Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Efficient gene targeting in Kluyveromyces lactis.

Rolf Kooistra1, Paul J J Hooykaas, H Yde Steensma

  • 1Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.

Yeast (Chichester, England)
|July 30, 2004
PubMed
Summary

Deleting the non-homologous end joining (NHEJ) pathway significantly enhances gene targeting efficiency in Kluyveromyces lactis. This study demonstrates that inhibiting NHEJ boosts homologous recombination, improving DNA integration accuracy.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

TONSL suppresses polymerase theta-dependent tandem duplications through chromatin-guided repair.

Nature communications·2026
Same author

Division of labor within polymerase theta in repair of CRISPR-induced DNA breaks in <i>Arabidopsis thaliana</i>.

PNAS nexus·2025
Same author

Crown Gall Induced by a Natural Isolate of <i>Brucella</i> (<i>Ochrobactrum</i>) <i>pseudogrignonense</i> Containing a Tumor-Inducing Plasmid.

Microorganisms·2025
Same author

Profiling Cas9- and Cas12a-induced mutagenesis in Arabidopsis thaliana.

The Plant journal : for cell and molecular biology·2024
Same author

Genetic dissection of mutagenic repair and T-DNA capture at CRISPR-induced DNA breaks in <i>Arabidopsis thaliana</i>.

PNAS nexus·2024
Same author

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.

The Plant journal : for cell and molecular biology·2024

Area of Science:

  • Molecular Biology
  • Yeast Genetics
  • DNA Repair Mechanisms

Background:

  • DNA integration into a host genome relies on double-strand break (DSB) repair pathways.
  • Homologous recombination (HR) facilitates targeted integration, while non-homologous end joining (NHEJ) leads to random integration.
  • Kluyveromyces lactis exhibits lower gene targeting efficiency compared to Saccharomyces cerevisiae.

Purpose of the Study:

  • To investigate and enhance gene targeting efficiency in Kluyveromyces lactis.
  • To understand the roles of specific genes (KlRAD51, KlRAD52, KlKU80) in DNA repair and integration.
  • To identify methods for improving targeted DNA integration in yeast.

Main Methods:

  • Isolation and deletion mutant construction of KlRAD51, KlRAD52, and KlKU80 genes in K. lactis.

Related Experiment Videos

  • Determination of gene targeting efficiency at the KlADE2 locus using constructs with varying homologous flanking sequences.
  • Transformation of wild-type and mutant K. lactis with targeting constructs, including those with short flanking sequences and in the presence of excess small DNA fragments.

    • Main Results:

    • Wild-type K. lactis showed gene targeting efficiency from 0% to 88% depending on flanking sequence length (50-600 bp).
    • The Klku80 deletion mutant exhibited >97% gene targeting efficiency, irrespective of flanking sequence length.
    • Co-transformation with excess small DNA fragments enhanced targeting efficiency in wild-type K. lactis, even with 50 bp flanks.

    Conclusions:

    • Deletion of the NHEJ pathway significantly increases gene targeting efficiency in K. lactis.
    • Inhibiting NHEJ is a viable strategy to improve targeted DNA integration.
    • Optimized transformation methods can achieve efficient targeted gene replacement with minimal homologous sequences.