Large DNA cloning system based on yeast artificial chromosomes

DETAILED DESCRIPTION OF THE INVENTION While the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the present invention, it is believed that the invention will be better understood from the following description of preferred embodiments taken in connection with the accompanying drawings in which briefly: FIG.
1 is a diagrammatic representation which shows the yeast plasmid vector pYAC2 and its use in the construction of a linear yeast artificial chromosome (YAC) in one embodiment of the invention.
FIG.
2 shows the results of electrophoresis of 5 YAC clones derived from the pYAC2 plasmid of FIG.
1.
FIG.
3 shows the results of electrophoresis of a YAC human/yeast clone HY1 of FIG.
2, using the yeast host strain AB1380, demonstrating that the DNA insert is a single large Sma I fragment.
FIG.
4 shows the results of electrophoresis of a YAC yeast/yeast clone YY1 of FIG.
2 demonstrating that the large yeast Sma I fragment of the clone is also present in the genome of the source yeast.
FIG.
5 shows the results of electrophoresis demonstrating the indirect-end-label mapping of the clone of FIG.
4 with BamH I.
FIG.
6 is a diagrammatic representation which shows the yeast plasmid vector pYAC4 in another embodiment of the invention.
FIG.
7 shows the results of electrophoresis demonstrating the sizing of yeast artificial chromosomes present in ten transformants generated by cloning human DNA fragments into the yeast plasmid vector pYAC4 of FIG.
6.
Plasmids pYAC2 and pYAC4 are on deposit without restriction at the American Type Culture Collection, Rockville, Md.
, under accession numbers ATCC 67380 and ATCC 67379, respectively.
Saccharomyces cerevisiae strain AB1380 is similarly on deposit without restriction at said depository under accession number ATCC 20843.
The preferred embodiments of the invention described herein employ the use of a number of commonly available restriction endonucleases which are identified below with their corresponding recognition sequences and (indicated by arrow) cleavage patterns