Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast

The complete synthetic Mycoplasma genitalium genome (∼583 kb) has been assembled and cloned as a circular plasmid in the yeast Saccharomyces cerevisiae. Attempts to engineer the cloned genome by standard genetic methods involving the URA3/5-fluoroorotic acid (5-FOA) counter-selection have shown a high background of 5-FOA resistant clones derived from spontaneous deletions of the bacterial genome maintained in yeast. Here, we report a method that can seamlessly modify the bacterial genome in yeast with high efficiency. This method requires two sequential homologous recombination events. First, the target region is replaced with a mutagenesis cassette that consists of a knock-out CORE (an18-bp I-SceI recognition site, the SCEI gene under the control of the GAL1 promoter, and the URA3 marker) and a DNA fragment homologous to the sequence upstream of the target site. The replacement generates tandem repeat sequences flanking the CORE. Second, galactose induces the expression of I-SceI, which generates a double-strand break (DSB) at the recognition site. This DSB promotes intra-molecular homologous recombination between the repeat sequences, and leads to an excision of the CORE. As a result, a seamless modification is generated. This method can be adapted for a variety of genomic modifications and may provide an important tool to modify and design natural or synthetic genomes propagated in yeast

Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA([Ser]Sec) as substrates to generate selenocysteyl-tRNA([Ser]Sec). Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA([Ser]Sec), seryl-tRNA synthetase, O-phosphoseryl-tRNA([Ser]Sec) kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA([Ser]Sec) kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins

Schreier type theorems for bicrossed products

We prove that the bicrossed product of two groups is a quotient of the pushout of two semidirect products. A matched pair of groups $(H, G, \alpha, \beta)$ is deformed using a combinatorial datum $(\sigma, v, r)$ consisting of an automorphism $\sigma$ of $H$, a permutation $v$ of the set $G$ and a transition map $r: G\to H$ in order to obtain a new matched pair $\bigl(H, (G,*), \alpha', \beta' \bigl)$ such that there exist an $\sigma$-invariant isomorphism of groups $H {}_{\alpha} \bowtie_{\beta} G \cong H {}_{\alpha'} \bowtie_{\beta'} (G,*)$. Moreover, if we fix the group $H$ and the automorphism \sigma \in \Aut(H) then any $\sigma$-invariant isomorphism $H {}_{\alpha} \bowtie_{\beta} G \cong H {}_{\alpha'} \bowtie_{\beta'} G'$ between two arbitrary bicrossed product of groups is obtained in a unique way by the above deformation method. As applications two Schreier type classification theorems for bicrossed product of groups are given.Comment: 21 pages, final version to appear in Central European J. Mat

Exploring user experience and technology acceptance for a fall prevention system: results from a randomized clinical trial and a living lab

Background: Falls are common in older adults and can result in serious injuries. Due to demographic changes, falls and related healthcare costs are likely to increase over the next years. Participation and motivation of older adults in fall prevention measures remain a challenge. The iStoppFalls project developed an information and communication technology (ICT)-based system for older adults to use at home in order to reduce common fall risk factors such as impaired balance and muscle weakness. The system aims at increasing older adults’ motivation to participate in ICT-based fall prevention measures. This article reports on usability, user-experience and user-acceptance aspects affecting the use of the iStoppFalls system by older adults. Methods: In the course of a 16-week international multicenter study, 153 community-dwelling older adults aged 65+ participated in the iStoppFalls randomized controlled trial, of which half used the system in their home to exercise and assess their risk of falling. During the study, 60 participants completed questionnaires regarding the usability, user experience and user acceptance of the iStoppFalls system. Usability was measured with the System Usability Scale (SUS). For user experience the Physical Activity Enjoyment Scale (PACES) was applied. User acceptance was assessed with the Dynamic Acceptance Model for the Re-evaluation of Technologies (DART). To collect more detailed data on usability, user experience and user acceptance, additional qualitative interviews and observations were conducted with participants. Results: Participants evaluated the usability of the system with an overall score of 62 (Standard Deviation, SD 15.58) out of 100, which suggests good usability. Most users enjoyed the iStoppFalls games and assessments, as shown by the overall PACES score of 31 (SD 8.03). With a score of 0.87 (SD 0.26), user acceptance results showed that participants accepted the iStoppFalls system for use in their own home. Interview data suggested that certain factors such as motivation, complexity or graphical design were different for gender and age. Conclusions: The results suggest that the iStoppFalls system has good usability, user experience and user acceptance. It will be important to take these along with factors such as motivation, gender and age into consideration when designing and further developing ICT-based fall prevention systems

Yeast Bax Inhibitor, Bxi1p, Is an ER-Localized Protein That Links the Unfolded Protein Response and Programmed Cell Death in Saccharomyces cerevisiae

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that Δbxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death

Functional Expression of Human Adenine Nucleotide Translocase 4 in Saccharomyces Cerevisiae

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells

Minimal state models for ionic channels involved in glucagon secretion

Pancreatic alpha cells synthesize and release glucagon. This hormone along with insulin, preserves blood glucose levels within a physiological range. During low glucose levels, alpha cells exhibit electrical activity related to glucagon secretion. In this paper, we introduce minimal state models for those ionic channels involved in this electrical activity in mice alpha cells. For estimation of model parameters, we use Monte Carlo algorithms to fit steadystate channel currents. Then, we simulate dynamic ionic currents following experimental protocols. Our aims are 1) To understand the individual ionic channel functioning and modulation that could affect glucagon secretion, and 2) To simulate ionic currents actually measured in voltage-clamp alpha-cell experiments in mice. Our estimations indicate that alpha cells are highly permeable to sodium and potassium which mainly manage action potentials. We have also found that our estimated N-type calcium channel population and density in alpha cells is in good agreement to those reported for L-type calcium channels in beta cells. This finding is strongly relevant since both, L-type and N-type calcium channels, play a main role in insulin and glucagon secretion, respectively

Cloning whole bacterial genomes in yeast

Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation

Roles for H2A.Z and Its Acetylation in GAL1 Transcription and Gene Induction, but Not GAL1-Transcriptional Memory

H2A.Z does not appear to have a role in GAL1 transcriptional memory, but it does have both acetylation-dependent and acetylation-independent roles in GAL1 induction and expression