scholarly journals A Tn 10-lacZ-kanR-URA3 Gene Fusion Transposon for Insertion Mutagenesis and Fusion Analysis of Yeast and Bacterial Genes

Genetics ◽  
1987 ◽  
Vol 116 (2) ◽  
pp. 191-199 ◽  
Author(s):  
Olivier Huisman ◽  
Wendy Raymond ◽  
Kai-Uwe Froehlich ◽  
Patrick Errada ◽  
Nancy Kleckner ◽  
...  
Keyword(s):  
Region Of Interest ◽  
Single Step ◽  
Insertion Mutagenesis ◽  
Lacz Gene ◽  
Ura3 Gene ◽  
New Variant ◽  
Fusion Analysis ◽  
Bacterial Genes ◽  
Yeast Genes ◽  
Insertion Mutations

ABSTRACT We describe here a new variant of transposon Tn 10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes. We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae. This transposon, nicknamed mini-Tn 10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant. The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene. Other yeast genes can easily be substituted for URA3 in the available constructs. The mini-Tn 10-LUK system has several important advantages. (1) Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA. It is easy to obtain enough insertions to sensitively define the functional limits of a gene. (2) Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E. coli. (3) The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene. (4) Under favorable circumstances, usable lacZ protein fusions are created. (5) Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.

scholarly journals A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

Genetics ◽  
1987 ◽  
Vol 117 (1) ◽  
pp. 5-12
Author(s):  
Eric Alani ◽  
Nancy Kleckner
Keyword(s):  
Gene Fusion ◽  
Enzyme Assay ◽  
Decarboxylase Activity ◽  
Lacz Gene ◽  
Coding Region ◽  
Ura3 Gene ◽  
Promoter Sequences ◽  
E Coli ◽  
New Type ◽  
Fusion Analysis

ABSTRACT We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5′-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.

A Kalman filtering fuzzy logic algorithm for recognition of lane departure

2021 ◽  
pp. 1-8
Author(s):  
Kai Ren
Keyword(s):  
Decision Making ◽  
Fuzzy Logic ◽  
Kalman Filtering ◽  
Traffic Accidents ◽  
Region Of Interest ◽  
Fuzzy Logic Algorithm ◽  
The Road ◽  
Departure Decision ◽  
Lane Departure ◽  
Fusion Analysis

In all kinds of traffic accidents, the unconscious departure of the vehicle from the lane is one of the most important reasons leading to the occurrence of these accidents. In view of the specific problem of lane departure, a lane departure decision-making method is established without calibration relying on the Kalman filtering fuzzy logic algorithm, according to the characteristics of expressway lanes, based on the machine vision and hearing fusion analysis of lane departure, integrating the extraction of the linear lane line model and the region of interest (ROI) in this paper to judge the degree of vehicle departure from the lane by integrating the slope values of the 2 lane lines in the road image. The results show that the system has good lane recognition capabilities and accurate departure decision-making capabilities, and meet the lane departure warning requirements in the expressway environment.

Interaction of GAL4 and GAL80 gene regulatory proteins in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3446-3451
Author(s):  
N F Lue ◽  
D I Chasman ◽  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Single Step ◽  
Regulatory Proteins ◽  
Mobility Shift ◽  
Ura3 Gene ◽  
Upstream Activation Sequence ◽  
Chromatographic Procedure ◽  
Gene Regulatory ◽  
Dna Complex ◽  
Activation Sequence

The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

scholarly journals Interaction of GAL4 and GAL80 gene regulatory proteins in vitro.

1987 ◽  
Vol 7 (10) ◽  
pp. 3446-3451 ◽  
Author(s):  
N F Lue ◽  
D I Chasman ◽  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Single Step ◽  
Regulatory Proteins ◽  
Mobility Shift ◽  
Ura3 Gene ◽  
Upstream Activation Sequence ◽  
Chromatographic Procedure ◽  
Gene Regulatory ◽  
Dna Complex ◽  
Activation Sequence

The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

scholarly journals A New Pathotype of Puccinia striiformis f. sp. tritici on Wheat in South Africa

Plant Disease ◽  
1999 ◽  
Vol 83 (6) ◽  
pp. 591-591 ◽  
Author(s):  
W. H. P. Boshoff ◽  
Z. A. Pretorius
Keyword(s):  
South Africa ◽  
Stripe Rust ◽  
Puccinia Striiformis ◽  
Single Step ◽  
Western Cape ◽  
Monogenic Resistance ◽  
Major Resistance Gene ◽  
New Variant ◽  
Production Areas ◽  
Wheat Lines

Following the detection of Puccinia striiformis f. sp. tritici for the first time on wheat (Triticum aestivum) in the Western Cape in August 1996, stripe rust has spread to all the important wheat production areas in South Africa. Only the introduced pathotype (pt. 6E16) was detected in surveys of these areas during 1996 and 1997. In 1998, a severe stripe rust epidemic occurred in the eastern Free State on the extensively grown cultivars Hugenoot and Carina, both which are resistant to pt. 6E16. Stripe rust severities of 100% were common on flag and lower leaves, and widespread applications of fungicides were necessary. Avirulence/virulence characteristics of P. striifomis f. sp. tritici isolates collected from Hugenoot and Carina were determined on 17 standard stripe rust differential wheat lines and 11 supplementary testers. The latter testers included the wheat lines TP981 and TP1295 (supplied by R. Johnson, Cambridge, UK), both of which have a major resistance gene in common with the differentials Heines Peko, Reichersberg 42, Strubes Dickkopf, Clement, and Heines VII (1). Isolates obtained from Hugenoot and Carina differed from pt. 6E16 based on virulence to Reichersberg 42 (Yr7,25), Heines Peko (Yr2,6,25), TP981 (Yr25), and TP1295 (Yr25). The new variant, designated as 6E22, was also identified in collections from the province KwaZulu-Natal. Seedling tests with 6E16 and 6E22 have shown that Hugenoot, Carina, and Tugela-DN are the only local cultivars affected by the new pathotype. The occurrence of pt. 6E22, which appears to be a single-step adaptation from 6E16 adding virulence to Yr25, emphasizes the vulnerability of monogenic resistance to this disease. Reference: (1) R. A. McIntosh et al. Wheat Inform. Serv. 85:56, 1997.

scholarly journals Iodine-Enhanced Micro-CT Imaging of Soft Tissue on the Example of Peripheral Nerve Regeneration

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Patrick Heimel ◽  
Nicole Victoria Swiadek ◽  
Paul Slezak ◽  
Markus Kerbl ◽  
Cornelia Schneider ◽  
...  
Keyword(s):  
Peripheral Nerve ◽  
Nerve Regeneration ◽  
Soft Tissues ◽  
Ex Vivo ◽  
Peripheral Nerve Regeneration ◽  
Region Of Interest ◽  
Staining Technique ◽  
Regenerative Process ◽  
Single Step ◽  
Staining Solution

Microcomputed tomography (μCT) is widely used for the study of mineralized tissues, but a similar use for soft tissues is hindered by their low X-ray attenuation. This limitation can be overcome by the recent development of different staining techniques. Staining with Lugol’s solution, a mixture of one part iodine and two parts potassium iodide in water, stands out among these techniques for its low complexity and cost. Currently, Lugol staining is mostly used for anatomical examination of tissues. In the present study, we seek to optimize the quality and reproducibility of the staining for ex vivo visualization of soft tissues in the context of a peripheral nerve regeneration model in the rat. We show that the staining result not only depends on the concentration of the staining solution but also on the amount of stain in relation to the tissue volume and composition, necessitating careful adaptation of the staining protocol to the respective specimen tissue. This optimization can be simplified by a stepwise staining which we show to yield a similar result compared to staining in a single step. Lugol staining solution results in concentration-dependent tissue shrinkage which can be minimized but not eliminated. We compared the shrinkage of tendon, nerve, skeletal muscle, heart, brain, and kidney with six iterations of Lugol staining. 60 ml of 0.3% Lugol’s solution per cm3 of tissue for 24 h yielded good results on the example of a peripheral nerve regeneration model, and we were able to show that the regenerating nerve inside a silk fibroin tube can be visualized in 3D using this staining technique. This information helps in deciding the region of interest for histological imaging and provides a 3D context to histological findings. Correlating both imaging modalities has the potential to improve the understanding of the regenerative process.

scholarly journals Iodine-enhanced micro-CT imaging of soft tissue on the example of peripheral nerve regeneration

2018 ◽  
Author(s):  
Patrick Heimel ◽  
Nicole Victoria Swiadek ◽  
Paul Slezak ◽  
Markus Kerbl ◽  
Cornelia Schneider ◽  
...  
Keyword(s):  
Peripheral Nerve ◽  
Nerve Regeneration ◽  
Soft Tissues ◽  
Ex Vivo ◽  
Peripheral Nerve Regeneration ◽  
Region Of Interest ◽  
Staining Technique ◽  
Regenerative Process ◽  
Single Step ◽  
Staining Solution

AbstractMicrocomputed tomography (μCT) is widely used for the study of mineralized tissues but a similar use for soft tissues is hindered by their low X-ray attenuation. This limitation can be overcome by the recent development of different staining techniques. Staining with Lugol’s solution, a mixture of one part iodine and two parts potassium iodide in water, stands out among these techniques for its low complexity and cost. Currently, Lugol staining is mostly used for anatomical examination of tissues. In the present study we seek to optimize the quality and reproducibility of the staining for ex vivo visualization of soft tissues in the context of a peripheral nerve regeneration model in the rat.We show that the staining result not only depends on the concentration of the staining solution, but also on the amount of stain in relation to the tissue volume and composition, necessitating careful adaptation of the staining protocol to the respective specimen tissue. This optimization can be simplified by a stepwise staining which we show to yield a similar result compared to staining in a single step. Lugol staining solution results in concentration dependent tissue shrinkage which can be minimized but not eliminated. We compared the shrinkage of tendon, nerve, skeletal muscle, heart, brain and kidney with six iterations of Lugol staining.60 ml of 0.3% Lugol’s solution per cm3 of tissue for 24h yielded good results on the example of a peripheral nerve regeneration model and we were able to show that the regenerating nerve inside a silk fibroin tube can be visualized in 3D using this staining technique. This information helps in deciding the region of interest for histological imaging and provides a 3D context to histological findings. Correlating both imaging modalities has the potential to improve the understanding of the regenerative process.

scholarly journals “Polar mutagenesis of bacterial transcriptional units using Cas12a”

2020 ◽  
Author(s):  
Antoine Graffeuil ◽  
Bernt Eric Uhlin ◽  
David A. Cisneros
Keyword(s):  
Single Step ◽  
Base Substitution ◽  
Type I ◽  
Mrna Levels ◽  
E Coli ◽  
Expression Of Genes ◽  
Type I Fimbriae ◽  
Transcriptional Units ◽  
Bacterial Genes ◽  
Terminator Sequence

AbstractBacterial genes are often organized in functionally related transcriptional units or operons. One such example is the fimAICDFGH operon, which codes for type I fimbriae in Escherichia coli. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in the fim operon. Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 30%, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Our results showed that some of the obtained mutants, including one with a single base substitution at the fim locus, had decreased mRNA levels of fimA, suggesting that the regulation of the fim operon was disrupted. We corroborated the polar effect of these mutants by phenotypic assays and quantitative PCR, showing up to a 43 fold decrease in expression of genes downstream fimA. We believe this strategy could be useful in engineering the transcriptional shut-down of multiple genes in one single step. For bio-production in E. coli, this opens the possibility of inhibiting competing metabolic routes.

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