Margit Burmeister

Physical mapping of yeast artificial chromosomes containing sequences from the human β-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.

Construction of Lambda Libraries from Large PFGE Fragments

Pulsed-field gel electrophoresis (PFGE) has the capacity to fractionate large fragments of DNA up to thousands of kilobases in size. This aspect of the technique has been exploited for constructing long-range restriction maps of chromosomes from many different species including humans (see Chapters 14 , 15 , and 18 ). Besides its use for analytical purposes, PFGE has also been used as a preparative tool. Intact DNA obtained from preparative PFGE gels has been used for cloning into yeast artificial chromosome (MC) vectors (see Chapter 16 ) and for constructing jumping libraries (1). In addition, DNAeluted from PFGE gels has been used for generating libraries with a smaller insert size (2-7). In this latter procedure, DNA from a somatic cell hybrid is digested with a rare-cutting restriction enzyme, separated by PFGE, and the DNA from a particular PFGE fragment is eluted, digested, and cloned into a plasmid or phage vector. The resulting library is then screened with a species-specific probe to identify DNA segments from the donor chromosome of the hybrid. This use of preparative PFGE has had widespread application in the cloning of DNA close to several important disease genes, namely cystic fibrosis (4,6), Duchenne muscular dystrophy (2), choroideremia (7), polycystic kidney disease (3), and Huntington disease (5).