华山姜中钙调素基因的克隆及其RNA原位杂交

本文报道了华山姜中钙调素cDNA的核苷酸序列以及由此推导的氨基酸序列。并用原化杂交的方法检测其在花器官中的时间表达模式。用[^32P]d-CTP标记的小草蔻-Alpinia hainanensis AGAMOUS（AG）cDNA的MADS-domain作为探针筛选华山姜的cDNA文库．得到一个钙调素蛋白相关克隆．命名为AoCAM。华山姜钙调素AoCAM的cDNA全长518bp．有一个包含149个氨基酸的开放读码框．编码区起始于第54个核苷酸,终止于第501个核苷酸。AoCAM与拟南芥、小麦、大豆、矮牵牛、玉米的钙调素氨基酸序列比较同源性高达95％。RNA原位杂交表明钙凋素基因和花瓣、雄蕊、雌蕊细胞中大量表达。钙调素基因的表达强度随不同的发育阶段而变化：花发育早期在花的各器官中部表达强烈,以后逐渐减弱并向特定部位集中．如花粉囊、唇瓣、花柱和胚珠等分生能力较强的细胞中表达较强。     

钙调素mRNA和蛋白在水稻花药和雌蕊发育过程中的原位定位

利用ＲＮＡ原位杂交和免疫组织化学定位技术分别检测了钙调素ｍＲＮＡ和钙调素蛋白在水稻（ＯｒｙｚａｓａｔｉｖａＬ．）花药和雌蕊发育过程中的时空分布特征。钙调素基因在绒毡层、柱头、花粉管生长途径、退化助细胞以及维管薄壁细胞中大量表达,也可在小孢子母细胞、小孢子、花粉、反足细胞、卵细胞以及中央细胞中检测到。钙调素基因的表达强度随不同的发育阶段而变化：花药发育早期表达强,以后逐渐减弱并向特定部位集中,如绒毡层和花粉萌发孔等。胚胎发育早期,钙调素基因在胚乳细胞中的表达比原胚中强,而后期则在分化胚中比胚乳细胞中强。推测在有性生殖过程中,钙调素可能通过Ｃａ２＋ＣａＭ信号途径调节小孢子发育、花粉萌发、花粉管生长、受精以及物质运输等生理过程     

细胞内信号分子传导的研究进展

近年来有关细胞内信号传导的研究,着重体现在Ca²⁺信号传导途径及相应的蛋白质分子如蛋白激酶C(PKC)、钙调素(CaM)、钙调素激酶Ⅱ(CaMKⅡ),同时也对Ras途径中出现的Vav、Rap、Crk、C3G等蛋白质分子以及cAMP和NF-κB途径作了有益的补充与修改.细胞外信号分子通过以上4种途径及其相互通讯(cross-talk),激活了某些蛋白激酶,调控了基因转录及其他相关功能,其中磷酸化对蛋白激酶及转录因子活性的调节起到了非常重要的作用.     

钙调素及钙调素相关蛋白在植物细胞中的研究进展

植物对一系列生物和非生物刺激所产生的反应都与细胞内Ca2+信号转导有关,而钙调素、钙调素相关蛋白则是Ca2+信号转导的下游靶蛋白。该文介绍了钙调素的结构及其在植物细胞中的分布,钙调素及钙调素相关蛋白在植物细胞中的表达等方面的最近研究进展。     

多功能的钙／钙调素依赖的蛋白激酶II介导的细胞信号传导

钙调素（Calmodulin,CaM）是一个特别的对钙敏感的蛋白,在钙信号传导通路中扮演重要角色钙／钙调素依赖性蛋白激酶（Calcium／calmodulin-dependent kinases（CaMKs））与荷尔蒙、神经迷质及其他信号引起的细胞反应相关、作为重要的第二信使,钙／钙调素依赖的蛋白激酶Ⅱ（CaM—KⅡ）是一类在细咆中无所不在的表达的蛋白激酶,能维持细胞内的钙浓度在很低的水平,再增加后续的特定的钙激动刺激。钙／钙调素依赖的簧白激酶Ⅱ独特的全酶结构和自我调节的性质使其对短暂的钙信号和胞内钙的变化能做出延长反应：本文从结构、合成、细胞分布、反应底物、生理功能等方面介绍了钙／钙调素依赖的蛋白激酶Ⅱ的激活对细胞信号传导的作用。     

光敏核不育水稻叶片中钙调素mRNA原位杂交分析(简报)

采用组织RNA原位杂交技术研究钙调素基因在光敏核不育水稻接受光周期信号后时空表达特征的结果表明,钙调素基因在不同光周期诱导后,叶片在育性转换对光周期敏感的不同发育时期中,mRNA表达量和空间分布上没有明显的差异,与光敏核不育水稻育性不存在直接的关系;叶肉细胞中钙调素mRNA的大量表达,可能与光敏色素信号系统调节叶绿体发育及光合作用有关。     

花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响

以烟草为材料,通过半体内实验,就花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响进行了观察。发现用EGTA及钙调素抗血清处理柱头或花粉均可抑制花粉在柱头上的萌发;向花柱引导组织中显微注射纯化钙调素可促进花粉管束伸长,而注射钙调素抗血清可抑制花粉管束伸长;同时证实玉米花柱和花粉细胞壁中均存在钙调素及钙调素结合蛋白,而且花粉和花柱细胞壁中钙调素结合蛋白的种类有差异。结果表明存在于花粉和花柱细胞外的钙调素对花粉萌发和花粉管伸长均有促进作用。     

玉米根尖质膜的受钙激活蛋白激酶的特性

以生长3－4d的玉米（ZeamaysL．）根尖为材料,用两相法得到高纯度的质膜,鉴定了质膜上存在一受钙激活的蛋白激酶。该类激酶主要位于质膜内侧;钙的半激活浓度为50μmol／L;外源钙调素对激酶没有明显的活化作用;钙调素拮抗剂三氟拉嗪（TFP）可明显抑制激酶活性,半抑制浓度为75μmol／L;该类激酶对外源底物组蛋白ⅢS型有较高的特异性。结果显示此激酶属于钙依赖钙调素不依赖蛋白激酶。质膜上33kD和58kD两种蛋白可能是这种钙依赖蛋白激酶的内源底物。     

钙调素依赖型蛋白激酶在植物开花调控中的作用

钙调素依赖型蛋白激酶已被证明在诸多信号传导过程中起重要作用.研究了这一类激酶在植物开花调控中的可能作用.将分离自玉米的钙调素依赖型蛋白激酶(maize calmodulin-dependent protein kinase 1,MCK1),经过基因羧基末端(此羧基末端含有该基因的钙调素结合区)缺失突变成为MCKt,该基因的表达不再受钙调素的调控,通过农杆菌转化法获得转基因烟草植株.结果表明,MCKt的表达显著影响烟草的开花发育程序, 导致植物主枝的花原基败育以及侧枝营养生长期的延长.败育过程首先开始于正在发育的花药,然后是整个花的衰老.侧芽营养生长期的延长表征为产生成簇的伸长、窄小而扭曲的非典型叶片.这些结果显示,钙调素依赖型蛋白激酶同源物在开花调控中起着重要作用.     

花柱和花胞外钙调素对花粉萌发和花粉管伸长的影响

以烟草为材料,通过半体内实验,就花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响进行了观察。发现用ＥＧＴＡ及钙调素抗血表处理柱头或花粉均可抑制花粉在柱头上的萌发;向花柱引导组织中显微注射纯化钙调素可促进花粉管束伸长,面注射钙调素抗血清可抑制花粉管束伸长;同时证实玉米花柱和花粉细胞壁中均存在钙调纱及钙调素结合蛋白,而且花粉和花柱细胞壁中钙调素结合蛋白的种类有差异。结果表明存在于花粉和花柱细胞外的钙调     

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [³⁵S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 M KN-93, but binding is not affected by 5 M KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 M KN-93, but not by 5 M KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.Abbreviations CaM calmodulin - CaMK (II) Ca²⁺/calmodulin-dependent protein kinase (II) - CBP CaM-binding protein - CDPK Ca²⁺-dependent protein kinase - MCK1 maize homolog of mamalian CaMK This work is supported by the National Aeronautics and Space Administration grant No: NAGW 238.     

A consensus CaMK IV-responsive RNA sequence mediates regulation of alternative exons in neurons

Neurons make extensive use of alternative pre-mRNA splicing to regulate gene expression and diversify physiological responses. We showed previously in a pituitary cell line that the Ca(++)/calmodulin-dependent protein kinase CaMK IV specifically repressed splicing of the BK channel STREX exon. This repression is dependent on a CaMK IV-responsive RNA element (CaRRE) within the STREX 3' splice site. Here, we report that similar Ca(++) regulation of splicing, mediated by L-type calcium channels and CaM kinase IV, occurs in cultured neurons and in the brain. We identify a critical CaRRE motif (CACATNRTTAT) that is essential for conferring CaMK IV repression on an otherwise constitutive exon. Additional Ca(++)-regulated exons that carry this consensus sequence are also identified in the human genome. Thus, the Ca(++)/CaMK IV pathway in neurons controls the alternative splicing of a group of exons through this short CaRRE consensus sequence. The functions of some of these exons imply that splicing control through the CaMK IV pathway will alter neuronal activity.     

Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.)

Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca^2 +/H⁺ antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca^2 + ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca^2 + ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet.     

日本七鳃鳗钙调蛋白分子克隆、组织定位及功能分析

钙调蛋白（calmodulin,CaM）是高度保守的钙离子结合蛋白质,可形成Ca ²⁺-CaM复合体,从而调节细胞代谢以及靶酶的功能。日本七鳃鳗（Lampetra japonica）作为原始的无颌类脊椎动物,对研究脊椎动物分子起源进化及器官发育分化具有重要的研究价值。通过提取日本七鳃鳗髓组织总RNA,利用RT-PCR方法获得日本七鳃鳗CaM（简称Lj-CaM）基因并进行生物信息学分析。将Lj-CaM基因分别构建到原核表达载体pColdⅠ和真核表达载体pEGFP-N1中,利用亲和层析技术纯化得到Lj-CaM蛋白。圆二色谱分析结果表明,Lj-CaM属于典型的α-螺旋结构型蛋白质。免疫印迹和免疫组化结果表明,CaM主要存在于日本七鳃鳗的肠、鳃、髓、肾组织中,在心和肝组织中几乎不表达。细胞免疫荧光结果显示,CaM定位于细胞核中。qPCR和免疫印迹方法检测发现,当293T细胞中Lj-CaM过表达时,对下游靶基因CaMKⅡ作用不明显,但促进PLA2G2A表达。本研究报道了日本七鳃鳗CaM结构、细胞组织定位分布以及基因调控研究,对其结构、分子起源与进化、分子调控及功能方面的研究奠定了基础。     

Developmentally regulated organ-, tissue-, and cell-specific expression of calmodulin genes in common wheat

Recently, we reported on the characterization of the calmodulin (CaM) gene family in wheat [44]. We classified wheat CaM genes into four subfamilies (SFs) designated SF-1 to SF-4, each representing a series of homoeoallelic loci on the homoeologous chromosomes of the three genomes of common wheat. Here we studied the expression of these wheat CaM genes in the course of wheat development. Northern blot analysis using SF-specific probes revealed differences in SF expression levels in different organs and stages of development. Subsequently, cell-specific expression of CaM SFs was investigated by in situ RNA hybridization. In developing seeds, all CaM SFs showed highest expression in the embryo and less in the aleurone and in the starchy endosperm. In primary roots, all four CaM SFs were expressed in the root cap, meristematic regions and in differentiating cells. During development of the roots, expression gradually decreased. The wheat glutenin gene, which was used as a control throughout our experiments, was found to be expressed in the starchy endosperm but not in the aleurone, embryos or vegetative tissues. In stems, at advanced stages of growth, differences in cell-specific expression of CaM SFs were found. For example, SF-2 was highly expressed in differentiating phloem fibers. Thus, CaM genes in common wheat exhibit a developmentally regulated organ-, tissue-, cell- and SF-specific expression patterns.     

Roles of Calmodulin and Calcium/Calmodulin-Dependent Protein Kinase in Flagellar Motility Regulation in the Coral Acropora Digitifera

In the corals Acropora spp., eggs secrete substances that induce sperm motility regulation. An elevation of intracellular pH ([pH]i) and a regulation of intracellular Ca²⁺ concentration ([Ca²⁺]) are involved in the sperm motility regulation cascade. However, the detailed molecular aspects of flagellar motility regulation have not been fully demonstrated in Acropora. In this study, we determined the presence and roles of both calmodulin (CaM) and calcium/calmodulin dependent-protein kinase (CaMK) in the sperm flagellar motility regulation of Acropora. A ⁴⁵Ca²⁺-overlay assay and an immunoblot analysis showed that sperm contain an acidic 16-kDa protein that was CaM, and an immunoblot analysis revealed the presence of CaMK in coral sperm. In addition, a specific inhibitor of CaMK, KN-93, and a CaM antagonist, W-7, inhibited sperm motility activation induced by NH₄Cl treatment. NH₄Cl treatment causes an increase in intracellular [pH]i of sperm, suggesting that CaM and CaMK are involved in sperm motility initiation caused by an increase in [pH]i. The involvement of CaM and CaMK in motility regulation in coral highlights the importance of these molecules throughout the animal kingdom.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.     

Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Neurite Outgrowth of PC12 Cells

Abstract: To investigate the physiological role of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca²⁺/calmodulin-independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP-dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.