Restriction digests of the clone give the following sizes (kb): HindIII--5.0, 3.1, 2.6, 1.3; BamHI/EcoRI--8.6, 1.9, 1.8; EcoRI--8.6, 3.3. Homologous recombination between pRAN4 and a clone in pYAC4 results in replacement of sequences in the right arm of pYAC4 with those of pRAN4 encoding ampR, G418R, and ADE2. The 1.8 kb BamHI-EcoRI fragment of pRAN4 is homologous to the SmaI-ApaI fragment of pYAC4. Transform S. cerevisiae containing YAC clones with BamHI-linearized pRAN4 and select for recombinants that grow in the absence of adenine (Ura- Ade+ Trp+). Maintenance of the E. coli ori, ampR and kanR genes allows plasmid rescue of YAC insert ends. Restriction sites available for right-end plasmid rescue include XhoI, XbaI, BglII and HindIII. Modification shuttle vector to construct larger yeast artificial chromosomes (YACs) by mitotic recombination. Also facilitates transfer to mammalian cells. The order of the major features of the plasmid is: BamHI - 1.8 kb pYAC4 homologous sequence - EcoRI - pMB1 ori/ampR - HindIII - SV40 ori - BglII - neo (kanR, G418R) - SmaI - ADE2 - EcoRI - HindIII - Tetrahymena telomere. |
