A single mutation in the core domain of the lac repressor reduces leakiness.

BACKGROUND: The lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements triggered by binding of the lactose isomer allolactose to the core domain of the repressor impede DNA binding and lift repression. In Nature, the ability to detect and respond to environmental conditions comes at the cost of the encoded enzymes being constitutively expressed at low levels. The readily-switched regulation provided by LacI has resulted in its widespread use for protein overexpression, and its applications in molecular biology represent early examples of synthetic biology. However, the leakiness of LacI that is essential for the natural function of the lac operon leads to an increased energetic burden, and potentially toxicity, in heterologous protein production. RESULTS: Analysis of the features that confer promiscuity to the inducer-binding site of LacI identified tryptophan 220 as a target for saturation mutagenesis. We found that phenylalanine (similarly to tryptophan) affords a functional repressor that is still responsive to IPTG. Characterisation of the W220F mutant, LacIWF, by measuring the time dependence of GFP production at different IPTG concentrations and at various incubation temperatures showed a 10-fold reduction in leakiness and no decrease in GFP production. Cells harbouring a cytotoxic protein under regulatory control of LacIWF showed no decrease in viability in the early phases of cell growth. Changes in responsiveness to IPTG observed in vivo are supported by the thermal shift assay behaviour of purified LacIWF with IPTG and operator DNA. CONCLUSIONS: In LacI, long-range communications are responsible for the transmission of the signal from the inducer binding site to the DNA binding domain and our results are consistent with the involvement of position 220 in modulating these. The mutation of this single tryptophan residue to phenylalanine generated an enhanced repressor with a 10-fold decrease in leakiness. By minimising the energetic burden and cytotoxicity caused by leakiness, LacIWF constitutes a useful switch for protein overproduction and synthetic biology.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

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MEN1 Gene mutation and reduced expression are associated with poor prognosis in pulmonary carcinoids

Context: MEN1 gene alterations have been implicated in lung carcinoids, but their effect on gene expression and disease outcome is unknown. Objective: Our objective was to analyze MEN1 gene and expression anomalies in lung neuroendocrine neoplasms and their correlations with clinicopathologic data and disease outcome. Design: We examined 74 lung neuroendocrine neoplasms including 58 carcinoids and 16 high-grade neuroendocrine carcinomas (HGNECs) for MEN1 mutations (n = 70) and allelic losses (n = 69), promoter hypermethylation (n = 65), and mRNA (n = 74) expression. Results were correlated with disease outcome. Results: MEN1 mutations were found in 7 of 55 (13%) carcinoids and in 1 HGNEC, mostly associated with loss of the second allele. MEN1 decreased expression levels correlated with the presence of mutations (P = .0060) and was also lower in HGNECs than carcinoids (P = .0024). MEN1 methylation was not associated with mRNA expression levels. Patients with carcinoids harboring MEN1 mutation and loss had shorter overall survival (P = .039 and P = .035, respectively) and low MEN1 mRNA levels correlated with distant metastasis (P = .00010) and shorter survival (P = .0071). In multivariate analysis, stage and MEN1 allelic loss were independent predictors of prognosis. Conclusion: Thirteen percent of pulmonary carcinoids harbor MEN1 mutation associated with reduced mRNA expression and poor prognosis. Also in mutation-negative tumors, low MEN1 gene expression correlates with an adverse disease outcome. Hypermethylation was excluded as the underlying mechanism

The carbon balance of South America: A review of the status, decadal trends and main determinants

Copyright © 2012 European Geosciences Union. This is the published version available at http://www.biogeosciences-discuss.net/9/627/2012/bgd-9-627-2012.htmlWe attempt to summarize the carbon budget of South America and relate it to its dominant controls: population and economic growth, changes in land use practices and a changing atmospheric environment and climate. Flux estimation methods which we consider sufficiently reliable are fossil fuel emission inventories, biometric analysis of old-growth rainforests, estimation of carbon release associated with deforestation based on remote sensing and inventories, and finally inventories of agricultural exports. Other routes to estimating land-atmosphere CO2 fluxes include atmospheric transport inverse modelling and vegetation model predictions but are hampered by the data paucity and the need for improved parameterisation. The available data we analyze suggest that South America was a net source to the atmosphere during the 1980s (∼0.3–0.4 Pg C yr−1) and close to neutral (∼0.1 Pg C yr−1) in the 1990s with carbon uptake in old-growth forests nearly compensating carbon losses due to fossil fuel burning and deforestation. Annual mean precipitation over tropical South America measured by Amazon River discharge has a long-term upward trend, although over the last decade, dry seasons have tended to be drier and longer (and thus wet seasons wetter), with the years 2005 and 2010 experiencing strong droughts. It is currently unclear what the effect of these climate changes on the old-growth forest carbon sink will be but first measurements suggest it may be weakened. Based on scaling of forest census data the net carbon balance of South America seems to have been an increased source roughly over the 2005–2010 period (a total of ∼1 Pg C of dead tree biomass released over several years) due to forest drought response. Finally, economic development of the tropical forest regions of the continent is advancing steadily with exports of agricultural products being an important driver and witnessing a strong upturn over the last decade