Integrative elements for Bacillus subtilis yielding tetracycline-dependent growth phenotypes

We describe the construction and application of elements for random insertion of promoter containing DNA into the genome of Bacillus subtilis. The outward-facing promoter of these integrative elements termed InsTet(G+) is inducible by tetracycline so that conditional mutants are generated. We constructed three InsTet(G+) variants using different regulatory windows. In the first, the regulator gene tetR is located within the element, allowing one-step mutagenesis. The second contains tetR in the chromosome and yields the best regulation efficiency. The third exploits xylose-dependent tetR expression from a plasmid, enabling induction of TetR synthesis so that distinct expression levels of an affected gene can be adjusted. We have obtained mutant strains with all three variants. For some of them, growth can be modulated by the presence of effectors. Most growth defects occur in the presence of inducers, presumably due to regulated expression of antisense RNA

Improved single-chain transactivators of the Tet-On gene expression system

BACKGROUND: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. RESULTS: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used. CONCLUSION: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used

Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange

Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70–80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro

Summer-drought constrains the phenology and growth of two coexisting Mediterranean oaks with contrasting leaf habit: implications for their persistence and reproduction

13 páginas, 9 figuras, 5 tablas.-- El PDF es la versión post-print.This study analyses how coexisting evergreen and deciduous oaks adjust their phenology to cope with the stressful Mediterranean summer conditions. We test the hypothesis that the vegetative and reproductive growth of the winter deciduous (Quercus faginea Lam.) is more affected by summer drought than that of the evergreen [Quercus ilex L. subsp. ballota (Desf.) Samp.]. First, we assessed the complete aboveground phenology of both species during two consecutive years. Shoot and litter production and bud, acorn and secondary growth were monitored monthly. Second, we identified several parameters affected by summer conditions: apical bud size, individual leaf area (LA), leaf mass per area (LMA) and acorn yield in both species, and leaf-fall in Q. faginea; and analysed their variation over 10 years. Q. ilex performed up to 25% of shoot growth and most leaf development during summer, whereas Q. faginea completed most of both phenophases during spring. Secondary growth was arrested in summer under drought conditions. Approximately, 30–40% of bud and 40–50% of acorn growth was undertaken during summer in both species. Summer drought related to differences in LA, LMA and leaf senescence, but not to acorn yield. Both species had similar year-to-year patterns of acorn production, though yields were always lower in Q. faginea. Bud size decreased severely in both species during extremely dry years. In Q. ilex, bud size tended to alternate between years of large and small buds, and these patterns were followed by opposite trends in stem length. In Q. faginea, bud size was more stable through time. Q. ilex was more phenologically active during summer than Q. faginea, indicating a higher tolerance to drought. Furthermore, bud and fruit growth (the only two phenophases that both species performed during summer) were more severely affected by summer drought in Q. faginea than in the evergreen. The differential effects of summer drought on key phenophases for the persistence (bud growth) and colonization ability (fruit production) of both species may have consequences for their coexistence.This study was possible thanks to the collaboration within the GLOBIMED network (Ministerio de Educación y Ciencia, Spain) and it was supported by the MEC-CICyT projects AGF96-0399, CGL2007-66066-C04/BOS and CGL2008- 04847-C02-01, DGA projects P-038/96 and GA-LC-011/2008, and INIA projects RTA2005-00100-C02-00 and SUM2006-00025-00-00. JJC acknowledges the support of the ‘‘Fundación Aragón I+D’’. SP and RM were funded by MEC by a postdoc (SEUI-FECYT) and a Juan de la Cierva contract, respectively. JA was funded by DGA.Peer reviewe

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1

Impact of everolimus plus calcineurin inhibitor on formation of non-HLA antibodies and graft outcomes in kidney transplant recipients: 12-month results from the ATHENA substudy

BackgroundNon-human leukocyte antigen (non-HLA) antibodies including antibodies targeting Angiotensin II type 1 (AT1R) and Endothelin-1 type A (ETAR) receptors represent a topic of interest in kidney transplantation (KTx). This exploratory substudy evaluated the impact of everolimus (EVR) or mycophenolic acid (MPA) in combination with tacrolimus (TAC) or cyclosporine A (CsA) in patients with preformed non-HLA antibodies, potentially associated rejections and/or their impact on renal function over 1 year.MethodsAll eligible patients were randomized (1:1:1) before transplantation to receive either EVR/TAC, EVR/CsA, or MPA/TAC regimen. The effect of these regimens on the formation of non-HLA antibodies within one year post de novo KTx and the association with clinical events was evaluated descriptively in randomized (n = 268) population.ResultsAt Month 12, in EVR/TAC group, higher incidence of patients negative for AT1R- and ETAR-antibodies (82.2% and 76.7%, respectively) was noted, whereas the incidence of AT1R- and ETAR-antibodies positivity (28.1% and 34.7%, respectively) was higher in the MPA/TAC group. Non-HLA antibodies had no influence on clinical outcomes in any treatment group and no graft loss or death was reported.ConclusionsThe studied combinations of immunosuppressants were safe with no influence on clinical outcomes and suggested minimal exposure of calcineurin inhibitors for better patient management.Clinical Trial Registrationhttps://clinicaltrials.gov/ (NCT01843348; EudraCT number: 2011-005238-21)

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder–order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates

Rohlfs (Gerhard). Romanische Sprachgeographie. Geschichte und Grundlagen, Aspekte und Probleme mit dem Versuch eines Sprachatlas der romanischen Sprachen

Hillen Wolfgang. Rohlfs (Gerhard). Romanische Sprachgeographie. Geschichte und Grundlagen, Aspekte und Probleme mit dem Versuch eines Sprachatlas der romanischen Sprachen. In: Revue belge de philologie et d'histoire, tome 52, fasc. 1, 1974. Antiquité - Oudheid. pp. 86-89