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Related Concept Videos

Reproductive Cloning01:27

Reproductive Cloning

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The first successfully cloned mammal was Dolly, a sheep, born on 5th July 1996 at Roslin Institute, Scotland. The cloned sheep was named after the American singer Dolly Parton. Dolly lived for seven years and died of respiratory complications, which is speculated to be due to the actual age of her DNA. Because the DNA in cloned cells belongs to an older individual,  the cloned individual’s life expectancy may be affected. Indeed, analysis of Dolly’s DNA revealed shorter...
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Proteins can form homomeric complexes with another unit of the same protein or heteromeric complexes with different types.  Most protein complexes self-assemble spontaneously via ordered pathways, while some proteins need assembly factors that guide their proper assembly. Despite the crowded intracellular environment, proteins usually interact with their correct partners and form functional complexes.
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Spindle assembly occurs through three, often coexisting, pathways – the centrosome-mediated pathway, the chromatin-mediated pathway, and the microtubule-mediated pathway – collectively contributing to form a robust spindle apparatus.
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Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
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Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun
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An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly.

Chenhao Li1,2, Chonghui Ji2, José C Huguet-Tapia3

  • 1Department of Plant Pathology, Nanjing Agricultural University, Nanjing, 210095, Jiangsu Providence, P.R. China.

Molecular Plant Pathology
|August 16, 2019
PubMed
Summary

We developed an efficient method to clone Transcription Activator-Like effectors (TALes) from pathogenic bacteria. This new technique significantly speeds up the cloning process for these complex infection-related genes.

Keywords:
XanthomonasGibson assemblyTAL effectorsrice

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Plant Pathology

Background:

  • Transcription Activator-Like effectors (TALes) are crucial type III bacterial effectors involved in plant pathogen infection.
  • Xanthomonas oryzae pv. oryzae (Xoo) possesses numerous TALe genes, posing cloning challenges due to high copy numbers and repetitive sequences.
  • Limited TALe gene characterization hinders understanding of their roles in pathogenicity.

Purpose of the Study:

  • To develop an efficient and rapid method for cloning Transcription Activator-Like effector (TALe) genes.
  • To overcome the technical difficulties associated with cloning multiple, highly repetitive TALe genes from bacterial genomes.
  • To facilitate the functional analysis of TALe genes in plant-pathogen interactions.

Main Methods:

  • Utilized in vitro homologous recombination and single-strand annealing for selective TALe gene cloning.
  • Employed Gibson assembly strategy with two complementary vectors designed to capture all TALe genes.
  • Tested the cloning approach on four different Xoo strains.

Main Results:

  • Successfully cloned the full complement of TALe genes from four Xoo strains using the novel method.
  • The new technique reduced the cloning and functional analysis timeline to approximately one month, a significant improvement over previous methods.
  • Demonstrated the feasibility and efficiency of the developed cloning strategy.

Conclusions:

  • The developed cloning technique is a robust tool for advancing TALe biology research.
  • This approach offers a potential solution for cloning multiple copies of highly competitive DNA elements from various genomes.
  • Facilitates broader functional analysis of TALes and their role in bacterial pathogenesis.