Amplification of Large Artificial Chromosomes

Yeast artificial chromosome cloning is an attractive technology for genomic mapping studies because very large DNA segments can be readily propagated. However, detailed analyses often require the extensive application of blotting-hybridization techniques because artificial chromosomes are normally p...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1990-11, Vol.87 (21), p.8242-8246
Main Authors: Smith, Douglas R., Smyth, Adrienne P., Moir, Donald T.
Format: Article
Language:English
Subjects:
Artificial chromosomes
Base Sequence
Biological and medical sciences
Biotechnology
Centromeres
Chromosomes
Chromosomes, Fungal
Cloning, Molecular - methods
DNA
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Gels
Genes
Genetic engineering
Genetic technics
Genetic Vectors
Methods. Procedures. Technologies
Molecular cloning
Molecular Sequence Data
Nucleic Acid Amplification Techniques
Nucleic Acid Hybridization
Oligonucleotide Probes
Plasmids
Restriction Mapping
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - growth & development
Sulfanilamides
Yeasts
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Summary:Yeast artificial chromosome cloning is an attractive technology for genomic mapping studies because very large DNA segments can be readily propagated. However, detailed analyses often require the extensive application of blotting-hybridization techniques because artificial chromosomes are normally present at only one copy per haploid genome. We have developed a cloning vector and host strain that alleviate this problem by permitting copy number amplification of artificial chromosomes. The vector includes a conditional centromere that can be turned on or off by changing the carbon source. Strong selective pressure for extra copies of the artificial chromosome can be applied by selecting for the expression of a heterologous thymidine kinase gene. When this system was used, artificial chromosomes ranging from about 100 to 600 kilobases in size were readily amplified 10- to 20-fold. The selective conditions did not induce obvious rearrangements in any of the clones tested. Reactivation of the centromere in amplified artificial chromosome clones resulted in stable maintenance of an elevated copy number for 20 generations. Applications of copy number control to various aspects of artificial chromosome analysis are addressed.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.87.21.8242