Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2253-2257, May 1979

Biochemistry

The ovalbumin gene: Cloning and molecular organization of the entire natural gene* (genomic DNA cloning/gene library screening/restriction mapping/electron microscopic mapping)

ACHILLES DUGAICZYKt, SAVIo L. C. Woof, DONALD A. COLBERTt, EUGENE C. LAIt, MYLES L. MACE, JR.t, AND BERT W. O'MALLEYt tDepartment of Cell Biology and tHoward Hughes Medical Institute Laboratory at Baylor College of Medicine, Houston, Texas 77030

Communicated by Klaus Hofmann, March 5, 1979

We report the analyses of recently cloned reABSTRACT striction fragments of the natural ovalbumin gene that overlap in part with previously cloned DNA fragments but extend further into the flanking sequences of the gene. These clones now permit us to identify the DNA sequence that codes for the 5' end of ovalbumin mRNA. Based on these and previous results, the molecular organization of the entire ovalbumin gene was established. The entire gene is composed of eight structural DNA sequences separated by seven intervening sequences that are not present in the mature mRNA. In addition, an ovalbumin gene clone has been obtained from a chicken gene library. Analysis of DNA isolated from this particular clone by molecular hybridization and electron microscopic mapping revealed that it contains the entire ovalbumin gene in a single segment of DNA and its structure was consistent with that predicted from our physical map constructed from individually cloned fragments of the gene. The structure of the natural ovalbumin gene has proven to be particularly complex due to the presence of multiple intervening DNA sequences within this gene. From restriction mapping of genomic DNA (1-4) it was originally concluded that there were at least two intervening DNA regions, each containing an EcoRI site that separated the structural gene sequences into three noncontiguous portions. The entire ovalbumin gene was thought to be contained in three EcoRI DNA fragments 2.4, 1.8, and 9.2 kilobases (kb) long. Subsequent molecular cloning and characterization of these EcoRI restriction fragments led us to the conclusion that there are seven intervening sequences within the natural ovalbumin gene (5-7). Other laboratories have reported the presence of only six intervening sequences in this gene (8-11). The discrepancy in the observed number of intervening sequences within this gene was due to the absence from the three cloned EcoRI DNA fragments of a short DNA sequence (7) coding for the 5'-terminal 45 nucleotides of ovalbumin mRNA (12). Our observation of seven intervening sequences has subsequently been confirmed by Breathnach et al. (13). In an effort to determine the structure of the missing 5' end of the gene, we have cloned additional overlapping restriction DNA fragments from genomic chicken DNA. Analyses of these cloned DNA fragments have enabled us to establish the molecular organization of the entire natural ovalbumin gene.

MATERIALS AND METHODS Enrichment of Ovalbumin DNA. About 2 mg of chicken liver DNA were digested with EcoRI, HindIll, or Pst I and subjected to preparative slab gel electrophoresis (14). Electrophoretic elution of DNA fractions of 9.2 kb (EcoRI-digested),

3.2 kb (HindIll-digested), and 4.5 kb (Pst I-digested) in size gave an approximately 20-fold enrichment of ovalbumin sequences from total chicken DNA. Bacteria and Phages. Escherichia coli LE392/ThyA, E. coli DP50/supF, and the certified EK2 bacteriophage vector A\gtWES-AB were kindly provided by Philip Leder (National Institutes of Health). The vector DNA was digested with EcoRI, and the XB fragment was separated from XgtWES-AB DNA by agarose gel electrophoresis. E. coli KH802, NS428, and Xdg805 and the bacteriophage vectors Charon 4A and 21A were kindly provided by Frederick Blattner (University of Wisconsin, Madison, WI). Charon 4A DNA was digested with EcoRI, and the middle DNA fragments were again separated from phage DNA arms by preparative agarose gel electrophoresis. Charon 21A DNA is cleaved by HindIII only once, and the resulting DNA was directly ligated with target DNA for cloning. Packaging Components. The protein A, sonic extract, and the freeze-thaw lysate were prepared from E. coli Xgt805 and NS428 strains by the procedure of Blattner et al. (15). Cloning of Ovalbumin DNA Fragments. All cloning experiments were performed in a P3 facility, and the procedures were carried out in accordance with the National Institutes of Health guidelines for recombinant DNA research. Ten micrograms of the 9.2-kb DNA fraction recovered after preparative gel electrophoresis were ligated (16) with 30 ,tg of Charon 4A DNA arms in 500 Ml and the entire mixture was used for transfection of host cells (17). About 20,000 recombinant phage plaques were amplified according to a described procedure (5) and screened with a 32P-labeled OVR DNA probe (labeled by nick translation) that contains sequences corresponding to the 3' terminus of ovalbumin mRNA with respect to the single Hae III site present in ovalbumin cDNA (4). Two positive phage plaques were obtained in this experiment. One microgram of the enriched 3.2-kb ovalbumin DNA was ligated with 1 Mg of HindIII-digested Charon 21A DNA in a total volume of 20 Ml. The phage DNA was packaged in vitro according to Blattner et al. (15). The ligation mixture was incubated for 15 min at room temperature with 150 Ml of buffer A (20 mM Tris-HCl, pH 8/3 mM MgCl2/0.05% 2 mercaptoethanol/i mM EDTA), 20,ul of buffer MI (6 mM Tris-HCI, pH 7.4/50 mM spermidine/60 mM putrescine/18 mM MgCl2/15 mM ATP/0.2% 2-mercaptoethanol), 100 ml of sonic extract, and 10 Ml of protein A. After the addition of 750 Mi of the freezethaw lysate, the mixture was further incubated for 1 hr at room temperature. Two to 5 Mi of this material was used to infect 0.2 Abbreviation: kb, kilobase. * This paper is no. 12 in a series dealing with the structure, organization, function, and regulation of this gene in chicken oviduct. Paper 11 is Lai, E. C., Woo, S. L. C., Dugaiczyk, A., Catterall, J. F. & O'Malley, B. W. (1979) Cell 16,201-211.

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.

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FIC. 1. Agarose gel electrophoresis of the cloned 9.2-kb EcoRI ovalbumin DNA after restriction digestion. (Left) Restriction DNA fragments in the gel after staining with ethidium bromide; (Right) corresponding radioautogram after Southern hybridization with :12P-labeled OVR DNA, which is specific for the 3'-structural ovallumin gene sequences (4), as probe. Lane a, 9.2-kb EcoRI fragment; lane b, after Bam HI digestion; lane c, after HindIII digestion; lane d, after Hae III digestion; lane e, after Pst I digestion.

ml of host cells and plated in soft agar. Plaques were screened with 32P-labeled OV2.4 DNA (5) as the hybridization probe. Three individual phage plaques yielded positive signals and were subcultured. Because no phage vectors are available to clone DNA fragments generated by Pst I digestion of DNA, the 4.5-kb Pst I ovalbumin DNA (0.66 gg, isolated after preparative gel electrophoresis) was ligated with EcoRI-digested XgtWES DNA arms (2.2 ,ug) in the presence of a natural 165-base-pair EcoRI/Pst I DNA linker (46 ng). The DNA linker was obtained from digestion of the previously cloned 1.8-kb ovalbumin DNA (7) with EcoRI and Pst I and isolated by preparative gel electrophoresis. Ligation was again carried out at 12°C in a volume of 20 ,ul, and the DNA in the ligation mixture was packaged in vitro as outlined above. An average of 400 plaques was obtained kb 3.5

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FIc. 2. Agarose gel electrophoresis of the cloned 3.2-kb HindIII ovalbumin DNA after restriction digestion. (Left) DNA fragments in the gel were visualized by ethidium bromide staining; (Right) the corresponding radioautogram after Southern hybridization with :32P-labeled 2.4-kb EcoRI ovalbumin DNA as probe. The 3.2-kb HindIll DNA (lane a) and its digestion products with Taq I, EcoRI, Sst I, and Hpa I are shown in lanes b, c, f, and h, respectively. The 2.4-kb EcoRI DNA (lane d) and its digestion products with HindIII and Sst I are shown in lanes e and g, respectively.

FIC. 3. Agarose gel electrophoresis of the cloned 4.5-kb Pst I ovalbumin DNA and its Hpa I digestion products. (Left) Ethidium bromide staining of the gel; (Right) corresponding radioautogram after Southern hybridization with 32P-Iabeled OVI, DNA, which is specific for the 5'-structural ovalbumin DNA sequences (4), as probe. Lane a, 4.5-kb Pst 1 DNA; lane b, partial Hpa I digestion. Note the weak but discernible hybridization of the 2.8-kb DNA fragment in addition to the expected intense 1.7-kb fragment.

per plate. Without the linkers in the ligation mixture only 50-100 plaques were obtained, which corresponds to background when the vector arms were used alone. Under identical conditions, an EcoRI fragment of similar size yielded 2000 plaques per plate. Plaques were screened by using the cloned 32P-labeled OV3.2 DNA as the hybridization probe. Four positive plaques were obtained. Propagation of Cloned Ovalbumin DNA. The cloned ovalbumin DNA fragments were subsequently transferred to the EcoRI, HindIII, or Pst I site of pBR322 (18) and propagated in E. coli X1776. Screening of Chicken Gene Library. We have recently obtained (from R. Axel, D. Engel, and J. Dodgson) a chicken gene library generated by the method of Lawn et al. (19) by use of Charon 4A XDNA vector and chicken DNA that was partially digested by Hae III and Alu I and ligated to synthetic EcoRI DNA linkers. The phage titer of the chicken gene library was first determined by serial dilution and plating. About 8000 phages were then spread in soft agar on each square agar plate (9 X 9 cm). At this concentration, the lysis of host cells on the agar lawn was slightly less than confluent. A total of 600 such plates were screened for clones containing the ovalbumin gene by using OV3.2 [32P]DNA as the hybridization probe. Some 20 phage plaques yielded positive signals, and DNA from each of these clones was analyzed. RESULTS Cloned 9.2-kb EcoRI Ovalbumin DNA (OV9.2). We previously published a characterization of a 9.7-kb fragment of the ovalbumin gene (7), which we now refer to as pOV9.2. The cloned' 9.2-kb EcoRI DNA was isolated from the recombinant

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Proc. Natl. Acad. Sci. USA 76 (1979)

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Kilobases FIG. 4. Physical map of the entire natural ovalbumin gene displaying some of the key restriction cleavage sites, the locations of the initiation and termination codons, and regions of interspersed structural and intervening sequences. This map is constructed from detailed analyses of various gene fragments cloned in our laboratory: 2.4-kb EcoRI DNA, 1.8-kb E(oRI DNA, 9.2-kb EcoRI DNA, 3.2-kb HindIII DNA, 4.5-kb Pst I DNA. Thin solid line, flanking gene sequence; thick solid line, intervening gene sequence; thick open line, structural gene sequence.

plasmid pOV9.2 and analyzed by restriction mapping and Southern hybridization (Fig. 1, lane a). BamHI digested this DNA into two fragments (4.7 and 4.5 kb) but only the 4.7-kb DNA fragment hybridized with the OVR [32P]DNA probe (Fig. 1, lane b). The location of this BamHI site within the 9.2-kb EcoRI DNA fragment is in accordance with our previous mapping of the ovalbumin gene within genomic chicken DNA (4). There are three HindIII cleavage sites within the 9.2-kb DNA, and only the left-terminal 2.4-kb DNA fragment was capable of hybridizing with OVR [32P]DNA (Fig. 1, lane c), as predicted from our previous restriction map of the gene (4). The remaining fragments did not hybridize because they are located outside of the DNA region that codes for the mRNA. Hae III cleaved the 9.2-kb DNA to some 11 fragments, but only one (2.7 kb) hybridized with the OVR [32P]DNA probe (Fig. 1, lane d). Similarly, Pst I cleaved the 9.2-kb DNA to six fragments, but only one (2.8 kb) showed sequence homology to the OVR probe (Fig. 1, lane e). These results identify the cluster of HindIII, Hae III, and Pst I sites present in genomic chicken DNA about 2.4 kb from the 5' end of the 9.2-kb EcoRI DNA fragment (4). This cluster of restriction sites is already beyond the DNA sequence coding for mRNA (7). The presence of the 3' end of the ovalbumin gene within this 2.7-kb fragment obtained from the 9.2-kb DNA was verified subsequently by direct determination of the DNA sequence (unpublished data). Cloned 3.2-kb HindIlI Ovalbumin DNA (OV3.2). According to our previous mapping of genomic DNA (4), a 3.2-kb HindIII DNA fragment should contain 2.1 kb of DNA sequence in common with the 2.4-kb EcoRI DNA and an additional 1.1 kb of DNA located toward the 5' end of the gene. EcoRI digestion of this cloned DNA fragment did give rise to a 2. 1-kb fragment that hybridized with the OV2.4 [32P]DNA probe (Fig. 2, lane c), and the same fragment could be generated from the cloned 2.4-kb EcoRI DNA by HindIII digestion (Fig. 2, lane e). In addition, there were two EcoRI sites located 0.48 and 0.93 kb to the left of the 2.4-kb EcoRI DNA fragment. Both the 3.2-kb HindlII and the 2.4-kb EcoRI DNA fragments yielded a common 1.6-kb Sst I fragment that hybridized with the probe (Fig. 2, lanes f and g). A Hpa I site unique to the entire ovalbumin gene was located within the 2.4-kb DNA, and this site was also present within the 3.2-kb HindIII DNA fragment (Fig. 2, lane h). These and other restriction mapping analyses and crosshybridization data (not shown) have confirmed the presence of a common 2.1-kb DNA sequence between these two

DNA fragments and the fact that the 3.2-kb HindIII DNA contains an additional 1.1 kb of DNA located further toward the 5' end of the ovalbumin gene. Because our previous data have demonstrated a Taq I site at position 41 of the structural gene (7, 12), the DNA fragment containing the 5'-terminal

be cleaved by this enzyme. I, however, failed to reveal a cleavage site for this enzyme (Fig. 2, lane b). Thus, it appeared that the 5' end of the gene was still not present within the 3.2-kb HindIII ovalbumin DNA. Cloned 4.5-kb Pst I Ovalbumin DNA (OV4.5). Mapping of the ovalbumin gene in total chicken DNA has shown that there is a 4.5-kb Pst I fragment that contains most of the 3.2-kb HindIII fragment plus an additional 1.3 kb of DNA on the 5' end of this fragment (4). The Pst I DNA fragment was isolated from a recombinant phage clone containing this DNA fragment and was analyzed by restriction mapping and Southern hybridization. The 4.5-kb DNA fragment hybridized with OVL [32P]DNA (Fig. 3, lane a), which is a probe that contains the left half of the structural ovalbumin gene (4). As expected, this 4.5-kb Pst I fragment was not cleaved by BamHI and was digested to a 3.2-kb fragment by HindIl (data not shown). A partial digestion of this 4.5-kb DNA by Hpa I generated a 1.8-kb fragment that hybridized with OVL (Fig. 3, lane b), indicating that the unique Hpa I site common to both the 2.4-kb EcoRtI and 3.2-kb HindIII ovalbumin DNA fragments was also present in the 4.5-kb Pst I fragment. More importantly, the other 2.7-kb Hpa I digestion product also showed a weak hybridization band (Fig. 2, lane b), indicating the presence of additional structural gene sequence within this DNA fragment. Furthermore, this 4.5-kb Pst I DNA contained at least one Taq I site and was cleaved by this enzyme to a 3.4-kb DNA fragment that hybridized with the probe (not shown). Because this Taq I site in the 4.5-kb Pst I DNA appeared to be the Taq 1 (41) site in the structural gene (7, 12), the structure of the entire ovalbumin gene could then be constructed based on the analyses of all the overlapping cloned fragments of the gene (Fig. 4). The overall length of the entire natural gene for ovalbumin was 7.6 structure gene sequences should

Digestion of the 3.2-kb HindIII DNA with Taq

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A Single Clone of Chicken Genomic DNA Containing the

Natural Ovalbumin Gene. Several million phage plaques from the chicken gene library were screened for the ovalbumin gene, and one of the positive clones, designated X4A-OV, appeared to contain the natural ovalbumin gene. Upon EcoRI digestion,

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FIG. 6. Histogram of 10 individual hybrid molecules formed between X4A-OV DNA and ovalbumin mRNA under conditions described in legend of Fig. 5. The molecules were measured with a Numonics Electronic Planimeter and the lengths and positions of the loops were plotted on a linear scale. Ovalbumin structural DNA sequences (SS); m, intervening DNA sequences (IVS); -, flanking DNA sequences. D,

ments, and loop G contained the junction between the OV1.8 and OV9.2 DNA fragments. Loop A was evidently to the left

FIG. 5. Electron micrograph and line drawing of a hybrid molecule formed between the ovalbumin gene (A4A-OV) and ovalbumin mRNA(20). Hybridization was carried out with 10 pg of A4A-OV DNA and 20 pg of purified ovalbumin mRNA per ml in 70% deionized formamide containing 100 mM Tris-HCl (pH 7.6), 10 mM Na2EDTA, and 150 mM NaCl. The mixture was heated at 80°C for 5 min to denature the DNA and hybridization was carried out at 54°C for 3 hr. Samples were immediately prepared for electron microscopy as follows: The hybridization mixture containing 0.1-0.5 ,g of nucleic acids was diluted into 100 p1 of a solution containing 70% formamide, 0.1 M Tris.HCl (pH 8.4), 10 mM Na2EDTA, and 100 pig of cytochrome ( per ml. The mixture was spread onto a hypophase of distilled water. Samples were collected on collodion-coated 300-mesh copper grids, stained with uranyl acetate, rotary shadowed with platinum/palladium, and examined at 80 kV on a Joel 100 C electron microscope. (-) X4A.OV DNA; (--- -) ovalbumin mRNA.

X4A-OV DNA yielded an 8.0- and a 5.5-kb DNA fragment in addition to the 2.4-, 1.8-, and 0.5-kb fragments (data not shown). HindIII and Pst I digestion of this DNA generated the expected 3.2- and 4.5-kb bands, indicating the presence of additional chicken DNA sequences to the left of the 2.4- and 0.5-kb EcoRI DNA fragments of this ovalbumin gene clone. X4A-OV DNA was also analyzed by electron microscopic mapping in order to determine whether it contains the entire ovalbumin gene. A typical molecule as seen after hybridizing single-stranded X4A.OV DNA with purified ovalbumin mRNA and its corresponding line drawing are shown in Fig. 5. A total of seven single-stranded loops corresponding to the seven intervening DNA sequences was evident. Loops B-D and loop F were identical to those observed previously with cloned 2.4-kb and 1.8-kb EcoRI DNA fragments, respectively (7). Loop E contained the junction between OV2.4 and OV1.8 DNA frag-

of OV2.4 DNA and could only be formed by the presence of additional structural gene sequences. Fig. 6 shows a histogram of several of these hybrid molecules. In addition to the fact that the sizes of loops B-G were in good agreement with the gene map generated by analyses of the various cloned gene fragments shown in Fig. 4, the histogram also indicated that loop A is approximately 1.6 kb long. This would place the 5'-structural gene sequences to the left of the 3.2-kb HindIII DNA but within the 4.5-kb Pst I fragment. This observation was again in precise agreement with our analyses of the cloned 3.2-kb HindIII and 4.5-kb Pst I fragments (Figs. 2 and 3).

DISCUSSION We have previously reported that all but 45 base pairs of DNA at the 5' terminus of the structural ovalbumin gene were present within EcoRI DNA fragments of 2.4, 1.8, and 9.2 kb (7). The cloning and restriction mapping analysis of the 9.2-kb EcoRI DNA fragment described in the present study supported the notion that the DNA sequence coding for the 3' terminus of the structural ovalbumin gene resides within this DNA fragment. The 3'-terminal DNA sequence of the structural gene has recently been localized between the Hinfl (1732) and the Hph I site, as shown in Fig. 4 and confirmed by direct determination of DNA sequences (unpublished data). We have attempted to obtain the 5' end of the structural ovalbumin gene by cloning a 3.2-kb HindIII DNA fragment. Although this DNA fragment contained an additional 1.1 kb of DNA to the left of the 2.4-kb EcoRI DNA fragment-it was still devoid of the 5'-terminal DNA sequence of the gene. This conclusion was reached because the diagnostic Taq I site at nucleotide 41 of the structural gene was not present within the cloned 3.2-kb HindIII DNA. However, this Taq I site was present in the cloned 4.5-kb Pst I DNA fragment which contained an additional 1.3 kb of DNA located to the left (5' end) of the 3.2-kb HindIII DNA. The presence of additional structural gene sequences within the 4.5-kb Pst I DNA was verified by the observation that the 2.7-kb Hpa I digestion product of the 4.5-kb Pst I fragment also hybridized with a structural gene

Biochemistry: Dugaiczyk et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

probe. Thus, our characterizations of previously and presently cloned overlapping DNA fragments of the ovalbumin gene have enabled us to construct a complete physical map of the natural gene (Fig. 4). Subsequently, we obtained a clone containing the entire ovalbumin gene from a chicken gene library. Analyses of this cloned DNA by restriction and electron microscopic mapping confirmed the location of the 5' end of the gene as shown in Fig. 4.§ The exact location of 5' end of the structural gene must eventually be obtained from direct determination of the DNA sequence. The ovalbumin gene is therefore composed of eight structural gene segments separated by seven intervening sequences of various lengths, as we have concluded previously (7). The first intervening sequence, separating the Taq I site at nucleotide 41 from the initiation codon AUG at nucleotide 65 in the structural gene, is 1.6 kb long. The size of the entire ovalbumin gene is thus approximately 7.6 kb of DNA in order to code for a mRNA of 1859 nucleotides (12). The size of the natural ovalbumin gene agrees well with our estimated size of 7800 nucleotides for the largest ovalbumin precursor RNA able to be isolated from oviduct nuclei (21). Given this complex gene structure and the existence of precursor RNAs of approximately the same length as the gene, we have postulated that the entire ovalbumin gene is transcribed into a large primary transcript. The precursor is then processed into mature mRNA molecules by eliminating the intervening RNA transcripts through splicing in a manner similar to the processing of mRNAs of several animal viruses (22, 26), precursor tRNAs in yeast (27, 28), and other eukaryotic genes such as globin (29) and immunoglobin (30, 31). We have recently reported also that the chicken ovomucoid gene contains at least seven intervening sequences (32). Although intervening sequences have been observed in several other cloned eukaryotic genes, the frequency of occurrence of intervening sequences appears to be the highest in the chicken genes coding for the egg-white proteins. Further studies of these interesting chicken genes should enhance our understanding of the expression and regulation of genetic information inherent to eukaryotic cells. We thank Dr. P. Leder and Dr. F. Blattner for making available to

us the A host-vector cloning systems. We are indebted to Drs. R.

Axel,

D. Engel, and J. Dodgson for providing us with the chicken gene library. We also thank Mses. Martha Ray, Olivia Dennison, Sulaf Skory, Jill Lahti, and Yvonne Hodges and Mr. Lawrence Day for their expert technical assistance and dedicated effort which has made the present study possible. This work was supported by National Institutes of Health Grant HD-08188 and the Baylor Population Center for Reproductive Biology. S.L.C.W. is an Associate Investigator of the Howard Hughes Medical Institute. D.A.C. is the recipient of a National Institutes of Health Fellowship (GM-06350-01). § A recent analysis of a complete ovalbumin gene clone from the same chicken gene library by P. Chambon and coworkers has yielded a similar conclusion (reported at the 1979 Miami Winter Symposia From Gene to Protein, eds. Russell,I T. R., Brew, K., Schultz, J. & Faber, H. (Academic, New York), in press.

Doel, M. T., Houghton, M., Cook, E. A. & Carey, N. H. (1977) Nucleic Acids Res. 4, 3701-3713. 2. Breathnach, R., Mandel, J. L. & Chambon, P. (1977) Nature (London) 270, 314-319. 3. Weinstock, R., Sweet, R., Weiss, M., Cedar, H. & Axel, R. (1978) Proc. Natl. Acad. Sci. USA 75, 1299-1303. 1.

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328-333. 8. Garapin, A. C., Lepennec, J. P., Roskan, W., Perrin, F., Comi, B., Krust, A., Breathnach, R., Chambon, P. & Kourilsky, P. (1978) Nature (London) 273,349-354. 9. Breathnach, R., Mandel, J. L., Gerlinger, P., Krust, A., LeMeur, M., Humphries, P., Cochet, M., Gannon, F. & Chambon, P. (1978) in Genetic Engineering, eds. Boyer, H. W. & Nicosia, S. (Elsevier/North-Holland, New York), pp. 77-88. 10. Garapin, A. C., Comi, B., Roskam, W., Kourilsky, P., LePennec, J. P., Perrin, F., Gerlinger, P., Cochet, M. & Chambon, P. (1978) Cell 14,629-639. 11. Mandel, J. L., Breathnach, R., Gerlinger, P., LeMeur, M., Gannon, F. & Chambon, P. (1978) Cell 14,641-653. 12. McReynolds, L., O'Malley, B. W., Nisbet, A. D., Fothergill, J. E., Givol, D., Fields, S., Robertson, M. & Brownlee, G. G. (1978) Nature (London) 273,723-728. 13. Breathnach, R., Benoist, C., O'Hare, K., Gannon, F. & Chambon, P. (1978) Proc. Natl. Acad. Sci. USA 75, 4853-4857. 14. Helling, R. B., Goodman, H. M. & Boyer, H. W. (1974) J. Virol. 14, 1235-1244. 15. Blattner, F. R., Blechl, A. E., Denniston-Thompson, K., Faber, H. E., Richards, J. E., Slightom, J. L., Tucker, P. W. & Smiethies, Q. (1979) Science 202, 1279-1284. 16. Dugaiczyk, A., Boyer, H. W. & Goodman, H. M. (1975) J. Mol. Biol. 96, 171-184. 17. Mandel, M. & Higa, A. (1970) J. Mol. Biol. 53, 159-162. 18. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H. & Falkov, S. (1977) Cene 2,95-113. 19. Lawn, R. M., Fritsch, E. F., Parker, R. C., Blake, G. & Maniatis, T. (1978) Cell 15, 1157-1174. 20. Woo, S. L. C., Rosen, J. M., Liarakos, C. D., Choi, Y. C., Busch, H., Means, A. R. & O'Malley, B. W. (1975) J. Biol. Chem. 250, 7027-7039. 21. 1Roop, D. R., Nordstrom, J. L., Tsai, S. Y., Tsai, M.-J. & O'Malley, B. W. (1978), Cell 15,671-685. 22. Bratosin, S., Horowitz, M. & Laub, 0. (1978) Cell 13, 783790. 23. Haegeman, G. & Fiers, W. (1978) Nature (London) 273, 70-

73. 24. Chow, L. T., Gelinas, R. E., Broker, T. R. & Roberts, R. J. (1977) Cell 12, 1-8. 25. Dunn, A. R. & Hassell, J. A. (1977) Cell 12,23-36. 26. !Evans, R. M., Fraser, N., Ziff, E., Weber, J., Wilson, M. & Darnell, J. E. (1978) Cell 12, 733-739. 27. O'Farrell, P. Z., Cordell, B., Valenzuella, P., Rutter, W. J. & Goodman, H. M. (1978) Nature (London) 274,438-445. 28. Knapp, G., Beckmann, J. S., Johnson, P. F., Fuhrman, S. A. & Abelson, J. (1978) Cell 14, 221-236. 29. 7'ilghman, S. M., Curtis, P. J., Tiemeier, D. C., Leder, P. & Weissman, C. (1978) Proc. Natl. Acad. Sci. USA 75, 1309-

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The ovalbumin gene: cloning and molecular organization of the entire natural gene.

Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2253-2257, May 1979 Biochemistry The ovalbumin gene: Cloning and molecular organization of the entire...
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