Ribozymes were first discovered in viral-like RNA pathogens of plants (hammerhead and hairpin) or of humans (HDV), and sporadically in metazoan genomes. They catalyze site-specific cleavage of RNA, and in some cases, the reverse ligation reaction via a concerted general acid–base mechanism. Nucleolytic ribozymes (hereafter referred to as ribozymes for short) are small RNAs (less than 200 nt) that function independently of proteins, of which nine classes are known to exist (hammerhead, hairpin, Varkud satellite, hepatitis delta virus (HDV), glmS, twister, twister-sister, pistol and hatchet) each with distinctive folding patterns. Distinct classes of RNA catalyse biochemical reactions (ribozymes), although the large subunit rRNA and RNaseP ribozymes common to all organisms require protein partners to function. The molecular functions of ribonucleic acid (RNA) are diverse and essential in all organisms. Expected size of RNaseJ1-6xHis protein is 63 kDa. Lane M indicates Kaledioscope™ prestained protein ladder (Bio-Rad). Lane 1 and 4 indicates protein lysates from pEGFP-hDHFR_iRJ1, lane 2 and 5 indicates protein lysates from pRzI_iRJ1, lane 3 and 6 indicates protein lysates from pRzII_iRJ1, lane 7, 10, 13 and 18 indicates protein lysates from iEGFP-hDHFR_iRJ1, lane 8, 11, 16 and 21 indicates protein lysates from iRzI_iRJ1, lane 9, 12, 17 and 22 indicates protein lysates from iRzII_iRJ1, lane 14 and 19 indicates protein lysates from i glmS_iRJ1, lane 15 and 20 indicates protein lysates from iM9_iRJ1, respectively. (A and C) Total protein-stained membrane (B and D) Immunodetection of RNaseJ1-6xHis using Anti-6X His IgG, CF™680 (Sigma-Aldrich, Merck KGaA, Germany). Western blot analysis of RNaseJ1-6xHis protein in single and double integrants All protein samples were extracted from integrants cultured in the absence (lane 1–3, 7–9, and 13–17) or presence of 0.0012% (w/v) arabinose (lane 4–6, 10–12, and 18–22) harvested after 8 h induction time. P-values from single value two-tailed t-tests comparing group mean to 1 of EGFP-hDHFR_33, RzI_33, RzII_33, glmS_33, and M9_33 are 0.8902, 0.9995, 0.9644, 0.8562, and 0.8483 respectively.Īdditional file 5: Figure S5. Dots indicate relative fluorescence data from individual experiments, and median values are indicated by the bold black line. Box plots show relative fluorescence data distribution. (B) Fluorescence intensity of i33 double integrants cultured in 0.0012% (w/v) arabinose relative to untreated control. Points represent the mean of 4–12 experiments and error bars represent 95% confidence intervals. Data are shown for each cell type grown in the presence or absence of 0.0012% (w/v) arabinose. OD 600 was measured every hour from 0 to 8 h cultivation time and data were plotted using the Growthcurve package in R software. (A) Growth analysis of double integrants ΔarsB::EGFP-hDHFR ΔlacZ::33 (iEGFP-hDHFR_i33), ΔarsB::RzIEGFP-hDHFR ΔlacZ::33 (iRzI_i33), ΔarsB::RzIIEGFP-hDHFR ΔlacZ::33 (iRzII_i33), ΔarsB:: glmSEGFP-hDHFR ΔlacZ::33 (i glmS_i33), and ΔarsB::M9EGFP-hDHFR ΔlacZ::33 (iM9_i33). Growth analysis and fluorescence intensity of iBAD33 double integrants. The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Īdditional file 4: Figure S4. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
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