PET112, a Saccharomyces cerevisiae nuclear gene required to maintain rho+ mitochondrial DNA.

The nuclear gene PET112 was originally identified by a mutation (pet112-1) that specifically blocked accumulation of cytochrome c oxidase subunit II. The mutation causes a post-transcriptional defect since the level of COX2 mRNA in the mutant is the same as in the wild-type. However, PET112 does not have a function similar to that of PET111, a COX2 mRNA-specific translational activator: while pet111 mutations are suppressed by chimeric COX2 mRNAs bearing 5' leaders of other mitochondrial mRNAs, pet112-1 is not. The PET112 gene was isolated and shown to code a protein of 541 residues (62 kDa) with no significant homology to known amino-acid sequences. By hybridization to defined genomic clones the gene was mapped to chromosome II between cdc25 and ils1. Disruption of the PET112 open reading frame destabilized the mitochondrial genome, causing cells to become rho-. This finding suggests that PET112 has an important general function in mitochondrial gene expression, probably in translation.

Differential growth of neonatal WKY and SHR ventricular myocytes within sympathetic co-cultures.

Sympathetic innervation is known to increase heart size in the immature animal, yet the mechanism for this growth remains to be established. This comparative study stereologically quantified the volume of cultured neonatal ventricular myocytes with and without in vitro sympathetic innervation to isolate the mechanisms regulating cardiac growth. Since ventricular myocyte size at birth differs between the spontaneously hypertensive rat (SHR) and the normotensive Wistar-Kyoto (WKY), we questioned whether SHR myocytes respond differently than WKY myocytes to innervation. Four groups of ventricular myocytes from each strain were compared: myocytes grown alone, myocytes innervated by cultured sympathetic neurons, innervated myocytes exposed to adrenoceptor blockade, and non-innervated myocytes in co-culture dishes. Volumes for the myocyte, nucleus, cytoplasm, mitochondria, sarcomeres and other cellular organelles were assessed within each population and between populations. Relative volumes were determined for the mitochondria, sarcomeres, and other cellular components within the cytoplasm. Innervated WKY myocytes were 38% larger than control myocytes (P < 0.0004). This growth was not blocked by adrenoceptor blockade (P = 0.89 vs. innervated) and was present in the non-innervated myocytes distant from the neurons in the co-cultures (P = 0.39 vs. innervated). SHR myocytes were 36% larger than WKY myocytes (P < 0.009) but did not increase with innervation (P = 0.48). SHR myocyte size was also unaffected by adrenoceptor blockade (P = 0.39) or presence of the neurons in the culture dish (P = 0.53). Neonatal WKY ventricular myocyte growth can be provoked in vitro by sympathetic innervation via regulatory mechanisms independent of neuroeffector transmission or anatomic contact, whereas volume of neonatal SHR myocytes is unaltered by sympathetic coculture. These findings are significant for understanding normal as well as aberrant cardiomyocyte growth.

Application of stereological analysis of cell volume to isolated myocytes in culture with and without adrenergic innervation.

A three-dimensional analysis to evaluate structural changes in cultured cardiac myocytes following adrenergic innervation was performed using stereological techniques formerly limited to cells in tissue and organs. Cell volumes were calculated for two groups of cells at 96 hours in culture: isolated myocytes and myocytes innervated with adrenergic neurons. Relative and absolute volumes of the nucleus, cytoplasm, and cell were quantified by systematically sampling sections throughout the cell and by point count sampling techniques. Volumetric estimates were similarly determined for the mitochondria, sarcomeres, and other cellular components in the cytoplasm. Data were analyzed with ANOVA and randomized block design to control for variation among the cultures. Adrenergic innervation produced a 44% increase in cell volume, X +/- SEM, (3,344 +/- 196 microns3 to 4,816 +/- 400 microns3, P = 0.007). The absolute volume of mitochondria significantly increased after innervation (521 +/- 42 microns3 to 744 +/- 54 microns3, P less than 0.01). Absolute sarcomere volume did not change significantly (750 +/- 92 microns3 to 642 +/- 1061 microns3, P = 0.14). Other cellular components, defined as all cytoplasmic components except mitochondria and sarcomeres, significantly increased with innervation (1,739 +/- 166 microns3 to 3,097 +/- 338 microns3, P = 0.02). The relative volume of the nucleus and the cytoplasm in the cell remained unchanged following innervation. However, the relative volume of mitochondria decreased by 6%, the percent of the cytoplasm occupied by the sarcomeres decreased by 44%, and the volume occupied by the other cellular components increased by 22%. These findings support the use of stereological analysis as a means to quantify cell volumes of cultured myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Translational regulation of mitochondrial gene expression by nuclear genes of Saccharomyces cerevisiae.

We describe several yeast nuclear mutations that specifically block expression of the mitochondrial genes encoding cytochrome c oxidase subunits II (COXII) and III (COXIII). These recessive mutations define positive regulators of mitochondrial gene expression that act at the level of translation. Mutations in the nuclear gene PET111 completely block accumulation of COXII, but the COXII mRNA is present in mutant cells at a level approximately one-third of that of the wild type. Mitochondrial suppressors of pet111 mutations correspond to deletions in mtDNA that result in fusions between the coxII structural gene and other mitochondrial genes. The chimeric mRNAs encoded by these fusions are translated in pet111 mutants; this translation leads to accumulation of functional COXII. The PET111 protein probably acts directly on coxII translation, because it is located in mitochondria. Translation of the mitochondrially coded mRNA for COXIII requires the action of at least three nuclear genes, PET494, PET54 and a newly discovered gene, provisionally termed PET55. Both the PET494 and PET54 proteins are located in mitochondria and therefore probably act directly on the mitochondrial translation system. Mutations in all three genes are suppressed in strains that contain chimeric coxIII mRNAs with the 5'-untranslated leaders of other mitochondrial transcripts fused to the coxIII coding sequence. The products of all three nuclear genes may form a complex and carry out a single function.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolated sinoatrial node cell in primary culture from the newborn rat.

We prepared primary cell cultures of the sinus node region from newborn rat hearts. Sinoatrial node cells were easily distinguished from the other cardiac muscle cells and nonmuscle cells in culture by size, configuration, and rapid, attenuated spontaneous contractions (185.0 +/- 8/min, mean +/- SEM). The spontaneously contracting sinoatrial node cells were extremely sensitive to acetylcholine and norepinephrine, responding to concentrations at least 1000-fold less than other cardiac muscle cells. These same sinoatrial node cells in culture were fixed and precisely relocated by either subsequent scanning or transmission electron microscopy. The ultrastructural features of these sinoatrial node cells in culture were similar to those observed in the cells of intact sinus node sections from the source hearts. This study is the first to present single, spontaneously active, neonatal sinoatrial node cells maintained in vitro with morphological and functional properties desirable for physiological investigations.