Dataset Information

Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii.

ABSTRACT: The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with gluthatione S-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca(2+), B. emersonii CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in B. emersonii, with CaM mRNA and protein concentrations increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit B. emersonii sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca(2+) channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that the Ca(2+)-CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii.

SUBMITTER: Simao RC

PROVIDER: S-EPMC95135 | biostudies-literature | 2001 Apr

REPOSITORIES: biostudies-literature

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Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii.

Simão R C RC Gomes S L SL

Journal of bacteriology 20010401 7

The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with gluthatione S-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific ...[more]

PMID: 11244068

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Project description:To investigate global changes in gene expression during B. emersonii germination process, we constructed cDNA microarrays containing 3,563 putative unique genes. Analyses were carried out during germination induced under various environmental conditions. Microarray data revealed that 26% of them are differentially expressed during germination in nutrient medium, in at least one of the time points investigated. Over 500 genes are up regulated during the time course of germination under these conditions, most of them being related to cell growth, including genes involved in protein biosynthesis, DNA transcription, energetic metabolism, carbohydrate and oligopeptide transport, and cell cycle control. On the other hand, several transcripts stored in the zoospores are down regulated during germination in nutrient medium, such as genes involved in signal transduction, amino acid transport and chromosome organization. We also compared germination induced in the presence of nutrients with that triggered either by adenine or potassium ions in inorganic salt solution. Several genes involved in cell growth that are induced during germination in nutrient medium do not show increased expression when B. emersonii zoospores germinate in inorganic solution, indicating that nutrients exert a positive regulatory effect on the transcription of these genes. Differentially expressed genes during germination were investigated in a time-course experiment, using RNA samples isolated from cells collected 30, 60 and 90 min after inoculation of zoospores in nutrient medium DM3. The reference sample was zoospore RNA, which was considered the time zero of germination. We also compared germination in nutrient medium with germination induced in inorganic salt solution, triggered either by adenine or potassium ions. In these experiments we considered germination in nutrient medium as the reference sample. There are at least 3 biological replicates (independent harvest) and 2 technical replicates for each array (L-left and R-right).

Dataset Information

Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii.

Publications

Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii.

Similar Datasets

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