Nucleoprotein gene tracking: localization of specific HIV-1 genes to subchromatin nucleoprotein complexes containing endonuclease activity in HIV-1-infected human cells.
We developed a technique with which to isolate specific subchromatin deoxyribonucleoprotein/ribonucleoprotein precursor complexes containing discrete genes from intact mammalian nuclei by direct restriction enzyme treatment with MspI. These nucleoprotein complexes can be further fractionated and purified by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electroelution and removal of detergent, virtually thousands of nucleoprotein complexes can be examined for the presence of tightly bound genes and enzymatic activities. Nucleoprotein gene tracking procedures were applied to study the acidic nucleoprotein complexes from steady-state human H9 cells uninfected or infected with human immunodeficiency virus type 1 (HIV-1) virus. The purified nucleoprotein complexes were screened for the presence of loosely and tightly associated HIV-1 gene sequences (pol, env, tat, and rev) using a DNA hybridization protocol. In HIV-1-infected cells, four specific nucleoprotein complexes out of several hundred were found to contain tightly bound HIV-1 pol gene sequences after purification. By contrast, the other HIV-1 gene sequences (env, tat, and rev) were not tightly bound to any of the nucleoprotein complexes in HIV-infected cells. The observations suggest that certain HIV-1 genes associate with specific chromatin nucleoprotein complexes, regardless of their pattern of DNA integration into the human genome. At least two of the HIV-1 pol-containing nucleoprotein complexes of apparent M(r) approximately 94,000, pI approximately 6.5, and M(r) approximately 47,000, pI approximately 5.1 contain DNA endonuclease activity. This was confirmed in the present study, using linearized pUC19 plasmid substrate. The technique can be used to study a variety of problems concerning the association of specific genes and enzymes with specific nucleoprotein complexes J. Cell. Biochem. Suppls. 32/33:158-165, 1999.
Nicolson
N L
NL
Institute for Molecular Medicine, Huntington Beach, California 92649, USA.
Nicolson
G L
GL
D002458:Cell Fractionation; D002460:Cell Line; D002843:Chromatin / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D003852:Deoxyribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015684:Gene Products, pol / Q000235:genetics; D015694:Gene Products, rev / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics* / Q000502:physiology; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007526:Isoelectric Point; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D009698:Nucleoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism / Q000821:virology; D054302:pol Gene Products, Human Immunodeficiency Virus; D054321:rev Gene Products, Human Immunodeficiency Virus
D002843:Chromatin; D003852:Deoxyribonucleoproteins; D015684:Gene Products, pol; D015694:Gene Products, rev; C060467:HIV pol protein p65; D009698:Nucleoproteins; D012261:Ribonucleoproteins; D054302:pol Gene Products, Human Immunodeficiency Virus; D054321:rev Gene Products, Human Immunodeficiency Virus; D004706:Endodeoxyribonucleases; D019003:Deoxyribonuclease HpaII