PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5′ termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories.

Fuel ethanol is produced by the yeast Saccharomyces cerevisiae mainly from corn starch in the United States and from sugarcane sucrose in Brazil, which together manufacture ∼85% of a global yearly production of 109.8 million m3 (in 2019). While in North America genetically engineered (GE) strains account for ∼80% of the ethanol produced, including strains that express amylases and are engineered to produce higher ethanol yields; in South America, mostly (>90%) non-GE strains are used in ethanol production, primarily as starters in non-aseptic fermentation systems with cell recycling. In spite of intensive research exploring lignocellulosic ethanol (or second generation ethanol), this option still accounts for <1% of global ethanol production. In this mini-review, we describe the main aspects of fuel ethanol production, emphasizing bioprocesses operating in North America and Brazil. We list and describe the main properties of several commercial yeast products (i.e., yeast strains) that are available worldwide to bioethanol producers, including GE strains with their respective genetic modifications. We also discuss recent studies that have started to shed light on the genes and traits that are important for the persistence and dominance of yeast strains in the non-aseptic process in Brazil. While Brazilian bioethanol yeast strains originated from a historical process of domestication for sugarcane fermentation, leading to a unique group with significant economic applications, in U.S.A., guided selection, breeding and genetic engineering approaches have driven the generation of new yeast products for the market.

The action of testosterone on the 45Ca2+ uptake and insulin secretion was studied in short-term experiments using isolated pancreatic islets of Langerhans. Testosterone (1 microM) stimulated 45Ca2+ uptake within 60 seconds of incubation on similar proportion than tolbutamide. Also, the hormone rapidly increased insulin release (34%; 180 seconds) on the presence of non-stimulatory concentrations of glucose (3 mM). Impermeant testosterone-BSA significantly stimulated the secretion of insulin to a lower percentage (10%). The action of the hormone is specific--neither 17beta-E2 nor progesterone stimulated insulin secretion in the presence of 3 mM glucose. The action of testosterone on insulin secretion was dose-dependent, and at rat plasma physiological concentrations (25 nM), stimulus was 17% (p < 0.05). In conclusion, in isolated pancreatic islets experiments, physiological concentration of testosterone rapidly stimulate insulin secretion and 45Ca2+ uptake through a membrane bound mechanism.

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