BIOCHEMICAL
Vol. 168, No. 2, 1990 April 30, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 580-588
NUCLEAR PROTEINS FROM HeLa CELLS ARB IMHUNOREACTIVE WITH ANTI-YEAST Channa
Shalitin'l"
Department
Received
March
15,
Yp20 ras
RELATED ANTIBODIES
and Valerie
Stanik
of Biochemistry and Biophysics Oregon State University Corvallis, Oregon 97331
1990
Xmmunoblot analysis using antibody specific to yeast protein Yp20 revealed a 2lkDa protein associated with chromatin which immunologically cross-reacted with this protein. The 21kDa protein can be rapidly released from the by micrococcal nuclease digestion. It seems to be associated mononucleosomes as determined by separation of chromatin We suggest that the PlkDa HeLa protein sucrose gradients. 0 1990 Academic Press, Inc. human homologue of yeast Yp20.
ras-related HeLa cell ras-related HeLa nuclei with long subunits on may be the
We have recently generated polyclonal and monoclonal antibodies against Yp20 (a yeast ras-related protein) which are capable to immunoreact with ~21 ras proteins in tissue culture cell lines and in fresh tissue sections (l-3). The polyclonal anti-Yp20 antibodies were shown to immunoreact with bacterially expressed cHras and T24 Hras genuine ras products (4). The Yp20 is distinct from other ras-related proteins previously described (5) in localization in the cell. While most ras-related gene products are localized in the inner surface of the plasma membrane, or cytoplasm (6), Yp20 protein is the first yeast chromatin-associated DNA binding protein which was shown to be rasrelated (4,7). In view of these findings, we have examined herein associated with HeLa chromatin in an attempt to search protein which could cross react with anti-Yp20 antibodies represent a human nuclear ras-related protein. +To whom correspondence should Geiduschek, Center for Molecular San Diego, La Jolla, California * On sabbatical leave Institute of Technology,
be addressed Genetics, 92093-0634.
in care University
from the Department of Haifa 32000, Israel.
the for
proteins a 21kDa and could
of Prof. E. Peter of California,
Biology,
Technion-Israel
Abbreviations used: 20kDa yeast protein; EGTA, YP20, ethyleneglycol-bis-(fi amino-ethyl ether); N, N'-tetra-acetic acid; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; EDTA, ethylenediamine-tetraacetic acid; BSA, bovine serum albumin; HMGl and HMGZ, high mobility group proteins 1 and 2; rho, ras homologue. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
580
Vol.
BIOCHEMICAL
166, No. 2, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALSAND METHODS Preparation
of HeLa S3 nuclei
Nuclei were prepared by a procedure modified from Strauss and Cells were grown in suspension culture in minimum Varshavsky (8). essential medium (Joklik-modified, Gibco, Lab supplemented with 10% fetal bovine serum to a concentration of 5x104' cells/ml. 500 ml cells were collected by centrifugation at 4000 rpm 10 min in Sorvall GSA rotor. The cell pellet was resuspended, and washed 3 times with cold O.lM NaCl, 5OmM KCl, 1OmM Na-phosphate, pH 7.2. The pellet was resuspended in 10 vol of 0.23M sucrose in buffer A (60mM KCl, 15mM NaCl, 0.25mM MgC12, 0.5mM Na EGTA, 0.5mM spermine, 0.15mM spermidine, 14mM 2mercaptoethanol, 15mM Tris-HCl (pH 7.4), containing freshly added proteinase inhibitors: phenylmethylsulfonyl fluoride (PMSF 0.2mM) and 5pg/ml each of antipain, leupeptin, chymostatin and pepstatin A (Sigma). Cells were disrupted in a Potter Elvehjem homogenizer by 12 strokes, then Triton X-100 was added to a final concentration of 1%. Phase contrast microscop showed only nuclei and no intact cells. The lysate ?i nuclei) (10ml -2.5-4.0x10 was diluted 3-fold with 2.OM sucrose in buffer A, layered onto 9ml cushion of 1.7M sucrose in buffer A and spun in SS34 rotor 13000rpm for 45 min at 4OC in RC-5B centrifuge (Sorvall). A 0.5ml sample of the supernatant containing the cytoplasm corresponding to 5~10~ cells was dialyzed vs. O.lM acetic acid and lyophilized. The nuclei were gently resuspended, washed once in lOm1 of 0.23M sucrose in buffer A plus protease inhibitors, and resuspended again in 0.23M sucrose in buffer A to a final DNA concentration of 2.5mg DNA/ml. The nuclei were frozen quickly in liquid nitrogen and stored at -7O'C for 6 weeks without a noticeable change in results. Prenaration
of HeLa chromatin
The nuclei were resuspended and washed four times in lml digestion buffer (0.23M sucrose, l.OmM CaC12, l.OmM Tris-HCl, pH 8.0, O.lmM PMSF). Extensive washes were necessary to remove the protease inhibitors which inhibit micrococcal nuclease activity. 900u/ml micrococcal nuclease (Cooper Biomedical - 19,00Ou/mg) was added to the nuclei at a concentration of l-2xlO*/ml. Digestion proceeded for 2 min at 37'C. The reaction was stopped by the addition of 2mM EDTA, pH 7.5 and cooling on ice. After mixing the suspension on a vortex, the nuclei were pelleted in a microfuge for 10 min. The supernatant contained more than 50% of the DNA and was used for the isolation of nucleosomes. Isolation
of nucleosomes
-1.5mg of DNA [A260'1.0 corresponds to %pg/ml DNA, assuming the absorbance of light by DNA is unaffected by histones - this value was used to calculate nucleosome concentration (9)] were loaded onto a linear 5-20% sucrose gradient in 1OmM Tris-HCl, pH 7.5, 1mM EDTA, 1mM PMSF, 1mM NaHSO and 14mM 2-mercaptoethanol. Gradients were run for 18h, 30,000 rpm at 4a C in SW40 rotor. One ml fractions were collected from the top of the gradient. The fractions were dialyzed overnight in a Spectrapor tubing (cutoff
Vol. 168, No. 2, 1990 April 30, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 580-588
NUCLEAR PROTEINS FROM HeLa CELLS ARB IMHUNOREACTIVE WITH ANTI-YEAST Channa
Shalitin'l"
Department
Received
March
15,
Yp20 ras
RELATED ANTIBODIES
and Valerie
Stanik
of Biochemistry and Biophysics Oregon State University Corvallis, Oregon 97331
1990
Xmmunoblot analysis using antibody specific to yeast protein Yp20 revealed a 2lkDa protein associated with chromatin which immunologically cross-reacted with this protein. The 21kDa protein can be rapidly released from the by micrococcal nuclease digestion. It seems to be associated mononucleosomes as determined by separation of chromatin We suggest that the PlkDa HeLa protein sucrose gradients. 0 1990 Academic Press, Inc. human homologue of yeast Yp20.
ras-related HeLa cell ras-related HeLa nuclei with long subunits on may be the
We have recently generated polyclonal and monoclonal antibodies against Yp20 (a yeast ras-related protein) which are capable to immunoreact with ~21 ras proteins in tissue culture cell lines and in fresh tissue sections (l-3). The polyclonal anti-Yp20 antibodies were shown to immunoreact with bacterially expressed cHras and T24 Hras genuine ras products (4). The Yp20 is distinct from other ras-related proteins previously described (5) in localization in the cell. While most ras-related gene products are localized in the inner surface of the plasma membrane, or cytoplasm (6), Yp20 protein is the first yeast chromatin-associated DNA binding protein which was shown to be rasrelated (4,7). In view of these findings, we have examined herein associated with HeLa chromatin in an attempt to search protein which could cross react with anti-Yp20 antibodies represent a human nuclear ras-related protein. +To whom correspondence should Geiduschek, Center for Molecular San Diego, La Jolla, California * On sabbatical leave Institute of Technology,
be addressed Genetics, 92093-0634.
in care University
from the Department of Haifa 32000, Israel.
the for
proteins a 21kDa and could
of Prof. E. Peter of California,
Biology,
Technion-Israel
Abbreviations used: 20kDa yeast protein; EGTA, YP20, ethyleneglycol-bis-(fi amino-ethyl ether); N, N'-tetra-acetic acid; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; EDTA, ethylenediamine-tetraacetic acid; BSA, bovine serum albumin; HMGl and HMGZ, high mobility group proteins 1 and 2; rho, ras homologue. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
580
Vol.
BIOCHEMICAL
166, No. 2, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALSAND METHODS Preparation
of HeLa S3 nuclei
Nuclei were prepared by a procedure modified from Strauss and Cells were grown in suspension culture in minimum Varshavsky (8). essential medium (Joklik-modified, Gibco, Lab supplemented with 10% fetal bovine serum to a concentration of 5x104' cells/ml. 500 ml cells were collected by centrifugation at 4000 rpm 10 min in Sorvall GSA rotor. The cell pellet was resuspended, and washed 3 times with cold O.lM NaCl, 5OmM KCl, 1OmM Na-phosphate, pH 7.2. The pellet was resuspended in 10 vol of 0.23M sucrose in buffer A (60mM KCl, 15mM NaCl, 0.25mM MgC12, 0.5mM Na EGTA, 0.5mM spermine, 0.15mM spermidine, 14mM 2mercaptoethanol, 15mM Tris-HCl (pH 7.4), containing freshly added proteinase inhibitors: phenylmethylsulfonyl fluoride (PMSF 0.2mM) and 5pg/ml each of antipain, leupeptin, chymostatin and pepstatin A (Sigma). Cells were disrupted in a Potter Elvehjem homogenizer by 12 strokes, then Triton X-100 was added to a final concentration of 1%. Phase contrast microscop showed only nuclei and no intact cells. The lysate ?i nuclei) (10ml -2.5-4.0x10 was diluted 3-fold with 2.OM sucrose in buffer A, layered onto 9ml cushion of 1.7M sucrose in buffer A and spun in SS34 rotor 13000rpm for 45 min at 4OC in RC-5B centrifuge (Sorvall). A 0.5ml sample of the supernatant containing the cytoplasm corresponding to 5~10~ cells was dialyzed vs. O.lM acetic acid and lyophilized. The nuclei were gently resuspended, washed once in lOm1 of 0.23M sucrose in buffer A plus protease inhibitors, and resuspended again in 0.23M sucrose in buffer A to a final DNA concentration of 2.5mg DNA/ml. The nuclei were frozen quickly in liquid nitrogen and stored at -7O'C for 6 weeks without a noticeable change in results. Prenaration
of HeLa chromatin
The nuclei were resuspended and washed four times in lml digestion buffer (0.23M sucrose, l.OmM CaC12, l.OmM Tris-HCl, pH 8.0, O.lmM PMSF). Extensive washes were necessary to remove the protease inhibitors which inhibit micrococcal nuclease activity. 900u/ml micrococcal nuclease (Cooper Biomedical - 19,00Ou/mg) was added to the nuclei at a concentration of l-2xlO*/ml. Digestion proceeded for 2 min at 37'C. The reaction was stopped by the addition of 2mM EDTA, pH 7.5 and cooling on ice. After mixing the suspension on a vortex, the nuclei were pelleted in a microfuge for 10 min. The supernatant contained more than 50% of the DNA and was used for the isolation of nucleosomes. Isolation
of nucleosomes
-1.5mg of DNA [A260'1.0 corresponds to %pg/ml DNA, assuming the absorbance of light by DNA is unaffected by histones - this value was used to calculate nucleosome concentration (9)] were loaded onto a linear 5-20% sucrose gradient in 1OmM Tris-HCl, pH 7.5, 1mM EDTA, 1mM PMSF, 1mM NaHSO and 14mM 2-mercaptoethanol. Gradients were run for 18h, 30,000 rpm at 4a C in SW40 rotor. One ml fractions were collected from the top of the gradient. The fractions were dialyzed overnight in a Spectrapor tubing (cutoff
