The Ovalbumin Gene INSERTION OF OVALBUMIN GENE SEQUENCES IN CHIMERIC BACTERIAL PLASMIDS”

Double-stranded ovalbumin DNA was amplified and purified by the cloning of bacterial transformants. The double-stranded DNA was synthesized from a complete complementary DNA transcript of ovalbumin mRNA using Escherichia coli DNA polymerase I and the self-priming ability of the initial transcript. After S, nuclease treatment, poly(dA) was added to the 3’ termini with terminal deoxynucleotidyltransferase and the ovalbumin gene was hybridized to a linear plasmid DNA, pMB9, containing 3’-poly(dT) termini. This hybrid molecule was used to transform the E. coli strain X1849. The cloned transformants contained from 30 to 53% of the complete ovalbumin DNA as determined by hybridization with full length cDNA,,,. The length of the inserts was confirmed by treatment of the isolated plasmids with the restriction enzyme Hha I. Separation


Double-stranded
ovalbumin DNA was amplified and purified by the cloning of bacterial transformants.
The doublestranded DNA was synthesized from a complete complementary DNA transcript of ovalbumin mRNA using Escherichia coli DNA polymerase I and the self-priming ability of the initial transcript.
After S, nuclease treatment, poly(dA) was added to the 3' termini with terminal deoxynucleotidyltransferase and the ovalbumin gene was hybridized to a linear plasmid DNA, pMB9, containing 3'-poly(dT) termini. This hybrid molecule was used to transform the E. coli strain X1849. The cloned transformants contained from 30 to 53% of the complete ovalbumin DNA as determined by hybridization with full length cDNA,,,. The length of the inserts was confirmed by treatment of the isolated plasmids with the restriction enzyme Hha I. Separation of the fragments by agarose gel electrophoresis showed that the amount of inserted DNA in the clones tested varied from 680 to 1090 base pairs. Bacterial Cloning of &albumin DNA involves the synthesis of double-stranded cDN&,, with Escherichia coli DNA polymerase I. No primer is necessary since the cDN&,, is self-priming (lo), as has been previously observed with globin cDNA (7-9). After cleavage with S1, poly(dA) was added to both ends of the cDN&,, molecule with terminal deoxynucleotidyltransferase.
After exposure of the plasmid pMB9 to Eco Rl, the same enzyme was used to add poly(dT) to the 3' end of the plasmid DNA molecule. The double-stranded cDN&,,-(dA),,,, was then hybridized to pMB9-(dT),,, and used to transform the bacterial strain X1849. This technique has been used previously to obtain plasmids containing rabbit globin DNA (8, 13). The plasmid pMB9 has a dual advantage; it confers bacterial resistance to tetracycline, and has the ability to continue to replicate when bacterial growth is inhibited by chloramphenicol.
When cell protein synthesis is inhibited the plasmid can comprise up to 40% of the total cellular DNA (36). Fig. 2 shows the autoradiograph of the 26 clones that were tetracycline-resistant.
The bacteria were grown on a lined Millipore filter, lysed with alkali, then hybridized with fv2P]RNA transcribed from cDNA,,,. The lined filter, which aids in numbering the clones, has been redrawn over the X-ray film. Thirteen clones hybridized to the probe. Four or five colonies which were strongly positive and were observed on a duplicate filter were studied in more detail. Plasmid DNA was purified for additional analysis from four clones which were designated, pOv1, pOv2, pOv3, and pOv4. The size of the inserted DNA was determined by agarose gel electrophoresis of the plasmids which had been digested with the restriction enzyme, Hha I (Fig. 3). SV40 DNA was digested with the restriction enzyme, Hpa I, into three fragments of 2075NTP, 1925NTP, and 1000. The size of the experimental bands was calculated from their mobilities relative to the three SV40 standards. The restriction enzyme, Hha I, has been shown by Maniatis et al. (13) to cut pMB9 into several fragments, with the largest fragment (950 NTP) containing the Eco Rl site for insertion of foreign DNA. As predicted, all four pOv clones lost this DNA band and another larger DNA band is observed (Fig. 3).
The size of the inserted DNA was calculated by subtracting the length of the plasmid DNA band (950 NTP) from the length of the new larger Hha I band. Table I shows that the inserts vary in size from 680 NTP to 1,090 NTP. These sizes were determined within an accuracy of 4 to 5% using the SV40 standards. The size of the inserted ovalbumin DNA has been overestimated slightly because it is joined to the plasmid by two poly(dA:dT) linkers.
The size of the ovalbumin DNA insert was also determined by a chimeric plasmid DNA excess hybridization to a complete copy of ["HlcDN&,.
The per cent of the probe hybridized represented the percentage of the total structural ovalbumin gene sequence (1750 NTP) present in the chimeric plasmid. Table I shows that the size determined is less than the estimate of the size of the inserted DNA obtained by gel electrophoresis. This difference, when divided by two, gives an estimate of the average length of the oligo(dA:dT) linkers (Table  I) Fig. 2. In addition, the purified plasmid DNA from transformants hybridized to cDNA,,, while the plasmid DNA did not. The correlation between the physical size of the inserted DNA as determined by gels and the length determined by hybridization to cDNA,,, argues that the larger size plasmids are produced by inserted ovalbumin DNA. The small differences between these two values probably reflects the oligo(dA:dT) regions that connect the plasmid and ovalbumin DNAs. Finally, using a ["H]cDNA,,. hybridization probe, we can detect the presence of mRNA,,, sequences in RNA synthesized from plasmid DNA in uiuo and in uitro.2 None of the initial four clones characterized contain the entire sequence complementary to mRNA,, The clones contain from 31 to 53% of the complete sequence of the ovalbumin structural gene. The partial inserts are easily explained by the omission of a sizing procedure during the preparation of the double-stranded cDNA,,, A complete copy of the doublestranded cDNA,, has been synthesized with viral RNA-dependent DNA polymerase (10). This DNA is presently being used to obtain a plasmid containing the entire mRN&, sequence Bacterial cloning of eukaryotic DNA allows the study of both the expression and the structure of purified genes. Since the ovalbumin gene is hormonally responsive, amplification and purification of milligram amounts of the complete DNA sequence via the use of bacterial plasmids should facilitate the discussion of the steps involved in steroid hormone regulation of gene transcription.
The role of DNA sequences in binding steroid receptor proteins, RNA polymerase, and chromosomal proteins can be evaluated. In addition, cleavage of the DNA by restriction enzymes has the potential of allowing the isolation and sequencing of specific regions of interest in the gene.