Enzymatic synthesis of a segment of bacteriophage Qbeta coat protein gene.
The oligoribonucleotide, A-A-A-C-U-U-U-Gp, constituting a segment of RNA bacteriophage Qbeta coat protein gene was efficiently synthesized at a milligram scale by a combination of enzymatic methods using bacteriophage T4 RNA ligase and the thermophilic polynucleotide phosphorylase. A-A-A-Cp was synthesized from A-A-A and pCp by the newly developed mononucleotide addition method using T4 RNA ligase in a yield of 83%, followed by dephosphorylation with bacterial alkaline phosphatase to obtain A-A-A-C. pU-U-U-Gp was synthesized from pU-U-U and GDP by the simultaneous action of polynucleotide phosphorylase and RNase T1 in a yield of 32%. finally, the two oligonucleotides (A-A-A-C and pU-U-U-Gp) were ligated with T4 RNA ligase and the octanucleotide, A-A-A-C-U-U-U-Gp, was obtained in a yield of 85%.
D000469:Alkaline Phosphatase / Q000378:metabolism; D001435:Bacteriophages; D003090:Coliphages / Q000201:enzymology; D005814:Genes, Viral; D009841:Oligonucleotides / Q000138:chemical synthesis*; D009843:Oligoribonucleotides / Q000138:chemical synthesis*; D011118:Polynucleotide Ligases / Q000378:metabolism*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D012367:RNA, Viral / Q000138:chemical synthesis* / Q000235:genetics; D006163:Ribonuclease T1 / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics*
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012367:RNA, Viral; D014764:Viral Proteins; D011117:Polyribonucleotide Nucleotidyltransferase; D006163:Ribonuclease T1; D000469:Alkaline Phosphatase; D011118:Polynucleotide Ligases; D011130:RNA Ligase (ATP)