CaM Gene Research Articles

First Report of Fusarium Wilt on Philotheca myoporoides Caused by Fusarium oxysporum in Italy.

Philotheca myoporoides (DC.) M.J. Bayly (previously known as Eriostemon myoporoides), commonly called long-leaf waxflower and native to eastern Australia (Rutaceae family), is a hardy compact shrub or small tree occurring in subtropical to cool temperate regions. P. myoporoides is cultivated in Sicily (Italy) for its ornamental appeal. During April of 2010, a widespread wilting was observed on approximately 80% of 2,000 1-year-old, potted long-leaf waxflower plants grown in a commercial nursery near Catania (eastern Sicily, Italy). Internally, symptomatic plants had conspicuous vascular brown discoloration from the crown to the canopy. Diseased crown and stem tissues of 20 plants were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissues. Colonies with white or light purple aerial mycelia and violet pigmentation on the underside of the cultures developed after 9 days. On carnation leaf agar, 20 single-spore isolates produced microconidia on short monophialides, macroconidia that were three to five septate with a pedicellate base, and solitary and double-celled or aggregate chlamydospores. A PCR assay was conducted on one representative isolate (ITEM 13490) by analyzing sequences of the benA gene (coding β-tubulin protein) and CaM gene (coding calmodulin protein) using the primers reported by O'Donnell et al. (1). The benA gene sequences of ITEM 13490 (GenBank No. FR828825) exhibited an identity of 100% to Fusarium oxysporum f. sp. radicis-lycopersici strain ATCC 52429 (GenBank No. DQ092480). CaM gene sequences of ITEM 13490 (GenBank No. FR828826) exhibited an identity of 99.6% to F. oxysporum strain ITEM 2367 (GenBank No. AJ560774). Morphological characteristics of the 20 isolates, as well as the PCR assay on a representative strain, identified the isolates associated with disease symptoms as F. oxysporum Schlechtend.:Fr. A pathogenicity test was performed by placing two 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 2-month-old cuttings of P. myoporoides. Thirty plants were inoculated with strain ITEM 13490 and the same number of plants served as noninoculated controls. All plants were enclosed for 4 days in plastic bags and placed in a growth chamber at 25 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. First symptoms, which were identical to those observed in the nursery, developed on one plant 15 days after inoculation. Wilting was detected on all plants after 30 days. Control plants remained symptomless. F. oxysporum was successfully reisolated from symptomatic crown and stem tissues and identified as described above, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum causing disease of P. myoporoides worldwide. Moreover, this pathogen was recently reported in the same nursery on Eremophila sp. (2), confirming the presence of Fusarium wilt as a potential threat to ornamental plant production in this area, and necessitates the innovation and development of disinfection methods for alveolar trays, greenhouses, and various propagation materials to reduce future disease outbreaks.

First Report of Fusarium Wilt on Eremophila spp. Caused by Fusarium oxysporum in Italy.

Eremophila spp. (Myoporaceae family), endemic to Australia, are evergreen shrubs or small trees occurring in arid, semi-arid, tropical, or temperate regions. In Europe, Eremophila spp. are grown for their horticultural appeal. During 2009 and 2010, extensive wilting was observed on 2-month to 1-year-old potted plants of Eremophila laanii F. Muell., E. glabra subsp. carnosa Chinnock, and E. maculata (Ker Gawl.) F. Muell. grown in a commercial nursery near Catania (southern Italy). Internally, symptomatic plants had conspicuous vascular discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissues. Colonies with purple mycelia and violet reverse colors developed after 9 days. On carnation leaf agar, single-spore isolates produced microconidia on short monophialides, macroconidia that were three to five septate with a pedicellate base, and solitary and double-celled or aggregated chlamydospores. A PCR assay was conducted on two representative isolates (ITEM 12591 and ITEM 12592) by analyzing sequences of the partial CaM gene (coding calmodulin protein) and benA (coding beta-tubulin protein) using the primers as reported by O'Donnell et al. (1). Calmodulin sequences of ITEM 12951 and ITEM 12952 isolates (GenBank Nos. FR671157 and FR671158) exhibited 99.8 and 99.5% identity with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), respectively, and had 99.5% homology between them. BenA gene sequences of ITEM 12951 (GenBank No. FR671426) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain CC-612-3 (GenBank No. AY714092.1), and benA gene sequences of ITEM 12952 (GenBank No. FR671427) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain LA 140 (GenBank No. FJ466740.1), whereas the homology between the two strains is 99.5%. Morphological characteristics, as well as CaM and benA sequences, identified the isolates as F. oxysporum Schlechtend:Fr. Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 3-month-old cuttings of E. laanii, E. glabra subsp. carnosa, and E. maculata. Twenty plants for each species were inoculated with each isolate. The same number of plants served as noninoculated controls. All plants were enclosed for 4 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. Symptoms identical to those observed in the nursery developed 20 days after inoculation with both strains. Crown and stem discoloration was detected in all inoculated plants after 45 days. Wilting was detected on 15% of plants. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously above. To our knowledge, this is the first report of F. oxysporum causing disease of Eremophila spp. worldwide. Reference: (1) K. O'Donnell et al. Mycoscience 41:61, 2000.

Developmental expression pattern of calmodulin gene in amphioxus Branchiostoma belcheri tsingtauense

We investigated the developmental expression pattern of AmphiCaM in cephalochordate amphioxus (Branchiostoma belcheri tsingtauense). We cultured and sampled the animals at different developmental stages (eggs and larvae), and used in-situ hybridization and northern blotting to document the spatial and temporal changes in AmphiCaM expression. The alimentary tract dominates the development from the late neurula stage to the adult stage. AmphiCaM expression increased significantly in the alimentary tract during the late neurula stage and remained elevated in the adults. Our results indicate that AmphiCaM is involved in the differentiation of the alimentary tract in amphioxus; and furthermore, provide an insight into the change in function of CaM genes during evolution.

Transcriptional effect of a calmodulin inhibitor, W-7, on the ligninolytic enzyme genes in Phanerochaete chrysosporium

We investigated the effects of a calmodulin (CaM) inhibitor, W-7, on the expression of lignin peroxidase (LiP) and manganese peroxidase (MnP) genes in Phanerochaete chrysosporium to consider the role of cam gene, which was upregulated in parallel with the total activities of LiP and MnP in our previous transcriptomic analysis. The addition of 100μMW-7 to the fungal cultures repressed the total activities of LiP and MnP, whereas the addition of 100μMW-5, which is a control drug of W-7, retained approximately half of them, indicating that the effect of W-7 was attributable to CaM inhibition. Real-time reverse transcription polymerase chain reaction analysis revealed that most of lip and mnp isozyme genes predicted from whole-genome data were significantly inhibited by W-7 at the transcription level (P≤0.05). These results suggest that CaM has an important role for the expression of isozyme genes of LiP and MnP at the transcription level.

First Report of Fusarium Wilt of Paper Flower (Bougainvillea glabra) Caused by Fusarium oxysporum in Italy.

Paper flower (Bougainvillea glabra Choisy), native to Brazil, is the most widely and intensively cultivated species of bougainvillea as a potted plant in Sicily (Italy). During 2008 and 2009, a wilting of vegetatively produced B. glabra cv. Sanderiana was observed in several nurseries in eastern Sicily (Catania and Messina provinces). Disease incidence was higher (~10 to 30%) in the tree-shaped potted plants (standards). Occasionally, wilting was detected on plants that were not tree shaped. Internally, symptomatic plants showed conspicuous vascular orange discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissue. Colonies with light purple or purple mycelia and violet reverse colony colors developed after 10 days. On carnation leaf agar, single-spore isolates produced microconidia in false heads on short monophialides, macroconidia that were 3-septate with a pedicellate base, and solitary and double-celled or aggregate chlamydospores. A PCR assay was conducted on two representative strains (DISTEF-BGS1 and DISTEF-BGS2) by analyzing sequences of the parzial translation elongation factor alpha gene (TEF-1α) and CaM gene (coding calmodulin protein). The primers used are previously used by O'Donnell et al. (1,2). Calmodulin sequences of BGS1 and BGS2 strains (GenBank Nos. FN645740 and FN645741, respectively) exhibited 99% homology with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), and have homology of 99.6% between them. TEF-1 gene sequences of BGS1 (GenBank No. FN645739) exhibited an identity of 100% to F. oxysporum f. sp. lycopersici MUCL 22544 GenBank No. EF056785.1) and TEF-1α gene sequences of BGS2 (GenBank No. FN655742) exhibited an identity of 100% to F. oxysporum strain NRRL 45954 (GenBank No. FJ985431.1), whereas the homology between the two strains is 98.5%. Both PCR approaches established the identity of the isolates to the F. oxysporum Schlechtend:Fr (1,2). Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 10-day-old mycelial cultures near the crown on 40 potted, healthy, 6-month-old cuttings of paper flower. Twenty plants for each isolate were used. The same number of plants served as noninoculated controls. All plants were enclosed for 5 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 24 to 26°C. Symptoms identical to those observed in nurseries developed 1 month after inoculation with both strains. Crown and stem orange discoloration was detected in all inoculated plants after 2 months. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously described. To our knowledge, F. oxysporum was previously reported on paper flower in Ghana (3). However, this is the first demonstration of the pathogenicity of F. oxysporum on paper flower and it is the first report in Europe of the disease. The presence of Fusarium wilt in Sicily is a potential threat to paper flower production in nurseries.

Molecular Cloning and Comparative In silico Analysis of Calmodulin Genes from Cereals and Millets for Understanding the Mechanism of Differential Calcium Accumulation

Calmodulin gene (CaM) - a calcium sensor was cloned by the technique of polymerase chain reaction (PCR) from cereals and millets using single set of primer designed from Eleusine coracana ESTs available in dbEST database. The PCR amplicons of 613bp were consistently observed in all cereals and millets except Setaria italica. The amplicons from these cereals were further gel eluted, cloned, sequenced and subjected to homology search, multiple sequence alignment, phylogenetic tree construction and motif analysis. The homology search con fi rmed their identity to CaM genes except for Triticum aestivum. Multiple sequence alignment of the translated CaM amino acid sequences con fi rmed the presence of conserved consensus sequences of 110 amino acids, which was uniformly observed across different cereals and millets except Sorghum bicolor. Phylogenetic tree constructed based on protein sequences of cloned CaM genes revealed closed evolutionary relationship between three varieties of Eleusine coracana (PRM-1, PRM-701 and PRM-801) and Hordeum vulgare as compared to other cereals and millets which also possess higher concentration of calcium in seeds. Thus indicates that structural variation in CaM has some role in the differential accumulation of calcium. Three EF-hands associated with a CaM gene were present consistently except Sorghum bicolor, having only two EF-hands. The in silico 3D-structural analysis of cloned sequences showed similar pattern and reveals high degree of conservation in CaM in terms of structure and interaction with calcium ions, thus re fl ecting to further investigate the role of CaM isoforms, their expression patterns and downstream interaction with transport machinery involved in calcium accumulation.

Cloning and analysis of calmodulin gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.

Structure and expression analysis of the OsCam1-1 calmodulin gene from Oryza sativa L.

Calmodulin (CaM) proteins, members of the EF-hand family of Ca(2+)- binding proteins, represent important relays in plant calcium signals. Here, OsCam1-1 was isolated by PCR amplification from the rice genome. The gene contains an ORF of 450 base pairs with a single intron at the same position found in other plant Cam genes. A promoter region with a TATA box at position-26 was predicted and fused to a gus reporter gene, and this construct was used to produce transgenic rice by Agrobacterium-mediated transformation. GUS activity was observed in all organs examined and throughout tissues in cross-sections, but activity was strongest in the vascular bundles of leaves and the vascular cylinders of roots. To examine the properties of OsCaM1-1, the encoding cDNA was expressed in Escherichia coli. The electrophoretic mobility shift when incubated with Ca(2+) indicates that recombinant OsCaM1-1 is a functional Ca(2+)-binding protein. In addition, OsCaM1-1 bound the CaMKII target peptide confirming its likely functionality as a calmodulin.

OKCAM: an ontology-based, human-centered knowledgebase for cell adhesion molecules

‘Cell adhesion molecules’ (CAMs) are essential elements of cell/cell communication that are important for proper development and plasticity of a variety of organs and tissues. In the brain, appropriate assembly and tuning of neuronal connections is likely to require appropriate function of many cell adhesion processes. Genetic studies have linked and/or associated CAM variants with psychiatric, neurologic, neoplastic, immunologic and developmental phenotypes. However, despite increasing recognition of their functional and pathological significance, no systematic study has enumerated CAMs or documented their global features. We now report compilation of 496 human CAM genes in six gene families based on manual curation of protein domain structures, Gene Ontology annotations, and 1487 NCBI Entrez annotations. We map these genes onto a cell adhesion molecule ontology that contains 850 terms, up to seven levels of depth and provides a hierarchical description of these molecules and their functions. We develop OKCAM, a CAM knowledgebase that provides ready access to these data and ontologic system at http://okcam.cbi.pku.edu.cn. We identify global CAM properties that include: (i) functional enrichment, (ii) over-represented regulation modes and expression patterns and (iii) relationships to human Mendelian and complex diseases, and discuss the strengths and limitations of these data.

Cloning of artemisinin biosynthetic cDNAs and novel ESTs and quantification of low temperature-induced gene overexpression

To isolate and verify novel genes from qinghao (Artemisia annua) based on the development-specific and environment-induced transcriptomics, leaves have been harvested from the flowering A. annua plants and exposed to low temperature for isolation of total RNAs and cloning of full-length cDNAs and cDNA fragments, or expressed sequence tags (ESTs). After being sequenced and browsed for homology, these sequences have been submitted to GenBank. Among the accessed 75 sequences, 4 full-length cDNAs are highly homologous to the known A. annua genes, but 71 ESTs are absent in the sequence records of A. annua genes, in which 34 sequences are homologous to other plant genes, including 24 identified protein-coding sequences and 10 unidentified protein-coding sequences, while other 37 sequences are not present in the sequence records of any plant genes, representing the first cloned plant genes. In order to investigate the responsive patterns of A. annua genes to extreme environmental stresses, especially low temperature, the expression levels of 3 critical qinhaosu (artemisinin) biosynthetic genes, ADS, CYP71AV1 and CPR, have been measured in pre-and post-chilling A. annua seedlings cultured in vitro by semi-quantitative PCR (SQ-PCR). Consequently, ADS and CYP71AV1 genes are strongly induced by chilling, but CPR gene is not significantly affected by such treatment. Furthermore, induction of these genes by chilling can be potently suppressed by Ca2+ channel inhibitor LaCl3 or Ca2+ chelator EGTA, suggesting a putative involvement of Ca2+-CaM signal transduction pathway in chilling-induced overexpression of ADS and CYP71AV1 genes. The real-time fluorescent quantitative PCR (RFQ-PCR) assay of A. annua seedlings exposed to chilling has shown that the expression level of CaM gene is up-regulated for more than 2.5 folds, thereby confirming our above inference on the relevance of Ca2+-CaM-mediated signal transduction to chilling-induced gene overexpression. Finally, 7 newly isolated A. annua ESTs have been functionally annotated by RFQ-PCR, which indicates that the chilling stress-induced overexpression levels of D/LTSRP, UBE, AR/DAP and POD1 genes are augmented approximately for 8, 5, 2.3, and 1.5 folds, respectively, but those of CHI and RGP genes are not predominantly up-or down-regulated. The present study has preliminarily explored the chilling-induced overexpression patterns of 3 artemisinin biosynthetic genes and 7 novel A. annua ESTs at the transcriptional level, which should further facilitate our understanding of the intrinsic rule and mechanism underlying the coordinative regulation manner of artemisinin biosynthesis and accumulation, and yield substantial insight into improvement of A. annua by the metabolic engineering-guided breeding strategy.

Topoisomerase Action on Short DNA Duplexes Reveals Requirements for Gate and Transfer DNA Segments

Type II topoisomerases change DNA topology by passage of one DNA duplex (the transfer, T-segment) through a transient double-stranded break in another (the gate, G-segment). Here we monitor the passage between short double-stranded DNA segments within long single-stranded DNA circles that leads to catenation of the circles. To facilitate catenation, the circles were brought into close proximity using a tethering oligonucleotide, which was removed after the reaction was complete. We varied the length and the composition of the reacting DNA segments. The minimal DNA duplex length at which we detected catenation was 50-60 bp for DNA gyrase and 40 bp for topoisomerase IV (Topo IV). For Topo IV, catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex. Topo IV cleaved the DNA-DNA duplex, but not the DNA-RNA duplex implying that the DNA-RNA duplex can be a T-segment but not a G-segment.

Ca2+ and AtCaM3 are involved in the expression of heat shock protein gene in Arabidopsis

ABSTRACTThe involvement of calcium and different calmodulin isoforms (Ca2+‐CaM) in heat shock (HS) signal transduction in Arabidopsis (Arabidopsis thaliana) was investigated. Using transgenic Arabidopsis plants which have the AtHsp18.2 promoter/GUS fusion gene, it was found that the level of β‐glucuronidase (GUS) activity was up‐regulated by the addition of CaCl2 and down‐regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl3 and verapamil, or the CaM antagonists N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W7), chlorpromazine (CPZ) and trifluoperazine (TFP). CaCl2 not only increased the GUS activity after HS, but also up‐regulated the GUS activity under non‐HS conditions. These results provide additional support for the involvement of the Ca2+‐CaM signalling system in HSP gene expression. The expression of nine CaM genes (AtCaM1–9) from Arabidopsis was differentially regulated by HS at 37 °C. The expression of AtCaM3 and AtCaM7 genes increased during HS. The temporal expression of the AtCaM3, AtCaM7 and hsp18.2 genes demonstrated that up‐regulation of AtCaM3 expression occurred earlier than that of AtCaM7 or hsp18.2.

Modulation of Calmodulin Gene Expression as a Novel Mechanism for Growth Hormone Feedback Control by Insulin-like Growth Factor in Grass Carp Pituitary Cells

Calmodulin (CaM), the Ca2+ sensor in living cells, is essential for biological functions mediated by Ca2+-dependent mechanisms. However, modulation of CaM gene expression at the pituitary level as a means to regulate pituitary hormone synthesis has not been characterized. In this study we examined the functional role of CaM in the feedback control of GH by IGF using grass carp pituitary cells as a cell model. To establish the structural identity of CaM expressed in the grass carp, a CaM cDNA, CaM-L, was isolated from the carp pituitary using 3'/5' rapid amplification of cDNA ends. The open reading frame of this cDNA encodes a 149-amino acid protein sharing the same primary structure with CaMs reported in mammals, birds, and amphibians. This CaM cDNA is phylogenetically related to the CaM I gene family, and its transcripts are ubiquitously expressed in the grass carp. In carp pituitary cells, IGF-I and IGF-II induced CaM mRNA expression with a concurrent drop in GH transcript levels. These stimulatory effects on CaM mRNA levels were not mimicked by insulin and appeared to be a direct consequence of IGF activation of CaM gene transcription without altering CaM transcript stability. CaM antagonism and inactivation of calcineurin blocked the inhibitory effects of IGF-I and IGF-II on GH gene expression, and CaM overexpression also suppressed the 5' promoter activity of the grass carp GH gene. These results, as a whole, provide evidence for the first time that IGF feedback on GH gene expression is mediated by activation of CaM gene expression at the pituitary level.

Characterization of adrenomedullin in birds

Adrenomedullin (AM) is a multifunctional evolutionarily highly conserved peptide. Although its genomic and amino acid (aa) sequences are known in several mammalian species and in fish, the structure of the AM gene remains unknown in intermediate phyla, including birds. Here, we report the structure and aa sequence of the chicken (c) AM ortholog. The cAM gene is located at the short arm of chromosome 5, which shows high synteny with the short arm of human (h) chromosome 11, where hAM is located. Key sequences in the third intron have been conserved which allow for an alternative splicing mechanism, similar to the one found in mammals. The preprohormone contains two peptides with high homology to human proadrenomedullin N-terminal 20 peptide (PAMP) and hAM. We found through real-time PCR and immunocytochemistry cAM mRNA and peptide expression in a variety of chicken tissues, which parallel patterns observed for mammals, with the exception that cAM levels are almost non-detectable in brain. Similarly to mammals, cAM expression is upregulated under hypoxic conditions and following dexamethasone treatment. These data demonstrate a high degree of homology between the cAM gene and its mammalian ortholog and evolutionary conservation of the regulatory mechanisms controlling its expression.

Molecular cloning and characterization of calmodulin genes in young oak seedlings ( Quercus petraea L.) during early flooding stress

As part of an integrated study on the molecular response of woody plants to flooding, three CaM genes were isolated from oak seedlings ( Quercus petraea Liebl.) and characterized. QpCaM-1 was almost exclusively expressed in roots, whereas QpCaM-2 and -3 were more evenly distributed throughout the plant. The present paper documents the differential expression of these genes during hypoxia.

Goldfish calmodulin: molecular cloning, tissue distribution, and regulation of transcript expression in goldfish pituitary cells.

Calmodulin (CaM) is a Ca(2+)-binding protein essential for biological functions mediated through Ca(2+)-dependent mechanisms. In the goldfish, CaM is involved in the signaling events mediating pituitary hormone secretion induced by hypothalamic factors. However, the structural identity of goldfish CaM has not been established, and the neuroendocrine mechanisms regulating CaM gene expression at the pituitary level are still unknown. Here we cloned the goldfish CaM and tested the hypothesis that pituitary expression of CaM transcripts can be the target of modulation by hypothalamic factors. Three goldfish CaM cDNAs, namely CaM-a, CaM-bS, and CaM-bL, were isolated by library screening. These cDNAs carry a 450-bp open reading frame encoding the same 149-amino acid CaM protein, the amino acid sequence of which is identical with that of mammals, birds, and amphibians and is highly homologous (>/=90%) to that in invertebrates. In goldfish pituitary cells, activation of cAMP- or PKC-dependent pathways increased CaM mRNA levels, whereas the opposite was true for induction of Ca(2+) entry. Basal levels of CaM mRNA was accentuated by GnRH and pituitary adenylate cyclase-activating polypeptide but suppressed by dopaminergic stimulation. Pharmacological studies using D1 and D2 analogs revealed that dopaminergic inhibition of CaM mRNA expression was mediated through pituitary D2 receptors. At the pituitary level, D2 activation was also effective in blocking GnRH- and pituitary adenylate cyclase-activating polypeptide-stimulated CaM mRNA expression. As a whole, the present study has confirmed that the molecular structure of CaM is highly conserved, and its mRNA expression at the pituitary level can be regulated by interactions among hypothalamic factors.

Detection of L1 CAM mutation in a male child with mental retardation

Recent studies have presented evidence for the involvement of L1CAM gene mutations in various X-linked mental retardation syndromes. The neural cell adhesion molecule, L1CAM is a transmembrane protein belonging to the super family of the immunoglobulins that play a key role in embryonic development of the nervous system and is involved in memory and learning. No studies were carried out from India on L1 CAM gene in X-linked mental retardation syndromes. Hence, an investigation was taken up to delineate the role of L1CAM gene in mental retardation.Two families (Family I and Family II) having only two members affected with mental retardation in each family were studied for mutations in L1CAM gene. In family II, the younger sibling showed deletion involving region between the nucleotide 13,773 (intron 25) and 14,158 (intron 27) region. The mutation what we observed in younger sibling of the family II is a novel mutation which was not hitherto reported in the world literature.

Biochemistry and the Sciences of Recognition

Biochemistry and the Sciences of Recognition

Intracellular targeting of calmodulin mRNAs in primary hippocampal cells.

We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling.

Expression of different calmodulin genes in bean (Phaseolus vulgaris L.): role of nod factor on calmodulin gene regulation.

Three calmodulin (PvCaM-1, PvCaM-2, and PvCaM-3) clones were isolated from a Phaseolus vulgaris nodule cDNA library. All clones contain the complete coding region and are 62 to 74% homologous within this region. Compared to plant CaM consensus sequences, PvCaM-2 has a novel tyrosine118 residue, representing a putative phosphorylation site. Southern analysis suggested that calmodulin is encoded by a gene family. These three CaM clones are expressed mainly in young tissues and meristems. The expression pattern of PvCaM-2 and PvCaM-3 is almost identical but different from that of PvCaM-1, suggesting that PvCaM-1 is a well-defined CaM gene, whereas PvCaM-2 and PvCaM-3 could be alleles. PvCaM clones are expressed early in nodules, and transcript levels increase from nodule primordia to nodule-like structures induced by the Nod factor. Conversely, in roots, Nod factor lowers mRNA levels of all three PvCaM clones, but especially of PvCaM-1. Inhibition of PvCaM-1 expression also is observed when 2,3,5-triiodobenzoic acid is added and is prevented when roots are treated with indole-3-acetic acid, suggesting that PvCaM-1 regulation is related to the Nod factor inhibition of polar auxin transport. These results could suggest that CaM clones do not participate in the early signaling generated by the Nod factor but do participate in early events of nodule formation.