BIOCHIMIE, 1975, 57, 315-323.
Properties of chromosomal proteins of human leukemic cells (1)(2)o) L. S. DESA], U. C. WULFF (4) and G. E. FOLEY (5). (1) Sidney Farber Cancer Center and Department of Pathology,
Harvard Medical School, 8oston, Massachusetts 02115. (4-11-197~). S u m m a r y . - Purified chromatin isolated from lymphocytic cells derived from patients with acute leukemia, or other lymphoproliferative disorders has been compared with chromatin isolated from normal hum.an lymphocytic cells, by gel eleetrophoresis and. differential gradient ultracentrifugation. Thermal denaturation studies showed higher Tm values for chromatin from leukemic cells, as compared to that o,f lymphocytic cells from normal donors or patients with infectious mononucleosis, reflecting the diverse complexity of these ehromatins with respect to their varying chemical compositions. There are significant differences in the ratios of DNA:R.N'A:protein, as well as in the ratios of chromatin-associated histone and non-histone proteins; althoug~h chromatin-associated histones were more homogeneous than were the non-histone proteins, as adjudged by amino acid analyses and acrylamide gel electrophoresis. T.hese differences in chromatin structure may relate to the differences in gene expression characteristic of these lymphocytic cells. The chromosomal acidic proteins isolated from the purified ehromatin of human leukemic cells greatly stimulated the template activity of the ehromatin in in oitro RNA synthesis. The non-histone proteins selectively interact with ehromatJins and influence the RNA polymerase reactions, indicating that there is selective tissue specificity of non-histone proteins. INTRODUCTION. T h e m a j o r c o m p o n e n t s of c h r o m o s o m e s in e u k a r y o t i c cells are DNA and histones. These, and the non-hislone n u c l e a r (aoidic) p r o t e i n s eonstitue nearly the w h o l e o r g a n i z e d f r a m e w o r k of the c h r o m o s o m e . If the c h r o m o s o m a l p r o t e i n s p a r t i c i pate in the regulatio.n of gene function, k n o w ledge of the ratios of the i n d i v i d u a l c o m p o n e n t s of c h r o m a t i n , and of t h e i r b i o c h e m i c a l and biop h y s i c a l p r o p e r t i e s m a y c o n t r i b u t e to u n d e r s t a n d i n g the differences in r e g u l a t o r y m e c h a n i s m s w h i c h are i m p l i c i t in over d i f f e r e n c e s in the biological p r o p e r t i e s of different k i n d s of e u k a r y o t i c cells. It is possible that different kinds of such cells m a y c o n t a i n different structural or configur a t i o n a l types of hisfone and n o n - h i s t o n e protein, or that c h r o m o s o m a l p r o t e i n s : DNA i n t e r a c t i o n s differ in different k i n d s of such cells. R e c e n t stu(2) Presented in part at the 56th Annual Meeting, Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 1972. (3) These studies were supported in part by research grants G-6'516 from the National Cancer Institute, and FR-0.55~2L6 from the Division of Research Facilities and Resources, Nfational, Institutes of Health. (4) Present Address : Kinderklinik der Medizinischen Akademie, 24, Liibeck, W. Germany. (5). Holds Research Career Award K6-CA-22,150 from the National Cancer Institute, National Institutes of Health.
dies [1] for example, i n d i c a t e that in the case of h u m a n l y m p h o c y t i c cells, isologous histones inhibit or repress RNA synthesis m o r e effectively than do homologous histones ; suggesting differences in the affinities b e t w e e n histones and the r e c e p t o r sites on the DNA in different k i n d s of cells. Biological f u n c t i o n s h a v e been a s c r i b e d to some n o n - h i s t o n e n u c l e a r p r o t e i n s [2]. The non-histone c h r o m o s o m a l p r o t e i n s are g e n e r a l l y m o r e active than histones in the i n c o r p o r a t i o n of a m i n o acids [2, 3~ and p h o s p h a t e [4, 5], and the level of such m e t a b o l i s m a p p e a r s to be related to n u c l e a r activity [6, 7]. Although it has been r e p o r t e d that the n u c l e a r non-histone p r o t e i n s are i n v o l v e d in the activation of t r a n s c r i p t i o n m e c h a n i s m s by r e s t o r i n g h i s t o n e - i n h i b i t e d RNA synthesis [6-8], only limited i n f o r m a t i o n is available w i t h r e s p e c t to the non-histone (acidic) p r o t e i n s in r e l a t i o n to chrom o s o n m l s t r u c t u r e and f u n c t i o n in h u m a n lymp h o c y t i c cells. The p r e s e n t studies d e s c r i b e the b i o p h y s i c a l and b i o c h e m i c a l c h a r a c t e r i s t i c s of isolated c h r o m a t i n and i n d i v i d u a l c h r o m o s o m a l c o m p o n e n t s of h u m a n l y m p h o c y t i c cells d e r i v e d from n o r m a l d o n o r s and patients w i t h i n f e c t i o u s m o n o n u c l e o sis or acute l y m p h o b l a s t i c leukemia.
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MAT.ERIALS AND METHO,DS.
Cells : The d e r i v a t i o n of the cell lines used in these studies (table I) has been d e s c r i b e d e l s e w h e r e in detail. The CCRF-CEM cells w e r e isolated d i r e c t l y in s u s p e n s i o n culture from the p e r i p h e r a l b l o o d buffy coat of a female p e d i a t r i c patient whose presenting lymphosarcoma had p r o g r e s s e d to acute l y m p h o b l a s t i c l e u k e m i a [9]. The CC,RF-SB cells w e r e d e r i v e d in p r i m a r y stat i o n a r y cultures of the p e r i p h e r a l b l o o d buffy coat of a n o t h e r (male) p e d i a t r i c p a t i e n t w i t h s i m i l a r disease [10] ; a n d the CCRF-H-SB2 ce'lls w e r e TABLE I.
Thermal Denaturation Characteristics o[ Chromatins Isolated from Human Lymphocytic Cells Derived from Different Diagnostic Categories. t
Isolated chromatin
I Normal
Tm (Midpoint (herren] denaturation) :
CCRF-SLT I 76.8 -4- 0.2 CCRF-TOH 1 77.1 -~- 0.6 Infectious mononucleosis : CCRF-RKB 81.4 -t- 0.7 CCRF-SB 82.2 ~___0.4 Acute lymphoblastic leukemia : CCRF-H-SB 84.7 ~___0.8 CCRF-CEM 84.3 + 0.5 DNA : CCRF_TOH
70.1 --t- 0.5
d e r i v e d by d i r e c t i m p l a n t a t i o n o,f the same specim e n of p e r i p ' h e r a l b l o o d buffy coat into neonatal S y r i a n h a m s t e r s [11]. The CCRF-RKB cells w e r e d e r i v e d from the p e r i p h e r a l blood buffy coat of a y o u n g adult w i t h infectious m o n o i m c l e o s i s [12], a n d the C~fi,RF-TOH a n d C~C~RF-SLT cells w e r e d e r i v e d from the p e r i p h e r a l b l o o d huffy coat of normal, h e a l t h y d o n o r s [13]. These h u m a n l y m p h o c y t e s w e r e m a i n t a i n e d in c o n t i n u o u s log-phase g r o w t h at p o p u l a t i o n densities of 3-4 )< 106 cells/ml, w i t h a g e n e r a t i o n time of circa 24 h r s in large-volume s u s p e n s i o n cultures in Eagle's m i n i m a l essential m e d i u m mod i f i e d for s p i n n e r culture [14] a n d s u p p l e m e n t e d w i t h 10 p e r c e n t w h o l e fetal calf s e r u m b y successive w i t h d r a w a l a n d r e p l a c e m e n t of substrate, as d e s c r i b e d p r e v i o u s l y [9, 10].
Isolation of Chromatin : C]womatin was extracted from large quantities (5,0-1.0.0 gin) of cells by the m e t h o d s d e s c r i b e d by Marushige and Bonner [15]. In brief, the cells w e r e h o m o g e n i z e d in BIOCHIMIE, 1975, 57, n ° 3.
saline-EDTA buffer and 4 times in 0.01 M Tris buffer (pH 8.0). The gelatinous crude c h r o m a t i n was further purified by centrifugation through 1.7 M sucrose (buffered w i t h 0.01 M Tris at p H 8.0) for 2.5 h r s at 5.0,0.0,0 g. This p u r i f i e d chrom a t i n "was 'w~shed, r e s u s p e n d e d a n d d i a l y z e d o v e r n i g h t against 0.01 M Tris buffer, p H 8.0, then s h e a r e d and c e n t r i f u g e d ; the resulting s u p e r n a tant c o n t a i n i n g the p u r i f i e d c h r o m a t i n .
Preparation of Chromosomal Proteins : Histones (basic p r o t e i n s ) w e r e e x t r a c t e d from the p u r i f i e d c h r o m a t i n w i t h 0.4 N H2SO ~ at 4°C for 30 rain. The pellet w a s w a s h e d once w i t h 0.4 N HeSO 4 a n d b r i e f l y w i t h 0.01 M Tris buffer (pH 8.0). More than 95 p e r c e n t of a c i d soluble p r o t e i n (histones) are e x t r a c t e d by this p r o c e d u r e [16]. The pellet w h i c h c o n t a i n e d a c i d insoluble p r o t e i n s (acidic) w a s r e c o v e r e d by centrifugation, dissolved by gentle h o m o g e n i z a t i o n in 1 p e r c e n t SDS-0.05M Tris buffer (pH 8.0), and s t i r r e d o v e r n i g h t at 37"C. This solution ~,as t h e n d i a l y z e d against 0.01 M T r i s buffer (pH 8) c o n t a i n i n g 0.1 p e r c e n t SDS at 37°C for 24 hrs, a n d the DNA r e m o v e d by centrifugation at 3.6,000 r p m for 18 hrs at 25°C in a Spinco SW-50 rotor. After centrifugation, the u p p e r t w o - t h i r d s of the s u p e r n a t a n t was p i p e t t e d a n d chilled in an ice bath. The s o d i u m d o d e e y l s u l p h a t e (S,DS) was p r e c i p i t a t e d by the a d d i t i o n of s a t u r a t e d solution of K'C1 (1-2 drops) and the r e s u l t i n g p r e c i p i t a t e of potassiunl d o d e c y l sulp h a t e w a s r e m o v e d b y c e n t r i f u g a t i o n at 12,000 g for 20 rain. The p r e c i p i t a t e c o n t a i n i n g non-histone p r o t e i n s was f n r t h e r subjected to column c h r o m a t o g r a p h y (1 × 12 cm) of D o w e x 2 × 10 (A.G. 2 × 10, 20,0-4,0,0 mesh, Biorad, l:lichmond, CA) as d e s c r i b e d b y L e n a r d [171. This p r o c e d u r e removes most of the SDS from n o n - h i s t o n e proteins. The non-histone p r o t e i n w a s f u r t h e r p r e c i p i D a t e d w i t h s a t u r a t e d a m m o n i u m sulphate to a final 35 p e r c e n t saturation. The no n-histoue p r o t e i n was then Ere c i p i t a t e d in the cold and collected by c e n t r i f u g a tion at 12,000 g for 20 rain. The p r o t e i n pellet was d i s s o l v e d in 0.01 M Ti'is buffer (pH 8) and p r e c i p i t a t i o n ~-ith a m m o n i u m s u l p h a t e was r e p e a t e d . The final p r o t e i n preci.pitate was dissolved in T r i s buffer (0.01 M, p H 8) and d i a l y z e d o v e r n i g h t against the same buffer [183. Characterization of Nucleic Acids and Proteins of Chromatin : The n o n - h i s t o n e content was determ i n e d b y the m e t h o d of L o w r y el al. [19] using b o v i n e s e r u m a l b u m i n (Sigma) as a s t a n d a r d . Histone c o n c e n t r a t i o n a'lso w a s d e t e r m i n e d b y the same p r o c e d u r e [19] c a l i b r a t e d against calf thymus histones. The c h r o m a t i n RNA was s e p a r a t e d from DNA by the m o d i f i e d S e h m i d t - P a n n h a u s e r
C h r o m o s o m a l proteins of h u m a n le~tkemic cells. p r o c e d u r e described by Ts'o a n d Sato [20]. RNA was d e t e r m i n e d by the Orcinol method I21], using yeast RNA (Sigma) as a standard. DNA was determ i n e d by the d i p h e n y l a m i n e m e t h o d [221, using calf t h y m u s DNA (Sigma) as a standard.
Thermal Denaturation and Ultraviolet .4bsorption : Melting profiles of p u r i f i e d DNA a n d the various c o m p o n e n t s of the c h r o m a t i n isolated from these h u m a n l y m p h o c y t i c cells were determ i n e d in a Gifford Model 2800 Multiple Sample Absorbance R e c o r d i n g I n s t r u m e n t for the recording of melting profiles. A p p r o p r i a t e c o n c e n t r a tions of samples were taken i n 0.1 X SSC ~SSC = 0.15 M s o d i u m chloride - - 0.015 M sodium citrate (pH 7.0)3 and heated i n a thermosta'tically controlled w a t e r bath. The rate of t e m p e r a t u r e increase was 0.5 - - l ° C / m i n . Ultraviolet a b s o r p t i o n spectra at 260 n m are r e c o r d e d by a Beckman DU Spectrophotometer.
Amino Acid Analyses : Chromosomal proteins (histone and n o n - h i s t o n c proteins) were dissolved i n c o n s t a n t b o i l i n g (glass-distilled) HCI with 1 p e r c e n t p h e n o l to p r e v e n t degradation of tyrosine. The tube was then flushed w i t h nitrogen, sealed u n d e r r e d u c e d pressure, and h y d r o l y z e d at 110°C in a toluene bath for 20 hrs. The HC1 was diluted w i t h deionized w a t e r and r e m o v e d in a r o t a r y evaporator. The hydrolysates were then dissolved in 1 ml of sodium citrate buffer at pH 2.2 [23], filtered t h r o u g h a Millipore filter (0.22 a), a n d analyzed i n a Beckman Spinco 120 Automatic Amino Acid Analyzer [24]. Gel Electrophoresis : Analytical gels were composed of 10 p e r c e n t acrylamide, 0.2 p e r c e n t N.N'm e t h y l e n e b i s a c r y l a m i d e (BIS), dissolved i n 0.1 M sodium phosphate buffer, pH 7.4, c o n t a i n i n g 0.5 p e r c e n t SDS~ The solution was filtered a n d deaerated before use. P o l y m e r i z a t i o n was accomplished by the a d d i t i o n of 0.3 ml of 10 p e r c e n t a m m o n i u m persulfate (W/V) a n d 0.0.3 ml of N,N,N',N'-tetramethylethylenediamine (Temed) to 40 ml of a c r y l a m i d e solution [25]. Gels 9 cm i n length w i t h an i n t e r n a l diameter of 6 m m were allowed to form b y p o l y m e r i z a t i o n for 30 rain, and then subjected to a p r e - r u n at 4.5 V / t u b e for 30 rain with 0.1 M sodium phosphate buffer, pH 7,4, c o n t a i n i n g 0.1 p e r c e n t SDS in both u p p e r and l o w e r electrode chambers. Samples were then applied to the gels a n d electrophoresis was c a r r i e d out for 4-5 hrs. F o l l o w i n g electrophoresis, the gels were fixed and stained for 3-8 hrs i n a solution of 0.25 p e r c e n t Coomassie b r i l l i a n t blue in methan o l : a c e t i c a c i d : w a t e r (5:1:5). De-staining was accomplished by s o a k i n g the gels with gentle BIOCHIMIE, 1975, 57, n" 3.
317
s h a k i n g in a solution c o n t a i n i n g m e t h a n o l : acetic acid : water (2:3:35).
In Vitro Synthesis of RNA : The in vitro synthesis of RNA utilized highly p u r i f i e d h u m a n leukemic DNA (CCRF-CEM) or h u m a n leukemic c h r o m a t i n (CCRF-CEM) as a template a n d bacterial (Micrococcus lysodeikticus) RNA polymerase as the enzyme [26]. The reaction mixture, contained in a final volume of 0.25 ml, was : 0.04 M Tris buffer at pH 7.9, 0.01 M MgCI~, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.15 mM ATP, GTP, CTP, a n d 0.15 mM 3H-UTP (10 !~Ci/~!~mole,New E n g l a n d Nuclear, Boston, Ma.). W h e n the enzyme was used for m e a s u r i n g template activity of c h r o m a t i n , the DNA in the system was replaced by c h r o m a t i n c o n t a i n i n g 20 ~g of DNA. The m i x t u r e was i n c u bated at 37°C for 10 rain and the r e a c t i o n was stopped by the a d d i t i o n of 10 p e r c e n t TCA. The precipitate was collected on a Millipore m e m b r a n e filter (0.2.2 ~), w a s h e d 5 times with 5 ml of cold 10 p e r c e n t TCA, and dried at 80°C. Radioactivity was d e t e r m i n e d in a toluene scintillation fluid, u s i n g a Beckman Model LS 250 Scintillation Counter. ]~ESULTS.
Spectrophotometric Study of Chromatin : A typical ultraviolet a b s o r p t i o n s p e c t r m n of c h r o m a t i n isolated from h u m a n leukemic (CCRF-CEM) cells is s h o w n in figure 1. Essentially i d e n t i c a l absorp0.5
/,. N
O5
\
z w c~
',,,,/
uz
\
.,\
/
\ \
o
0 t L_L
225
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i
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t
i
~" I
~ t
~
9_55 245 255 265 275 285 295 505 5t.5 WAVELENGTH (A}
FIG. 1.
tion p a t t e r n s were obtained with the c h r o m a t i n isolated from n o r m a l (SLT, TOH), infectious mononucleosis (RKB, SB), and leukemic (GEM, HSB) l y m p h o c y t i c cells. The purified c h r o m a t i n isolated from all these cells showed no t u r b i d i t y , indicating little or no ultraviolet a b s o r p t i o n (0.0l) at 320 nin. A h i g h e r absorption at 320 niu would
L. S. Desai, U. C. W u l f f and G. E. Foley.
318
h a v e i n d i c a t e d e i t h e r a g g r e g a t i o n of c h r o m a t i n o r t h e p r e s e n c e of c o n t a n f i n a t i n g a g g r e g a t e s of n o n chromosomal proteins.
Tm Studies : T h e r m a l d e n a t u r a t i o n s t u d i e s o n t h e s e t o t a l c h r o m a t i n s t r u c t u r e s i n d i c a t e d a reelring temperature (Tm) several degrees higher than 20--
).. ~ 0
15
-
"ros--~-.Tf
Chemical Analysis of Chromatin : T h e c h e m i c a l c o m p o s i t i o n s of t h e s e isol'ated c h r o m a t i n s a r e s u m m a r i z e d i n t a b l e II. C o n s p i c u o u s d i f f e r e n c e s a r e e v i d e n t i n t h e m a s s r a t i o s of h i s t o n e , n o n histone and RNA conten.t ; for example, somewhat higher values are apparent in the leukemic and i n f e c t i o u s m o n o n u c l e o s i s l y m p h o c y t i c cells (CEM ; RKB, H-S,B) as c o m p a r e d to t h o s e f r o m n o r m a l d o n o r s (.S~LT, T O H ) . T h e r a t i o o.f h i s t o n e to n o n h i s t o n e p r o t e i n s a p p e a r s to b e circa 1.0 i n l e u k e m i c cells, ~ v h e r e a s c h r o m a t i n d e r i v e d f r o m n o r mal lymphocytes contains only a small quantity of n o n - h i s t o n e p r o t e i n s ( t a b l e t I ) .
""
o t,r ""
rrLO I 0 >.. ""
R K B ~
EM
S ~ ~
HSB
5
0
20
40
60
80
I00
Amino Acid Composition of Chromosomal Proteins : A m i n o a c i d a n a l y s e s of a c i d s o l u b l e p r o -
TEMPERATURE(*'C} ~ S L T . . . . TON ............. R K B
f r o m l y m p h o c y t i c cells (>f n o r m a l d o n o r s (SLT, T O H ) o r p,atients w i t h i n f e c t i o u s m o n o n u c l e o s i s (RKB, SB) p r o b a b l y r e f l e c t i n g t h e d i v e r s e c o m p o s i t i o n s of t h e s e c h r o m a t i n , as d e t e r m i n e d b y t h e p r e s e n c e of d i f f e r e n t (fig. 2) k i n d s of c o m p l e x e s w i t h n u c l e a r p r o t e i n s . T h u s , t h e r e is s o m e evidence that the chromosomal proteins participate i n t h e s t a b i l i t y of t h e c h r o m a t i n s t r u c t u r e i n t h e s e t h e r m a l d e n a t u r a t i o n s t u d i e s ( t a b l e I).
t e i n s ( h i s t o n e s ) of t h e c h r o m a t i n s i s o l a t e d f r o m these lymphocytic cells are summarized in t a b l e III. T h e s e a n a l y s e s a r e c o m p a r a b l e to t h o s e
IP4P4b-OSB O - ~ C E M ¢-,¢"¢'~DHSB
Fm. 2.
TABLE JI.
Chemical Characteristics of the Chromatins Isolated [rom Normal Human (SLT and TOH), Infectious Mononucleosis (RKB and SB), and Leukemic (HSB and CEM) Ceils. Chemical composition of chromatin Cells (')
CCRF-SLT . . . . . . . . . . . . . . -TO H . . . . . . . . . . . . . -RKB ............. -SB . . . . . . . . . . . . . . . -HSB . . . . . . . . . . . . . -CEM . . . . . . . . . . . . . .
No.
Preps
Histone
Non-histone protein
RNA
1, O0 9.93 ~ 0.06 0.49 __~ 0.04 0.06 ~ 0.05 1 . 0 0 0.86 ~- 0.04 0.58 ~- 0.03 0.08 ~- 0.07 1 . 0 0 1.02 -+- 0.12 0.86 ~- 0.08 0.10 -~ 0.06
1. O0 1.16 -4- 0.09 0.91 ~- 0.06 0.09 ' ~ 0.04 1 . 0 0 1.14 ___+0.13 1.17 -¢- 0.07 0.13 ~___0.07 1 . 0 0 1.22 - b 0.05 1 . 2 6 _~ 0.02 0.15 _____0.03
The nucleic acids a n d c h r o m a t i n - a s s o c i a t e d basic (histones) a n d acidic (nonhistone) p r o t e i n s were extracted f r o m isolated c h r o m a t i n a n d t h e values are normalized a g a i n s t the DNA content. (*) Cf. t a b l e I. T h e v a l u e s are m a s s ratios of c h r o m a t i n s is olated~ f r o m h u m a n l y m p h o c y t i c cells ; ~ are s t a n d a r d errors.
t h a t of t h e p u r i f i e d D N A i s o l a t e d f r o m h u m a n l y m p h o c y t i c cells (TO,H) ( t a b l e I). I n t e r e s t i n g l y , m e l ting profiles revealed higher melting temperatur e s (Tin) f o r c h r o m a t i n d e r i v e d f r o m h u m a n leuk e m i c cells (CEM, H S B ) t h a n c h r o m a t i n d e r i v e d
BIOCHIMIE, 1975, 57, n ° 3.
of t h e t o t a l h i s t o n e s iso,lated f r o m w h o l e c e l l s [27]. T h e r e a p p e a r s to b e a d e g r e e of s i m i l a r i t y i n t h e a n l i n o a c i d c o m p o s i t i o n of t h e h i s t o n e s i s o l a t e d , i n d i c a t i n g t h e p r o b a b l e h o m o g e n e i t y of t h e acid soluble proteins.
C h r o m o s o m a l proteins of h u m a n leukemic cells. The non-histone chromosomal proteins appear to b e m o r e h e t e r o g e n e o u s i n t h e i r a m i n o a c i d c o m p o s i t i o n s . T h e h i g h r a t i o o f a s p a r t i c p l u s gluTABLE IIL
Amino Acid Composition of Histories Isolated [rom Chromatin. Chromosomal histones from cells('):
Amino acids
SLT 7C H RKB! $3 HSB CEM Lysine . . . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . . Arginine . . . . . . . . . . . . . . . Aspartie acid . . . . . . . . . . . Threonine . . . . . . . . . . . . . . Serine . . . . . . . . . . . . . . . . . Glutamic acid . . . . . . . . . . Proline . . . . . . . . . . . . . . . . . Glycine . . . . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . . . Cystine/2 . . . . . . . . . . . . . . Valine . . . . . . . . . . . . . . . . . Methionine . . . . . . . . . . . . . . lsoleucine . . . . . . . . . . . . . . . Lencine . . . . . . . . . . . . . . . . . Tyrosine . . . . . . . . . . . . . . . Phenylalanine . . . . . . . . . . .
319
r a m i e a c i d s , w h i c h e x c e e d s t h a t of t h e b a s i c a m i n o a c i d s , l y s i n e , a r g i n i n e , a n d h i s t i d i n e (table IV), i d e n t i f y t h e s e n o n - h i s t o n e p r o t e i n s to b e a c i d i c c h r o m o s o m a l p r o t e i n s . T h e r e a r e d i f f e r e n c e s in the a m i n o acid c o n t e n t a n d c o m p o s i t i o n of these a c i d i c p r o t e i n s , as c o m p a r e d to t h e n o n - h i s t o n e p r o t e i n s i s o l a t e d f r o m tlle v a r i o u s l y m p h o c y t i c cells c o n s i d e r e d h e r e i n (cf. t e x t ) .
Heterogeneity and Cell Specificity : T h e nuc l e a r n o n - h i s t o n e p r o t e i n s a r e a m i x t u r e of p r o t e i n s w h i c h d i f f e r i n size a n d e l e c t r o p h o r e t i c m o b i l i t y . E l e c t r o p h o r e s i s i n t h e p r e s e n c e of s o d i u m
10.2[ 1 8 1 2 . 1 11.4 12.5 13.0 1.2 1 9 2 1 1 6 2.1] 2.6 8,2 9.01 7.8 5,t I 4.9 7 (7 6.3[ 6.0 5.0 5616.31 5 8 6.1 6.7 5.3 4 7 / 5 . 1 5,G 5.41 5.6 5.5 4.9] 7.9 6.7 7.5] 8.3 4.8 4 . 9 1 5 2 42 5.1] 5 4 9.0 9.2 8.2 8.8 8.7j 8.4 13.21 2 . 4 / 1 4 2 I14 6 14.3 13 8 o0j o.oi 0.~3 0.O I 0.0 6.8 5.6 6.2 6.3 t 6.5 1.0 0 7 1 . 1 I.~ 1.2] 0.9 5.2 5.(,I 4 2 4.8 4 7 / 4 0 7.0 7.2 / 7.6 6.,q 7.2 7.9 2.0 2 6 / 2 5 I £ 2.2 2.2 1.6 1.St 1.9 2.1 2.4[ 2 1
9o!
A
B
(*) Cf. t a m e I. The values are moles of each a m i n o acid,/100 moles of amino acids recovered.
C
TABLE IV.
Amino Acid Composition of Non-Histone Protein Isolated From Chromatin. Chromosomal non histone (acidic) proteins from cells(')
Amino acids
Fla. 3.
H RI:B[ SB HSB CEM Lysine . . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . . Argininc . . . . . . . . . . . . . . . . Aspartic acid . . . . . . . . . . . . Threonine . . . . . . . . . . . . . . . Serine . . . . . . . . . . . . . . . . . Glutamic acid . . . . . . . . . . . Proline . . . . . . . . . . . . . . . . . . Glycine . . . . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . . . . Cystine/2 . . . . . . . . . . . . . . . Valine . . . . . . . . . . . . . . . . . . Methionine . . . . . . . . . . . . . ]soleucine . . . . . . . . . . . . . . Leucine . . . . . . . . . . . . . . . . Tyrosine . . . . . . . . . . . . . . . PhenyIalanine . . . . . . . . . .
5 3/ .2 6.8 6.5 5.8 7.3 2.4 .1 1 2 1 2.0 2.2 5.2 .6 5 5.8 5 9 6.0 11.4 1 .2115 U.912.3136 13.4 4.6 o 5.5 4.2 .7 7 5.~ 6 ,q 6.0 13.41 9 1 4 2.~ 15 8 18.3 3.2 5.5~ 4.7, 4 5 5 l 6.7 8.8 .1 9 2.3 12.3 6.5 8 .1 8..q 7.3 6 8 1.0 0.8[ 0 o.e 0.9 1.1 5.1 .2 6.1 6.6 6 3 .8 2.4 2 2 2 0 1.8 I 1 7.4 4 8 5 . 2 5.0 36,i. 25 13 4. 11 4.5 7.8 9.4 6, 9.(: 8 6 8 . 9 2.4 3 .0 2.6 2.1 3 6 2 . 4 2.6 4 3.4 4.313.8
-- --ii ....
(*) Cf. table I. The values are moles of each amino acid/100 moles of a m i n o acids recovered. BIOCHIMIE, 1975, 57, n ° 3.
d o d e c y l s u l p h a t e s e p a r a t e s the p r o t e i n s on the b a s i s of m o l e c u l a r size [28, 29] ; s u b s e q u e n t stain i n g w i t h a c o m b i n a t i o n of C o o m a s s i e b l u e a n d A m i d o B l a c k r e v e a l e d t h e p r e s e n c e of s e v e r a l s p e c i e s of p r o t e i n s w i t h v a r y i n g m o l e c u l a r size as e v i d e n c e d by d e n s i t o m e t e r l r a c i n g s (fig. 3). U n l i k e hisiones, non-histone proteins cover a wide range o f m o l e c u l a r w e i g h t s ; i.e., j u d g i n g f r o m t h e m o b i l i t i e s of t h e b a n d s in gel e l e c t r o p h o r e s i s as c o m p a r e d to m a r k e r p r o t e i n s w i t h k n o w n m o l e c u l a r w e i g h t s . T h e o b s e r v e d m o l e c u l a r w e i g h t of s e v e ral a c i d i c p r o t e i n s f r o m t h e h u m a n l e u k e m i c c e l l s s t u d i e d fall i n t h e r a n g e of 15,000 to 80,000 as r e p o r t e d b y S h e l t o n a n d A l l f r e y [7]. T h e d e n s i t o m e t e r t r a c i n g s of a c i d i c p r o t e i n a n a l y s e s o n p o l y a c r y l a m i d e gels r e v e M e d c o n s i d e r a b l e h e t e r o g e neity ; suggesting different characteristics of these
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c h r o m a t i n - a s s o e i a t e d p r o t e i n s w h i c h nlav be conc e r n e d w i t h differences in the eontroI of transc r i p t i o n in these different k i n d s of h u m a n lymp h o c y t i c cells.
Effect of Acidic or Non-histone Protein (NHP) on Template Activity : It is w e l l - k n o w n that DNAd e p e n d e n t RNA synthesis in vitro is i n h i b i t e d by the a d d i t i o n of h i s to n e to a cell-free system cont a i n i n g RNA p o l y m e r a s e and e i t h e r DNA, or DNA
×
A
20
rase r e a c t i o n exhibits only 10-20 p e r c e n t of the tempIate activity as c o m p a r e d to the t e m p l a t e activity of n ak ed CEM.DNA (fig. 4A). The a d d i t i o n of non-histone isolated f r o m CEM cells to the RNA p o l y m e r a s e r e a c t i o n act i v at ed the t e m p l a t e activity by 4-5 fold (fig. 4B). Th e RNA t r a n s c r i b e d f r o m the c h r o m a t i n act i v at ed by n o n - h i st o n e proteins is p e r h a p s due to the u n m a s k i n g of the genome, w h i c h m ay have s i m i l a r or different nucleotide sequences as the e x p r e s s e d genome. It has been suggested p r e v i o u s l y that n u c l e a r a c i d i c proteins r est o r e h i s t o n e - i n h i b i t e d RNA synthesis [6, 8]. Other e x p e r i m e n t s (tables V and VI) e m p l o y i n g e h r o m a t i n s and non-histone p r o t e i n s (NHP) isola-
5 DNA
Properties of chromosomal proteins of human leukemic cells (1)(2)o) L. S. DESA], U. C. WULFF (4) and G. E. FOLEY (5). (1) Sidney Farber Cancer Center and Department of Pathology,
Harvard Medical School, 8oston, Massachusetts 02115. (4-11-197~). S u m m a r y . - Purified chromatin isolated from lymphocytic cells derived from patients with acute leukemia, or other lymphoproliferative disorders has been compared with chromatin isolated from normal hum.an lymphocytic cells, by gel eleetrophoresis and. differential gradient ultracentrifugation. Thermal denaturation studies showed higher Tm values for chromatin from leukemic cells, as compared to that o,f lymphocytic cells from normal donors or patients with infectious mononucleosis, reflecting the diverse complexity of these ehromatins with respect to their varying chemical compositions. There are significant differences in the ratios of DNA:R.N'A:protein, as well as in the ratios of chromatin-associated histone and non-histone proteins; althoug~h chromatin-associated histones were more homogeneous than were the non-histone proteins, as adjudged by amino acid analyses and acrylamide gel electrophoresis. T.hese differences in chromatin structure may relate to the differences in gene expression characteristic of these lymphocytic cells. The chromosomal acidic proteins isolated from the purified ehromatin of human leukemic cells greatly stimulated the template activity of the ehromatin in in oitro RNA synthesis. The non-histone proteins selectively interact with ehromatJins and influence the RNA polymerase reactions, indicating that there is selective tissue specificity of non-histone proteins. INTRODUCTION. T h e m a j o r c o m p o n e n t s of c h r o m o s o m e s in e u k a r y o t i c cells are DNA and histones. These, and the non-hislone n u c l e a r (aoidic) p r o t e i n s eonstitue nearly the w h o l e o r g a n i z e d f r a m e w o r k of the c h r o m o s o m e . If the c h r o m o s o m a l p r o t e i n s p a r t i c i pate in the regulatio.n of gene function, k n o w ledge of the ratios of the i n d i v i d u a l c o m p o n e n t s of c h r o m a t i n , and of t h e i r b i o c h e m i c a l and biop h y s i c a l p r o p e r t i e s m a y c o n t r i b u t e to u n d e r s t a n d i n g the differences in r e g u l a t o r y m e c h a n i s m s w h i c h are i m p l i c i t in over d i f f e r e n c e s in the biological p r o p e r t i e s of different k i n d s of e u k a r y o t i c cells. It is possible that different kinds of such cells m a y c o n t a i n different structural or configur a t i o n a l types of hisfone and n o n - h i s t o n e protein, or that c h r o m o s o m a l p r o t e i n s : DNA i n t e r a c t i o n s differ in different k i n d s of such cells. R e c e n t stu(2) Presented in part at the 56th Annual Meeting, Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 1972. (3) These studies were supported in part by research grants G-6'516 from the National Cancer Institute, and FR-0.55~2L6 from the Division of Research Facilities and Resources, Nfational, Institutes of Health. (4) Present Address : Kinderklinik der Medizinischen Akademie, 24, Liibeck, W. Germany. (5). Holds Research Career Award K6-CA-22,150 from the National Cancer Institute, National Institutes of Health.
dies [1] for example, i n d i c a t e that in the case of h u m a n l y m p h o c y t i c cells, isologous histones inhibit or repress RNA synthesis m o r e effectively than do homologous histones ; suggesting differences in the affinities b e t w e e n histones and the r e c e p t o r sites on the DNA in different k i n d s of cells. Biological f u n c t i o n s h a v e been a s c r i b e d to some n o n - h i s t o n e n u c l e a r p r o t e i n s [2]. The non-histone c h r o m o s o m a l p r o t e i n s are g e n e r a l l y m o r e active than histones in the i n c o r p o r a t i o n of a m i n o acids [2, 3~ and p h o s p h a t e [4, 5], and the level of such m e t a b o l i s m a p p e a r s to be related to n u c l e a r activity [6, 7]. Although it has been r e p o r t e d that the n u c l e a r non-histone p r o t e i n s are i n v o l v e d in the activation of t r a n s c r i p t i o n m e c h a n i s m s by r e s t o r i n g h i s t o n e - i n h i b i t e d RNA synthesis [6-8], only limited i n f o r m a t i o n is available w i t h r e s p e c t to the non-histone (acidic) p r o t e i n s in r e l a t i o n to chrom o s o n m l s t r u c t u r e and f u n c t i o n in h u m a n lymp h o c y t i c cells. The p r e s e n t studies d e s c r i b e the b i o p h y s i c a l and b i o c h e m i c a l c h a r a c t e r i s t i c s of isolated c h r o m a t i n and i n d i v i d u a l c h r o m o s o m a l c o m p o n e n t s of h u m a n l y m p h o c y t i c cells d e r i v e d from n o r m a l d o n o r s and patients w i t h i n f e c t i o u s m o n o n u c l e o sis or acute l y m p h o b l a s t i c leukemia.
L. S. Desai, U. C. W u l f f and G. E. Foley.
316
MAT.ERIALS AND METHO,DS.
Cells : The d e r i v a t i o n of the cell lines used in these studies (table I) has been d e s c r i b e d e l s e w h e r e in detail. The CCRF-CEM cells w e r e isolated d i r e c t l y in s u s p e n s i o n culture from the p e r i p h e r a l b l o o d buffy coat of a female p e d i a t r i c patient whose presenting lymphosarcoma had p r o g r e s s e d to acute l y m p h o b l a s t i c l e u k e m i a [9]. The CC,RF-SB cells w e r e d e r i v e d in p r i m a r y stat i o n a r y cultures of the p e r i p h e r a l b l o o d buffy coat of a n o t h e r (male) p e d i a t r i c p a t i e n t w i t h s i m i l a r disease [10] ; a n d the CCRF-H-SB2 ce'lls w e r e TABLE I.
Thermal Denaturation Characteristics o[ Chromatins Isolated from Human Lymphocytic Cells Derived from Different Diagnostic Categories. t
Isolated chromatin
I Normal
Tm (Midpoint (herren] denaturation) :
CCRF-SLT I 76.8 -4- 0.2 CCRF-TOH 1 77.1 -~- 0.6 Infectious mononucleosis : CCRF-RKB 81.4 -t- 0.7 CCRF-SB 82.2 ~___0.4 Acute lymphoblastic leukemia : CCRF-H-SB 84.7 ~___0.8 CCRF-CEM 84.3 + 0.5 DNA : CCRF_TOH
70.1 --t- 0.5
d e r i v e d by d i r e c t i m p l a n t a t i o n o,f the same specim e n of p e r i p ' h e r a l b l o o d buffy coat into neonatal S y r i a n h a m s t e r s [11]. The CCRF-RKB cells w e r e d e r i v e d from the p e r i p h e r a l blood buffy coat of a y o u n g adult w i t h infectious m o n o i m c l e o s i s [12], a n d the C~fi,RF-TOH a n d C~C~RF-SLT cells w e r e d e r i v e d from the p e r i p h e r a l b l o o d huffy coat of normal, h e a l t h y d o n o r s [13]. These h u m a n l y m p h o c y t e s w e r e m a i n t a i n e d in c o n t i n u o u s log-phase g r o w t h at p o p u l a t i o n densities of 3-4 )< 106 cells/ml, w i t h a g e n e r a t i o n time of circa 24 h r s in large-volume s u s p e n s i o n cultures in Eagle's m i n i m a l essential m e d i u m mod i f i e d for s p i n n e r culture [14] a n d s u p p l e m e n t e d w i t h 10 p e r c e n t w h o l e fetal calf s e r u m b y successive w i t h d r a w a l a n d r e p l a c e m e n t of substrate, as d e s c r i b e d p r e v i o u s l y [9, 10].
Isolation of Chromatin : C]womatin was extracted from large quantities (5,0-1.0.0 gin) of cells by the m e t h o d s d e s c r i b e d by Marushige and Bonner [15]. In brief, the cells w e r e h o m o g e n i z e d in BIOCHIMIE, 1975, 57, n ° 3.
saline-EDTA buffer and 4 times in 0.01 M Tris buffer (pH 8.0). The gelatinous crude c h r o m a t i n was further purified by centrifugation through 1.7 M sucrose (buffered w i t h 0.01 M Tris at p H 8.0) for 2.5 h r s at 5.0,0.0,0 g. This p u r i f i e d chrom a t i n "was 'w~shed, r e s u s p e n d e d a n d d i a l y z e d o v e r n i g h t against 0.01 M Tris buffer, p H 8.0, then s h e a r e d and c e n t r i f u g e d ; the resulting s u p e r n a tant c o n t a i n i n g the p u r i f i e d c h r o m a t i n .
Preparation of Chromosomal Proteins : Histones (basic p r o t e i n s ) w e r e e x t r a c t e d from the p u r i f i e d c h r o m a t i n w i t h 0.4 N H2SO ~ at 4°C for 30 rain. The pellet w a s w a s h e d once w i t h 0.4 N HeSO 4 a n d b r i e f l y w i t h 0.01 M Tris buffer (pH 8.0). More than 95 p e r c e n t of a c i d soluble p r o t e i n (histones) are e x t r a c t e d by this p r o c e d u r e [16]. The pellet w h i c h c o n t a i n e d a c i d insoluble p r o t e i n s (acidic) w a s r e c o v e r e d by centrifugation, dissolved by gentle h o m o g e n i z a t i o n in 1 p e r c e n t SDS-0.05M Tris buffer (pH 8.0), and s t i r r e d o v e r n i g h t at 37"C. This solution ~,as t h e n d i a l y z e d against 0.01 M T r i s buffer (pH 8) c o n t a i n i n g 0.1 p e r c e n t SDS at 37°C for 24 hrs, a n d the DNA r e m o v e d by centrifugation at 3.6,000 r p m for 18 hrs at 25°C in a Spinco SW-50 rotor. After centrifugation, the u p p e r t w o - t h i r d s of the s u p e r n a t a n t was p i p e t t e d a n d chilled in an ice bath. The s o d i u m d o d e e y l s u l p h a t e (S,DS) was p r e c i p i t a t e d by the a d d i t i o n of s a t u r a t e d solution of K'C1 (1-2 drops) and the r e s u l t i n g p r e c i p i t a t e of potassiunl d o d e c y l sulp h a t e w a s r e m o v e d b y c e n t r i f u g a t i o n at 12,000 g for 20 rain. The p r e c i p i t a t e c o n t a i n i n g non-histone p r o t e i n s was f n r t h e r subjected to column c h r o m a t o g r a p h y (1 × 12 cm) of D o w e x 2 × 10 (A.G. 2 × 10, 20,0-4,0,0 mesh, Biorad, l:lichmond, CA) as d e s c r i b e d b y L e n a r d [171. This p r o c e d u r e removes most of the SDS from n o n - h i s t o n e proteins. The non-histone p r o t e i n w a s f u r t h e r p r e c i p i D a t e d w i t h s a t u r a t e d a m m o n i u m sulphate to a final 35 p e r c e n t saturation. The no n-histoue p r o t e i n was then Ere c i p i t a t e d in the cold and collected by c e n t r i f u g a tion at 12,000 g for 20 rain. The p r o t e i n pellet was d i s s o l v e d in 0.01 M Ti'is buffer (pH 8) and p r e c i p i t a t i o n ~-ith a m m o n i u m s u l p h a t e was r e p e a t e d . The final p r o t e i n preci.pitate was dissolved in T r i s buffer (0.01 M, p H 8) and d i a l y z e d o v e r n i g h t against the same buffer [183. Characterization of Nucleic Acids and Proteins of Chromatin : The n o n - h i s t o n e content was determ i n e d b y the m e t h o d of L o w r y el al. [19] using b o v i n e s e r u m a l b u m i n (Sigma) as a s t a n d a r d . Histone c o n c e n t r a t i o n a'lso w a s d e t e r m i n e d b y the same p r o c e d u r e [19] c a l i b r a t e d against calf thymus histones. The c h r o m a t i n RNA was s e p a r a t e d from DNA by the m o d i f i e d S e h m i d t - P a n n h a u s e r
C h r o m o s o m a l proteins of h u m a n le~tkemic cells. p r o c e d u r e described by Ts'o a n d Sato [20]. RNA was d e t e r m i n e d by the Orcinol method I21], using yeast RNA (Sigma) as a standard. DNA was determ i n e d by the d i p h e n y l a m i n e m e t h o d [221, using calf t h y m u s DNA (Sigma) as a standard.
Thermal Denaturation and Ultraviolet .4bsorption : Melting profiles of p u r i f i e d DNA a n d the various c o m p o n e n t s of the c h r o m a t i n isolated from these h u m a n l y m p h o c y t i c cells were determ i n e d in a Gifford Model 2800 Multiple Sample Absorbance R e c o r d i n g I n s t r u m e n t for the recording of melting profiles. A p p r o p r i a t e c o n c e n t r a tions of samples were taken i n 0.1 X SSC ~SSC = 0.15 M s o d i u m chloride - - 0.015 M sodium citrate (pH 7.0)3 and heated i n a thermosta'tically controlled w a t e r bath. The rate of t e m p e r a t u r e increase was 0.5 - - l ° C / m i n . Ultraviolet a b s o r p t i o n spectra at 260 n m are r e c o r d e d by a Beckman DU Spectrophotometer.
Amino Acid Analyses : Chromosomal proteins (histone and n o n - h i s t o n c proteins) were dissolved i n c o n s t a n t b o i l i n g (glass-distilled) HCI with 1 p e r c e n t p h e n o l to p r e v e n t degradation of tyrosine. The tube was then flushed w i t h nitrogen, sealed u n d e r r e d u c e d pressure, and h y d r o l y z e d at 110°C in a toluene bath for 20 hrs. The HC1 was diluted w i t h deionized w a t e r and r e m o v e d in a r o t a r y evaporator. The hydrolysates were then dissolved in 1 ml of sodium citrate buffer at pH 2.2 [23], filtered t h r o u g h a Millipore filter (0.22 a), a n d analyzed i n a Beckman Spinco 120 Automatic Amino Acid Analyzer [24]. Gel Electrophoresis : Analytical gels were composed of 10 p e r c e n t acrylamide, 0.2 p e r c e n t N.N'm e t h y l e n e b i s a c r y l a m i d e (BIS), dissolved i n 0.1 M sodium phosphate buffer, pH 7.4, c o n t a i n i n g 0.5 p e r c e n t SDS~ The solution was filtered a n d deaerated before use. P o l y m e r i z a t i o n was accomplished by the a d d i t i o n of 0.3 ml of 10 p e r c e n t a m m o n i u m persulfate (W/V) a n d 0.0.3 ml of N,N,N',N'-tetramethylethylenediamine (Temed) to 40 ml of a c r y l a m i d e solution [25]. Gels 9 cm i n length w i t h an i n t e r n a l diameter of 6 m m were allowed to form b y p o l y m e r i z a t i o n for 30 rain, and then subjected to a p r e - r u n at 4.5 V / t u b e for 30 rain with 0.1 M sodium phosphate buffer, pH 7,4, c o n t a i n i n g 0.1 p e r c e n t SDS in both u p p e r and l o w e r electrode chambers. Samples were then applied to the gels a n d electrophoresis was c a r r i e d out for 4-5 hrs. F o l l o w i n g electrophoresis, the gels were fixed and stained for 3-8 hrs i n a solution of 0.25 p e r c e n t Coomassie b r i l l i a n t blue in methan o l : a c e t i c a c i d : w a t e r (5:1:5). De-staining was accomplished by s o a k i n g the gels with gentle BIOCHIMIE, 1975, 57, n" 3.
317
s h a k i n g in a solution c o n t a i n i n g m e t h a n o l : acetic acid : water (2:3:35).
In Vitro Synthesis of RNA : The in vitro synthesis of RNA utilized highly p u r i f i e d h u m a n leukemic DNA (CCRF-CEM) or h u m a n leukemic c h r o m a t i n (CCRF-CEM) as a template a n d bacterial (Micrococcus lysodeikticus) RNA polymerase as the enzyme [26]. The reaction mixture, contained in a final volume of 0.25 ml, was : 0.04 M Tris buffer at pH 7.9, 0.01 M MgCI~, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.15 mM ATP, GTP, CTP, a n d 0.15 mM 3H-UTP (10 !~Ci/~!~mole,New E n g l a n d Nuclear, Boston, Ma.). W h e n the enzyme was used for m e a s u r i n g template activity of c h r o m a t i n , the DNA in the system was replaced by c h r o m a t i n c o n t a i n i n g 20 ~g of DNA. The m i x t u r e was i n c u bated at 37°C for 10 rain and the r e a c t i o n was stopped by the a d d i t i o n of 10 p e r c e n t TCA. The precipitate was collected on a Millipore m e m b r a n e filter (0.2.2 ~), w a s h e d 5 times with 5 ml of cold 10 p e r c e n t TCA, and dried at 80°C. Radioactivity was d e t e r m i n e d in a toluene scintillation fluid, u s i n g a Beckman Model LS 250 Scintillation Counter. ]~ESULTS.
Spectrophotometric Study of Chromatin : A typical ultraviolet a b s o r p t i o n s p e c t r m n of c h r o m a t i n isolated from h u m a n leukemic (CCRF-CEM) cells is s h o w n in figure 1. Essentially i d e n t i c a l absorp0.5
/,. N
O5
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\
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225
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i
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t
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9_55 245 255 265 275 285 295 505 5t.5 WAVELENGTH (A}
FIG. 1.
tion p a t t e r n s were obtained with the c h r o m a t i n isolated from n o r m a l (SLT, TOH), infectious mononucleosis (RKB, SB), and leukemic (GEM, HSB) l y m p h o c y t i c cells. The purified c h r o m a t i n isolated from all these cells showed no t u r b i d i t y , indicating little or no ultraviolet a b s o r p t i o n (0.0l) at 320 nin. A h i g h e r absorption at 320 niu would
L. S. Desai, U. C. W u l f f and G. E. Foley.
318
h a v e i n d i c a t e d e i t h e r a g g r e g a t i o n of c h r o m a t i n o r t h e p r e s e n c e of c o n t a n f i n a t i n g a g g r e g a t e s of n o n chromosomal proteins.
Tm Studies : T h e r m a l d e n a t u r a t i o n s t u d i e s o n t h e s e t o t a l c h r o m a t i n s t r u c t u r e s i n d i c a t e d a reelring temperature (Tm) several degrees higher than 20--
).. ~ 0
15
-
"ros--~-.Tf
Chemical Analysis of Chromatin : T h e c h e m i c a l c o m p o s i t i o n s of t h e s e isol'ated c h r o m a t i n s a r e s u m m a r i z e d i n t a b l e II. C o n s p i c u o u s d i f f e r e n c e s a r e e v i d e n t i n t h e m a s s r a t i o s of h i s t o n e , n o n histone and RNA conten.t ; for example, somewhat higher values are apparent in the leukemic and i n f e c t i o u s m o n o n u c l e o s i s l y m p h o c y t i c cells (CEM ; RKB, H-S,B) as c o m p a r e d to t h o s e f r o m n o r m a l d o n o r s (.S~LT, T O H ) . T h e r a t i o o.f h i s t o n e to n o n h i s t o n e p r o t e i n s a p p e a r s to b e circa 1.0 i n l e u k e m i c cells, ~ v h e r e a s c h r o m a t i n d e r i v e d f r o m n o r mal lymphocytes contains only a small quantity of n o n - h i s t o n e p r o t e i n s ( t a b l e t I ) .
""
o t,r ""
rrLO I 0 >.. ""
R K B ~
EM
S ~ ~
HSB
5
0
20
40
60
80
I00
Amino Acid Composition of Chromosomal Proteins : A m i n o a c i d a n a l y s e s of a c i d s o l u b l e p r o -
TEMPERATURE(*'C} ~ S L T . . . . TON ............. R K B
f r o m l y m p h o c y t i c cells (>f n o r m a l d o n o r s (SLT, T O H ) o r p,atients w i t h i n f e c t i o u s m o n o n u c l e o s i s (RKB, SB) p r o b a b l y r e f l e c t i n g t h e d i v e r s e c o m p o s i t i o n s of t h e s e c h r o m a t i n , as d e t e r m i n e d b y t h e p r e s e n c e of d i f f e r e n t (fig. 2) k i n d s of c o m p l e x e s w i t h n u c l e a r p r o t e i n s . T h u s , t h e r e is s o m e evidence that the chromosomal proteins participate i n t h e s t a b i l i t y of t h e c h r o m a t i n s t r u c t u r e i n t h e s e t h e r m a l d e n a t u r a t i o n s t u d i e s ( t a b l e I).
t e i n s ( h i s t o n e s ) of t h e c h r o m a t i n s i s o l a t e d f r o m these lymphocytic cells are summarized in t a b l e III. T h e s e a n a l y s e s a r e c o m p a r a b l e to t h o s e
IP4P4b-OSB O - ~ C E M ¢-,¢"¢'~DHSB
Fm. 2.
TABLE JI.
Chemical Characteristics of the Chromatins Isolated [rom Normal Human (SLT and TOH), Infectious Mononucleosis (RKB and SB), and Leukemic (HSB and CEM) Ceils. Chemical composition of chromatin Cells (')
CCRF-SLT . . . . . . . . . . . . . . -TO H . . . . . . . . . . . . . -RKB ............. -SB . . . . . . . . . . . . . . . -HSB . . . . . . . . . . . . . -CEM . . . . . . . . . . . . . .
No.
Preps
Histone
Non-histone protein
RNA
1, O0 9.93 ~ 0.06 0.49 __~ 0.04 0.06 ~ 0.05 1 . 0 0 0.86 ~- 0.04 0.58 ~- 0.03 0.08 ~- 0.07 1 . 0 0 1.02 -+- 0.12 0.86 ~- 0.08 0.10 -~ 0.06
1. O0 1.16 -4- 0.09 0.91 ~- 0.06 0.09 ' ~ 0.04 1 . 0 0 1.14 ___+0.13 1.17 -¢- 0.07 0.13 ~___0.07 1 . 0 0 1.22 - b 0.05 1 . 2 6 _~ 0.02 0.15 _____0.03
The nucleic acids a n d c h r o m a t i n - a s s o c i a t e d basic (histones) a n d acidic (nonhistone) p r o t e i n s were extracted f r o m isolated c h r o m a t i n a n d t h e values are normalized a g a i n s t the DNA content. (*) Cf. t a b l e I. T h e v a l u e s are m a s s ratios of c h r o m a t i n s is olated~ f r o m h u m a n l y m p h o c y t i c cells ; ~ are s t a n d a r d errors.
t h a t of t h e p u r i f i e d D N A i s o l a t e d f r o m h u m a n l y m p h o c y t i c cells (TO,H) ( t a b l e I). I n t e r e s t i n g l y , m e l ting profiles revealed higher melting temperatur e s (Tin) f o r c h r o m a t i n d e r i v e d f r o m h u m a n leuk e m i c cells (CEM, H S B ) t h a n c h r o m a t i n d e r i v e d
BIOCHIMIE, 1975, 57, n ° 3.
of t h e t o t a l h i s t o n e s iso,lated f r o m w h o l e c e l l s [27]. T h e r e a p p e a r s to b e a d e g r e e of s i m i l a r i t y i n t h e a n l i n o a c i d c o m p o s i t i o n of t h e h i s t o n e s i s o l a t e d , i n d i c a t i n g t h e p r o b a b l e h o m o g e n e i t y of t h e acid soluble proteins.
C h r o m o s o m a l proteins of h u m a n leukemic cells. The non-histone chromosomal proteins appear to b e m o r e h e t e r o g e n e o u s i n t h e i r a m i n o a c i d c o m p o s i t i o n s . T h e h i g h r a t i o o f a s p a r t i c p l u s gluTABLE IIL
Amino Acid Composition of Histories Isolated [rom Chromatin. Chromosomal histones from cells('):
Amino acids
SLT 7C H RKB! $3 HSB CEM Lysine . . . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . . Arginine . . . . . . . . . . . . . . . Aspartie acid . . . . . . . . . . . Threonine . . . . . . . . . . . . . . Serine . . . . . . . . . . . . . . . . . Glutamic acid . . . . . . . . . . Proline . . . . . . . . . . . . . . . . . Glycine . . . . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . . . Cystine/2 . . . . . . . . . . . . . . Valine . . . . . . . . . . . . . . . . . Methionine . . . . . . . . . . . . . . lsoleucine . . . . . . . . . . . . . . . Lencine . . . . . . . . . . . . . . . . . Tyrosine . . . . . . . . . . . . . . . Phenylalanine . . . . . . . . . . .
319
r a m i e a c i d s , w h i c h e x c e e d s t h a t of t h e b a s i c a m i n o a c i d s , l y s i n e , a r g i n i n e , a n d h i s t i d i n e (table IV), i d e n t i f y t h e s e n o n - h i s t o n e p r o t e i n s to b e a c i d i c c h r o m o s o m a l p r o t e i n s . T h e r e a r e d i f f e r e n c e s in the a m i n o acid c o n t e n t a n d c o m p o s i t i o n of these a c i d i c p r o t e i n s , as c o m p a r e d to t h e n o n - h i s t o n e p r o t e i n s i s o l a t e d f r o m tlle v a r i o u s l y m p h o c y t i c cells c o n s i d e r e d h e r e i n (cf. t e x t ) .
Heterogeneity and Cell Specificity : T h e nuc l e a r n o n - h i s t o n e p r o t e i n s a r e a m i x t u r e of p r o t e i n s w h i c h d i f f e r i n size a n d e l e c t r o p h o r e t i c m o b i l i t y . E l e c t r o p h o r e s i s i n t h e p r e s e n c e of s o d i u m
10.2[ 1 8 1 2 . 1 11.4 12.5 13.0 1.2 1 9 2 1 1 6 2.1] 2.6 8,2 9.01 7.8 5,t I 4.9 7 (7 6.3[ 6.0 5.0 5616.31 5 8 6.1 6.7 5.3 4 7 / 5 . 1 5,G 5.41 5.6 5.5 4.9] 7.9 6.7 7.5] 8.3 4.8 4 . 9 1 5 2 42 5.1] 5 4 9.0 9.2 8.2 8.8 8.7j 8.4 13.21 2 . 4 / 1 4 2 I14 6 14.3 13 8 o0j o.oi 0.~3 0.O I 0.0 6.8 5.6 6.2 6.3 t 6.5 1.0 0 7 1 . 1 I.~ 1.2] 0.9 5.2 5.(,I 4 2 4.8 4 7 / 4 0 7.0 7.2 / 7.6 6.,q 7.2 7.9 2.0 2 6 / 2 5 I £ 2.2 2.2 1.6 1.St 1.9 2.1 2.4[ 2 1
9o!
A
B
(*) Cf. t a m e I. The values are moles of each a m i n o acid,/100 moles of amino acids recovered.
C
TABLE IV.
Amino Acid Composition of Non-Histone Protein Isolated From Chromatin. Chromosomal non histone (acidic) proteins from cells(')
Amino acids
Fla. 3.
H RI:B[ SB HSB CEM Lysine . . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . . Argininc . . . . . . . . . . . . . . . . Aspartic acid . . . . . . . . . . . . Threonine . . . . . . . . . . . . . . . Serine . . . . . . . . . . . . . . . . . Glutamic acid . . . . . . . . . . . Proline . . . . . . . . . . . . . . . . . . Glycine . . . . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . . . . Cystine/2 . . . . . . . . . . . . . . . Valine . . . . . . . . . . . . . . . . . . Methionine . . . . . . . . . . . . . ]soleucine . . . . . . . . . . . . . . Leucine . . . . . . . . . . . . . . . . Tyrosine . . . . . . . . . . . . . . . PhenyIalanine . . . . . . . . . .
5 3/ .2 6.8 6.5 5.8 7.3 2.4 .1 1 2 1 2.0 2.2 5.2 .6 5 5.8 5 9 6.0 11.4 1 .2115 U.912.3136 13.4 4.6 o 5.5 4.2 .7 7 5.~ 6 ,q 6.0 13.41 9 1 4 2.~ 15 8 18.3 3.2 5.5~ 4.7, 4 5 5 l 6.7 8.8 .1 9 2.3 12.3 6.5 8 .1 8..q 7.3 6 8 1.0 0.8[ 0 o.e 0.9 1.1 5.1 .2 6.1 6.6 6 3 .8 2.4 2 2 2 0 1.8 I 1 7.4 4 8 5 . 2 5.0 36,i. 25 13 4. 11 4.5 7.8 9.4 6, 9.(: 8 6 8 . 9 2.4 3 .0 2.6 2.1 3 6 2 . 4 2.6 4 3.4 4.313.8
-- --ii ....
(*) Cf. table I. The values are moles of each amino acid/100 moles of a m i n o acids recovered. BIOCHIMIE, 1975, 57, n ° 3.
d o d e c y l s u l p h a t e s e p a r a t e s the p r o t e i n s on the b a s i s of m o l e c u l a r size [28, 29] ; s u b s e q u e n t stain i n g w i t h a c o m b i n a t i o n of C o o m a s s i e b l u e a n d A m i d o B l a c k r e v e a l e d t h e p r e s e n c e of s e v e r a l s p e c i e s of p r o t e i n s w i t h v a r y i n g m o l e c u l a r size as e v i d e n c e d by d e n s i t o m e t e r l r a c i n g s (fig. 3). U n l i k e hisiones, non-histone proteins cover a wide range o f m o l e c u l a r w e i g h t s ; i.e., j u d g i n g f r o m t h e m o b i l i t i e s of t h e b a n d s in gel e l e c t r o p h o r e s i s as c o m p a r e d to m a r k e r p r o t e i n s w i t h k n o w n m o l e c u l a r w e i g h t s . T h e o b s e r v e d m o l e c u l a r w e i g h t of s e v e ral a c i d i c p r o t e i n s f r o m t h e h u m a n l e u k e m i c c e l l s s t u d i e d fall i n t h e r a n g e of 15,000 to 80,000 as r e p o r t e d b y S h e l t o n a n d A l l f r e y [7]. T h e d e n s i t o m e t e r t r a c i n g s of a c i d i c p r o t e i n a n a l y s e s o n p o l y a c r y l a m i d e gels r e v e M e d c o n s i d e r a b l e h e t e r o g e neity ; suggesting different characteristics of these
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c h r o m a t i n - a s s o e i a t e d p r o t e i n s w h i c h nlav be conc e r n e d w i t h differences in the eontroI of transc r i p t i o n in these different k i n d s of h u m a n lymp h o c y t i c cells.
Effect of Acidic or Non-histone Protein (NHP) on Template Activity : It is w e l l - k n o w n that DNAd e p e n d e n t RNA synthesis in vitro is i n h i b i t e d by the a d d i t i o n of h i s to n e to a cell-free system cont a i n i n g RNA p o l y m e r a s e and e i t h e r DNA, or DNA
×
A
20
rase r e a c t i o n exhibits only 10-20 p e r c e n t of the tempIate activity as c o m p a r e d to the t e m p l a t e activity of n ak ed CEM.DNA (fig. 4A). The a d d i t i o n of non-histone isolated f r o m CEM cells to the RNA p o l y m e r a s e r e a c t i o n act i v at ed the t e m p l a t e activity by 4-5 fold (fig. 4B). Th e RNA t r a n s c r i b e d f r o m the c h r o m a t i n act i v at ed by n o n - h i st o n e proteins is p e r h a p s due to the u n m a s k i n g of the genome, w h i c h m ay have s i m i l a r or different nucleotide sequences as the e x p r e s s e d genome. It has been suggested p r e v i o u s l y that n u c l e a r a c i d i c proteins r est o r e h i s t o n e - i n h i b i t e d RNA synthesis [6, 8]. Other e x p e r i m e n t s (tables V and VI) e m p l o y i n g e h r o m a t i n s and non-histone p r o t e i n s (NHP) isola-
5 DNA
