Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour

Water vapour in the atmosphere of a transiting extrasolar planet

Water is predicted to be among, if not the most abundant molecular species after hydrogen in the atmospheres of close-in extrasolar giant planets (hot-Jupiters) Several attempts have been made to detect water on an exoplanet, but have failed to find compelling evidence for it or led to claims that should be taken with caution. Here we report an analysis of recent observations of the hot-Jupiter HD189733b taken during the transit, where the planet passed in front of its parent star. We find that absorption by water vapour is the most likely cause of the wavelength-dependent variations in the effective radius of the planet at the infrared wavelengths 3.6, 5.8 and 8 microns. The larger effective radius observed at visible wavelengths may be due to either star variability or the presence of clouds/hazes. We explain the most recent thermal infrared observations of the planet during secondary transit behind the star, reporting a non-detection of water on HD189733b, as being a consequence of the nearly isothermal vertical profile of the planet.s atmosphere. Our results show that water is detectable on extrasolar planets using the primary transit technique and that the infrared should be a better wavelength region than the visible, for such searches

Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase

DNA and RNA polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. We previously showed, for the reverse transcriptase (RT) encoded by the yeast retrotransposon Ty1, that one of the three conserved active site aspartates (D211) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. Transposition is partially restored by second site suppressor mutations in the RNAse H domain. The novel properties of this amino acid substitution led us to express the WT and D211N mutant enzymes, and study their pre-steady state kinetic parameters. We found that the kpol was reduced by a factor of 223 in the mutant, although the Kd for nucleotide binding was unaltered. Further, the mutant enzyme had a marked preference for Mn2+ over Mg2+. To better understand the functions of this residue within the Ty1 RT active site, we have now examined the in vitro properties of WT and D211N mutant Ty1 RTs in carrying out pyrophosphorolysis, the reverse reaction to polymerization, where pyrophosphate is the substrate and dNTPs are the product. We find that pyrophosphorolysis is efficient only when the base-paired primer template region is >14 bases, and that activity increases when the primer end is blunt-ended or recessed by only a few bases. Using pre-steady state kinetic analysis, we find that the rate of pyrophosphorolysis (kpyro) in the D211N mutant is nearly 320 fold lower than the WT enzyme, and that the mutant enzyme has an ∼170 fold lower apparent Kd for pyrophosphate. These findings indicate that subtle substrate differences can strongly affect the enzyme's ability to properly position the primer-end to carry out pyrophosphorolysis. Further the kinetic data suggests that the D211 residue has a role in pyrophosphate binding and release, which could affect polymerase translocation, and help explain the D211N mutant's transposition defect

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ

QUANTITATIVE APPROACH TO ASSESSMENT OF THERAPEUTIC RESPONSE ON EARLY STAGES OF ULCERATIVE COLITIS TREATMENT

Research is devoted to the design of a quantitative approach to the assessment of therapeutic response to therapy of patients with ulcerative colitis in the first 5 days of treatment. The study included 48 patients with ulcerative colitis with mild and moderate-severe current disease. Quantitative changes in the severity of the condition of the patient were analyzed using a specially designed questionnaire, including laboratory values and clinical symptoms. It was found that the standard qualitative approach to evaluating the effectiveness of the therapy in patients with ulcerative colitis with multidirectional dynamics of individual clinical symptoms and laboratory parameters in the first 5 days of treatment in most cases does not allow to give an integral characteristic of the general condition of the patient changes. Designed quantitative approach to evaluating therapeutic response to the treatment allow not only identify the minimal changes in patient condition in the first 5 days, but also to predict the effect obtained by the treatment of two weeks

NSAID-induced gastro-duodenal bleeding: risk of development and patient management

A.P. Rebrov, E.l. Koshkina, A.A. Antonyan, R.V. Lyakisheva NSAID-induced gastro-duodenal bleeding: risk of development and patient management. Retrospective analysis of gastro-duodenal bleeding (GDB) report forms was performed. Pts admitted to the department of internal diseases of Saratov regional clinical hospital from 1999 to 2004 were included. 354 cases of GDB were recorded ( 282 from 1999 to 2001 and 72 from 2002 to 2004). Cause ofbleeding, influence of NSAID administration on appearance and relapse ofbleeding, source ofbleeding and pt management were assessed. NSAJD administration was considered trigger factor of GDB in 24% in 1999-2001 and in 18% in 2002-2004. The results of the study show some tendency to decrease of NSAID- induced gastropathy frequency among causes of GDB. The main risk factors ofbleeding during NSAID treatment were age of more than 30 years, ulcer history, the first month of NSAID administration. GDB frequency did not depend on regularity of treatment and NSAID dosage

Template misalignment in multisubunit RNA polymerases and transcription fidelity.

Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity

Nonequilibrium carbon black suspensions used in synthesis of polymer composite material

Nowadays polymer matrix-based composite material with various carbon fillers are widely used to protect radioequipment from different interference, to improve characteristics of radar absorbing coatings. Current synthesis processes are sophisticated and rather costly. The challenge is to develop new methods of producing composite materials by efficient knowledge intensive technologies to reduce the cost of products. The paper studies possibility of producing composite material on the basis of elastic polyurethane foam with carbon fillers using polyurethane impregnation in nonequilibrium black carbon suspension. Suspension composition: running water and carbon-bearing powders of nanometer range: fullerene black carbon, Taunite (multi-layer carbon nano tube material), technical carbon T900, wood soot. Nonequilibrium suspension was produced by treatment in hydrodynamic generator of rotor type (cavitation mode). Angular rotation speed of the rotor ω = 10000 rpm