43 research outputs found
Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e
Mercury has a long history as an antimicrobial agent effective against eukaryotic and prokaryotic organisms.
Despite its prolonged use, the basis for mercury toxicity in prokaryotes is not well understood. Archaea, like
bacteria, are prokaryotes but they use a simplified version of the eukaryotic transcription apparatus. This
study examined the mechanism of mercury toxicity to the archaeal prokaryote Sulfolobus solfataricus. In vivo
challenge with mercuric chloride instantaneously blocked cell division, eliciting a cytostatic response at
submicromolar concentrations and a cytocidal response at micromolar concentrations. The cytostatic response
was accompanied by a 70% reduction in bulk RNA synthesis and elevated rates of degradation of several
transcripts, including tfb-1, tfb-2, and lacS. Whole-cell extracts prepared from mercuric chloride-treated cells
or from cell extracts treated in vitro failed to support in vitro transcription of 16S rRNAp and lacSp promoters.
Extract-mixing experiments with treated and untreated extracts excluded the occurrence of negative-acting
factors in the mercury-treated cell extracts. Addition of transcription factor B (TFB), a general transcription
factor homolog of eukaryotic TFIIB, to mercury-treated cell extracts restored \u3e50% of in vitro transcription
activity. Consistent with this finding, mercuric ion treatment of TFB in vitro inactivated its ability to restore
the in vitro transcription activity of TFB-immunodepleted cell extracts. These findings indicate that the toxicity
of mercuric ion in S. solfataricus is in part the consequence of transcription inhibition due to TFB-1 inactivation
Regulation of Mercury Resistance in the Crenarchaeote \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e
Mercuric ion, Hg(II), inactivates generalized transcription in the crenarchaeote Sulfolobus solfataricus. Metal
challenge simultaneously derepresses transcription of mercuric reductase (merA) by interacting with the
archaeal transcription factor aMerR. Northern blot and primer extension analyses identified two additional
Hg(II)-inducible S. solfataricus genes, merH and merI (SSO2690), located on either side of merA. Transcription
initiating upstream of merH at promoter merHp was metal inducible and extended through merA and merI,
producing a merHAI transcript. Northern analysis of a merRA double mutant produced by linear DNA
recombination demonstrated merHp promoter activity was dependent on aMerR to overcome Hg(II) transcriptional
inhibition. Unexpectedly, in a merA disruption mutant, the merH transcript was transiently induced after
an initial period of Hg(II)-mediated transcription inhibition, indicating continued Hg(II) detoxification. Metal
challenge experiments using mutants created by markerless exchange verified the identity of the MerR binding
site as an inverted repeat (IR) sequence overlapping the transcription factor B binding recognition element of
merHp. The interaction of recombinant aMerR with merHp DNA, studied using electrophoretic mobility shift
analysis, demonstrated that complex formation was template specific and dependent on the presence of the IR
sequence but insensitive to Hg(II) addition and site-specific IR mutations that relieved in vivo merHp repression.
Despite containing a motif resembling a distant ArsR homolog, these results indicate aMerR remains
continuously DNA bound to protect and coordinate Hg(II)-responsive control over merHAI transcription. The
new genetic methods developed in this work will promote experimental studies on S. solfataricus and other
Crenarchaeota
Epigenetic manipulation of gene expression: a toolkit for cell biologists
Cell biologists have been afforded extraordinary new opportunities for experimentation by the emergence of powerful technologies that allow the selective manipulation of gene expression. Currently, RNA interference is very much in the limelight; however, significant progress has also been made with two other approaches. Thus, antisense oligonucleotide technology is undergoing a resurgence as a result of improvements in the chemistry of these molecules, whereas designed transcription factors offer a powerful and increasingly convenient strategy for either up- or down-regulation of targeted genes. This mini-review will highlight some of the key features of these three approaches to gene regulation, as well as provide pragmatic guidance concerning their use in cell biological experimentation based on our direct experience with each of these technologies. The approaches discussed here are being intensely pursued in terms of possible therapeutic applications. However, we will restrict our comments primarily to the cell culture situation, only briefly alluding to fundamental differences between utilization in animals versus cells
Cellular Delivery and Biological Activity of Antisense Oligonucleotides Conjugated to a Targeted Protein Carrier
Targeted delivery can potentially improve the pharmacological effects of antisense and siRNA oligonucleotides. Here we describe a novel bioconjugation approach to the delivery of splice-shifting antisense oligonucleotides (SSOs). The SSOs are linked to albumin via reversible S-S bonds. The albumin is also conjugated with polyethylene glycol (PEG) chains that terminate in an RGD ligand that selectively binds the αvβ3 integrin. As a test system we utilized human melanoma cells that express the αvβ3 integrin and that also contain a luciferase reporter gene that can be induced by delivery of SSOs to the cell nucleus. The RGD-PEG-SSO-albumin conjugates were endocytosed by the cells in an RGD-dependent manner; using confocal fluorescence microscopy evidence was obtained that the SSOs accumulate in the nucleus. The conjugates were able to robustly induce luciferase expression at concentrations in the 25−200nM range. At these levels little short-term or long-term toxicity was observed. Thus the RGD-PEG-Albumin conjugates may provide an effective tool for targeted delivery of oligonucleotides to certain cells and tissues
Regulation of Mercury Resistance in the Crenarchaeote \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e
Mercuric ion, Hg(II), inactivates generalized transcription in the crenarchaeote Sulfolobus solfataricus. Metal
challenge simultaneously derepresses transcription of mercuric reductase (merA) by interacting with the
archaeal transcription factor aMerR. Northern blot and primer extension analyses identified two additional
Hg(II)-inducible S. solfataricus genes, merH and merI (SSO2690), located on either side of merA. Transcription
initiating upstream of merH at promoter merHp was metal inducible and extended through merA and merI,
producing a merHAI transcript. Northern analysis of a merRA double mutant produced by linear DNA
recombination demonstrated merHp promoter activity was dependent on aMerR to overcome Hg(II) transcriptional
inhibition. Unexpectedly, in a merA disruption mutant, the merH transcript was transiently induced after
an initial period of Hg(II)-mediated transcription inhibition, indicating continued Hg(II) detoxification. Metal
challenge experiments using mutants created by markerless exchange verified the identity of the MerR binding
site as an inverted repeat (IR) sequence overlapping the transcription factor B binding recognition element of
merHp. The interaction of recombinant aMerR with merHp DNA, studied using electrophoretic mobility shift
analysis, demonstrated that complex formation was template specific and dependent on the presence of the IR
sequence but insensitive to Hg(II) addition and site-specific IR mutations that relieved in vivo merHp repression.
Despite containing a motif resembling a distant ArsR homolog, these results indicate aMerR remains
continuously DNA bound to protect and coordinate Hg(II)-responsive control over merHAI transcription. The
new genetic methods developed in this work will promote experimental studies on S. solfataricus and other
Crenarchaeota
The Biological Effect of an Antisense Oligonucleotide Depends on Its Route of Endocytosis and Trafficking
We demonstrate that the biological effect of an oligonucleotide is influenced by its route of cellular uptake. Utilizing a splice-switching antisense oligonucleotide (SSO) and a sensitive reporter assay involving correction of RNA splicing, we examined induction of luciferase in cells treated either with various concentrations of an unconjugated (“free”) SSO or an SSO conjugated to a bivalent RGD ligand that promotes binding to the αvβ3 integrin (RGD-SSO). Under conditions of equal accumulation in cells, the RGD-SSO consistently had a greater effect on luciferase induction than the unconjugated SSO. We determined that the RGD-SSO and the unconjugated SSO were internalized by distinct endocytotic pathways, suggesting that the route of internalization affects the magnitude of the biological response
Biological effects of hexitol and altritol-modified siRNAs targeting B-Raf
Increasing the effectiveness of siRNAs through chemical modification is an important task. Here we describe altritol and hexitol modified oligonucleotides targeting the B-Raf oncogene that is critical for the growth and survival of melanoma cells. Using assays for apoptosis, DNA synthesis, colony formation and B-Raf protein and message levels, we demonstrate that certain hexitol modifications can improve the effectiveness of B-Raf siRNAs and also increase duration of action. Altritol modified siRNAs were similar to or slightly less effective than unmodified B-Raf siRNA. Modifications at the 3′ or 5′ end of the sense strand, at the 3′ end of the antisense strand, or within either strand were well tolerated. The basis for the increased effectiveness of the hexitol-modified siRNAs is not fully understood but may be partly due to increased stability to nucleases
Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
We describe the synthesis and characterization of a 5′ conjugate between a 2′-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine–glycine–aspartic acid) peptide that is a high-affinity ligand for the αvβ3 integrin. We used αvβ3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD–623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while ‘free’ 623 did not. However, the kinetics of luciferase expression was distinct; the RGD–623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD–623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD–623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide–oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the αvβ3 integrin
Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e
Mercury has a long history as an antimicrobial agent effective against eukaryotic and prokaryotic organisms.
Despite its prolonged use, the basis for mercury toxicity in prokaryotes is not well understood. Archaea, like
bacteria, are prokaryotes but they use a simplified version of the eukaryotic transcription apparatus. This
study examined the mechanism of mercury toxicity to the archaeal prokaryote Sulfolobus solfataricus. In vivo
challenge with mercuric chloride instantaneously blocked cell division, eliciting a cytostatic response at
submicromolar concentrations and a cytocidal response at micromolar concentrations. The cytostatic response
was accompanied by a 70% reduction in bulk RNA synthesis and elevated rates of degradation of several
transcripts, including tfb-1, tfb-2, and lacS. Whole-cell extracts prepared from mercuric chloride-treated cells
or from cell extracts treated in vitro failed to support in vitro transcription of 16S rRNAp and lacSp promoters.
Extract-mixing experiments with treated and untreated extracts excluded the occurrence of negative-acting
factors in the mercury-treated cell extracts. Addition of transcription factor B (TFB), a general transcription
factor homolog of eukaryotic TFIIB, to mercury-treated cell extracts restored \u3e50% of in vitro transcription
activity. Consistent with this finding, mercuric ion treatment of TFB in vitro inactivated its ability to restore
the in vitro transcription activity of TFB-immunodepleted cell extracts. These findings indicate that the toxicity
of mercuric ion in S. solfataricus is in part the consequence of transcription inhibition due to TFB-1 inactivation