The ovalbumin gene. In vitro enzymatic synthesis and characterization.
Using purified single-stranded ovalbumin complementary DNA (cDNAov) as a template for avian myeloblastosis (AM) virus reverse transcriptase, we have enzymatically synthesized a complete double-stranded cDNAov sequence. Our data suggests that many single-stranded cDNAov molecules contain short double-stranded sequences (hairpins) at their 3' termini capable of acting as primers for synthesis of complete double-stranded cDNAs. Optimum conditions for synthesis of the double-stranded cDNAov were found to be a high temperature (46 degrees) and a low salt concentration. Nevertheless, in all cases 40% of the initial single-stranded cDNAov molecules fail to prime for synthesis of a complementary double strand. Following synthesis, the second DNA strand is covalently linked to the first cDNAov strand as shown by analysis on alkaline sucrose gradients. The two strands have a high Tm on hydroxylapatite (89 degrees). These intact double-stranded cDNAov structures have a bouyant density in CsCl gradients of 1.700 g/cm3 and rapidly renature after heat denaturation with a C0t1/2 value of less than 2 X 10(-6) mol s liter(-1). All size classes of cDNAs, i.e. partial as well as complete transcripts of the mRNA, are capable of forming double-stranded structures. The closed loop of the double-stranded cDNAov could be opened with S1 nuclease. The denatured complementary strands of the cDNAov then renatured with the appropriate second order kinetics at a C0t1/2 value of 1.89 X 10(-3) mol s liter(-1). Using the enzyme terminal deoxyribonucleotidyltransferase to label to free 3'-terminal end of double-stranded [32P]cDNAov with 3H, we demonstrate a convenient procedure to study the site for restriction endonuclease cleavage within the ovalbumin gene.
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009695:Nucleic Acid Renaturation; D009994:Osmolar Concentration; D010047:Ovalbumin / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D012965:Sodium Chloride; D010047:Ovalbumin; D012194:RNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes