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First published online September 9, 2003

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Mitochondrial mRNA stability and polyadenylation during anoxia-induced quiescence in the brine shrimp Artemia franciscana

Brian D. Eads^1,2,* and Steven C. Hand^1,

¹ Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA
² Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder, CO 80303-0334 USA

View larger version (31K): [in a new window]   Fig. 1. (A-D) Low pH (pH 6.4) and anoxia in organello increase the stability of mRNA pools during 6 h incubations relative to controls (normoxic, pH 7.8). Isolated mitochondria given both treatments were incubated for the indicated times and RNA was extracted and hybridized on dot blots. Filled circles, controls; filled squares, experimental groups. Data were normalized on a nanogram basis to exogenously added HK standards, and homoskedasticity of standards and samples was assessed using Levene's test. Values are means ± S.E.M. (N=3). HK, hexokinase; CYB, cytochrome b; ND1, NADH dehydrogenase; ATP6, ATP synthase; COX1, cytochrome oxidase. (E-J) Example of a northern blot of total mitochondrial RNA (2 µg) at time 0 (E-G) and 6 h (H-J) for controls (E,H) vs. low pH (F,I) and anoxia (G,J), showing the specificity of the COXI probe against mitochondrial RNA under our experimental conditions.

View larger version (16K): [in a new window]   Fig. 2. Conditions of anoxia (filled squares), low pH (6.4; filled triangles), anoxia plus low pH (+ symbols) or anoxia plus 1.5 mmol l-1 ATP (open diamonds) in organello cause decreased transcription in A. franciscana embryos compared to controls (filled circles; normoxic, pH 6.4).Incorporation of 32P-UTP was followed by scintillation counting of TCA-precipitated RNA. Mitochondria were incubated at room temperature with 100 µg ml-1 actinomycin D to measure RNA synthesis under conditions identical to those used to assess mRNA half-life. All values are means ± S.E.M. (N=3).

View larger version (24K): [in a new window]   Fig. 3. The effects of either anoxia or low pH (pH 6.4) in organello on mitochondrial CYB (A), ND1 (B), ATP6 (C) and COX1 (D) mRNA levels are similar to their combined effects. Filled circles, control incubations (normoxic, pH 7.8); filled squares, incubations at low pH; open squares, anoxic incubations. Data were evaluated and expressed as in Fig. 1.

View larger version (21K): [in a new window]   Fig. 4. Anoxia plus 1.5 mmol l-1 ATP (filled squares) has a stabilizing effect on mitochondrial mRNA levels. Filled circles, control incubations (normoxic, pH 7.8).Data were analyzed and expressed as in Fig. 1.

View larger version (58K): [in a new window]   Fig. 5. Polyadenylation of mitochondrial mRNAs of A. franciscana embryos under control and various experimental treatments in organello. Following RACE-PAT, products were electrophoresed on 6% polyacrylamide at 100 V and stained with ethidium bromide. The fluorescence intensities for polyadenylated bands in each lane were quantified using ImageQuant software and normalized to the intensity of the upstream, non-adenylated band (uppermost band) and used in Figs 6 and 7. COXI (A), CYB (B), ND1 (C) and ATP6 (D); lane 1, control (30 min); lane 2, control (6 h); lane 3, low pH (30 min); lane 4, low pH (6 h); lane 5, low pH plus anoxia (1 h); lane 6, low pH plus anoxia (6 h); lane 7, anoxia (1 h); lane 8, anoxia (6 h); lane 9, ATP plus anoxia (0 h); lane 10, ATP plus anoxia (1 h); lane 11, ATP plus anoxia (6 h). M, markers (nucleotides). For CYB (B) and ND1 (C), lanes from two different gels are combined, and in all cases contrast has been adjusted among lanes to enhance visualization. For presentation purposes in this figure, the high intensity of the reference bands in B and D were uniformly reduced so the polyadenylated bands could be viewed at proper intensity.

View larger version (35K): [in a new window]   Fig. 6. Size-class-specific changes in amount of poly(A)+ tail (PAT) over time as a function of in organello treatment. Mitochondria were incubated under the indicated conditions, RNA extracted and PAT analysis performed. Increases represent the ratio of PAT amounts present after 6 h compared to 1 h in the size classes indicated. (A-D) The four mRNA species studied: CYB (A); COXI (B); ND1 (C); ATP6 (D). Asterisks indicate significant differences from controls. Values without error bars are data taken directly from gels shown in Fig. 5; values with error bars represent mean changes in total polyadenylation from 1-6 h for N=3 separate experiments.

View larger version (25K): [in a new window]   Fig. 7. Levels of total polyadenylation in all size classes combined are lower under all experimental treatments relative to control conditions (normoxia, pH 7.8). The amounts of fluorescence in poly(A)+ tails (PAT) from the gels in Fig. 5 were normalized and 6 h time points compared. Asterisks indicate significant differences from controls for the mean of N=3 separate experiments.