Identification of extracellular enzyme producing alkalophilic bacilli from Izmir province by 16S-ITS rDNA RFLP

WOS: 000223817600012PubMed ID: 15357726Aims: To screen industrially important extracellular enzymes from the newly isolated alkalophilic bacilli and to characterize them by phenotypic and 16S-internal transcribed spacer (ITS) rDNA restriction pattern analysis. Methods and Results: Three different environmental samples, soil, leather and horse faeces, were collected within the province of Izmir. Isolates grown on Horikoshi-I medium for 24 h at 37degreesC were screened for extracellular enzyme activity by using eight different substrates: birchwood xylan, carboxymethylcellulose, casein, citrus pectin, polygalacturonic acid, soluble starch, and Tween 20 and 80. In total, 115 extracellular enzyme-producing bacilli were obtained. Casein was hydrolysed by 78%, soluble starch by 67%, citrus pectin by 63%, polygalacturonic acid by 62%, Tween 20 by 34%, birchwood xylan by 16%, Tween 80 by 12%, and carboxymethylcellulose by 3% of the isolates. The isolates were differentiated into 19 distinct homology groups by the 16S-ITS rDNA restriction pattern analysis. Conclusions: Eight different extracellular enzyme activities were determined in 115 endospore forming bacilli. The largest 16S-ITS rDNA homology group (HT1) included 36% of the isolates, 98% of which degraded casein, polygalacturonic acid, pectin and starch. Significance and Impact of the Study: This study is the first report on the characterization of the industrial enzyme-producing alkalophilic bacilli by 16S-ITS rDNA restriction fragment length polymorphism (RFLP). Restriction profiles of 64% of the isolates were found to be different from those of five reference strains used

Visualization of newly synthesized neuronal RNA in vitro and in vivo using click-chemistry

The neuronal transcriptome changes dynamically to adapt to stimuli from the extracellular and intracellular environment. In this study, we adapted for the first time a click chemistry technique to label the newly synthesized RNA in cultured hippocampal neurons and intact larval zebrafish brain. Ethynyl uridine (EU) was incorporated into neuronal RNA in a time- and concentration-dependent manner. Newly synthesized RNA granules observed throughout the dendrites were colocalized with mRNA and rRNA markers. In zebrafish larvae, the application of EU to the swim water resulted in uptake and labeling throughout the brain. Using a GABA receptor antagonist, PTZ (pentylenetetrazol), to elevate neuronal activity, we demonstrate that newly transcribed RNA signal increased in specific regions involved in neurogenesis

Activity-dependent spatially localized miRNA maturation in neuronal dendrites

MicroRNAs (miRNAs) regulate gene expression by binding to target messenger RNAs (mRNAs) and preventing their translation. In general, the number of potential mRNA targets in a cell is much greater than the miRNA copy number, complicating high-fidelity miRNA-target interactions. We developed an inducible fluorescent probe to explore whether the maturation of a miRNA could be regulated in space and time in neurons. A precursor miRNA (pre-miRNA) probe exhibited an activity-dependent increase in fluorescence, suggesting the stimulation of miRNA maturation. Single-synapse stimulation resulted in a local maturation of miRNA that was associated with a spatially restricted reduction in the protein synthesis of a target mRNA. Thus, the spatially and temporally regulated maturation of pre-miRNAs can be used to increase the precision and robustness of miRNA-mediated translational repression

Visualization of newly synthesized neuronal RNA in vitro and in vivo using click-chemistry

The neuronal transcriptome changes dynamically to adapt to stimuli from the extracellular and intracellular environment. In this study, we adapted for the first time a click chemistry technique to label the newly synthesized RNA in cultured hippocampal neurons and intact larval zebrafish brain. Ethynyl uridine (EU) was incorporated into neuronal RNA in a time- and concentration-dependent manner. Newly synthesized RNA granules observed throughout the dendrites were colocalized with mRNA and rRNA markers. In zebrafish larvae, the application of EU to the swim water resulted in uptake and labeling throughout the brain. Using a GABA receptor antagonist, PTZ (pentylenetetrazol), to elevate neuronal activity, we demonstrate that newly transcribed RNA signal increased in specific regions involved in neurogenesis

Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity

Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function