STREAMLINED APPROACH TO CREATING YEAST ARTIFICIAL CHROMOSOME LIBRARIES FROM SPECIALIZED CELL SOURCES

The study of tumor-specific chromosomal abnormalities has been severely impeded by an inability to link cytogenetic to molecular data. Restriction fragment length polymorphism mapping of any particular chromosomal rearrangement to the resolution limit of genetic methodology generates sets of probes that frequently are still too widely spaced to render the rearrangement breakpoints accessible to molecular isolation. The stable propagation of genomic fragments of up to one million base pairs in size as yeast artificial chromosomes (YACs) represents an important development in this regard. However, existing YAC libraries have been made from karyotypically normal sources making the localization and cloning of specific rearrangement breakpoints much more difficult. As a solution to this problem, we present an improved method for creating YAC libraries that can utilize specialized tumor-derived materials and that can be executed effectively in a small laboratory setting. Procedures that enabled more consistent DNA insert size selection and enhanced yeast transformation frequency were employed to generate a human YAC library from a neuroepithelioma cell line containing a characteristic t(11;22) chromosomal translocation. Approximately 40,000 colonies with an average insert size of 330 kilobase pairs were created. This library was screened with two single-copy probes that bracket the translocation breakpoint. YAC clones ranging from 370 to 550 kilobase pairs that were specific for each single-copy probe were identified. Specialized YAC libraries will make many more tumor-specific chromosomal abnormalities accessible to molecular isolation.