Journal: Molecular and Cellular Biology

Article Title: UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

doi:

Figure Lengend Snippet: Degradation kinetics of mitochondrial RNAs labeled with [α- 32 P]CTP. Mitochondrial vesicles were labeled with [α- 32 P]CTP for 8.5 min and chased for 65 min in transcription buffer containing 2 mM unlabeled CTP (no UTP) or in transcription buffer containing 2 mM unlabeled CTP plus 2 mM unlabeled UTP (+ UTP). At the times indicated, incorporation of [α- 32 P]CTP into total RNA (total) and poly(A) + RNA (A+) was measured.

Article Snippet: [α-32 P]UTP- and [α-32 P]CTP-labeled total and poly(A)+ RNA was spotted onto DE81 paper (Whatman), washed three times for 2 min at room temperature with 0.5 M Na2 HPO4 , and rinsed with water and then 95% ethanol.

Techniques: Labeling

Journal: Molecular and Cellular Biology

Article Title: UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

doi:

Figure Lengend Snippet: UTP-independent and UTP-dependent degradation of RPS12 RNA. (A) Mitochondrial vesicles were labeled with [α- 32 P]CTP for 8.5 min (column 1) and chased for 65 min in buffer containing 2 mM CTP (column 2) or buffer containing 2 mM CTP plus 2 mM UTP (column 3). Labeled mitochondrial RNA was isolated and separated into poly(A) − and poly(A) + fractions by oligo(dT) chromatography. Labeled RNAs were used to probe filters containing a control pBS plasmid, a plasmid containing unedited RPS12 DNA (pRPS12-U), and a plasmid containing fully edited RPS12 DNA (pRPS12-E). Labeled poly(A) − RNA (A−) was used to probe the top filters, and labeled poly(A) + RNA (A+) was used to probe the bottom filters. Hybridization signals were detected by autoradiography. (B) The percentage of unedited RPS12 RNA remaining under different chase conditions was determined by densitometry and displayed in the bar graph. The amount of RNA in each pulse sample was designated 100% RNA remaining.

Article Snippet: [α-32 P]UTP- and [α-32 P]CTP-labeled total and poly(A)+ RNA was spotted onto DE81 paper (Whatman), washed three times for 2 min at room temperature with 0.5 M Na2 HPO4 , and rinsed with water and then 95% ethanol.

Techniques: Labeling, Isolation, Chromatography, Plasmid Preparation, Hybridization, Autoradiography

Journal: Molecular and Cellular Biology

Article Title: UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

doi:

Figure Lengend Snippet: Kinetics of [α- 32 P]UTP and [α- 32 P]CTP incorporation into mitochondrial RNA. Mitochondrial vesicles were labeled with [α- 32 P]UTP (U) or [α- 32 P]CTP (C) for the indicated times. Total RNA (total) and poly(A) + RNA (A+) were isolated, and incorporation of radioisotope was measured by scintillation counting. The maximal amount of labeled nucleotide incorporation observed for each sample was designated 100% incorporation.

Article Snippet: [α-32 P]UTP- and [α-32 P]CTP-labeled total and poly(A)+ RNA was spotted onto DE81 paper (Whatman), washed three times for 2 min at room temperature with 0.5 M Na2 HPO4 , and rinsed with water and then 95% ethanol.

Techniques: Labeling, Isolation

Journal: Molecular and Cellular Biology

Article Title: UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

doi:

Figure Lengend Snippet: Gel electrophoresis analysis of mitochondrial RNAs synthesized in organello. Mitochondrial vesicles were labeled with [α- 32 P]UTP or [α- 32 P]CTP for 25 min. Total RNA (T), poly(A) − RNA (A−), and poly(A) + ) are also labeled. Guide RNAs were labeled weakly by [α- 32 P]UTP and are not indicated. RNA size standards are indicated on the left (nt, nucleotides).

Article Snippet: [α-32 P]UTP- and [α-32 P]CTP-labeled total and poly(A)+ RNA was spotted onto DE81 paper (Whatman), washed three times for 2 min at room temperature with 0.5 M Na2 HPO4 , and rinsed with water and then 95% ethanol.

Techniques: Nucleic Acid Electrophoresis, Synthesized, Labeling

Journal: Molecular and Cellular Biology

Article Title: UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

doi:

Figure Lengend Snippet: Degradation kinetics of mitochondrial RNAs labeled with [α- 32 P]UTP. Mitochondrial vesicles were labeled with [α- 32 P]UTP for 25 min and chased for 305 min in transcription buffer containing 2 mM unlabeled UTP. At the times indicated, incorporation of [α- 32 P]UTP into total RNA (total), poly(A) + RNA (A+), 9S rRNA (9S), and 12S rRNA (12S) was measured.

Article Snippet: [α-32 P]UTP- and [α-32 P]CTP-labeled total and poly(A)+ RNA was spotted onto DE81 paper (Whatman), washed three times for 2 min at room temperature with 0.5 M Na2 HPO4 , and rinsed with water and then 95% ethanol.

Techniques: Labeling

Journal: Molecular and Cellular Biology

Article Title: UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

doi:

Figure Lengend Snippet: Effects of transcriptional inhibitors on RNA synthesis and UTP-dependent turnover of mitochondrial polyadenylated RNAs. To measure the effects of transcriptional inhibitors on in organello poly(A) + RNA synthesis (trans.), mitochondrial vesicles were labeled with [α- 32 P]CTP for 10 min in the absence or presence of actinomycin D (50 μg/ml) or ethidium bromide (20 μg/ml). The inhibitors were added 10 min prior to the addition of nucleoside triphosphates. Incorporation of [α- 32 P]CTP into poly(A) + RNA was determined. To measure the effect of transcriptional inhibitors on UTP-dependent poly(A) + RNA turnover (turnover), mitochondrial vesicles were labeled with [α- 32 P]CTP for 10 min and chased for 65 min in transcription buffer containing 2 mM CTP and 0.1 mM UTP. Actinomycin D (50 μg/ml) or ethidium bromide (20 μg/ml) was added directly to the chase reaction mixtures. Incorporation of [α- 32 P]CTP into poly(A) + RNA was determined. To calculate UTP-dependent turnover, the amount of poly(A) + RNA degradation observed in the absence of UTP was subtracted from the amount observed in the presence of UTP. The amount of transcription or UTP-dependent RNA turnover observed in the absence of drug treatment was defined as 100% activity. Error bars represent 1 standard deviation.

Article Snippet: [α-32 P]UTP- and [α-32 P]CTP-labeled total and poly(A)+ RNA was spotted onto DE81 paper (Whatman), washed three times for 2 min at room temperature with 0.5 M Na2 HPO4 , and rinsed with water and then 95% ethanol.

Techniques: Labeling, Activity Assay, Standard Deviation