Efficiency of the nuclease I-SceI in excising selectable marker genes from the plant genome

Gene stacking is a method used in biotechnology by which multiple genes can be placed at a single genomic site, thereby simplifying plant breeding. In this approach, DNA nucleases are used for excising selectable marker genes (SMG), which are the unneeded components of transgenic plants. The goal of this project is to evaluate the effectiveness of the nuclease I-SceI in excising DNA in plants. Specifically, this study tests heat-inducible I-SceI through the use of a heat-shock promoter (HS) in order to control SMG excision by heat application. The DNA plasmid containing a visual marker gene flanked by I-SceI target sites and the heat-inducible I-SceI gene has been created and confirmed. Arabidopsis thaliana plants have been transformed with the plasmid, which will be used for testing the efficiency of HS:I-SceI in excising DNA from plant genomes

Heat‐shock‐inducible CRISPR/Cas9 system generates heritable mutations in rice

Summary Transient expression of CRISPR/Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat‐shock‐inducible CRISPR/Cas9 for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using the soybean heat‐shock protein gene promoter and the rice U3 promoter to express Cas9 and sgRNA, respectively, we developed the heat‐shock (HS)‐inducible CRISPR/Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted in rice, and the presence of targeted mutations was determined before and after the HS treatment. Only a low rate of targeted mutagenesis was detected before HS (~16%), but an increased rate of mutagenesis was observed after the HS treatment among the transgenic lines (50–63%). Analysis of regenerated plants harboring HS‐CRISPR/Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS, which was detectable by Sanger sequencing after a few weeks of HS treatments. Most importantly, the HS‐induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutations that independently segregated from the Cas9 gene. Additionally, off‐target mutations were either undetectable or found at a lower rate in HS‐CRISPR/Cas9 lines as compared to the constitutive‐overexpression CRISPR/Cas9 lines. Taken together, this work shows that HS‐CRISPR/Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome‐wide off‐target effects and improving the precision of genome editing

Сравнително Изследване Върху Характеризирането На Три Стафилококови Изолата От Различен Произход

Two strains of Staphylococcus isolated from urine and sputum samples were compared with a laboratory strain of Staphylococcus. Since all three grew on the same media specific for S. aureus, they were studied for their relatedness by morphological, biochemical and molecular characterization. Variation in properties among the isolates pointed towards their different identity which was confirmed through 16S rRNA gene sequence based phylogenetic analysis. The isolate A from urine was found to be closely related to S. haemolyticus, while isolate P from sputum was S. aureus and isolate S from NICED culture collection was closest to S. epidermidis. Thus the Hicrome aureus agar (Himedia M1468) was not specific for the species S. aureus and could only screen at genus level. The antibiotic profile of the strains was evaluated using a spectrum of 18 antibiotics. The lecithinase activity was retained only in freshly isolated strains and totally ceased in subsequent subculturing.Два щама Staphylococcus , изолирани от проби на урина и слюнка, са срав- нени с лабораторен щам на Staphylococcus. Тъй като и трите изолата рас- тат на средата специфична за S. aureus, тяхното родство е изследвано по морфологични, биохимични и молекулярни характеристики. Вариацията в свойствата на изолатите, сочеща за различна идентичност, е потвърдена чрез филогенетичен анализ на база 16S рРНК. Установено е, че изолат А от урина е тясно родствен на S. haemolyticus, докато изолат Р от слюнка е S. aureus, а изолат S от колекцията NICED е най-близък до S. epidermidis. Следователно средата Hicrome aureus agar (Himedia M1468) не е специфична за вид S. aureus, а може да диференцира само до ниво род. Антибиотичният профил на щамовете е изпитан чрез спектър от 18 антибиотици. Лецити- назната активност се запазва само при прясно изолирани щамове и напълно се губи при последващо субкултивиране

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Oriented Total-Coloring of Oriented Graphs

A proper $n$-coloring of a graph $G$ is an assignment of colors from $\{1,\ldots,n\}$ to its vertices such that no two adjacent vertices get assigned the same color. The chromatic number of $G$, denoted by $\chi(G)$, refers to the smallest $n$ such that $G$ admits a proper $n$-coloring. This notion naturally extends to edge-colorings (resp. total-colorings) when edges (resp. both vertices and edges) are to be colored, and this provides other parameters of $G$: its chromatic index $\chi'(G)$ and its total chromatic number $\chi''(G)$.These coloring notions are among the most fundamental ones of the graph coloring theory. As such, they gave birth to hundreds of studies dedicated to several of their aspects, including generalizations to more general structures such as oriented graphs. They include notably the notions of oriented $n$-colorings and oriented $n$-arc-colorings, which stand as natural extensions of their undirected counterparts, and which have been receiving increasing attention.Our goal is to introduce a missing piece in this line of work, namely the oriented counterparts of proper $n$-total-colorings and total chromatic number. We first define these notions and show that they share properties and connections with oriented (arc) colorings that are reminiscent of those shared by their undirected counterparts. We then focus on understanding the oriented total chromatic number of particular types of oriented graphs,such as oriented forests, cycles, and some planar graphs. Finally, we establish a full complexity dichotomy for the problem of determining whether an oriented graph is totally $k$-colorable.Throughout this work, each of our results is compared to what is known regarding the oriented chromatic number and oriented chromatic index. We also disseminate some directions for further research on the oriented total chromatic number

Two-center of the convex hull of a point set: Dynamic model, and restricted streaming model

In this paper, we consider the dynamic version of covering the convex hull of a point set P in R2 by two congruent disks of minimum size. Here, the points can be added or deleted in the set P, and the objective is to maintain a data structure that, at any instant of time, can efficiently report two disks of minimum size whose union completely covers the boundary of the convex hull of the point set P. We show that maintaining a linear size data structure, we can report a radius r satisfying r 2ropt at any query time, where ropt is the optimum solution at that instant of time. For each insertion or deletion of a point in P, the update time of our data structure is O(log n). Our algorithm can be tailored to work in the restricted streaming model where only insertions are allowed, using constant work-space. The problem studied in this paper has novelty in two ways: (i) it computes the covering of the convex hull of a point set P, which has lot of surveillance related applications, but not studied in the literature, and (ii) it also considers the dynamic version of the problem. In the dynamic setup, the extent measure problems are studied very little, and in particular, the k-center problem is not at all studied for any k2

Targeting TOR and SnRK1 Genes in Rice with CRISPR/Cas9

Genome targeting with CRISPR/Cas9 is a popular method for introducing mutations and creating knock-out effects. However, limited information is currently available on the mutagenesis of essential genes. This study investigated the efficiency of CRISPR/Cas9 in targeting rice essential genes: the singleton TARGET OF RAPAMYCIN (OsTOR) and the three paralogs of the Sucrose non-fermenting-1 (SNF1)-related kinase 1 (OsSnRK1α), OsSnRK1αA, OsSnRK1αB and OsSnRK1αC. Strong activity of constitutively expressed CRISPR/Cas9 was effective in creating mutations in OsTOR and OsSnRK1α genes, but inducible CRISPR/Cas9 failed to generate detectable mutations. The rate of OsTOR mutagenesis was relatively lower and only the kinase domain of OsTOR could be targeted, while mutations in the HEAT region were unrecoverable. OsSnRK1α paralogs could be targeted at higher rates; however, sterility or early senescence was observed in >50% of the primary mutants. Additionally, OsSnRK1αB and OsSnRK1αC, which bear high sequence homologies, could be targeted simultaneously to generate double-mutants. Further, although limited types of mutations were found in the surviving mutants, the recovered lines displayed loss-of-function or knockdown tor or snrk1 phenotypes. Overall, our data show that mutations in these essential genes can be created by CRISPR/Cas9 to facilitate investigations on their roles in plant development and environmental response in rice

Virulence Characteristics and Molecular Epidemiology of Enteroaggregative Escherichia coli Isolates from Hospitalized Diarrheal Patients in Kolkata, India

Enteroaggregative Escherichia coli (EAEC) is an important diarrheal enteropathogen defined by aggregative adherence to cultured epithelial cells. We have detected EAEC from 121 (6.6%) of 1,826 hospitalized patients admitted with diarrhea to the Infectious Diseases Hospital in Kolkata, India. Watery diarrhea was recorded significantly (P = 0.0142) more often in children. The majority of the EAEC isolates were not serotypeable (62%) and showed resistance to five or more antibiotics (76%). We studied different virulence genes and the molecular epidemiology of 121 EAEC isolates recovered from diarrheal patients. A PCR assay for detection of virulence genes, an assay for determination of clump formation in liquid culture, and a HeLa cell adherence assay were carried out to characterize the EAEC isolates. Investigations were also conducted to correlate the virulence gene profiles with diarrheal symptoms and molecular epidemiology by pulsed-field gel electrophoresis (PFGE). Two or more virulence genes were detected in 109 (90.1%) EAEC isolates. In the cluster analysis, some isolates with specific gene profiles and phenotypes formed a group or subcluster. This study highlights the comparative distributions of three fimbrial adhesins and other virulence genes among EAEC isolates. The diverse virulence gene and PFGE profiles, along with the existence of diverse serotypes and antibiograms, suggests that the EAEC isolates are genetically heterogeneous in Kolkata