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Published on: January 26, 2017
Yeast artificial chromosome fragmentation vectors that utilize URA3 selection
J R Cook1, S L Emanuel, S Pestka
1Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635.
Genetic Analysis, Techniques and Applications
|January 1, 1993
Summary
Researchers developed fragmentation vectors to create smaller yeast artificial chromosomes (YACs) from large human DNA segments. This method aids in mapping human chromosomes and functional analysis of YACs in eukaryotic cells.
Area of Science:
- Molecular Biology
- Genetics
- Yeast Genetics
Background:
- Yeast artificial chromosomes (YACs) are crucial for maintaining and studying large human genomic DNA fragments.
- Efficient methods for YAC fragmentation are needed for detailed mapping and analysis.
Purpose of the Study:
- To develop novel fragmentation vectors (pSE1 and pSE2) for YACs containing human genomic DNA.
- To establish a method for generating YAC deletion sets for precise genomic region analysis.
Main Methods:
- Development of pSE1 and pSE2 fragmentation vectors.
- Transformation of Ura- yeast cells containing YACs with the vectors.
- Selection using 5-fluoro-orotic acid and subsequent selection for Ura+ phenotype.
- Generation of fragmented YACs with reduced molecular size.
Main Results:
- Over 80% of transformants yielded YACs of reduced molecular size.
- Successful generation of fragmented YACs using URA3 transformation.
- Linkage of a neomycin resistance gene to URA3 facilitates functional analysis.
Conclusions:
- The developed fragmentation vectors and URA3-based method enable efficient YAC deletion set generation.
- Fragmented YACs are valuable tools for mapping human chromosomal regions.
- This approach facilitates functional analysis of YACs in eukaryotic systems.
