Chemical synthesis and expression of a gene encoding HIV-1 TAT protein.
A gene encoding the human immunodeficiency virus-1 (HIV-1) TAT protein was chemically synthesized and expressed in HeLa cells and in a cell-free system. To facilitate both the assembly of the synthetic gene and further mutagenesis and gene fusion studies, several unique restriction endonuclease cleavage sites were included in the coding sequence without altering the encoded protein sequence. The synthetic TAT coding sequence was fused to a translation start signal and placed under SV40 early transcriptional control. Co-transfection of the TAT-encoding synthetic gene together with a reporter gene (chloramphenical acetyl transferase or beta-galactosidase) linked to an HIV LTR confirmed that the synthetic gene product exhibits similar activity to TAT expressed from HIV genomic DNA in the transactivation of the LTR. TAT mRNA prepared by cell-free transcription of the synthetic TAT coding sequence was also shown to produce functional TAT following microinjection into HeLa-derived cells containing an integrated reporter gene with the HIV LTR linked to beta-galactosidase.
Ferguson
B Q
BQ
Medical Products Department, E.I. du Pont de Nemours & Co., Inc., Wilmington, DE 19880.
Friedland
B K
BK
Hendrickson
E
E
Strehl
L
L
Petteway
S R
SR
Jonak
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000138:chemical synthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D015696:Gene Products, tat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D010944:Plants / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014170:Transformation, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015696:Gene Products, tat; D009856:Oncogene Proteins, Viral; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus