Detection of enhancer binding proteins recognizing the human immunodeficiency virus long terminal repeat by in situ gel retardation.
We have examined the formation of a DNA "enhancer" - protein complex occurring in situ. Oligonucleotides corresponding to the human immunodeficiency virus (HIV) core enhancer sequence were synthesized, annealed and radiolabeled. The DNA was electroporated either into Jurkat cells or into fresh human peripheral blood T lymphocytes. After the appropriate incubation time and stimulation with various mitogenic agents, cells were lysed and the lysates were electrophoresed on a native polyacrylamide gel. The specific protein-nucleic acid complexes which we obtained were apparently identical to those observed with the "classical" in vitro gel mobility shift assay: one complex seems to be constitutive and the other is induced by mitogens. Additionally competition experiments using "cold" oligonucleotides demonstrated binding specificity in situ. We recommend this novel method for studying DNA-binding proteins and their activation since it requires as few as 10(6) cells, may use primary tissue isolates, and furthermore, allows the rapid assessment of cellular activation signals involved in the post-transcriptional modification of trans-acting factors.
Brini
A T
AT
Laboratory of Molecular Immunoregulation, Frederick Cancer Research Facility, MD 21701-1013.
Harel-Bellan
A
A
Korner
M
M
Farrar
W L
WL
D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D006678:HIV / Q000235:genetics*; D006801:Humans; D015458:Leukemia, T-Cell; D012091:Repetitive Sequences, Nucleic Acid; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D004268:DNA-Binding Proteins