Proc. Natl. Acad. Sca. USA Vol. 76, No. 12, pp. 6196-6200, December 1979 Biochemistry Arrangement of coding and intervening sequences of chicken lysozyme gene (DNA library screening/gene isolation/gene organization) W. LINDENMAIEtt, M. C. NGUYEN-HUU*, R. LURZ, M. STRATMANN, N. BLIN, T. WURTZt, H. J. HAUSER, A. E. SIPPELt, AND G. SCHUTZ Max-Planck-Institut fur Molekulare Genetik, Berlin-Dahlem, West Germany Communicated by Wolfgang Beermann, September 13, 1979 ABSTRACT Hybrid phages that contain chicken lysozyme gene sequences have been isolated from a chicken DNA library. Two overlapping clones covering a region of 22 kilobase pairs around this gene have been studied by restriction mapping, Southern hybridization, and electron microscopic analysis of hybrids between lysozyme mRNA hnd the cloned cellular DNA. Three intervening sequences interrupt the lysozyme structural gene. The cellular gene is at least 3.9 kilobases long, about 6 times the length of the structural gene. The amino acid sequence and the three-dimensional structure of chicken egg-white lysdzyme have been established (for re- view, see ref. 1). Like the other major egg-white proteins, ovalbumin, conalbumin, and ovomucoid, it is synthesized in the chicken oviduct under the control of steroid hormones (2). The rate-limiting variable in the steroid induction of these proteins is the concentration of their mRNAs in the tubular gland cells of the oviduct (3-11). Changes in the rates of syn- thesis and degradation are responsible for the steroid-induced accumulation of the four egg-white protein mRNAs (10, 12- 18). The regulated expression of the egg-white protein genes should be based, at least in part, on the sequence and organi- zation of the DNA coding for these proteins. Therefore, we have studied the structure of the lysozyme and the ovomucoid gene. By Southern hybridization analysis (19) of cellular chicken DNA with a recombinant plasmid containing lysozyme mRNA se- quences (20), we found that the structural lysozyre gene is split by intervening sequences (21) as is the ovalbutnin gene (refs. 22 and 23 and references therein) and the ovomucoid gene (ref. 24 and unpublished data). For a more detailed analysis of the structure of this gene, we isolated hybrid clones that contain the entire lysozyme gene and the 3'- and 5'-flanking regions. MATERIALS AND METHODS DNAs. X Charon phage (25) were grown and purified and the DNA was prepared essentially as described by Blattner et al. (25, 26). HNL laying hen oviduct DNA and pls-1 plasmid DNA were prepared as described (20, 21). Hybridization Probes. pls-1 DNA (20) was labeled with [a-32P]dCTP to high specific activity (1-2 X 108 cpm/flg) by nick translation (27). [32P]cDNA was synthesized from oviduct poly(A)-RNA with avian myeloblastosis virus reverse tran- scriptase (1 1). Screening the Chicken DNA Library. The amplified chicken DNA library was screened by using the in situ plaque hybridization techniques of Benton and Davis (28). Plaques containing lysozyme structural gene sequences were purified as described by Maniatis et al. (29). Restriction Enzyme Mapping. The DNAs were digested with restriction enzymes and the fragments were separated by agarose gel electrophoresis (20). After visualization of the DNA bands by ethidium bromide staining, the DNA was transferred to nitrocellulose filters as described by Southern (19) and hy- bridized to labeled pls-1 DNA or cDNA synthesized from poly(A)-containing mRNA. XDNA digested with HindIII (30) and X Charon 4A-DNA fragments of known size were used as molecular weight markers. Electron Microscopy of RNA-DNA Hybrids. Heat-dena- tured cloned DNA was hybridized to partially pdrified mrRNA for 3 hr at 580C in 80% formamide/0.4 M NaCl/0.05 M pi- perazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), pH 6.8. The hybrids were prepared for electron microscopy by the car- bonate method of Paulson and Laemmli (31). Single-stranded 4X174 DNA and double-stranded ColEl DNA were used as length standards. Biosafety Conditions. Isolation and growth of recombinant phages were done under L3/B2 conditions as specified by the "Rithtlinien zum Schutz vor Gefahren durch in vitro rekom- binierte Nukleinsauren" of the Bundesministerium fur Forschung und Technologie of the Federal Republic of Ger- many. RESULTS Isolation of Clones Containing Lysozyme Gene Sequences. The chicken DNA library was constructed, according to the method of Maniatis et al. (29), by J. Dodgson, D. Engel, and R. Axel as follows. Chicken erythrocyte DNA was partially di- gested with Alu I and Hae III. The 15-to-20-kilobase (kb) fragments were ligated to the EcoRI end fragments of X Charon 4A by using synthetic EcoRI linkers. Due to the abundance of cleavage sites for Alu I and Hae III, a nearly random repre- sentation of chicken DNA in the clones should have been achieved. Because the genome length is 2 X 106 kb and 8 X 105 independent hybrid phages had been constructed, overlapping clones were expected. We isolated seven clones that hybridized to pls-l, a cDNA plasmid that contains almost the entire pre- lysozyme coding sequence, and the 3' nontranslated portion of chicken lysozyme mRNA (20). After EcoRI cleavage, two types of clones could be differentiated by the hybridization pattern. Five clones contained a single hybridizing EcoRI fragment of about 7 kb (e.g., Xlys3l; Fig. 1, lane 2); the two other clones had an additional hybridizing fragment of about 2.7 kb (e.g., Xlys3O; Fig. 1, lane 1). The size of these fragments corresponded to the Abbreviation: kb, kilobase. * Present address: Department of Biochemistry, University of Cali- fornia, San Francisco, CA 94143. t Swiss Institute for Experimental Cancer Research, Lausanne, Swit- zerland. t Institut fur Genetik, Universitat Koln, 5000 Koln, West Germany. 6196 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.