Search results for " cloning"

Variation in gene expression within clones of the earthworm Dendrobaena octaedra

2017

Gene expression is highly plastic, which can help organisms to both acclimate and adapt to changing environments. Possible variation in gene expression among individuals with the same genotype (among clones) is not widely considered, even though it could impact the results of studies that focus on gene expression phenotypes, for example studies using clonal lines. We examined the extent of within and between clone variation in gene expression in the earthworm Dendrobaena octaedra, which reproduces through apomictic parthenogenesis. Five microsatellite markers were developed and used to confirm that offspring are genetic clones of their parent. After that, expression of 12 genes was measured…

An isoleucine-leucine substitution in chloroplastic acetyl-CoA carboxylase from green foxtail (Setaria viridis L. Beauv.) is responsible for resistan…

2002

The cDNAs encoding chloroplastic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) from three lines of Setaria viridis (L. Beauv.) resistant or sensitive to sethoxydim, and from one sethoxydim-sensitive line of Setaria italica (L. Beauv.) were cloned and sequenced. Sequence comparison revealed that a single isoleucine-leucine substitution discriminated ACCases from sensitive and resistant lines. Using near-isogenic lines of S. italica derived from interspecific hybridisation, we demonstrated that the transfer of the S. viridis mutant ACCase allele into a sethoxydim-sensitive S. italica line conferred resistance to this herbicide. We confirmed this result using allele-specific polymerase chain rea…

Patented intelligence: Cloning human decision models for Industry 4.0

2018

Industry 4.0 is a trend related to smart factories, which are cyber-physical spaces populated and controlled by the collective intelligence for the autonomous and highly flexible manufacturing purposes. Artificial Intelligence (AI) embedded into various planning, production, and management processes in Industry 4.0 must take the initiative and responsibility for making necessary real-time decisions in many cases. In this paper, we suggest the Pi-Mind technology as a compromise between completely human-expert-driven decision-making and AI-driven decision-making. Pi-Mind enables capturing, cloning and patenting essential parameters of the decision models from a particular human expert making …

2017

Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-…

2020

Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a s…

A more efficient cutting planes approach for the green vehicle routing problem with capacitated alternative fuel stations

2021

AbstractThe Green Vehicle Routing Problem with Capacitated Alternative Fuel Stations assumes that, at each station, the number of vehicles simultaneously refueling cannot exceed the number of available pumps. The state-of-the-art solution method, based on the generation of all feasible non-dominated paths, performs well only with up to 2 pumps. In fact, it needs cloning the paths between every pair of pumps. To overcome this issue, in this paper, we propose new path-based MILP models without cloning paths, for both the scenario with private stations (i.e., owned by the fleet manager) and that with public stations. Then, a more efficient cutting plane approach is designed for addressing both…

Specific bovine antibody response against a new recombinant Cryptosporidium parvum antigen containing 4 zinc-finger motifs

2002

A Cryptosporidium parvum sporozoite and oocyst lambda gt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicity of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cryptosporidium immune calv…

Cloning and characterization of the phenylalanyl-tRNA synthetase β subunit gene fromCandida albicans

1998

A Candida albicans expression library was constructed from RNA isolated from regenerating protoplasts. A 1.4-kb cDNA clone was used to isolate a genomic fragment. Sequence analysis revealed an open reading frame of 593 amino acids with an overall identity of 63.6% with the phenylalanyl-tRNA synthetase beta subunit (FRS1) of Saccharomyces cerevisiae. We named it CaFRS1. It is located in a single copy in chromosome R, SfiI fragment M. Its expression showed a decrease during the cell wall regeneration process in protoplasts of both yeast and mycelial cells of C. albicans, suggesting its requirement thereof in initial steps of the cell wall synthesis.

Artificial chromosomes for antibiotic-producing actinomycetes.

2000

Bacteria belonging to the order Actinomycetales produce most microbial metabolites thus far described, several of which have found applications in medicine and agriculture. However, most strains were discovered by their ability to produce a given molecule and are, therefore, poorly characterized physiologically and genetically. Thus, methodologies for genetic manipulation of actinomycetes are not available and efficient tools have been developed for just a few strains. This constitutes a serious limitation to applying molecular genetics approaches to strain development and structural manipulation of microbial metabolites. To overcome this hurdle, we have developed bacterial artificial chrom…

Repetitive nucleotide sequencing of a dispensable DNA segment in a clonal population of African swine fever virus

1991

Abstract Repetitive nucleotide sequencing of a dispensable genomic segment of a clonal population of African swine fever (ASF) virus has been carried out to estimate the mutant frequency to neutral alleles. Since no mutations have been detected in a total of 54026 nucleotides screened, the maximum mutant frequency is 5.5 × 10 −5 substitutions/nucleotide (95% confidence level). The result renders very unlikely the occurrence of hypermutational events during ASF virus DNA replication, at least within the selected DNA fragment.