ABSTRACT
The translational enhancer (TREN) sequence of the phage T7 gene 10 (in full and also its proximal or distal parts) have been obtained by chemical-enzymatic synthesis and cloned into the plasmids immediately before the human interleukin 3 (hIL3) artificial gene. Expression levels of the hIL3 gene in E. coli in these constructions show that the region controlling the specific activity is placed in distal part of TREN more than 40 nucleotides upstream from the initiation codon.
Subject(s)
Enzymes/chemistry , Genes, Viral , Protein Biosynthesis , T-Phages/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enhancer Elements, Genetic , Humans , Interleukin-3/genetics , Molecular Sequence Data , PlasmidsABSTRACT
A modified H-phosphonate method was used to synthesize 32 oligodeoxyribonucleotides ranging in length from 23 to 28, which were enzymatically joined together to give the human interleukin 4 gene. The high degree of the oligonucleotide purity, achieved through the application of anion-exchange and reverse phase HPLC, ensures the high percentage of the desired sequence (about 75%) in the cloned DNA.
Subject(s)
Genes, Synthetic , Genes , Interleukin-4/genetics , Amino Acid Sequence , Base Sequence , Chemical Phenomena , Chemistry , Humans , Molecular Sequence Data , Oligonucleotide Probes , OrganophosphonatesABSTRACT
p-Nitrophenylethyl blocking group was used to protect the endocyclic imido groups of guanine and thymine nucleoside 3'-H-phosphonates employed in the H-phosphonate synthesis of a large number of oligodeoxyribonucleotides varying in length from 8 to 45 units. A combination of the fully protected monomers with a condensing agent, pivaloyl chloride or mesitylenesulphonyl-3-nitro-1,2,4-triazole, provides a rapid and effective synthesis of long oligonucleotides.