GENOMICS lo, 976-984 (1991) Physical Mapping of Yeast Artificial Chromosomes Containing Sequences from the Human @-Globin Gene Region KARIN M. L. GAENSLER,” MARGIT BURMEISTER,* BERNARD H. BRowrmEIN,t PATRICIA TAiLLor+MiLLER,t AND RICHARD M. MYERS*,* Departments of *Physiology and *Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California 94143-0444; and tDepartment of Genetics and Center for Genetics in Medicine, Washington University School of Medicine, St. Louis, Missouri 63 130 Received January 21, 1991; revised April 2, 1991 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 in- tegrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human @-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 @-globin gene as ex- pected. 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 @-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the reg- ulation of expression of genes in the @globin gene cluster. 0 1991 Academic Press, Inc. INTRODUCTION The human P-globin gene locus has been exten- sively mapped and has been studied as a model system for the regulation of tissue- and developmental stage- specific gene expression. It spans approximately 60 kilobases (kb) of DNA on the short arm of chromo- some 11 and contains five functional genes (see Col- lins and Weissman, 1984, for review). These genes (the embryonic gene, E, the two fetal genes, Ay and Gy, and the adult genes, 6 and /3) are sequentially ex- pressed in three stages during ontogeny. Mutations in this gene family are responsible for the fi thalasse- mias, among the most common human genetic dis- eases worldwide. Many &-acting DNA sequences that regulate glo- bin gene expression have been identified either by studying naturally occurring mutations, such as the thalassemias, or by gene transfer studies of globin gene expression in cell lines or transgenic mice. Analy- sis of yS@ thalassemias and structural analysis of the normal fi-globin region initially demonstrated the re- quirement for a series of four erythroid-specific DNase I hypersensitive sites approximately 11-18 kb 5’ to the E gene for activation of the locus (Wood et al., 1979, Kioussis et al., 1983, Tuan et al., 1985; Curtin et aZ., 1985; Taramelli et al., 1986, Forrester et aZ., 1987, Driscoll et al., 1989). This region has been designated the domain control region (DCR; Forrester et aZ., 1987), the locus-activating region (LAR; Blom van Assendelft et aZ., 1989), and, more recently, the locus control region (LCR, Orkin, 1990). While such gene transfer studies have been infor- mative, they have been limited in two important ways. First, it has not been feasible to study the regula- tory interactions of distant &-acting sequences within the LCR in their normal position with respect to the intact P-globin region, due to the insert size limitations of plasmid and cosmid vectors. &s-acting sequences such as the LCR may be integrated both 5’ and 3’ to genes whose order and orientation are also random. The level of globin gene expression in these experiments may in part reflect the relative positions of putative c&acting sequences in the test constructs, rather than delineating their normal role during on- togeny. To definitively establish the role of each of the cis- acting elements within the @-globin locus, it would be preferable to analyze these sequences in their normal position within the locus. Recently, techniques for cloning human genomic DNA fragments of several hundred kilobases or more into yeast artificial chro- mosomes (YACs) have been described (Burke et al., 1987). We have extensively analyzed a series of YACs containing sequences from the adult human ,B-globin gene that were identified by PCR screening of a hu- man genomic YAC library (Brownstein et al., 1989). 088%7543/91$3X@ Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved. 976
