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Organ discrimination through organ-specific nonhistone chromosomal proteins
ARCHIVES
OF
BIOCHEMISTRY
Organ
.2ND
161, 11-19 (1974)
BIOPHYSICS
Discrimination
Through
Organ-Specific
Chromosomal ISAAC BEKHOR,
Deparlmenl
LAKSHMI
of Biochemistry,
Nonhistone
Proteins
ANNE, JUNG KIM, JEAN-NUMA ROGER STAMBAUGH Sch,ool of Dentistry, University Los Angeles, California 90007 Received
November
of Southern
LAPEYRE,
AX’D
California,
12, 1973
Prefractionation of chromosomal proteins in 5 M urea with stepwise increase in NaCl molarity has been used to facilitate the examination of nonhistone chromosomal proteins isolated from various rabbit tissues. Electrophoretic analysis on polyacrylamide gels under denat,uring conditions of the protein fractions derived from brain, liver, heart, and submandibular salivary gland chromatins displays reproducible compositional differences in nonhistone chromosomal proteins. The enzymatic removal of 48’7; of protein-bound phosphate with alkaline phosphatase does not significantly alter the electrophoretic mobility of these proteins. With the present technique, it is estimated that chromatin polypeptides (of average M, 100,000) occurring in greater than 3 X lo* copies per genome can be detected. At this level of sensia significant fraction of total nonhistone chromosomal proteins manifests tivity, organ specificit.y.
The implication that nonhistone chromosomal proteins (NHCP)’ represent tissue specificity (1-3) requires additional experimentation on the significance of such a finding. Recently, Bckhor et al. (2) have established a fractionation procedure for NHCP which demonstrated the resolution of at least 99 electrophoretic bands of varying mobilities for rabbit liver chromosomal proteins. This procedure is now employed to look for tissue-specific chromosomal protein complements isolated from rabbit brain, heart, and submandibular salivary gland chromatins. It is concluded that these organs may be classified in terms of their NHCP as resolved by disc gel electrophoresis. This type of an analytical examination would
facilitate the isolation NHCP cither individually MATERIALS
11 @ 1974 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Materials. All procedures were carried out at 04” unless otherwise indicated. All chemicals and biochemicals were purchased from Calbiochem unless otherwise stated. The following solutions were used in the fractionation of chromosomal proteins : Medium 0: 5 M urea, 0.01 M Tris-HCl, 2.85 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0. Medium 0.5: 5 M urea, 0.01 M Tris-HCl, 0.5 M NaCl, 2.85 mM PMSF, pH 8.0. Medium 1.0: 5 M urea, 0.01 M Tris-HCl, 1.0 M NaCl, 2.85 mM PMSF, pH 8.0. Medium 2.0: 5 M urea 0.01 M Tris-HCl, 2.0 M NaCl, 2.85 mrvr PMSF, pH 8.0. Medium 3.0: 5 M urea, 0.01 M Tris-HCl, 3.0 M NaCl, 2.85 mM PMSF, pH 8.0. Isolation of nonhisto?le chromosomal protein. Purified chromatin was isolated from newborn (3to 5-day-old) New Zealand white rabbit brain, heart, liver, and submandibular salivary gland according to the procedure of Marushige and Bon-
1 Abbreviations used: NHCP, nonhistone chromosomal proteins; CP, chromosomal proteins; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; TCA, trichloroacetic acid; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; OD, optical density; ilf,, molecular weight; U, enzyme internationa.1 units.
Copyright All rights
AND
of tissue-specific or in a group.
12
BEKHOR
ner (4). Chromosomal proteins (CP) were isolated from purified chromatin as described by Bekhor et al. (2) in accompanying communication (2). Briefly, after the two washes in .Ol M Tris, pH 8.0, after purification on 1.7 M sucrose, chromatin was homogenized with Teflon-glass Potter-Elvehjem homogenizer in Medium 0, then centrifuged in
DIAGRAM
ET AL. SW 50.1 for 20 hr at 50,000 rpm to pellet urea-exA\ tracted chromatin. The supernatant (fraction was saved for analysis of urea-extracted NHCP on disc gel electrophoresis. Further fractionation of the chromatin proteins was carried out with Medium 0.5, Medium 1.0, and Medium 2.0 or 3.0 (Diagram 1).
1
FRACTIONATION OF NHCP Purified chromatin suspended in Med. 0 I Centrifuged at 50 krpm for 20 hr
Supernatanl Fraction A
Peliet 0 Suspended in Med. 0.5
I
Centrifuged at 50 krpm for 20 hr I Pellet 0.5 Suspended in Med.
1
Supernatant Fraction B
1 .O
I
I
Centrifuged at 50 krpm for 2Q hr I
Pellet 1 .O Suspended in Med.
Supernatant Fraction C
2.0
Centrifuged &t 50 krpm for 20 hr
Pellet 2.0 Saved for thermomelting studies
Superhatant Fraction D
NONHISTONE
CHROMOSOMAL
The various chromosomal protein fractions were dialyzed extensively against distilled water, lyophilized to a dry powder, and resolubilized with of l-2 mg 5 M urea, ,270 SDS to a concentration protein/ml (5-10 ODpso/ml). Electrophoretic analysis of 100-200 fig protein/gel was carried out in the Laemmli system (5) as described previously (2). Gels were stained with Coomassie brilliant blue (Sigma Chemical Co.) and banding pattern recorded by visual inspection, photographically, and by scanning at 600 nm with Guilford Model 240 recording spectrophotometer equipped with a linear transport. Elrzymatic dephosphorylation of chromosomal proteins. Chromatin proteins were enzymatically dephosphorylated with electrophoretically purified E. coli alkaline phosphatase (BAPF, 30 units/ mg, Worthington Biochemicals). One to five milligrams protein/ml were incubated for 1 hr at 37” C with 90 rg/ml of alkaline phosphatase in the presence of 1% SDS. During initial experiments on optimal conditions for phosphatase activity, it was found that the rate of phosphate hydrolysis was stimulated by SDS, reaching a 4-fold increase in lyO SDS relative to that found without SDS. The presence of 5 M urea decreased the rate of hydrolysis by about 2056 relative to the 1% SDS level (data not shown). Therefore, the incubation of alkaline phosphatase with CP was done in 10/OSDS in the absence of urea and at pH 7.0. After enzymatic digestion, the samples were adjusted to 5 M urea and examined by disc gel electrophoresis for possible effects of dephosphorylation on chromosomal protein gel electrophoretic mobilities. Determination of protein-bourld phosphate release from NHCP with alkaline phosphatase. Protein-free rabbit liver DNA, purified as described previously (6), was labeled in vitro with [3H]dimethylsulfate (New England Nuclear, sp act 384 mCi/mmole) (7), and purified by ethanol precipitation, and gel exclusion chromatography on 1.5 X 20-cm G-100 Sephadex column equilibrated with 0.1 M phosphate buffer, pH 6.8. An aliquot of irk vifro-labeled [3H]DNA (220,000 cpm, sp act 9200 cpm/pg) was mixed with 300 OD,,, of purified liver chromatin; resultant sp act 18.3 cpm/pg DNA. Residual DNA content in subsequent fractions was determined by measurement of acid-insoluble tritium counts. The chromatin solution was adjusted to 20/Oin Na dodecylsarcosinate and 4 M CsCl (p = 1.48 g/cc) and centrifuged for 20 hr at 50 krpm in SW 50.1. The floating protein skin was washed once in 70yo ethanol, twice in 5cj, TCA, and rinsed twice with 0.01 M Tris, pH 8.0. This material was dispersed by sonication for 1 min at maximum intensity with Branson Sonifier in TM buffer (0.01 M Tris, 1 mM MgC12, pH 8.0) to yield a protein concentration of
13
PROTEINS
2.8 mg/ml. Residual nucleic acid contamination was removed by incubation for 1 hr at 37°C with 50 pg RNase A (Worthington Biochemicals), 360 U/ml RNase Tl (Schwartz Bio-Research), 200 rg/ml DNase 1 (DN-EP) Sigma Chemical Co.), and 25 U/ml snake venom phosphodiesterase (Calbiochem). Alkaline phosphatase hydrolysis was carried out with 4-15 U/ml for 1 hr at 37” C in TM buffer containing lyO SDS. The protein was precipitated with 20y0 TCA and combusted with concentrated H,SO, in 0.1 1\’ nitric acid-washed Corex tubes. Blanks with appropriate concentration of enzymes and buffers were also analyzed to correct for introduction of phosphate in enzyme preparations and buffers. Phosphate analysis was done by the method of Rathbun and Betlach (8). DNA was determined by diphenylamine reaction according to Burton (9). RNA determination was done according to Lin and Schjeide (10) and corrected for l>NA reactivity. Protein was determined by the method of Lowry et al. (11). RESULTS
AND
DISCUSSION
Fractionation and analysis of nonhistone chromosomal proteins on polyacrylamide disc gel electrophoresis make possible the eventual “finger printing” of chromatin relative to a specific organ. It would, therefore, become possible to identify genetic aberration at the nonhistone chromosomal protein level once functions are assigned to NHCP. Amplification of the types of NHCP which may be related to tissue-specific gene activity is only the first step toward the ultimate functional characterization of the acidic proteins found associated to DNA in chromatin. The result’s in Fig. 1 show that organ specificity may be assigned to a part of all the fractions of NHCP examined for brain, heart, liver, and submandibular salivary glands. These same gels may also be compared by scanning at 600 nm in the Guilford Spectrophotometer Model 240. Figures 2 and 3 show such a comparison between brain and heart chromosomal proteins. It is, therefore, concluded that one can discriminate
the various
organs
under
study
on the
basis of their NHCP composition. That the liberation of the phosphates from NHCP does not cause appreciable variation in the mobility of these prot’eins is shown in Fig. 4 in the case for liver XHCP fract,ions A and B; the presence of alkaline
u
t
HEART
BRAIN
SALIVARY
LIVER
HEART
BRAIN
SALIVARY
LIVER
GLAND
GLAND
+
I //I II
I
BRAIN
SALIVARY
LIVER
HEART
BRAIN
SALIVARY
LIVER
GLAND
GLAND
NONHISTONE
CHROMOSOMAL
PROTEINS
16
BEKHOR
ET AL.
+
0
m
a
uu 009
'a.0
1.0
NONHISTONE
CHROMOSOMAL
PROTEINS
17
FIG. 4. Guilford spectrophotometer scans at 600 nm of liver NHCP fractions A and B (see Diagram 1) before (top) and after (bottom) hydrolysis of protein-bound phosphate with electrophoretically purified E. coli alkaline phosphatase in 1% SDS. The presence of alkaline phosphatase is shown by an arrow (bottom), and the histone peaks in fraction B are pointed out by the letter H. No marked differences are observed in the scans of alkaline phosphatase treated and untreated NHCP. Fraction A alkaline phosphatase peak corresponds to 9 pg and that in fraction B represents 1 pg of protein. A breakin the scan in A indicates a a-fold increase Z-fold decrease in scanning sensitivity. density scale is shown on the left.
in scanning sensitivity, Direction of scanning
phosphatase band is identified by an arrow. The molecular weight of the metallo-enzyme E. coli alkaline phosphatase was 86,000, and this enzyme gave a single band on electrophoresis on polyacrylamide gels in 4 M urea-O.2% SDS, pH 8.9. It is significant to note that, although E. coli alkaline phosphatase is known to be formed of two subunits of M, approx 40,000 (12), it electrophoresed only as a single band equivalent to M, approx SO,OOO-90,000. Thus, the conditions used for electrophoresis as indicated in this communication may not necessarily yield subunit mobility of NHCP.
and that in B indicates a is from left to right. Optical
The fractions of chromosomal proteins extracted at the high salt concentrations (medium 2.0 or 3.0) were found to be contaminated with DNA. Although RNA can be totally removed by treatment with RNase, DNA is more difficult to eliminate. Therefore, the experiment shown in Table I was performed to determine actual proteinbound phosphate released by hydrolysis with alkaline phosphatase. This type of a measurement was necessary to exclude any errors in the assessment of protein-bound phosphate which was not accessible to hydrolysis with alkaline phosphatase. It was
18
BEKHOR
found that treatment with alkaline phosphatase (under the conditions described in Methods) liberated about 48% of proteinbound phosphate (Table I) from total liver CP extracted from chromatin with 4 M CsCI. The electrophoretic pattern of this preparation is shown in Fig. 5. This type of an experiment indicated not only the reproducibility of the gels but also made it possible to identify the presence of a major foreign protein in that fraction. The alkaline phosphatase peak with an isoelectric point of pH 4.5 (13) represents a total of 9 pg in Fig. 4A and 1 kg in Fig. 4B. TABLE ANALYSIS
3H-DNA (cpm) per 300 OD260 chromatin
sarcosyl
The sensitivity of detecting Coomassie brilliant blue-stained bands may be of the order of 0.2 pg or less for NHCP (Fig. 4). We estimate from the size of the rabbit genome (5.3 pg/nucleus) (14) and the range of the molecular weights of polypeptides obtained on disc gel electrophoresis (lO,OOO200,000 daltons) that the minimum level of detectable copies of homogeneous NHCP per genome would be on the order of lo5 down to 1.5 X lo4 for the higher molecular weight NHCP. It would, therefore, be expected that we are either detecting nonhistone chromosomal proteins which are I
OF PROTEIN-BOUND PHOSPHATE OF DNA IN CP UNDER VARIOUS
Fraction
4 M C&1-2’%
ET AL.
chromatin
suspension 0.01 M Tris, pH 8.0, sonicated”skin”” after centrifugation from 4 M CsCl Nuclease-treated sonicated “skin” Actual Corrected for DNA* Nuclease and alkaline phosphatasetreated “skin” Actual Corrected for DNAb
DNA
AND DISTRIBUTION CONDITIONS
(a)
220,000
12,000
8,170
444
% DNA remaining
Phosphate bd
Phosphate bmole per mg protein)
100
-
3.7
101.85
0.235
0.51
1,131 0.0
61.5 0.0
0.0
57.45 51.30
0.132 0.118
1,131 0.0
61.5 0.0
0.51 0.0
33.20 27.05
0.076 0.062
0 See Methods, section on determination of protein-bound phosphate. * Corrected for DNA by assuming that all of DNA phosphate groups are susceptible alkaline phosphatase.
to hydrolysis
FIG. 5. Gel electrophoretic pattern of total liver NHCP isolated from chromatin upon centrifugation through 4 M CSCI-~~~ sarcosyl, and after washing in 70y0 ethanol, precipitation from 5’% TCA, and suspension by sonication in TM buffer (see Methods).
by
NONHISTONE
CHROMOSOMAL
found in many repeating copies, or that we are looking at classes of proteins similar in molecular weight. The latter would imply that the number of NHCP associated to DNA may fall in the hundreds if each electrophoretic band represent’s numerous structurally different proteins or protein subunits. Fen- conclusions may be invoked from the present study: (1) organs may be discriminated through their NHCP composition; (2) removal of 48% of protein-bound phosphate does not significantly alter the electrophoretic mobility of chromosomal proteins; (3) electrophoresis on polyacrylamide disc gels in 4 M urea-O.2 % SDS, pH 8.9, may not necessarily represent, mobility of protein subunits; (4) it is possible to identify the presence of a major abnormal acidic protein in NHCP by disc gel electrophoresis; and (5) it is now conceivable that we can examine genetic aberration at the level of nonhistone chromosomal proteins. ACKNOWLEDGMENTS The authors are grateful to Mr. P. Bringas, Jr., for the illustrations. This work was supported by Grant DE-03235-02 and by a Career Development Award 5K04DE47354-03 to I.B. from the National Institutes of Dental Research. JNL, predoctoral fellow and RS postdoctoral fellow are supported by Training Grant DE-00094.11 from
PROTEINS
19
the National Institutes of Dental Research. LA is a graduate student of this department supported by Grant DE-03235-02 from the National Institutes of Dental Research. REFERENCES
1.
SPELSBERG, T. C., WILHELM, J. A., BND HINILICA, L. S. (1972) Subcell. Biochem. 1, 107. 2. BEKHOR, I., L~PEYRE, J. N., MD KIM, J.
(1974) Arch. Biochem. Biophys. 161, l-10. 3. WAKABAYASHI, K., AND HINILICA, L. 8. (1973) Nature (London) New Biol. 242, 153. 4. MARUSHIGE, K., AND BONNER, J. (1966) J. Mol. Biol. 16, 160. 5. LAEMMLI, U. K. (1970) Nature (London) 227, 680. I. (1973) Arch. Biochem. Biophys. 6. BEKHOR, 166, 39. H., .~ND YAMAMOTO, N. (1970) Bio7. AKIYOSHI, them. Biophys. Res. Commun. 38, 915. W. B., AND BETLACH, M. V. (1969) 8. RATHBUN, Anal. Biochem. 28, 436. 9. BURTON, K. (1956) Biochem. J. 63, 615. 10. LIN, R. I., AND SCHJEIDE, 0. A. (1969) Anal. Biochem. 2’7, 473. 11. LOWRY, O., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 12. LAZDUNSKI, C., AND L~ZDUNSKI, M. (1969) Eur. J. Biochem. 7, 294. 13. GAREN, A., AND LEVINTHAL, C. (1960) Biochim. Biophys. Acta 38, 470. 14. VENDRELY, R., .~ND VENDRELY, C. (1949) Experientia 6, 327.
OF
BIOCHEMISTRY
Organ
.2ND
161, 11-19 (1974)
BIOPHYSICS
Discrimination
Through
Organ-Specific
Chromosomal ISAAC BEKHOR,
Deparlmenl
LAKSHMI
of Biochemistry,
Nonhistone
Proteins
ANNE, JUNG KIM, JEAN-NUMA ROGER STAMBAUGH Sch,ool of Dentistry, University Los Angeles, California 90007 Received
November
of Southern
LAPEYRE,
AX’D
California,
12, 1973
Prefractionation of chromosomal proteins in 5 M urea with stepwise increase in NaCl molarity has been used to facilitate the examination of nonhistone chromosomal proteins isolated from various rabbit tissues. Electrophoretic analysis on polyacrylamide gels under denat,uring conditions of the protein fractions derived from brain, liver, heart, and submandibular salivary gland chromatins displays reproducible compositional differences in nonhistone chromosomal proteins. The enzymatic removal of 48’7; of protein-bound phosphate with alkaline phosphatase does not significantly alter the electrophoretic mobility of these proteins. With the present technique, it is estimated that chromatin polypeptides (of average M, 100,000) occurring in greater than 3 X lo* copies per genome can be detected. At this level of sensia significant fraction of total nonhistone chromosomal proteins manifests tivity, organ specificit.y.
The implication that nonhistone chromosomal proteins (NHCP)’ represent tissue specificity (1-3) requires additional experimentation on the significance of such a finding. Recently, Bckhor et al. (2) have established a fractionation procedure for NHCP which demonstrated the resolution of at least 99 electrophoretic bands of varying mobilities for rabbit liver chromosomal proteins. This procedure is now employed to look for tissue-specific chromosomal protein complements isolated from rabbit brain, heart, and submandibular salivary gland chromatins. It is concluded that these organs may be classified in terms of their NHCP as resolved by disc gel electrophoresis. This type of an analytical examination would
facilitate the isolation NHCP cither individually MATERIALS
11 @ 1974 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Materials. All procedures were carried out at 04” unless otherwise indicated. All chemicals and biochemicals were purchased from Calbiochem unless otherwise stated. The following solutions were used in the fractionation of chromosomal proteins : Medium 0: 5 M urea, 0.01 M Tris-HCl, 2.85 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0. Medium 0.5: 5 M urea, 0.01 M Tris-HCl, 0.5 M NaCl, 2.85 mM PMSF, pH 8.0. Medium 1.0: 5 M urea, 0.01 M Tris-HCl, 1.0 M NaCl, 2.85 mM PMSF, pH 8.0. Medium 2.0: 5 M urea 0.01 M Tris-HCl, 2.0 M NaCl, 2.85 mrvr PMSF, pH 8.0. Medium 3.0: 5 M urea, 0.01 M Tris-HCl, 3.0 M NaCl, 2.85 mM PMSF, pH 8.0. Isolation of nonhisto?le chromosomal protein. Purified chromatin was isolated from newborn (3to 5-day-old) New Zealand white rabbit brain, heart, liver, and submandibular salivary gland according to the procedure of Marushige and Bon-
1 Abbreviations used: NHCP, nonhistone chromosomal proteins; CP, chromosomal proteins; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; TCA, trichloroacetic acid; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; OD, optical density; ilf,, molecular weight; U, enzyme internationa.1 units.
Copyright All rights
AND
of tissue-specific or in a group.
12
BEKHOR
ner (4). Chromosomal proteins (CP) were isolated from purified chromatin as described by Bekhor et al. (2) in accompanying communication (2). Briefly, after the two washes in .Ol M Tris, pH 8.0, after purification on 1.7 M sucrose, chromatin was homogenized with Teflon-glass Potter-Elvehjem homogenizer in Medium 0, then centrifuged in
DIAGRAM
ET AL. SW 50.1 for 20 hr at 50,000 rpm to pellet urea-exA\ tracted chromatin. The supernatant (fraction was saved for analysis of urea-extracted NHCP on disc gel electrophoresis. Further fractionation of the chromatin proteins was carried out with Medium 0.5, Medium 1.0, and Medium 2.0 or 3.0 (Diagram 1).
1
FRACTIONATION OF NHCP Purified chromatin suspended in Med. 0 I Centrifuged at 50 krpm for 20 hr
Supernatanl Fraction A
Peliet 0 Suspended in Med. 0.5
I
Centrifuged at 50 krpm for 20 hr I Pellet 0.5 Suspended in Med.
1
Supernatant Fraction B
1 .O
I
I
Centrifuged at 50 krpm for 2Q hr I
Pellet 1 .O Suspended in Med.
Supernatant Fraction C
2.0
Centrifuged &t 50 krpm for 20 hr
Pellet 2.0 Saved for thermomelting studies
Superhatant Fraction D
NONHISTONE
CHROMOSOMAL
The various chromosomal protein fractions were dialyzed extensively against distilled water, lyophilized to a dry powder, and resolubilized with of l-2 mg 5 M urea, ,270 SDS to a concentration protein/ml (5-10 ODpso/ml). Electrophoretic analysis of 100-200 fig protein/gel was carried out in the Laemmli system (5) as described previously (2). Gels were stained with Coomassie brilliant blue (Sigma Chemical Co.) and banding pattern recorded by visual inspection, photographically, and by scanning at 600 nm with Guilford Model 240 recording spectrophotometer equipped with a linear transport. Elrzymatic dephosphorylation of chromosomal proteins. Chromatin proteins were enzymatically dephosphorylated with electrophoretically purified E. coli alkaline phosphatase (BAPF, 30 units/ mg, Worthington Biochemicals). One to five milligrams protein/ml were incubated for 1 hr at 37” C with 90 rg/ml of alkaline phosphatase in the presence of 1% SDS. During initial experiments on optimal conditions for phosphatase activity, it was found that the rate of phosphate hydrolysis was stimulated by SDS, reaching a 4-fold increase in lyO SDS relative to that found without SDS. The presence of 5 M urea decreased the rate of hydrolysis by about 2056 relative to the 1% SDS level (data not shown). Therefore, the incubation of alkaline phosphatase with CP was done in 10/OSDS in the absence of urea and at pH 7.0. After enzymatic digestion, the samples were adjusted to 5 M urea and examined by disc gel electrophoresis for possible effects of dephosphorylation on chromosomal protein gel electrophoretic mobilities. Determination of protein-bourld phosphate release from NHCP with alkaline phosphatase. Protein-free rabbit liver DNA, purified as described previously (6), was labeled in vitro with [3H]dimethylsulfate (New England Nuclear, sp act 384 mCi/mmole) (7), and purified by ethanol precipitation, and gel exclusion chromatography on 1.5 X 20-cm G-100 Sephadex column equilibrated with 0.1 M phosphate buffer, pH 6.8. An aliquot of irk vifro-labeled [3H]DNA (220,000 cpm, sp act 9200 cpm/pg) was mixed with 300 OD,,, of purified liver chromatin; resultant sp act 18.3 cpm/pg DNA. Residual DNA content in subsequent fractions was determined by measurement of acid-insoluble tritium counts. The chromatin solution was adjusted to 20/Oin Na dodecylsarcosinate and 4 M CsCl (p = 1.48 g/cc) and centrifuged for 20 hr at 50 krpm in SW 50.1. The floating protein skin was washed once in 70yo ethanol, twice in 5cj, TCA, and rinsed twice with 0.01 M Tris, pH 8.0. This material was dispersed by sonication for 1 min at maximum intensity with Branson Sonifier in TM buffer (0.01 M Tris, 1 mM MgC12, pH 8.0) to yield a protein concentration of
13
PROTEINS
2.8 mg/ml. Residual nucleic acid contamination was removed by incubation for 1 hr at 37°C with 50 pg RNase A (Worthington Biochemicals), 360 U/ml RNase Tl (Schwartz Bio-Research), 200 rg/ml DNase 1 (DN-EP) Sigma Chemical Co.), and 25 U/ml snake venom phosphodiesterase (Calbiochem). Alkaline phosphatase hydrolysis was carried out with 4-15 U/ml for 1 hr at 37” C in TM buffer containing lyO SDS. The protein was precipitated with 20y0 TCA and combusted with concentrated H,SO, in 0.1 1\’ nitric acid-washed Corex tubes. Blanks with appropriate concentration of enzymes and buffers were also analyzed to correct for introduction of phosphate in enzyme preparations and buffers. Phosphate analysis was done by the method of Rathbun and Betlach (8). DNA was determined by diphenylamine reaction according to Burton (9). RNA determination was done according to Lin and Schjeide (10) and corrected for l>NA reactivity. Protein was determined by the method of Lowry et al. (11). RESULTS
AND
DISCUSSION
Fractionation and analysis of nonhistone chromosomal proteins on polyacrylamide disc gel electrophoresis make possible the eventual “finger printing” of chromatin relative to a specific organ. It would, therefore, become possible to identify genetic aberration at the nonhistone chromosomal protein level once functions are assigned to NHCP. Amplification of the types of NHCP which may be related to tissue-specific gene activity is only the first step toward the ultimate functional characterization of the acidic proteins found associated to DNA in chromatin. The result’s in Fig. 1 show that organ specificity may be assigned to a part of all the fractions of NHCP examined for brain, heart, liver, and submandibular salivary glands. These same gels may also be compared by scanning at 600 nm in the Guilford Spectrophotometer Model 240. Figures 2 and 3 show such a comparison between brain and heart chromosomal proteins. It is, therefore, concluded that one can discriminate
the various
organs
under
study
on the
basis of their NHCP composition. That the liberation of the phosphates from NHCP does not cause appreciable variation in the mobility of these prot’eins is shown in Fig. 4 in the case for liver XHCP fract,ions A and B; the presence of alkaline
u
t
HEART
BRAIN
SALIVARY
LIVER
HEART
BRAIN
SALIVARY
LIVER
GLAND
GLAND
+
I //I II
I
BRAIN
SALIVARY
LIVER
HEART
BRAIN
SALIVARY
LIVER
GLAND
GLAND
NONHISTONE
CHROMOSOMAL
PROTEINS
16
BEKHOR
ET AL.
+
0
m
a
uu 009
'a.0
1.0
NONHISTONE
CHROMOSOMAL
PROTEINS
17
FIG. 4. Guilford spectrophotometer scans at 600 nm of liver NHCP fractions A and B (see Diagram 1) before (top) and after (bottom) hydrolysis of protein-bound phosphate with electrophoretically purified E. coli alkaline phosphatase in 1% SDS. The presence of alkaline phosphatase is shown by an arrow (bottom), and the histone peaks in fraction B are pointed out by the letter H. No marked differences are observed in the scans of alkaline phosphatase treated and untreated NHCP. Fraction A alkaline phosphatase peak corresponds to 9 pg and that in fraction B represents 1 pg of protein. A breakin the scan in A indicates a a-fold increase Z-fold decrease in scanning sensitivity. density scale is shown on the left.
in scanning sensitivity, Direction of scanning
phosphatase band is identified by an arrow. The molecular weight of the metallo-enzyme E. coli alkaline phosphatase was 86,000, and this enzyme gave a single band on electrophoresis on polyacrylamide gels in 4 M urea-O.2% SDS, pH 8.9. It is significant to note that, although E. coli alkaline phosphatase is known to be formed of two subunits of M, approx 40,000 (12), it electrophoresed only as a single band equivalent to M, approx SO,OOO-90,000. Thus, the conditions used for electrophoresis as indicated in this communication may not necessarily yield subunit mobility of NHCP.
and that in B indicates a is from left to right. Optical
The fractions of chromosomal proteins extracted at the high salt concentrations (medium 2.0 or 3.0) were found to be contaminated with DNA. Although RNA can be totally removed by treatment with RNase, DNA is more difficult to eliminate. Therefore, the experiment shown in Table I was performed to determine actual proteinbound phosphate released by hydrolysis with alkaline phosphatase. This type of a measurement was necessary to exclude any errors in the assessment of protein-bound phosphate which was not accessible to hydrolysis with alkaline phosphatase. It was
18
BEKHOR
found that treatment with alkaline phosphatase (under the conditions described in Methods) liberated about 48% of proteinbound phosphate (Table I) from total liver CP extracted from chromatin with 4 M CsCI. The electrophoretic pattern of this preparation is shown in Fig. 5. This type of an experiment indicated not only the reproducibility of the gels but also made it possible to identify the presence of a major foreign protein in that fraction. The alkaline phosphatase peak with an isoelectric point of pH 4.5 (13) represents a total of 9 pg in Fig. 4A and 1 kg in Fig. 4B. TABLE ANALYSIS
3H-DNA (cpm) per 300 OD260 chromatin
sarcosyl
The sensitivity of detecting Coomassie brilliant blue-stained bands may be of the order of 0.2 pg or less for NHCP (Fig. 4). We estimate from the size of the rabbit genome (5.3 pg/nucleus) (14) and the range of the molecular weights of polypeptides obtained on disc gel electrophoresis (lO,OOO200,000 daltons) that the minimum level of detectable copies of homogeneous NHCP per genome would be on the order of lo5 down to 1.5 X lo4 for the higher molecular weight NHCP. It would, therefore, be expected that we are either detecting nonhistone chromosomal proteins which are I
OF PROTEIN-BOUND PHOSPHATE OF DNA IN CP UNDER VARIOUS
Fraction
4 M C&1-2’%
ET AL.
chromatin
suspension 0.01 M Tris, pH 8.0, sonicated”skin”” after centrifugation from 4 M CsCl Nuclease-treated sonicated “skin” Actual Corrected for DNA* Nuclease and alkaline phosphatasetreated “skin” Actual Corrected for DNAb
DNA
AND DISTRIBUTION CONDITIONS
(a)
220,000
12,000
8,170
444
% DNA remaining
Phosphate bd
Phosphate bmole per mg protein)
100
-
3.7
101.85
0.235
0.51
1,131 0.0
61.5 0.0
0.0
57.45 51.30
0.132 0.118
1,131 0.0
61.5 0.0
0.51 0.0
33.20 27.05
0.076 0.062
0 See Methods, section on determination of protein-bound phosphate. * Corrected for DNA by assuming that all of DNA phosphate groups are susceptible alkaline phosphatase.
to hydrolysis
FIG. 5. Gel electrophoretic pattern of total liver NHCP isolated from chromatin upon centrifugation through 4 M CSCI-~~~ sarcosyl, and after washing in 70y0 ethanol, precipitation from 5’% TCA, and suspension by sonication in TM buffer (see Methods).
by
NONHISTONE
CHROMOSOMAL
found in many repeating copies, or that we are looking at classes of proteins similar in molecular weight. The latter would imply that the number of NHCP associated to DNA may fall in the hundreds if each electrophoretic band represent’s numerous structurally different proteins or protein subunits. Fen- conclusions may be invoked from the present study: (1) organs may be discriminated through their NHCP composition; (2) removal of 48% of protein-bound phosphate does not significantly alter the electrophoretic mobility of chromosomal proteins; (3) electrophoresis on polyacrylamide disc gels in 4 M urea-O.2 % SDS, pH 8.9, may not necessarily represent, mobility of protein subunits; (4) it is possible to identify the presence of a major abnormal acidic protein in NHCP by disc gel electrophoresis; and (5) it is now conceivable that we can examine genetic aberration at the level of nonhistone chromosomal proteins. ACKNOWLEDGMENTS The authors are grateful to Mr. P. Bringas, Jr., for the illustrations. This work was supported by Grant DE-03235-02 and by a Career Development Award 5K04DE47354-03 to I.B. from the National Institutes of Dental Research. JNL, predoctoral fellow and RS postdoctoral fellow are supported by Training Grant DE-00094.11 from
PROTEINS
19
the National Institutes of Dental Research. LA is a graduate student of this department supported by Grant DE-03235-02 from the National Institutes of Dental Research. REFERENCES
1.
SPELSBERG, T. C., WILHELM, J. A., BND HINILICA, L. S. (1972) Subcell. Biochem. 1, 107. 2. BEKHOR, I., L~PEYRE, J. N., MD KIM, J.
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