Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair.
The synthesis of a gene for the HIV TAT protein is described using a novel approach that capitalises on the ability to synthesise oligonucleotides of greater than 100 bp in length. It involves the synthesis of large oligomers covering one strand of the desired gene in its entirety and the use of small complementary bridging and adapter oligonucleotides to direct the assembly and cloning of the large oligomers. After ligation to the cloning vector the partially single stranded intermediate is transformed directly into the recipient bacterial host where the plasmid is repaired. The synthetic tat gene has been expressed in HeLa cells and is shown to trans-activate TAR+ but not TAR- HIV LTR-CAT constructs.
Adams
S E
SE
Department of Molecular Biology, British Bio-technology Ltd, Cowley, Oxford, UK.
Johnson
I D
ID
Braddock
M
M
Kingsman
A J
AJ
Kingsman
S M
SM
Edwards
R M
RM
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015694:Gene Products, rev; D005796:Genes; D005813:Genes, Synthetic; D005814:Genes, Viral; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D012191:Retroviridae Proteins / Q000235:genetics*; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D012191:Retroviridae Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus