Increased Transcript Abundance Research Articles

Metabolic regulation of glyoxylate-cycle enzyme synthesis in detached cucumber cotyledons and protoplasts

When cotyledons are detached from cucumber (Cucumis sativus L. cv. Masterpiece) plants 14 d after seed imbibition, accumulation of the glyoxylate-cycle enzymes malate synthase (MS) and isocitrate lyase (ICL) occurs rapidly in the dark but not in the light. This is primarily due to an increase in transcript abundance. It has been proposed that glyoxylate-cycle enzymes are synthesised in response to lipid degradation. However, MS and ICL synthesis in detached cucumber cotyledons begins before a decline in lipid can be detected. Furthermore, the activation of MS and ICL gene expression does not correlate with loss of chlorophyll, carotenoid, protein or RNA. These results are discussed in terms of the activation of genes encoding glyoxylate-cycle enzymes and their possible function in detached and senescing organs. Previous work (Graham et al, 1992, Plant Cell 3, 349–357) has suggested that sucrose or other carbohydrates can repress the synthesis of MS and ICL. Consistent with this proposal, incubation of detached cotyledons in the light leads to a fivefold increase in sucrose content. However, when MS and ICL synthesis occurs in the dark, no change in the amount of sucrose is detected. Incubation of cotyledons in the dark in the presence of 25 mM sucrose leads to a complete inhibition of the accumulation of MS and ICL. Similarly, cotyledon protoplasts incubated in darkness accumulate MS and ICL, but not if 25 mM sucrose is included in the medium. These results collectively support the view that MS and ICL synthesis responds to the metabolic activities of plant cells.

Glucose repression of yeast mitochondrial transcription: kinetics of derepression and role of nuclear genes.

Yeast mitochondrial transcript and gene product abundance has been observed to increase upon release from glucose repression, but the mechanism of regulation of this process has not been determined. We report a kinetic analysis of this phenomenon, which demonstrates that the abundance of all classes of mitochondrial RNA changes slowly relative to changes observed for glucose-repressed nuclear genes. Several cell doublings are required to achieve the 2- to 20-fold-higher steady-state levels observed after a shift to a nonrepressing carbon source. Although we observed that in some yeast strains the mitochondrial DNA copy number also increases upon derepression, this does not seem to play the major role in increased RNA abundance. Instead we found that three- to sevenfold increases in RNA synthesis rates, measured by in vivo pulse-labelling experiments, do correlate with increased transcript abundance. We found that mutations in the SNF1 and REG1 genes, which are known to affect the expression of many nuclear genes subject to glucose repression, affect derepression of mitochondrial transcript abundance. These genes do not appear to regulate mitochondrial transcript levels via regulation of the nuclear genes RPO41 and MTF1, which encode the subunits of the mitochondrial RNA polymerase. We conclude that a nuclear gene-controlled factor(s) in addition to the two RNA polymerase subunits must be involved in glucose repression of mitochondrial transcript abundance.

Glucose repression of yeast mitochondrial transcription: kinetics of derepression and role of nuclear genes.

Yeast mitochondrial transcript and gene product abundance has been observed to increase upon release from glucose repression, but the mechanism of regulation of this process has not been determined. We report a kinetic analysis of this phenomenon, which demonstrates that the abundance of all classes of mitochondrial RNA changes slowly relative to changes observed for glucose-repressed nuclear genes. Several cell doublings are required to achieve the 2- to 20-fold-higher steady-state levels observed after a shift to a nonrepressing carbon source. Although we observed that in some yeast strains the mitochondrial DNA copy number also increases upon derepression, this does not seem to play the major role in increased RNA abundance. Instead we found that three- to sevenfold increases in RNA synthesis rates, measured by in vivo pulse-labelling experiments, do correlate with increased transcript abundance. We found that mutations in the SNF1 and REG1 genes, which are known to affect the expression of many nuclear genes subject to glucose repression, affect derepression of mitochondrial transcript abundance. These genes do not appear to regulate mitochondrial transcript levels via regulation of the nuclear genes RPO41 and MTF1, which encode the subunits of the mitochondrial RNA polymerase. We conclude that a nuclear gene-controlled factor(s) in addition to the two RNA polymerase subunits must be involved in glucose repression of mitochondrial transcript abundance.

Transcripts encoding protein kinase C-alpha, -delta, -epsilon, -zeta, and -eta are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells.

The protein kinase C (PKC) family of phospholipid-dependent serine-threonine kinases has been implicated in keratinocyte differentiation and neoplastic transformation. To determine if Ca(2+)-mediated keratinocyte differentiation is associated with changes in PKC isozyme gene expression, RNA was isolated from primary mouse keratinocytes grown in medium with 0.05, 0.12, or 1.4 mM Ca2+. Based on northern blot analysis, primary keratinocytes expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta, and -eta, but not PKC-beta or -gamma. Relatively little change was detected in the level of these transcripts in cells induced to differentiate by exposure to elevated extracellular Ca2+. Interestingly, the PKC-zeta transcripts detected in RNA isolated from keratinocytes were approximately 200 nucleotides longer than those from mouse brain, suggesting the existence of an alternative form of this isozyme. An early change in benign neoplastic transformation of keratinocytes is the inability to differentiate in response to Ca2+ or the PKC activator 12-O-tetradecanoylphorbol-13-acetate, which is consistent with altered PKC function in these cells. The PKC isozyme mRNA profile was examined in two benign neoplastic keratinocyte cell lines, 308 and SP-1, which contain an activating mutation of the c-Ha-ras gene. Like normal keratinocytes. 308 and SP-1 cells expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta, and -eta. However, the abundance of PKC-zeta transcripts in both cell lines was reduced by 74-89% when compared with normal keratinocytes at similar Ca2+ levels. In addition, SP-1 but not 308 cells exhibited a sevenfold increase in PKC-eta mRNA when cultured in medium with 1.4 mM Ca2+. To address whether these changes were related to the presence of an activated ras gene, RNA was isolated from primary keratinocytes transduced to a benign neoplastic phenotype with the v-Ha-ras oncogene. As with normal, 308, and SP-1 cells, v-Ha-ras keratinocytes expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta and -eta. The level of PKC-zeta transcripts was similar in normal and v-Ha-ras keratinocytes, indicating that reduction of this mRNA in both 308 and SP-1 cells was not a direct result of ras activation. As in SP-1 cells, PKC-eta in v-Ha-ras keratinocytes was responsive to extracellular Ca2+, with a four-fold increase in transcript abundance in 0.12 mM Ca2+ medium relative to 0.05 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 400 WORDS)

58 MOLECULAR BASIS OF INHERITED MYOADENYIATE DEAMINASE (AMP-D) DEFICIENCY

Inherited AMP-D deficiency is associated with a myopathy characterized by easy fatigability and myalgias. Deficiency of AMP-D is restricted to skeletal muscle and is associated with a decrease in the abundance of immunoreactive muscle-specific peptide. To define the molecular basis for this abnormality we have cloned cDNAs for the skeletal muscle-specific isoforms of human and rat AMP-D. We have made the following observations regarding AMP-D expression in a rodent model and in AMP-D deficient patients: 1) A single gene encodes AMP-D. 2) Two AMP-D transcripts are noted during muscle development in vivo and during myocyte differentiation in vitro. Rat fetal muscle contains an “embryonic” transcript (3.4 Kb) and peptide; postnatal skeletal muscle contains an “adult” transcript (2.5 Kb) and peptide. The latter are restricted to striated muscle. Production of the adult peptide is controlled by transcript switching, increase in transcript abundance during muscle development and possibly efficiency of translation. 3) Four patients with inherited deficiency of AMP-D have been evaluated with these probes. Although immunoreactive peptide is absent from their skeletal muscle, the abundance of the 2.5 Kb muscle-specific transcript is normal. We conclude from these studies that 1). AMP-D expression is controlled at multiple steps during myocyte development 2) Inherited deficiency of the muscle-specific isoform cannot be explained by a defect in transcript switching or rate of transcription, suggesting a defect in translation or a single base substitution.

Organ-Specific and Light-Induced Expression of Plant Genes

Light plays a pivotal role in the development of plants. The photoregulation of plant genes involves recognition of light quality and quantity by phytochrome and other light receptors. Two gene families, rbcS and Cab, which code for abundant proteins active in photosynthesis, the small subunit of ribulose bisphosphate carboxylase and the chlorophyll a/b binding protein, show a 20-to 50-fold increase in transcript abundance in the light. Analyses in calli and transgenic plants of deletions of the rbcS gene and of chimeric constructions has allowed localization of two regions involved in light-induced transcription. One element is confined to a 33-base pair region surrounding the TATA box. In addition, an enhancer-like element contained within a 240-base pair fragment can confer phytochrome-induced transcription and organ specificity on nonregulated promoters.