A novel RNA species from the turtle mitochondrial genome: induction and regulation of transcription and processing under anoxic and freezing stresses

The present study identifies a previously cloned cDNA, pBTaR914, as homologous to the mitochondrial WANCY (tryptophan, alanine, asparagine, cysteine, and tyrosine) tRNA gene cluster. This cDNA clone has a 304-bp sequence and its homologue, pBTaR09, has a 158-bp sequence with a long poly(A)+ tail (more than 60 adenosines). RNA blotting analysis using pBTaR914 probe against the total RNA from the tissues of adult and hatchling turtles revealed five bands: 540, 1800, 2200, 3200, and 3900 nucleotides (nt). The 540-nt transcript is considered to be an intact mtRNA unit from a novel mtDNA gene designated WANCYHP that overlaps the WANCY tRNA gene cluster region. This transcript was highly induced by both anoxic and freezing stresses in turtle heart. The other transcripts are considered to be the processed intermediates of mtRNA transcripts with WANCYHP sequence. All these transcripts were differentially regulated by anoxia and freezing in different organs. The data suggest that mtRNA processing is sensitive to regulation by external stresses, oxygen deprivation, and freezing. Furthermore, the fact that the WANCYHP transcript is highly induced during anoxic exposure suggests that it may play an important role in the regulation of mitochondrial activities to coordinate the physiological adaptation to anoxia.Key words: mitochondria, RNA processing, anoxia, freezing, Trachemys scripta.

Decreased total ventricular and mitochondrial protein synthesis during extended anoxia in turtle heart

The turtle heart provides a model system to study the effects of anoxia on protein synthesis without the potentially confounding factor of contractile failure and decreased ATP levels. Protein synthesis, as measured by 3H-labeled phenylalanine incorporation, was studied under conditions of normoxia and anoxia in isolated perfused turtle [Trachemys (= Pseudemys) scripta elegans] hearts at 15 degrees C. Heart rate, cardiac output, and ventricular pressure development were unaffected by 2 or 3 h of anoxia. Despite the anoxia, energy levels in the heart were presumably still high, since contractility was maintained. RNA content of ventricle decreased after anoxic perfusion. Rates of total protein synthesis rates in ventricle were threefold lower under anoxia than under normoxia. These findings suggest that the total level of RNA is one determinant of protein synthesis. Incorporation of label into protein extracted from mitochondria was also assessed. The ratio of mitochondrial to whole ventricular protein synthesis was significantly lower after anoxia, revealing preferential control mechanisms under anoxia between the synthesis of total cellular protein and protein destined for mitochondria. Isolated mitochondria were still coupled after 2 or 3 h of anoxia. In effect, the mitochondria enter into a state of hypometabolism in terms of rates of ATP synthesis and protein synthesis, but functional integrity is maintained. The decrease in protein synthesis in general and mitochondrial protein synthesis in particular may represent an adaptation to allow the partitioning of the available energy resources toward mechanical function during anoxia.

Analysis of cardiac shunting in the turtle Trachemys (Pseudemys) scripta: application of the three outflow vessel model.

Blood distribution within the ventricle was analysed in acutely prepared turtles Trachemys scripta by measuring the oxygen concentration and flow rates of blood in the central vessels. Pulmonary (Qp) and systemic (Qs) blood flow rates were similar when total cardiac output (Qtot) was below 40 ml min-1 kg-1. Above this value, increments of Qtot were directed to the pulmonary circuit, with Qs levelling off at approximately 20 ml min-1 kg-1. When Qtot was larger than 40 ml min-1 kg-1, the systemic circuit was almost exclusively perfused by left atrial blood and systemic venous return was almost all directed towards the lungs. Blood oxygen levels and flow rates were consistently higher in the right aorta than in the left aorta. Blood movement within the ventricle, coupled with differences in ejection timing, is probably the decisive factor determining this pattern of blood distribution in the turtle heart.