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Related Experiment Videos

Construction of stable episomes in Cryptococcus neoformans

A Varma1, K J Kwon-Chung

  • 1Molecular Microbiology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Building 10, Room 11C304, National Institutes of Health, Bethesda, MD 20892, USA.

Current Genetics
|July 31, 1998
PubMed
Summary

Researchers enhanced plasmid stability and transformation efficiency in Cryptococcus neoformans by incorporating DNA sequences from a stable minichromosome. These sequences improved uracil prototrophy stability and episomal maintenance, crucial for genetic engineering.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Mycology

Background:

  • Plasmid instability and low transformation frequencies are common challenges in genetic manipulation of microorganisms.
  • Stable minichromosomes are valuable resources for identifying DNA elements that confer stability and enhance transformation.

Purpose of the Study:

  • To identify and characterize DNA sequences from a stable minichromosome that enhance plasmid stability and transformation efficiency in Cryptococcus neoformans.
  • To investigate the role of specific DNA elements, including telomeric repeats and ARS-like sequences, in improving plasmid performance.

Main Methods:

  • Construction of plasmids by inserting DNA fragments from a stable minichromosome into unstable vectors.
  • Electroporative transformation of Cryptococcus neoformans with engineered plasmids.

Related Experiment Videos

  • Assessment of transformation frequencies and uracil prototrophy stability on non-selective media.

    • Main Results:

    • A 6-kb insert significantly increased URA5 transformant frequencies and uracil prototrophy stability.
    • A 1.5-kb fragment containing telomeric repeats enhanced plasmid transformation frequency.
    • A 1081-bp sequence (STAB) exhibited ARS-like function, promoting episomal plasmid maintenance with different selection markers (ADE2/URA5).

    Conclusions:

    • Specific DNA sequences derived from stable minichromosomes can be effectively used to engineer more stable and efficiently transformable plasmids.
    • Telomeric repeats and ARS-like elements play critical roles in enhancing both transformation efficiency and episomal stability of plasmids in Cryptococcus neoformans.