Expressing functional siRNAs in mammalian cells using convergent transcription

BACKGROUND: The use of small interfering RNAs (siRNAs) as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs) or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi)-mediated gene silencing. RESULTS: With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells. CONCLUSION: The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences. Furthermore, the cassette design reported is ideal for both routine use in controlling specific gene expression and construction of randomised RNAi expression libraries for use in unbiased forward genetic selections

TUF factor binds to the upstream region of the pyruvate decarboxylase structural gene ( PDC1 ) of Saccharomyces cerevisiae

The upstream activation site of the pyruvate decarboxylase gene, PDC1 , of Saccharomyces cerevisiae contains an RPG box, and mediates the increase in expression of a PDC1-lacZ fusion gene during growth on glucose. Oligonucleotide replacement experiments indicate that the RPG box functions as an absolute activator of expression, but other elements (possibly CTTCC repeats) are required for carbon source regulation, and maximal expression. Gel retardation and oligonucleotide competition experiments suggest that the DNA binding factor TUF interacts with the RPG box in the upstream region of PDC1 . Binding of TUF factor is not carbon source dependent in in vitro experiments, and is probably not responsible for glucose induction of PDC1 expression.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47573/1/438_2004_Article_BF00264453.pd

Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Protein-SH groups are amongst the most easily oxidized residues in proteins, but irreversible oxidation can be prevented by protein glutathionylation, in which protein-SH groups form mixed disulphides with glutathione. Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms. Glutaredoxins have been proposed to be the predominant deglutathionylase enzymes in many plant and mammalian species, whereas, thioredoxins have generally been thought to be relatively inefficient in deglutathionylation.</p> <p>Results</p> <p>We show here that the levels of glutathionylated proteins in yeast are regulated in parallel with the growth cycle, and are maximal during stationary phase growth. This increase in glutathionylation is not a response to increased reactive oxygen species generated from the shift to respiratory metabolism, but appears to be a general response to starvation conditions. Our data indicate that glutathionylation levels are constitutively high in all growth phases in thioredoxin mutants and are unaffected in glutaredoxin mutants. We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins. Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.</p> <p>Conclusions</p> <p>There is increasing evidence that the thioredoxin and glutathione redox systems have overlapping functions and these present data indicate that the thioredoxin system plays a key role in regulating the modification of proteins by the glutathione system.</p

Population structure of Helicobacter pylori among ethnic groups in Malaysia: recent acquisition of the bacterium by the Malay population

<p>Abstract</p> <p>Background</p> <p><it>Helicobacter pylori </it>is a major gastric bacterial pathogen. This pathogen has been shown to follow the routes of human migration by their geographical origin and currently the global <it>H. pylori </it>population has been divided into six ancestral populations, three from Africa, two from Asia and one from Europe. Malaysia is made up of three major ethnic populations, Malay, Chinese and Indian, providing a good population for studying recent <it>H. pylori </it>migration and admixture.</p> <p>Results</p> <p>Seventy eight <it>H. pylori </it>isolates, including 27 Chinese, 35 Indian and 16 Malay isolates from Malaysia were analysed by multilocus sequence typing (MLST) of seven housekeeping genes and compared with the global MLST data. STRUCTURE analysis assigned the isolates to previously identified <it>H. pylori </it>ancestral populations, hpEastAsia, hpAsia2 and hpEurope, and revealed a new subpopulation, hspIndia, within hpAsia2. Statistical analysis allowed us to identify population segregation sites that divide the <it>H. pylori </it>populations and the subpopulations. The majority of Malay isolates were found to be grouped together with Indian isolates.</p> <p>Conclusion</p> <p>The majority of the Malay and Indian <it>H. pylori </it>isolates share the same origin while the Malaysian Chinese <it>H. pylori </it>is distinctive. The Malay population, known to have a low infection rate of <it>H. pylori</it>, was likely to be initially <it>H. pylori </it>free and gained the pathogen only recently from cross infection from other populations.</p

Distinct redox regulation in sub-cellular compartments in response to various stress conditions in Saccharomyces cerevisiae

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (EGSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. EGSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (-340 to -350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H+/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H+/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions

Avoiding unscheduled transcription in shared promoters: Saccharomyces cerevisiae Sum1p represses the divergent gene pair SPS18-SPS19 through a midsporulation element (MSE)

The sporulation-specific gene SPS18 shares a common promoter region with the oleic acid-inducible gene SPS19. Both genes are transcribed in sporulating diploid cells, albeit unevenly in favour of SPS18, whereas in haploid cells grown on fatty acids only SPS19 is highly activated. Here, SPS19 oleate-response element (ORE) conferred activation on a basal CYC1-lacZ reporter gene equally in both orientations, but promoter analysis using SPS18-lacZ reporter constructs with deletions identified a repressing fragment containing a midsporulation element (MSE) that could be involved in imposing directionality towards SPS19 in oleic acid-induced cells. In sporulating diploids, MSEs recruit the Ndt80p transcription factor for activation, whereas under vegetative conditions, certain MSEs are targeted by the Sum1p repressor in association with Hst1p and Rfm1p. Quantitative real-time PCR demonstrated that in haploid sum1Δ, hst1Δ, or rfm1Δ cells, oleic acid-dependent expression of SPS18 was higher compared with the situation in wild-type cells, but in the sum1Δ mutant, this effect was diminished in the absence of Oaf1p or Pip2p. We conclude that SPS18 MSE is a functional element repressing the expression of both SPS18 and SPS19, and is a component of a stricture mechanism shielding SPS18 from the dramatic increase in ORE-dependent transcription of SPS19 in oleic acid-grown cells

Genetic basis of arsenite and cadmium tolerance in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Arsenic and cadmium are widely distributed in nature and pose serious threats to the environment and human health. Exposure to these nonessential toxic metals may result in a variety of human diseases including cancer. However, arsenic and cadmium toxicity targets and the cellular systems contributing to tolerance acquisition are not fully known.</p> <p>Results</p> <p>To gain insight into metal action and cellular tolerance mechanisms, we carried out genome-wide screening of the <it>Saccharomyces cerevisiae </it>haploid and homozygous diploid deletion mutant collections and scored for reduced growth in the presence of arsenite or cadmium. Processes found to be required for tolerance to both metals included sulphur and glutathione biosynthesis, environmental sensing, mRNA synthesis and transcription, and vacuolar/endosomal transport and sorting. We also identified metal-specific defence processes. Arsenite-specific defence functions were related to cell cycle regulation, lipid and fatty acid metabolism, mitochondrial biogenesis, and the cytoskeleton whereas cadmium-specific defence functions were mainly related to sugar/carbohydrate metabolism, and metal-ion homeostasis and transport. Molecular evidence indicated that the cytoskeleton is targeted by arsenite and that phosphorylation of the Snf1p kinase is required for cadmium tolerance.</p> <p>Conclusion</p> <p>This study has pin-pointed core functions that protect cells from arsenite and cadmium toxicity. It also emphasizes the existence of both common and specific defence systems. Since many of the yeast genes that confer tolerance to these agents have homologues in humans, similar biological processes may act in yeast and humans to prevent metal toxicity and carcinogenesis.</p

Saccharomyces cerevisiae genes involved in survival of heat shock

The heat-shock response in cells, involving increased transcription of a specific set of genes in response to a sudden increase in temperature, is a highly conserved biological response occurring in all organisms. Despite considerable attention to the processes activated during heat shock, less is known about the role of genes in survival of a sudden temperature increase. Saccharomyces cerevisiae genes involved in the maintenance of heat-shock resistance in exponential and stationary phase were identified by screening the homozygous diploid deletants in nonessential genes and the heterozygous diploid mutants in essential genes for survival after a sudden shift in temperature from 30 to 50. More than a thousand genes were identified that led to altered sensitivity to heat shock, with little overlap between them and those previously identified to affect thermotolerance. There was also little overlap with genes that are activated or repressed during heat-shock, with only 5% of them regulated by the heat-shock transcription factor. The target of rapamycin and protein kinase A pathways, lipid metabolism, vacuolar H+-ATPase, vacuolar protein sorting, and mitochondrial genome maintenance/translation were critical to maintenance of resistance. Mutants affected in l-tryptophan metabolism were heat-shock resistant in both growth phases; those affected in cytoplasmic ribosome biogenesis and DNA double-strand break repair were resistant in stationary phase, and in mRNA catabolic processes in exponential phase. Mutations affecting mitochondrial genome maintenance were highly represented in sensitive mutants. The cell division transcription factor Swi6p and Hac1p involved in the unfolded protein response also play roles in maintenance of heat-shock resistance

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases

A Role for Phosphatidic Acid in the Formation of “Supersized” Lipid Droplets

Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. Here, a genome-wide screen identifies ten yeast mutants producing “supersized” LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of “supersized” LDs. In summary, our results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism