for the treatment of Plasmodium falciparum malaria, yet their sustained efficacy is threatened by the potential spread of parasite resistance. Recent studies have provided evidence that artemisinins can inhibit the function of PfATP6, the P. falciparum ortholog of the ER calcium pump SERCA, when expressed in Xenopus laevis oocytes. Inhibition was significantly reduced in an L263E variant, which introduced the mammalian residue into a putative drug-binding pocket. To test the hypothesis that this single mutation could decrease P. falciparum susceptibility to artemisinins, we implemented an allelic-exchange strategy to replace the wild-type pfatp6 allele by a variant allele encoding L263E. Transfected P. falciparum clones were screened by PCR analysis for disruption of the endogenous locus and introduction of the mutant L263E allele under the transcriptional control of a calmodulin promoter. Expression of the mutant allele was demonstrated by reverse transcriptase (RT) PCR and verified by sequence analysis. Parasite clones expressing wild-type or L263E variant PfATP6 showed no significant difference in 50% inhibitory concentrations (IC(50)s) for artemisinin or its derivatives dihydroartemisinin and artesunate. Nonetheless, hierarchical clustering analysis revealed a trend toward reduced susceptibility that neared significance (artemisinin, P approximately = 0.1; dihydroartemisinin, P = 0.053 and P = 0.085; and artesunate, P = 0.082 and P = 0.162 for the D10 and 7G8 lines, respectively). Notable differences in the distribution of normalized IC(50)s provided evidence of decreased responsiveness to artemisinin and dihydroartemisinin (P = 0.02 for the D10 and 7G8 lines), but not to artesunate in parasites expressing mutant PfATP6.

Characterization and Structural Studies of the Plasmodium falciparum Ubiquitin and Nedd8 Hydrolase UCHL3

Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival. The crystal structures of both PfUCHL3 alone and in complex with the ubiquitin-based suicide substrate UbVME suggest a rather rigid active site crossover loop that likely plays a role in restricting the size of ubiquitin adduct substrates. Molecular dynamics simulations of the structures and a model of the PfUCHL3-PfNedd8 complex allowed the identification of shared key interactions of ubiquitin and PfNedd8 with PfUCHL3, explaining the dual specificity of this enzyme. Distinct differences observed in ubiquitin binding between PfUCHL3 and its human counterpart make it likely that the parasitic DUB can be selectively targeted while leaving the human enzyme unaffected.

Genomic structure and transcriptional regulation of grass carp calmodulin gene

A fish calmodulin (CaM) gene was characterized for the first time in grass carp. The CaM gene is about 12-Kb in size with identical intron/exon organization as that of mammalian CaM genes. When compared to mammalian counterparts, the 5′-promoter region of grass carp CaM gene contains a TATA box and has a much lower GC content and CpG dinucleotide frequency. Interestingly, the 5′-promoter of carp CaM gene is AT-rich with multiple IRS elements and putative binding sites for Pit-1, Sp1/Sp3 and AP1. Using luciferase reporter assay, a potent silencer region was identified in the distal region of grass carp CaM promoter. Besides, the CaM promoter activity could be upregulated by IGF but suppressed by PACAP, forskolin and over-expression of Sp1 and Sp3. These findings, taken together, indicate that grass carp CaM gene does not exhibit the typical features of housekeeping genes and its expression is under the control of hormone factors, presumably by coupling with the appropriate signaling pathways/transcription factors.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

상처에 의해서 유도되는 벼 calmodulin promoter의 transgenic 담배에서조직 특이적 발현

To study calmodulin (CaM) gene expression and its regulation, rice CaM promoter (RCaM-2) was isolated and fused to (GUS), reporter gene. X-Glue staining patterns revealed that GUS localization is high in meristemic tissues such as the stem apex, stolen tip, and vascular regions. GUS staining in the transverse sections of stem and petiole was restricted to the inside of the vascular system, and cortex and epidermis located outside of the vascular system usually did not show GUS staining even a plant that expressed strong activity. GUS activity was found to be tissue specific expressed and exhibited a dramatic transient increase in response to wounding. These results suggest that the 5`-flanking region of RCaM gene regulates wound-inducible expression.

Thioredoxin Reductase Is Essential for the Survival ofPlasmodium falciparum Erythrocytic Stages

The human malaria parasite Plasmodium falciparum poses an increasing threat to human health in the tropical regions of the world, and the validation and assessment of possible drug targets is required for the development of new antimalarials. It has been shown that the erythrocytic stages of the parasites, which are responsible for the pathology of the disease in humans, are under enhanced oxidative stress and are particularly vulnerable to exogenous challenges by reactive oxygen species. Therefore it is postulated that the disruption of the antioxidant and/or redox systems of the parasite is a feasible way to interfere with their development during erythrocytic schizogony. In order to test this suggestion thioredoxin reductase (TrxR), an enzyme heavily involved in maintenance of redox homeostasis and antioxidant defense, was knocked out in P. falciparum. It was impossible to generate parasites with a disrupted trxR gene suggesting that TrxR is essential for P. falciparum erythrocytic stages. Technical problems were excluded by transfecting a 3' replacement construct, which recombined correctly and transfectants did not show any phenotypic alterations. In order to prove that the trxR knockout was responsible for the lethal phenotype of the null mutants, a co-transfection with both the knockout construct and a construct containing the trxR coding region under the control of the calmodulin promoter was conducted. Despite the disruption of the trxR gene, parasites were viable. In a Southern blot analysis a complicated restriction pattern was obtained, but it was shown by pulse field gel electrophoresis and field inverse gel electrophoreses that only the trxR gene locus on chromosome 9 was targeted by the constructs. It was found that the co-transfected constructs form concatemeric structures prior to integration into the trxR gene locus, which is further supported by plasmid rescue followed by restriction analyses of the plasmids. Northern and Western blot analyses proved that the co-transfectants highly overexpress TrxR from the introduced gene. Our results demonstrate that TrxR is essential for the survival of the erythrocytic stages of P. falciparum.

Expression of a chimeric GUS gene construct as a tool to study nodule morphogenesis in chicory leaves

Leaves from micropropagated chicory plantlets were cultivated in vitro to regenerate organogenic nodules. The nodules were transformed with a 2.3 kb fragment of an apple calmodulin promoter region fused to the coding sequence of uidA reporter gene. Histochemical detection of β-glucuronidase expression in transgenic regenerants showed that vessel-associated cells were strongly stained. Previous histological investigations have shown that the differentiation of vascular elements is essential to nodule and bud-derived-nodule development. Therefore, β-glucuronidase activity was tested in a single transgenic chicory line during nodule morphogenesis and bud regeneration. The vascular connections between leaves and nodules, then between nodules and buds were stained blue, indicating an alternative system for determining the histological origin of nodules and adventitious buds from chicory leaf explants.

Members of the Sp transcription factor family regulate rat calmodulin gene expression

We have previously demonstrated that insulin positively regulates transcription of the rat calmodulin (CaM) I gene and that both basal and insulin stimulation of this gene are critically dependent on Sp1. Furthermore, a 392 bp CaM promoter was stimulated by insulin equal to the full promoter but lost activity with deletion of any of the three Sp1 sites (Solomon SS, Palazzolo MR, Takahashi T, Raghow R. Endocrinology 1997;138:5052-5054). Herein we document that Sp1 preferentially binds to the upstream sites Sp12 and Sp13 but not Sp11. Furthermore, gel-mobility super-shift assays demonstrate that both Sp1 and Sp3 protein are found in these complexes. When pPac-Sp1, pPac-Sp3, pPac-USp3, and pPac-Sp4 were cotransfected with rCaM 1-392 promoter into Drosophila SL2 cells and challenged with 10,000 μU/mL insulin, we discovered that (1) Sp1 enhanced both basal and insulin-stimulated CaM I gene expression; (2) USp3, a “long” form of the Sp3 molecule, had a stimulatory effect on CaM I gene expression; (3) Sp1 or USp3 is involved in mediating insulin-stimulation of the CaM I gene in SL2 cells; and (4) Sp3, a “short” form of the Sp3 molecule, and Sp4 inhibited Sp1-stimulated and insulin-stimulated Sp1-mediated CaM I gene expression. Together these data corroborate and extend our previous observations on Sp1 and elucidate that other members of the Sp family of transcription factors may also be involved in regulating the activity of the CaM promoter. (J Lab Clin Med 2000;136:157-63)

Stable transgene expression in Plasmodium falciparum

Plasmid vectors designed to express transgenes and a selectable marker in Plasmodium falciparum were constructed. These consist of a selectable gene cassette comprising the Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene mutated to confer pyrimethamine resistance flanked by either Plasmodium chabaudi DHFR-TS or P. falciparum calmodulin promoter sequences and the P. falciparum histidine rich protein 2 3′ region. Also, each vector includes a different expression cassette driven by various Plasmodium transcriptional control sequences. Initially, the chloramphenicol acetyl transferase (CAT) reporter gene was cloned into the expression site of two vectors, pCC6-CAT and pCC13-CAT, which were identical except for the orientation of the expression cassette with respect to the selectable gene cassette. Approximately 8-fold more CAT activity was detected when the direction of transcription of the expression cassettes was in a head to head, rather than a tail to head, orientation. Importantly, it was found that stable transfection could only be achieved when the gene cassettes were in the head to head direction suggesting that this orientation also has an effect on the level of expression of the selectable marker. All other plasmids were designed with the cassettes in a head to head orientation. With the exception of pCC6-CAT and a second vector pHC4-CAT, stable transfectants were obtained with each vector in which the CAT gene had been inserted into the expression cassette. This is the first time vectors for the stable expression in Plasmodium parasites of transgenes other than a selectable marker have been described.

Transcription Factor Sp1 Is Necessary for Basal Calmodulin Gene Transcription and for Its Selective Stimulation by Insulin

Insulin positively regulates transcription of rat calmodulin (CaM) I gene and activates the low Km cyclic AMP (cAMP) phosphodiesterase (PDE). To elucidate the mechanism of transcriptional regulation, rat hepatoma (H-411E) cells were transfected with DNA constructs containing the putative CaM promoters coupled to a luciferase reporter and challenged with insulin. Activation of the full length 1835 bp rat CaM I promoter containing all three Sp1 sites or truncated promoters with combinations of one to three of the Sp1 sites was studied in Sp1 deficient Drosophilia SL2 cells and in SL2 cells co-transfected with an Sp1 expression vector and re-challenged with insulin. Our results demonstrate that Sp1 is obligatory for basal activation of the CaM promoter. The maximal insulin stimulation of CaM promoter is elicited only if it contains at least two Sp1 sites.

Transcription factor Sp1 is necessary for basal calmodulin gene transcription and for its selective stimulation by insulin.

Insulin positively regulates transcription of rat calmodulin (CaM) I gene and activates the low Km cyclic AMP (cAMP) phosphodiesterase (PDE). To elucidate the mechanism of transcriptional regulation, rat hepatoma (H-411E) cells were transfected with DNA constructs containing the putative CaM promoters coupled to a luciferase reporter and challenged with insulin. Activation of the full length 1835 bp rat CaM I promoter containing all three Sp1 sites or truncated promoters with combinations of one to three of the Sp1 sites was studied in Sp1 deficient Drosophilia SL2 cells and in SL2 cells co-transfected with an Sp1 expression vector and re-challenged with insulin. Our results demonstrate that Sp1 is obligatory for basal activation of the CaM promoter. The maximal insulin stimulation of CaM promoter is elicited only if it contains at least two Sp1 sites.

Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase

Telomeric DNA at the ends of chromosomes consist of short, tandem repeat sequences. The telomeres of Paramecium tetraurelia are made up of variable repeats, whereas Paramecium caudatum telomeric repeats are largely invariant. To investigate variable repeat synthesis in P. tetraurelia, mutated telomerase RNA genes were expressed in vivo. We demonstrate that the P. caudatum telomerase RNA can participate in telomere synthesis when expressed in the P. tetraurelia macronucleus, despite 24% primary sequence divergence of the RNAs between the two species. De novo telomeric repeats from transformants indicate that P. tetraurelia telomerase fidelity is dramatically affected by template substitutions and that misincorporation at a single templating position is likely to account for the majority of P. tetraurelia telomeric DNA variability. Furthermore, we show that fidelity is not solely a function of the RNA moiety, as the P. caudatum telomerase RNA does not impart high fidelity to the chimeric enzyme.

Developmentally Regulated Expression of the Rice Calmodulin Promoter in Transgenic Tobacco Plants

Developmentally Regulated Expression of the Rice Calmodulin Promoter in Transgenic Tobacco Plants

Gene transfer through implantation of embryonal carcinoma cells in the brain

Mouse embryonal carcinoma cell line P19 could be differentiated to neurons or glial cells by appropriate induction. We introduced the lacZ gene driven by a calmodulin promoter into P19 cells, and cloned the transfectant CaM-Z that showed the strong expression of lacZ gene after the induction of differentiation. CaM-Z cells after differentiation were then implanted in newborn rats or mice intracranially. PCR amplification of the lacZ gene and histological examination of the recipient animals revealed that transplanted cells survived for 1 to 7 mo in the rat or mouse brains with lacZ gene expression and neuronal differentiation. The rate of the successful transplantation was higher in mice than in rats.

Calmodulins with deletions in the central helix functionally replace the native protein in yeast cells.

Deletion of Glu-84, Glu-83 and Glu-84, or Ser-Glu-Glu-Glu (residues 81-84) from the central helix of mammalian calmodulin is known to result in a 5-7 times decrease in its apparent in vitro affinities for three calmodulin-dependent enzymes. However, based on in vitro experiments, it is difficult to estimate how these deletions might affect in vivo cellular function. The yeast Saccharomyces cerevisiae, which requires calmodulin for growth, provides an excellent system to evaluate these deletion proteins in vivo. Based on its ability to restore normal growth characteristics to yeast cells, mammalian calmodulin is functionally identical to the yeast protein; herein we evaluate the effect of deleting residues 84, 83 and 84, or 81-84 from the central helix. Sequences encoding the deletion proteins and an unaltered control sequence were introduced by means of a yeast shuttle vector and were expressed under control of the yeast calmodulin promoter. The deletion and control calmodulins are produced at levels similar to that observed for the yeast protein, and they completely restore normal growth characteristics. This result suggests that the regions deleted from the central helix are not critical for activation of any yeast calmodulin target normally required for cell growth or division. It is likely that there are twisting and shortening motions associated with the deletions from the central helix that alter significantly the spatial relationship between the two lobes of calmodulin. The abilities of the deletion calmodulins to restore completely normal growth characteristics to yeast cells suggest that the lobes of all the deletion proteins can still be appropriately positioned in calmodulin-target complexes. This is consistent with the hypothesis that the central helix of calmodulin is analogous to a flexible tether rather than to a rigid connector between the two lobes of the molecule.

Effects of changes in calmodulin levels on cell proliferation.

Calmodulin (CaM) is one of several proteins regulated in a cell cycle-dependent manner. CaM is synthesized at the G1/S boundary and has been implicated in the regulation of cell cycle progression. To elucidate the role of calmodulin in cell cycle control, clonal mouse C127 cell lines transformed with one of four different bovine papilloma virus (BPV)-based vectors were studied. These vectors express a) a chicken CaM gene regulated by its own promoter (CM cells), b) the chicken CaM gene regulated by the inducible human metallothionein-IIa promoter (MCM cells), c) CaM antisense RNA using the Zn2+ inducible mouse metallothionein-I (mMT-I) promoter (AS cells), or d) a rat parvalbumin gene using the chicken CaM promoter (PV cells). C127 cells transformed by BPV-1 alone (BPV cells) are used as a control in each case. Previous studies showed that a 4-fold increase in CaM levels in CM cells shortened the cell cycle by reducing the length of the G1 period. Expression of parvalbumin in PV cells has no effect on cell cycle length, suggesting that increased CAM, and not simply increased Ca2(+)-binding protein, accelerates proliferation. Zn2(+)-induced expression of the chicken CaM gene in MCM cells increased the rate of proliferation, while Zn2(+)-induced expression of high levels of CaM anti-sense RNA stops proliferation at Zn2+ levels that do not affect the growth of BPV cells. In CM cells increased CaM affects cell cycle-dependent level of mRNAs for tubulin, vimentin, and c-myc relative to the levels in BPV cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Changes in Calmodulin Levels on Cell Proliferation

Calmodulin (CaM) is one of several proteins regulated in a cell cycle-dependent manner. CaM is synthesized at the G1/S boundary and has been implicated in the regulation of cell cycle progression. To elucidate the role of calmodulin in cell cycle control, clonal mouse C127 cell lines transformed with one of four different bovine papilloma virus (BPV)-based vectors were studied. These vectors express a) a chicken CaM gene regulated by its own promoter (CM cells), b) the chicken CaM gene regulated by the inducible human metallothionein-IIa promoter (MCM cells), c) CaM antisense RNA using the Zn2+ inducible mouse metallothionein-I (mMT-I) promoter (AS cells), or d) a rat parvalbumin gene using the chicken CaM promoter (PV cells). C127 cells transformed by BPV-1 alone (BPV cells) are used as a control in each case. Previous studies showed that a 4-fold increase in CaM levels in CM cells shortened the cell cycle by reducing the length of the G1 period. Expression of parvalbumin in PV cells has no effect on cell cycle length, suggesting that increased CAM, and not simply increased Ca2(+)-binding protein, accelerates proliferation. Zn2(+)-induced expression of the chicken CaM gene in MCM cells increased the rate of proliferation, while Zn2(+)-induced expression of high levels of CaM anti-sense RNA stops proliferation at Zn2+ levels that do not affect the growth of BPV cells. In CM cells increased CaM affects cell cycle-dependent level of mRNAs for tubulin, vimentin, and c-myc relative to the levels in BPV cells.(ABSTRACT TRUNCATED AT 250 WORDS)

48] Methods for analyzing bovine papilloma virus-based calmodulin expression vectors

Publisher Summary This chapter describes a method for the transfection and analysis of bovine papilloma virus (BPV) expression vector-containing cell lines. This method allows a rapid and simple way to identify cells that express calmodulin (CaM) gene. The approach used is generally applicable to the study of any BPV-based expression vector. Three different BPV-based CaM expression vectors were used to study the effect of changes in intracellular CaM levels on cellular function. The vectors BPV-CM and BPV-MCM II both contain a CaM minigene whose expression is regulated either by a CaM promoter or by the human metallothionein IIa promoter. Cells that retained unrearranged episomal plasmids are screened for expression of vector-derived mRNA by slot-blot and Northern blot analysis. Finally, cells are shown to be producing significantly higher levels of intact protein by techniques such as RIA or SDS-PAGE. The use of expression vectors has many applications in studying the biology of gene products in vivo. The synthesis of mutated proteins or the production of antisense RNA as a specific antagonist for a single gene product both hold promise in identifying physiologically relevant roles in systems where in vitro analysis has suggested an important function.