| The pufferfish, Fugu rubripes, has been established as an ideal model for the study of vertebrate genomes. Construction of a highy stable and faithfully representative library with large insert fragments is the prerequisite to genome analysis, comparative genomics study,and isolations of intact genes. The BAC library is the best library with large insert fragments to date.We have constructed an arrayed bacterial aitificial chromosome (BAC) library from Fugu rubripes sperm DNA using the second generation BAC vector, pBeloBACll. The library now consists of 23,040 clones arrayed in 240 96 -well miorotiter plates. Analysis of 100 randomly selected BAC clones indicates that 99% of the clones contain inserts ranging from 25 to 450 kb with an average size of approximately 100kb. Thus it is estimated to represent a nearly 6 - fold coverage of whole Fugu haploid genome and to provide 99. 7% probability to find any unique DNA fragment in the pufferfish genome. Stability test with 10 BAC clones containg inserts larger than 100kb showed that they were stable for at leastlOO cell generations of serial propagation. In the "freeze —thaw"test,more than 50% bacterial cells in sample wells were alive after 10 times "freeze —thaw". To our knowledge,this is the the first BAC library for this newly introduced model organism.We have also prepared middle —density hybridization filters for this BAC library using a tool designed by ourselves . The filters have been probed with radi-olabeled full length cDNA of human multi — drug resistance gene 1(MDR1)and its 5'sequence and 3'sequence amplified by PCR. Four candidate BAC clones containing full length pufferfish homologue of multi —drug resistance gene 1 have been obtained through screening. Southern blot analysis and partial sequencing showed that the candidate BAC clones actually contained pufferfish homologue of...
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