For the functional analysis of many genes investigators need to isolate and manipulate large DNA fragments. The advent of genomics and the study of genomic regions of DNA has generated a need for vectors capable of carrying large DNA regions.
In general, two types of yeast vector systems are presently available. The first type of vector is one capable of transferring small insert DNA between yeast and bacteria (33, 34). A second type of vector is a fragmenting vector which creates interstitial or terminal deletions in YACs (35, 36, 37). The small insert shuttle vectors are able to recombine with and recover homologous sequences. They are centromere-based and replicate stably and autonomously in yeast, but also contain a high-copy origin of replication for maintenance as bacterial plasmids. However, these vectors are limited by their small insert capacity.
The second type of vector, known as fragmenting vectors, also have recombinogenic sequences but are unable to transfer the recovered insert DNA to bacteria for large preparations of DNA.
Researchers use fragmentation techniques to narrow down the region of interest in YACs (1, 2). However, isolating sufficient quantities of YAC DNA from agarose gels for microinjection or electroporation remains cumbersome. Purification remains a problem when the YAC comigrates with an endogenous chromosome. In addition, YACs may be chimeric or contain additional DNA regions that are not required for the particular functional study.
Types of vectors available for cloning large fragments in bacteria are cosmids, P1s and BACS. These vectors are limited to bacteria and cannot be shuttled to yeast for modification by homologous recombination.
One object of the present invention relates to the identification of a new yeast-bacteria shuttle vector capable of accommodating large fragments of DNA.
Another object of the present invention relates to the use of the new yeast bacteria shuttle vector for cloning large DNAs.
Yet another object of the present invention relates to the use of the new yeast bacteria for site-specific targeting of genomic DNA.