Exceptional Chromosome Regions II



Exploring TAR cloning for isolation and analysis of exceptional regions of human genome

Natalay Kouprina, Carl Barrett, and Vladimir Larionov
Laboratory of Biosystems and Cancer, Center for Cancer Research, National Cancer Institute, NIH, Bethesda MD 20854

The TAR (Transformation-Associated Recombination) cloning technique allows a direct isolation of specific chromosomal regions and genes from mammalian genomes without a time-consuming step of construction of a representative library of random clones. The technique is based on homologous recombination between a vector containing a gene-specific sequence and a genomic DNA fragment during co-transformation into yeast spheroplasts. Using this technique, chromosomal regions up to 400 kb can be rescued in yeast as circular YACs. These YACs can be easily retrofitted into BACs and transferred into E. coli cells for further structural and functional studies. A new technology has several potential utilities for a human genome project. Among them: i) verification of the assembled contigs, ii) closing the gaps and iii) isolation of centromeric regions and other exceptional regions that can not be cloned by a routine technique based on in vitro ligation.

Isolation of exceptional regions that are not clonable in E. coli cells

During last two years a TAR cloning strategy was successfully applied in our lab for isolation of dozen of genes entire copies of those have not been found in BAC libraries. Most of the genes were structurally stable during propagation in yeast cells as YACs and in E. coli cells as BACs. However, some genes, including the metastasis-suppressor gene KAI1 and the mouse mucin gene MUC2 were stable in yeast cells but can not be stably transferred into E. coli cells. During electroporation, the YAC/BACs containing these genes exhibited a low efficiency of E. coli transformation (approximately 100 times lower compared to that of other randomly selected BACs with a similar size insert). Moreover, all the transformants obtained contained large deletions eliminating up to 90% of the insert. We demonstrated that such inserts represent a significant fraction of human genome (~6%). Analysis of such regions requires isolation of circular YACs directly from yeast cells.

Verification of the assembled contigs

End sequencing of YAC/BAC clones isolated from human genome by TAR allows to verify a quality of contigs assembling. Such analysis revealed several mistakes. Some YAC/BAC clones contained inserts with sizes bigger than that in contigs, suggesting a loss of internal sequences during assembling of the contigs. Other clones contained sequences mapped to different contigs. Thus, the TAR cloning technique can greatly simplify verification of contigs assembling.

Isolation of centromeric and pericentromeric regions

The TAR cloning technique has been also applied for isolation and characterization of human centromeric regions. These regions remain the most poorly mapped areas in the human genome and they are underrepresented in the libraries. Using the TAR technique we isolated and physically characterized centromeric and pericentromeric regions from seven human chromosomes (2, 5, 8, 15, 16, 22 and X).

Therefore, we conclude that TAR cloning provides a powerful tool for verification of the genome draft sequence and for development of continuous sequences of human chromosomes. Significance of TAR cloning may be even more important for mouse genome that has not been physically mapped beforehand.

Last modified: Wednesday, October 22, 2003

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