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.
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 hybridized 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-denatured cloned DNA was hybridized to partially pdrified mrRNA for 3 hr at 580C in 80% formamide/0.4 M NaCl/0.05 M piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), pH 6.8. The hybrids were prepared for electron microscopy by the carbonate 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 rekombinierte Nukleinsauren" of the Bundesministerium fur Forschung und Technologie of the Federal Republic of Germany.
The amino acid sequence and the three-dimensional structure of chicken egg-white lysdzyme have been established (for review, 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 synthesis and degradation are responsible for the steroid-induced accumulation of the four egg-white protein mRNAs (10, 1218). The regulated expression of the egg-white protein genes should be based, at least in part, on the sequence and organization 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 sequences (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 transcriptase (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).
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 digested 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 representation 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 prelysozyme 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 California, San Francisco, CA 94143. t Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland. t Institut fur Genetik, Universitat Koln, 5000 Koln, West Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6196
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Lindenmaier et al. 1 A
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Proc. Natl. Acad. Sci. USA 76 (1979)
3
fragments was common to both clones (see below). Therefore, Alys3O must contain fragments extending into the 3' direction from the 7.1-kb fragment. Both clones together cover a total length of 22 kb of the chicken genome, and the lysozyme gene is located approximately in the middle of this region. Restriction Enzyme Mapping of the Lysozyme Gene Clones. In order to define precisely the overlapping portion of Xlys30 and AlysSl and to localize the structural gene sequences in the cloned lysozyme gene region, we constructed a restriction map of these phage DNAs. Construction of this map was facilitated by the fact that restriction fragments derived from the A Charon 4A portion of the clones could be identified by their known molecular weights and by comparison with X Charon 4A DNA from corresponding digestions. In most cases, the fragments consisting of both chicken and vector DNA could be identified on the basis of the size of the vector portion contained in them. Thus, the first cleavage site for the corresponding enzyme on either end of the cloned DNA was established. Fragments containing structural gene sequences were identified by Southern hybridization. Furthermore, comparison of the restriction patterns of AlysSO and Xlys31 allowed the localization of some restriction sites. The position of all the EcoRI sites in the chicken DNA inserted in Alys3O and the orientation of the fragments with respect to the A arms were determined by analysis of EcoRI-, BamHI-, and BamHI/EcoRI-digests (Figs. 1 and 2A). BamHI digestion of Alys3O DNA produced fragments 19.5, 8.5, 5.05, and 1.95 kb long, in addition to fragments entirely derived from A Charon 4A-DNA. The 19.5- and 5.05-kb BamHI fragments hybridized to pls-i (Fig. 2A, lane 1). Upon digestion with EcoRI, the 19.5-kb BamHI fragment was divided into a 14.3-kb BamHI/EcoRI fragment expected from the A left arm and a 5.2-kb EcoRI/BamHI fragment that must have been part of the 7.1-kb EcoRI fragment because it hybridized to pls-1 DNA. Thus, the 7.1-kb fragment must be adjacent to the A Charon 4A left arm. The 2.7-kb EcoRI fragment must be adjacent in the 3' direction as we have shown previously (21). The 8.5-kb BamHI fragment should contain 5.0 kb of the right arm of the vector. Therefore, if the 3.7-kb EcoRI fragment was located adjacent to the right arm of the vector, a 3.5-kb fragment should arise in an EcoRI/BamHI double digestion, but no fragment of this size was found. Thus, the 3.7-kb EcoRI fragment must be located between the 2.7- and 2.2-kb EcoRI fragments, which are not cut by BamHI. The order of the 5.05- and 1.95-kb BamHI fragments was also deduced from EcoRI/BamHI
b
1.1-R
FIG. 1. Identification of EcoRI fragments containing lysozyme structural gene sequences. The EcoRI-digested DNA was separated on a 1% agarose gel, stained with ethidium bromide, and visualized under UV light. The DNA was transferred to a nitrocellulose filter and hybridized to 32P-labeled denatured pls-1 DNA. The stained gel (lanes a) and the corresponding autoradiograms (lanes b) are shown. Lanes: 1, Xlys3O; 2, Xlys3l; 3, cellular chicken DNA. The size of the fragments is given in kb. The smallest fragment of Xlys31 (0.5 kb) has run off this gel.
size of fragments hybridizing from EcoRI-digested cellular DNA (ref. 21; Fig. 1, lane 3). The clones Alys30 and Xlys31 were chosen for further analysis by restriction mapping and electron microscopy. The Two Overlapping Lysozyme Gene Clones Span 22 kb of the Chicken Genome. EcoRI digestion of the phage DNAs produced fragments 7.0, 3.1, 1.7, 1.1, and 0.5 kb long from XlysSi and 7.1, 3.7, 2.7, and 2.2 kb long from Xlys3O, in addition to the left and right arm of A Charon 4A (19.8 and 10.9 kb). Of these, the 7.0-kb fragment of Alys31 and the 7.1- and 2.7-kb fragments of AlysS0 hybridized to pls-1 DNA (Fig. 1). As we
have shown previously by hybridization of EcoRI-digested genomic DNA with 5'- and 3'-specific probes (21), the 7.1-kb fragment represents the 5' portion of the lysozyme gene and the 2.7-kb fragment, the 3' portion. The 7.0-kb EcoRI fragment of Xlys31 should be part of the 7. 1-kb fragment of AlysSO and probably results from an artificial EcoRI site introduced during the construction of the chicken DNA library. AlysS1 therefore contains most of the 5' hybridizing fragment and adjacent fragments extending into the 5' direction. None of the other A
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FIG. 2. Agarose gel electrophoresis of restriction fragments of Xlys3O and Xlys31 DNA. The DNA of the recombinant phages was digested, separated on a 1% agarose gel, and visualized by ethidium bromide staining (lanes a). The DNA was transferred to nitrocellulose filters and hybridized to 32P-labeled pls-l DNA (A and B, lanes b) or lysozyme cDNA (C and D, lanes b). (A and B) Xlys3O DNA (lanes 1 and 3) and Alys3l DNA (lanes 2 and 4) digested with BamHI (A, lanes 1 and 2), BamHI/EcoRI (A, lanes 3 and 4), Sac I (B, lanes 1 and 2), and Sac I/EcoRI (B, lanes 3 and 4). (C and D) Restriction fragments of Alys3O DNA (C) and Alys31-DNA (D) digested with Xba I (lanes 1), Xba I/HindIII (lanes 2), and HindIII (lanes 3). The size of fragments is given in kb.
6198
Biochemistry: Lindenmaier et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
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FIG. 3. Restriction endonuclease cleavage map of chicken lysozyme gene clones Mlys31 (A) and Xlys30 (C). B, the composite map of the lysozyme gene region is redrawn on a larger scale. The cleavage sites for EcoRI (E), BamHI (B), Sac I(S), Xba I (X), and HindIll (H), and Kpn I (K) are shown. The single line represents X Charon 4A-DNA; the bars represent chicken DNA; the hatched bars indicate chicken DNA fragments generated by digestion with EcoRI or double digestion with EcoRI and the indicated enzymes and hybridized with pls-1 DNA. The numbers indicate the length, in kb, of the fragments from single digestions. Bold numbers specify fragments hybridizing to pls-l DNA. The small numbers separated by slashes indicate HindIII restriction fragments, the order of which has not been determined. In B, the cleavage sites are indexed by numbers according to their relative order 5'-to-3'.
double digestions. BamHI site B5 (Fig. 3B) is located 1.3 kb from one end of the 3.7-kb EcoRI fragment. Because there was no 2.4-kb fragment generated in the double digestion, an additional BamHI site must be located in the 3.7-kb EcoRI fragment (Fig. 3C). Digestion of Xlys3O by Sac I produced fragments 23.45, 11.5, 2.4, 1.85, and 1.4 kb long which contained chicken DNA and 3.9- and 1.9-kb fragments derived from the X Charon 4A right arm (Fig. 2B, lane 1). The two largest Sac I fragments must contain X Charon tA left and right arm sequences, respectively. In a Sac I/EcoRI double digestion (Fig. 2B, lane 3) the 23.45-kb Sac I fragment was cut into a 19.8-kb fragment (X left arm) and a 3.65-kb fragment derived from the 7.1-kb EcoRI fragment. The 2.4-kb Sac I fragment must be located within the 7.1-kb EcoRI fragment because it was common to Xlys3O and XlysSl. The 1.1-kb Sac I/EcoRI fragment must be derived from the 1.85-kb Sac I fragment because the 1.4-kb Sac I fragment was not cut by EcoRI. Furthermore, the 2.4- and 1.4-kb fragments hybridized strongly to pls-1, whereas the 1.85-kb Sac I fragment and the corresponding 1.05-kb Sac I/EcoRI fragment hybridized only weakly. This suggests that only a small part of the coding sequence is contained within the 5' portion of the 1.85-kb Sac I fragment. Xba I cleaved Xlys3O DNA into fragments 26.1, 12.7, 6.95, and 0.65 kb long (Fig. 2C, lane 1). The 26.1- and 12.7-kb fragments must contain the left and right arm of the vector, respectively. The 0.65-kb fragment is common to Xlys3O and Alys3l and therefore should be located next to the left arm fragment. Thus, the 6.95-kb Xba I fragment was located between the 0.65- and 12.7-kb fragment. The 0.65-kb fragment
separates two hybridizing fragments and must therefore consist of intervening sequence DNA (Fig. 3C). HindIII produced fragments 20.0, 6.05, 5.7, 5.3, 3.2, 2.1, 1.65, 1.35, 0.8, and -0.45 kb long (Fig. 2C, lane 3). The 3.2-, 2.1-, and 0.8-kb fragments contained lysozyme structural gene sequences and their order is 2.1, 0.8, and 3.2 kb, starting from the 5' end (21). Analysis of a HindIII/Xba I double digestion (Fig. 2C, lane 2) allowed us to determine the location of most of the HindIII fragments. Kpn I did not cut within the chicken DNA inserted in Xlys3O (data not shown). Southern hybridization of Xlys3O and Xlys3l DNA with cDNA from total poly(A)-mRNA did not reveal any additional bands (Fig. 2, and data not shown). Similar experiments were carried out with Xlys3l DNA (Fig. 2). The results obtained from these experiments allowed us to construct the restriction maps shown in Fig. 3. Three separate regions containing mRNA sequences can be defined (Fig. 3B). The presence of a third intervening sequence which had been deduced from mapping of the lysozyme gene in cellular DNA (21) could not be demonstrated with the enzymes used. Electron Microscopic Mapping of the Chicken Lysozyme Gene. For a more detailed analysis of the arrangement of coding and intervening sequences we examined, by electron microscopy, hybrid molecules consisting of lysozyme mRNA and cloned DNA. Thermally denatured clone DNA was incubated with partially purified lysozyme mRNA under hybridization conditions that permitted formation of RNA-DNA hybrids but not of DNA-DNA duplexes. Thus, homologous regions between the mRNA and the DNA should appear as double-
Biochemistry:
Lindenmaier et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
stranded structures, whereas intervening sequences should form single-stranded loops. Hybrids between lysozyme mRNA and Xlys31 DNA (Fig. 4B) or the isolated 7.1-kb EcoRI fraginent of Xlys30 DNA (Fig. 4C) had similar structures. They showed a single-stranded loop of 1340 bases flanked by two doublestranded regions (about 200 and 150 base pairs) and a fork consisting of a short and a long single-stranded tail. The short tail (190 bases) probably represents the 3' part of the lysozyme mRNA, because Xlys3l and the EcoRI fragment of Xlys3O do not contain the 3' part of the lysozyme structural gene. The hybrids of Xlys30 DNA and lysozyme mRNA (Fig. 4A) displayed a double-stranded region of about 630 base pairs interrupted by two single-stranded loops 1300 and 1800 bases long. About 70% of the molecules examined clearly showed a small knob 94 base pairs from the 1.8-kb loop. The position and A
2
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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.
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 hybridized 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-denatured cloned DNA was hybridized to partially pdrified mrRNA for 3 hr at 580C in 80% formamide/0.4 M NaCl/0.05 M piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), pH 6.8. The hybrids were prepared for electron microscopy by the carbonate 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 rekombinierte Nukleinsauren" of the Bundesministerium fur Forschung und Technologie of the Federal Republic of Germany.
The amino acid sequence and the three-dimensional structure of chicken egg-white lysdzyme have been established (for review, 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 synthesis and degradation are responsible for the steroid-induced accumulation of the four egg-white protein mRNAs (10, 1218). The regulated expression of the egg-white protein genes should be based, at least in part, on the sequence and organization 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 sequences (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 transcriptase (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).
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 digested 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 representation 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 prelysozyme 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 California, San Francisco, CA 94143. t Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland. t Institut fur Genetik, Universitat Koln, 5000 Koln, West Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6196
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Lindenmaier et al. 1 A
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Proc. Natl. Acad. Sci. USA 76 (1979)
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fragments was common to both clones (see below). Therefore, Alys3O must contain fragments extending into the 3' direction from the 7.1-kb fragment. Both clones together cover a total length of 22 kb of the chicken genome, and the lysozyme gene is located approximately in the middle of this region. Restriction Enzyme Mapping of the Lysozyme Gene Clones. In order to define precisely the overlapping portion of Xlys30 and AlysSl and to localize the structural gene sequences in the cloned lysozyme gene region, we constructed a restriction map of these phage DNAs. Construction of this map was facilitated by the fact that restriction fragments derived from the A Charon 4A portion of the clones could be identified by their known molecular weights and by comparison with X Charon 4A DNA from corresponding digestions. In most cases, the fragments consisting of both chicken and vector DNA could be identified on the basis of the size of the vector portion contained in them. Thus, the first cleavage site for the corresponding enzyme on either end of the cloned DNA was established. Fragments containing structural gene sequences were identified by Southern hybridization. Furthermore, comparison of the restriction patterns of AlysSO and Xlys31 allowed the localization of some restriction sites. The position of all the EcoRI sites in the chicken DNA inserted in Alys3O and the orientation of the fragments with respect to the A arms were determined by analysis of EcoRI-, BamHI-, and BamHI/EcoRI-digests (Figs. 1 and 2A). BamHI digestion of Alys3O DNA produced fragments 19.5, 8.5, 5.05, and 1.95 kb long, in addition to fragments entirely derived from A Charon 4A-DNA. The 19.5- and 5.05-kb BamHI fragments hybridized to pls-i (Fig. 2A, lane 1). Upon digestion with EcoRI, the 19.5-kb BamHI fragment was divided into a 14.3-kb BamHI/EcoRI fragment expected from the A left arm and a 5.2-kb EcoRI/BamHI fragment that must have been part of the 7.1-kb EcoRI fragment because it hybridized to pls-1 DNA. Thus, the 7.1-kb fragment must be adjacent to the A Charon 4A left arm. The 2.7-kb EcoRI fragment must be adjacent in the 3' direction as we have shown previously (21). The 8.5-kb BamHI fragment should contain 5.0 kb of the right arm of the vector. Therefore, if the 3.7-kb EcoRI fragment was located adjacent to the right arm of the vector, a 3.5-kb fragment should arise in an EcoRI/BamHI double digestion, but no fragment of this size was found. Thus, the 3.7-kb EcoRI fragment must be located between the 2.7- and 2.2-kb EcoRI fragments, which are not cut by BamHI. The order of the 5.05- and 1.95-kb BamHI fragments was also deduced from EcoRI/BamHI
b
1.1-R
FIG. 1. Identification of EcoRI fragments containing lysozyme structural gene sequences. The EcoRI-digested DNA was separated on a 1% agarose gel, stained with ethidium bromide, and visualized under UV light. The DNA was transferred to a nitrocellulose filter and hybridized to 32P-labeled denatured pls-1 DNA. The stained gel (lanes a) and the corresponding autoradiograms (lanes b) are shown. Lanes: 1, Xlys3O; 2, Xlys3l; 3, cellular chicken DNA. The size of the fragments is given in kb. The smallest fragment of Xlys31 (0.5 kb) has run off this gel.
size of fragments hybridizing from EcoRI-digested cellular DNA (ref. 21; Fig. 1, lane 3). The clones Alys30 and Xlys31 were chosen for further analysis by restriction mapping and electron microscopy. The Two Overlapping Lysozyme Gene Clones Span 22 kb of the Chicken Genome. EcoRI digestion of the phage DNAs produced fragments 7.0, 3.1, 1.7, 1.1, and 0.5 kb long from XlysSi and 7.1, 3.7, 2.7, and 2.2 kb long from Xlys3O, in addition to the left and right arm of A Charon 4A (19.8 and 10.9 kb). Of these, the 7.0-kb fragment of Alys31 and the 7.1- and 2.7-kb fragments of AlysS0 hybridized to pls-1 DNA (Fig. 1). As we
have shown previously by hybridization of EcoRI-digested genomic DNA with 5'- and 3'-specific probes (21), the 7.1-kb fragment represents the 5' portion of the lysozyme gene and the 2.7-kb fragment, the 3' portion. The 7.0-kb EcoRI fragment of Xlys31 should be part of the 7. 1-kb fragment of AlysSO and probably results from an artificial EcoRI site introduced during the construction of the chicken DNA library. AlysS1 therefore contains most of the 5' hybridizing fragment and adjacent fragments extending into the 5' direction. None of the other A
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FIG. 2. Agarose gel electrophoresis of restriction fragments of Xlys3O and Xlys31 DNA. The DNA of the recombinant phages was digested, separated on a 1% agarose gel, and visualized by ethidium bromide staining (lanes a). The DNA was transferred to nitrocellulose filters and hybridized to 32P-labeled pls-l DNA (A and B, lanes b) or lysozyme cDNA (C and D, lanes b). (A and B) Xlys3O DNA (lanes 1 and 3) and Alys3l DNA (lanes 2 and 4) digested with BamHI (A, lanes 1 and 2), BamHI/EcoRI (A, lanes 3 and 4), Sac I (B, lanes 1 and 2), and Sac I/EcoRI (B, lanes 3 and 4). (C and D) Restriction fragments of Alys3O DNA (C) and Alys31-DNA (D) digested with Xba I (lanes 1), Xba I/HindIII (lanes 2), and HindIII (lanes 3). The size of fragments is given in kb.
6198
Biochemistry: Lindenmaier et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
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FIG. 3. Restriction endonuclease cleavage map of chicken lysozyme gene clones Mlys31 (A) and Xlys30 (C). B, the composite map of the lysozyme gene region is redrawn on a larger scale. The cleavage sites for EcoRI (E), BamHI (B), Sac I(S), Xba I (X), and HindIll (H), and Kpn I (K) are shown. The single line represents X Charon 4A-DNA; the bars represent chicken DNA; the hatched bars indicate chicken DNA fragments generated by digestion with EcoRI or double digestion with EcoRI and the indicated enzymes and hybridized with pls-1 DNA. The numbers indicate the length, in kb, of the fragments from single digestions. Bold numbers specify fragments hybridizing to pls-l DNA. The small numbers separated by slashes indicate HindIII restriction fragments, the order of which has not been determined. In B, the cleavage sites are indexed by numbers according to their relative order 5'-to-3'.
double digestions. BamHI site B5 (Fig. 3B) is located 1.3 kb from one end of the 3.7-kb EcoRI fragment. Because there was no 2.4-kb fragment generated in the double digestion, an additional BamHI site must be located in the 3.7-kb EcoRI fragment (Fig. 3C). Digestion of Xlys3O by Sac I produced fragments 23.45, 11.5, 2.4, 1.85, and 1.4 kb long which contained chicken DNA and 3.9- and 1.9-kb fragments derived from the X Charon 4A right arm (Fig. 2B, lane 1). The two largest Sac I fragments must contain X Charon tA left and right arm sequences, respectively. In a Sac I/EcoRI double digestion (Fig. 2B, lane 3) the 23.45-kb Sac I fragment was cut into a 19.8-kb fragment (X left arm) and a 3.65-kb fragment derived from the 7.1-kb EcoRI fragment. The 2.4-kb Sac I fragment must be located within the 7.1-kb EcoRI fragment because it was common to Xlys3O and XlysSl. The 1.1-kb Sac I/EcoRI fragment must be derived from the 1.85-kb Sac I fragment because the 1.4-kb Sac I fragment was not cut by EcoRI. Furthermore, the 2.4- and 1.4-kb fragments hybridized strongly to pls-1, whereas the 1.85-kb Sac I fragment and the corresponding 1.05-kb Sac I/EcoRI fragment hybridized only weakly. This suggests that only a small part of the coding sequence is contained within the 5' portion of the 1.85-kb Sac I fragment. Xba I cleaved Xlys3O DNA into fragments 26.1, 12.7, 6.95, and 0.65 kb long (Fig. 2C, lane 1). The 26.1- and 12.7-kb fragments must contain the left and right arm of the vector, respectively. The 0.65-kb fragment is common to Xlys3O and Alys3l and therefore should be located next to the left arm fragment. Thus, the 6.95-kb Xba I fragment was located between the 0.65- and 12.7-kb fragment. The 0.65-kb fragment
separates two hybridizing fragments and must therefore consist of intervening sequence DNA (Fig. 3C). HindIII produced fragments 20.0, 6.05, 5.7, 5.3, 3.2, 2.1, 1.65, 1.35, 0.8, and -0.45 kb long (Fig. 2C, lane 3). The 3.2-, 2.1-, and 0.8-kb fragments contained lysozyme structural gene sequences and their order is 2.1, 0.8, and 3.2 kb, starting from the 5' end (21). Analysis of a HindIII/Xba I double digestion (Fig. 2C, lane 2) allowed us to determine the location of most of the HindIII fragments. Kpn I did not cut within the chicken DNA inserted in Xlys3O (data not shown). Southern hybridization of Xlys3O and Xlys3l DNA with cDNA from total poly(A)-mRNA did not reveal any additional bands (Fig. 2, and data not shown). Similar experiments were carried out with Xlys3l DNA (Fig. 2). The results obtained from these experiments allowed us to construct the restriction maps shown in Fig. 3. Three separate regions containing mRNA sequences can be defined (Fig. 3B). The presence of a third intervening sequence which had been deduced from mapping of the lysozyme gene in cellular DNA (21) could not be demonstrated with the enzymes used. Electron Microscopic Mapping of the Chicken Lysozyme Gene. For a more detailed analysis of the arrangement of coding and intervening sequences we examined, by electron microscopy, hybrid molecules consisting of lysozyme mRNA and cloned DNA. Thermally denatured clone DNA was incubated with partially purified lysozyme mRNA under hybridization conditions that permitted formation of RNA-DNA hybrids but not of DNA-DNA duplexes. Thus, homologous regions between the mRNA and the DNA should appear as double-
Biochemistry:
Lindenmaier et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
stranded structures, whereas intervening sequences should form single-stranded loops. Hybrids between lysozyme mRNA and Xlys31 DNA (Fig. 4B) or the isolated 7.1-kb EcoRI fraginent of Xlys30 DNA (Fig. 4C) had similar structures. They showed a single-stranded loop of 1340 bases flanked by two doublestranded regions (about 200 and 150 base pairs) and a fork consisting of a short and a long single-stranded tail. The short tail (190 bases) probably represents the 3' part of the lysozyme mRNA, because Xlys3l and the EcoRI fragment of Xlys3O do not contain the 3' part of the lysozyme structural gene. The hybrids of Xlys30 DNA and lysozyme mRNA (Fig. 4A) displayed a double-stranded region of about 630 base pairs interrupted by two single-stranded loops 1300 and 1800 bases long. About 70% of the molecules examined clearly showed a small knob 94 base pairs from the 1.8-kb loop. The position and A
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