Regulation of mitochondrial cAMP-dependent protein kinase activity in yeast.

We have shown that transcription of the yeast (S. cerevisiae) mitochondrial (mt) genome is cAMP-sensitive, via a mt cAMP-dependent protein kinase (cAPK). In relation to that work, we examined whether the BCY 1 gene product functions as regulatory subunit for mt cAPK, as it does for the cytoplasmic enzyme. We demonstrate that mt protein extracts from a bcy 1 strain show no cAPK activity, whereas similar extracts from an otherwise isochromosomal BCY 1 strain show high levels of such activity. Partial purification of mt cAPK from each strain confirms this difference. Photoaffinity labeling with 8-N3[32P]cAMP and highly-purified mt protein extracts from the BCY 1 strain identifies one cAMP-binding protein (M(r) approximately 47000), while similar mt extracts from the bcy 1 strain lack all cAMP-binding proteins. These data suggest that BCY 1 regulates yeast mt cAPK, and that inactivation of BCY 1 removes that mt activity from cAMP control.

Regulation of stringent mitochondrial transcription in yeast following amino-acid deprivation.

In the yeast Saccharomyces cerevisiae, a phenotypically identical stringent response is induced by either nutritional downshift or starvation for a required auxotrophic amino acid (aa); in each case, the response selectively includes transcriptional curtailment for the mitochondrial (mt) genome. We have shown previously that the downshift-induced mt stringent response is governed by changing cellular cyclic AMP (cAMP) levels, via a mt cAMP-dependent protein kinase. In contrast, we demonstrate here that cAMP levels are not altered in yeast following starvation for a required aa, and we use in vitro mt transcription assays with organelles from wild-type and mutant strains to confirm that the aa starvation-induced mt stringent response is not governed by cAMP. Rather, such stringent organellar transcriptional attenuation may result from altered availability of an unidentified small molecule which is probably a product of the cytoplasmic and/or mt protein synthesis systems.

Transcription of the yeast mitochondrial genome requires cyclic AMP.

Using various mutant strains and nutritional manipulations, we investigated a potential role for cyclic AMP (cAMP) in the regulation of mitochondrial (mt) gene expression in the yeast Saccharomyces cerevisiae. In RAS mutants known to have either abnormally low or high cellular levels of this nucleotide, we show that both mt transcription rate and overall mt transcript levels vary directly with cellular cAMP levels. We further show that nutritional downshift of actively growing cells causes a severe, rapid fall in cAMP levels, and that this fall is concomitant with the stringent mt transcriptional curtailment that we and others have previously shown to follow this nutritional manipulation. In in vitro mt transcription assays using intact organelles from downshifted and actively growing cells, stringently curtailed mt gene expression can be restored to 75% of control levels by addition of cAMP to the assay mix. Consistent with these observations a RAS2vall9 mutant strain, which cannot adjust cAMP levels in response to external stimuli, shows no mt stringent response following nutritional downshift. We also demonstrate a significant but transient increase in both mt transcript levels and mt transcription rate following shift of actively respiring wild-type cells to glucose-based medium, a manipulation known to cause a short-lived pulse of cAMP in yeast; similar manipulation of the RAS2vall9 mutant strain generates no such response. Taken together all these observations indicate that cellular cAMP levels are involved in the regulation of mt transcription in yeast. Moreover, the lack of a mt stringent transcriptional response following downshift in a strain in which the BCY1 gene had been insertionally inactivated suggests that cAMP may influence mt transcription via a mt cAMP-dependent protein kinase. These results link mt gene expression with mechanisms governing growth control and nutrient adaptation in yeast, and they provide a means by which mt gene expression might be coordinated with that of related nuclear genes.

Inapparent ocular infection by Chlamydia trachomatis in experimental and human trachoma.

There is substantial indirect evidence which suggests that Chlamydia trachomatis can generate inapparent, persistent infections in human. To confirm this directly, we examined ocular chlamydial infection in both the cynomolgus monkey model of trachoma and in patient samples from a trachoma-endemic area. In monkeys, ocular infection was studied over time using direct immunofluorescence cytology (DFA) and a molecular hybridization screening system which targets chlamydial ribosomal RNA. In eleven animals infected once with B serovar, DFA and probe screening of parallel conjunctival swabs gave congruent results through day 42 post-infection. Thereafter, DFA showed clearing of chlamydia and was negative by day 70, as in previous studies. In contrast, hybridization analysis indicated a continuing presence of chlamydial RNA in all samples from all animals through the end of the experiment at day 84 post-infection. Similarly, analysis of swabs from trachoma patients showed that a number of DFA-negative samples gave clear positive signal for chlamydial RNA. Taken together these data indicate that ocular chlamydial infection persists for longer periods than previously thought, judging solely on the basis of DFA, and they support the idea that inapparent ocular chlamydial infection occurs in vivo.

Regulation of mitochondrial transcription during the stringent response in yeast.

In yeast (S. cerevisiae) the stringent response is known to include rapid, selective, and severe transcriptional curtailment for genes specifying cytoplasmic rRNAs and r-proteins. We have shown that transcription of the mitochondrial 21S rRNA gene is also congruently and selectively curtailed during the yeast stringent response. Using an in vitro transcription assay with intact organelles from both rho+ and rho- strains, we show here that the mitochondrial stringent response includes not only transcription of the 21S and 16S rRNA genes, but also that of organellar genes specifying non-mitoribosome-related products. Stringent organellar transcriptional curtailment is identical when cells are starved for a required (marker) amino acid or when they are subjected to nutritional downshift, and the relative level of that transcriptional curtailment following either perturbation is the same in cells growing on fermentative (repressing) or purely respiratory carbon sources. These results confirm that the mechanism governing mitochondrial gene expression during a stringent response is specified outside the organelle, and they show that this transcriptional control mechanism is not immediately subject to glucose repression. In all strains examined, stringent organellar gene expression requires a mitochondrial promoter, suggesting that the regulatory mechanism which functions during the stringent response operates primarily at transcriptional initiation.

Mitochondrial rRNA-containing petite strains of yeast (Saccharomyces cerevisiae) show a normal nuclear-mitochondrial stringent response.

The nuclear-mitochondrial stringent response was examined in isonuclear rho+, 21S rRNA-containing rho-, and rho o strains of S. cerevisiae. By 30 min after nutritional downshift, nuclear rDNA transcription falls to 15% of control levels congruently in all strains, as assayed via whole-cell RNA or by hybrid selection of specific double-labeled transcripts. Both in vivo and in vitro, the mitochondrial stringent response is identical between the rho- strain and its parental rho+ strain, and in both, the kinetics and magnitude of the organellar response mirror those of the nuclear response. The data show that mitochondrial transcription and protein synthesis are not required for stringent regulation of either nuclear or mitochondrial rDNA transcription.

Preparation of RNA from unspheroplasted yeast cells (Saccharomyces cerevisiae).

High-quality RNA can be prepared from up to 100-ml culture volumes of unspheroplasted yeast cells (Saccharomyces cerevisiae) via homogenization in high-temperature phenol:chloroform mixtures. The yield of RNA from this preparative method is equivalent to those of other methods requiring preliminary spheroplasting of cells. Quality and quantity of recovered RNA are independent of yeast strain and cell growth medium used, and the method works equally well on cells in either log phase growth or in stationary phase. Mitochondrial RNAs recovered as part of whole cell RNA mixtures may be slightly degraded. Analyses of individual transcripts in the recovered RNA mixtures suggest that there is no selection for or against any specific single transcript or any group of transcripts when RNA is prepared by this method.