BIOCHIMICAET BIOPHYSICAACTA
473
BBA 96359
S T U D I E S ON T H E Y E A S T NUCLEUS
I I I . P R O P E R T I E S OF A D E O X Y R I B O N U C L E O P R O T E I N COMPLEX
D E R I V E D FROM Y E A S T
P. CH. VAN DER VLIET, G. J. M. TONINO AND TH. H. ROZIJN
Laboratory/or Physiological Chemistry, The State University, Utrecht (The Netherlands)
(Received July i7th, 1969)
SUMMARY
I. A deoxyribonucleoprotein complex was isolated from Saccharomyces cerevisiae. It is composed of 36 % DNA, 4 % RNA and 60 % protein. About 7 ° % of the
protein is acid-extractable. The complex sediments as a single band with a s°20,~ of 27 S.
2. The yeast deoxylibonucleoprotein shows a biphasic melting profile. About
50 % of the deoxyribonucleoprotein melts at 71° which is close to the Tm of DNA
(68 °) and 50 % melts at 83 °. When protein, mostly histone, was dissociated from the
deoxyribonucleoprotein b y extraction with NaC1 of increasing concentrations, the
percentage of deoxyribonucleoprotein melting at 83 ° decreases gradually, while the
T m of the remainder shifts to that of DNA. After dissociation of deoxyribonucleoprotein at I M NaC1, the melting profile is identical to that of yeast DNA.
3. The template activity for RNA synthesis of yeast deoxyribonucleoprotein
was compared to that of yeast DNA using Escherichia coli RNA polymerase. The
template activity is dependent on the concentration ratio of enzyme to template.
A m a x i m u m template activity of 60 % was observed for yeast deoxyribonucleoprotein
at high concentration ratios of enzyme to template.
4. It is concluded that yeast deoxyribonucleoprotein resembles the deoxyribonucleoprotein of higher organisms in several aspects.
INTRODUCTION
In previous publications 1,~, we have described a procedure for the isolation of
nuclear chromatin from pressed baker's yeast. In yeast, as in higher eukaryotes, the
chromatin is composed of DNA complexed with proteins and some RNA. The DNA
in yeast chromatin is associated with acid-extractable basic proteins which can be
regarded as histones. The yeast histones resemble the histones of higher organisms
in several respects but are definitely less basic z,z.
Several studies have shown that histones from higher eukaryotes can act as
inhibitors of the DNA-directed synthesis of RNA 3,4. When chromatin from animal
tissues is used as a template for RNA synthesis in vitro, only part of the DNA is
transcribed. 80-90 % of the DNA in chromatin is prevented from acting as a template
by its association with histones ~,5. PAUL AND GILMOUR5,6 showed that this restriction
Biochim. Biophys. Acta, I95 (I969) 473-483
474
P. CH. VAN DER VLIET et al.
of template activity is organ-specific. The basis of this specificity is not yet understood,
although there are several indications that the specificity m a y reside in chromosomal
RNA ~,s or in acidic proteins 6. Anyway, this specificity is apparently an inherent
property of the chromatin that is preserved during isolation.
In a nondifferentiating organism, there seems to be no need to keep a large part of
the DNA permanently in an inactive state, as is the case in differentiated tissues.
Therefore we do not expect to find an extensive restriction of chromosomal template
activity in a unicellular organism like yeast. This implies that the histones in yeast
chromatin m a y not prevent DNA from acting as a template.
In the present study, the template activity in vitro of solubilized preparations of
chromosomal deoxyribonucleoprotein from yeast was examined. In addition, the
sedimentation properties and the melting behavior of yeast deoxyribonucleoprotein
and of partially deproteinized deoxyribonucleoprotein were studied in order to obtain
more information on the interaction between DNA and yeast histones.
MATERIALS AND METHODS
Chemicals
L8-14CIATP (specific activity 22 #C/#mole) was purchased from the Schwartz
Bioresearch, U.S.A.
Nonlabeled ribonucleoside triphosphates were obtained from the Sigma Biochemical Corp. (U.S.A.) and the Calbiochem., Switzerland.
Ribonuclease (i time crystallized) was a product of Worthington Biochemical
Corp., N.Y., U.S.A. The enzyme was made free of deoxyribonnclease activity b y
heating for IO min at 80 ° in o.ooi M NaC1.
Pronase was purchased from the Calbiochem., Switzerland and was preincubated for 2 h at 37 ° in 0.05 M Tris (pH 8.5) to destroy other enzymatic activities.
Isolation o/chromatin and deoxyribonucleoprotein
Yeast chromatin was isolated as previously described 1,2. Deoxyribonucleoprotein was prepared in the following way. Chromatin (purified b y sucrose gradient
centrifugation) was washed twice with o.15 M NaC1- 5 mM E D T A (pH 6.5) and suspended in 0.2 mM E D T A (pH 8.0) to a concentration of about 0.5 mg DNA per ml.
The suspension was homogenized during 2 min in a Biihler homogenizer and centrifuged for 30 min at 50 ooo xg. The snpernatant, containing about 7 ° % of the DNA
as soluble deoxyribonucleoprotein, was dialyzed overnight against 0.2 mM E D T A
(pH 8.0) and in some cases was pelletted by centrifugation for 22 h at lO 5 ooo xg.
All operations were performed at 0-4 °. The deoxyribonucleoprotein was stored at
0-4 ° in 0.2 mM E D T A (pH 8.0) and used within 2 days.
Isolation and puri/ication o/yeast D N A
DNA was isolated from chromatin essentially as described b y KAY et al. 9 for calfthymus chromatin; the chromatin was suspended in o.15 M NaC1 and sodium dodecyl
sulfate was added to 0.5 % after stirring for I h at 20 °. NaC1 was added to a final concentration of I M. The mixture was cooled, and centrifuged for 60 rain at 20 ooo xg. To the
supernatant was added I vol. of 96 % ethanol, DNA was spinned on a glass rod,
washed twice with 80 % ethanol and dissolved in I mM NaC1 to a concentration of
Biochim. Biophys. Acta, 195 (1969) 473-483
DEOXYRIBONUCLEOPROTEIN COMPLEX FROM YEAST
475
about o.5 mg/ml. The DNA was further purified subsequently b y treatment with
ribonuclease (50 #g/ml) for I h at 37 °, followed by incubation at 37 ° with pronase
(50 #g/ml) during one night, after which the sodium dodecyl sulfate-NaCl-ethanol
procedure was repeated. The DNA was again dissolved in I mM NaC1, reprecipitated
with ethanol and stored in 80 % ethanol at --20 °. The s20,, of this yeast DNA was
21 S. I t contained less than 1.5 % RNA and less than I ~o protein. According to the
buoyant density in CsC1 the DNA was contaminated with 5-t-2 ~o of mitochondrial
DNA.
Assay o~ template activity
RNA polymerase (nucleoside triphosphate:RNA nucleotidyl transferase, EC 2.7.7.6 )
was prepared from E. coli QI 3, lacking ribonuclease I and RNA phosphorylase l°,
essentially according to the method of CHAMBERLIN AND BERG11 up to Fraction 4.
The amount of protamine sulfate necessary to precipitate more than 80 % of the
activity was determined b y titration. The specific activity was about 2000 units per
mg protein. The enzyme was stored in o.oi M Tris-HC1 buffer (pH 8.o), I mM MgC12,
o.I M (NH4)2SO 4 and 5 mM glutathion in liquid N~. The reaction mixture (final
volume 0.25 ml) for measuring the incorporation of [14C]ATP in acid-insoluble
material contained 40 mM Tris-HC1 buffer (pH 7.9), 12 mM 2-mercaptoethanol,
4 mM MgC12, I mM MnCI~ and ribonucleoside triphosphates (o.I mM each). [8-14CJA T P in this reaction mixture had a specific activity of I #C//~mole. DNA or deoxyribonucleoprotein was added as indicated, and the reaction was started by adding RNA
polymerase. After incubation for IO min at 37 °, 0.2 ml of a solution containing o.I M
[12CJATP and 1 % albumin (pH 7.0) was added, the mixture was cooled and the
reaction was stopped with 3 ml of cold 0.5 M HC1Q. After 20 min in ice, the suspension was centrifuged, the pellet washed 3 times with cold 0.5 M HC1Q, dissolved
in 5 ml of 0. 5 M NH4OH and added to a 6.5-ml mixture of 3 ml of Triton X - I o o and
3.5 ml of toluene, containing 0. 5 % 2,5-diphenyloxazole, 0.005 % 1,4-bis-(5-phenyloxazolyl-2)benzene TM.
Radioactivity was counted in a Nuclear Chicago liquid scintillation counter.
The incorporation with enzyme alone (in all cases less than 20 counts/rain) was
subtracted.
Template activity is defined as the incorporation rate with deoxyribonucleoprotein as template, divided b y the incorporation rate with DNA as template, at the
same DNA concentration.
Melting pro/iles
Melting profiles of DNA and deoxyribonucleoprotein in 15 mM NaCI-I. 5 mM
sodium citrate-o.2 mM E D T A were obtained in a Beckman spectrophotometer
connected to a water bath. The temperature was changed discontinuously b y 2 ° in
each step.
The temperature was measured b y a thermocouple in one of the cuvettes. An
equilibration period of IO rain was used at each step. E v e r y value was corrected for
turbidity b y subtracting the absorbance at 320 nm from that at 260 nm.
Dissociation o/deoxyribonucleoprotein solutions
Deoxyribonucleoprotein was dialyzed against three changes of NaC1 solutions of the
desired concentrations, containing 0.2 mM EDTA. The remaining nucleoprotein was
Biochim. Biophys. Acta, 195 (1969) 473-483
P. CH. VAN DER VLIET et al.
476
separated from the dissociated protein by centrifugation (22 h at lO5 ooo ×g). The
sediment was suspended in 15 mM NaCI-I. 5 mM sodium citrate-o.2 mM EDTA (pH
7.0). The sedimented nucleoproteins redissolve rather slowly and were therefore
homogenized in a Btihler homogenizer (2 rain) and dialyzed overnight against
15 mM NaCI-I.5 mM sodium citrate-o.2 mM EDTA (pH 7.0). In this way complete
solution was effected.
General methods
DNA, RNA, total protein and histones were determined as previously
described 13.
Sedimentation coefficients of DNA and deoxyribonucleoprotein in 15 mM NaC11.5 mM sodium citrate-o.2 mM EDTA (pH 7.o) were obtained using a Spinco analytical ultracentrifuge, Model E, equipped with a photoelectric scanning system.
RESULTS
Chemical composition o/solubilized chromosomal deoxyribonucleoprotein and ultravioletabsorption spectrum
Table I shows the chemical composition of solubilized deoxyribonucleoprotein.
The chemical composition of the yeast deoxyribonucleoprotein resembles that of
similar preparations from animal and plant cells.
TABLE
I
CHEMIC&L COMPOSITION OF CHROMOSOMAL DEOXYRIBONUCLEOPROTEIN
Source o[
nucleoprotein
Ratio o] total
protein to DNA
Ratio o]
histone to DNA
Ratio o[
R N A to DNA
Yeast
Rat liver*
Pea embryo**
Calf t h y m u s * *
1.68
1.67
:.31
1.47
1.3o
I.OO
1.o 3
I. 14
o.1o
o.o 4
o.26
o.oo 7
* R e f . 14.
** R e f , 3.
The ultraviolet spectrum (Fig. I) is characteristic of a nucleoprotein; the
absorption minimum is shifted to higher wavelengths.
Partial deproteinization of deoxyribonucleoprotein
Proteins can be partially removed from the deoxyribonucleoprotein complex by
stepwise extraction with salt solutions of increasing ionic strength. Fig. 2 shows the
fraction of the original proteins which remains associated to DNA as a function of
the NaC1 concentration. Only a small fraction (about 5 %) of the histone protein
remains associated with DNA after extraction with 2 M NaC1.
However, a relatively large fraction (about 30 %) of the nonhistone proteins
remains bound to DNA even after extraction with 2 M NaC1.
Biochim. Biophys. Mcta, 195 ( I 9 6 9 ) 4 7 3 - 4 8 3
477
] ) E O X Y R I B O N U C L E O P R O T E I N C O M P L E X FROM Y E A S T
07
A
O6
o5
!
04
0.3
02
01
0
I
I
220
I
240
I
I
260
I
I
I
I
280
300
WaveJength (nm)
,deoxy-
F i g . I. Absorption spectra of yeast deoxyribonucleoprotein and D N A . - - - , D N A ;
ribonucleoprotein.
g
¢)
#o
~o
24
16
0.6
8
=
i
"6
0.2
0.4
f'
I
i
12
2.0
NaCI(M)
0
I
0
I
I
l
08
1.6
NaCi(M)
F i g . 2. The fraction of total histone and nonhistone proteins remaining bound to D N A after extraction of yeast deoxyribonucleoprotein with different NaC1 concentrations. 0 - O , histone; Q - Q ,
nonhistone protein.
F i g . 3. Effect of extraction of yeast deoxyribonucleoprotein with different NaC1 concentrations
on the sedimentation coefficient.
Sedimentation studies
Sedimentation velocity analysis shows that the deoxyribonucleoprotein sediments as a single broad band with a s°~0,~ = 27 S. The D N A isolated from deoxyribonucleoprotein by the same method that is used for the isolation of D N A from chromatin (see MATERIALS AND METHODS) has a sedimentation coefficient of 12 S. The
difference between the sedimentation coefficient of D N A isolated from deoxyribonucleoprotein (12 S) and from chromatin (21 S) can be explained readily by taking
into account the shearing forces that are employed in the isolation of deoxyribonucleoprotein.
The sedimentation coefficients of native deoxyribonucleoprotein and of partially deproteinized deoxyribonucleoprotein preparations are presented in Fig. 3. It
should be noted that all salt-extracted nucleoproteins sediment as a single band.
Biochim. Biophys. Aeta,
195 ( I 9 6 9 ) 4 7 3 - 4 8 3
478
p. CH. VAN DER VLIET et al.
Sedimentation behavior has not changed appreciably after dissociation with
0.4 M NaC1, which removes about 40 % of the protein. Removal of another 40 %,
however, (0.8 M NaC1) results in a sharp drop in the sedimentation coefficient to about
12 S, i.e., nearly that of completely deproteinized DNA. This indicates that the change
in sedimentation coefficient is not merely due to the loss of protein, but to a conformational change which accompanies the dissociation of the deoxyribonucleoprotein
molecules.
1.4
A
._u 1.4
E
1.3
.c
u
1.2
~' 1.2
1.1
1.1
1.0
1.0
%
,
I
I
I
60 °
40 °
I
I
1.3
J
J
I
I
80°
100 o
Temperature
40 °
I
60 °
I
I
80 °
Temperature
Fig. 4- T h e r m a l d e n a t u r a t i o n profiles of DNA, deoxyribonucleoprotein and partially deproteinized
y e a s t deoxyribonucleoprotein in 15 mM NaCI-I. 5 mM sodium citrate-o.2 mM EDTA. A. O - O ,
DNA; V]-fN, native deoxyribonucleoprotein; Q - O , deoxyribonucleoprotein dissociated b y 0. 4 M
NaC1. B. O - O , deoxyribonucleoprotein dissociated b y 0.6 M NaC1; A - A, deoxyribonucleoprotein
dissociated b y 0,8 M NaC1.
Thermal denaturation
The melting curves for yeast DNA, for deoxyribonucleoprotein and for partially
deproteinized deoxyribonucleoprotein preparations are shown in Fig. 4. The melting
curve for deoxyribonucleoprotein shows two distinguishable steps, the Tm ot which
differ markedly. The Tm of the first step is close to that of D N A (68°), while the Tm
of the second step is 83 °. The percentage of D N A melting at 83 ° as a function of the
amount of protein removed is shown in Fig. 5. The removal of about 4 ° % of the
proteins from the deoxyribonucleoprotein obviously has no effect on the melting
behavior. However, if more protein is removed the percentage of deoxyribonucleoprotein melting at 83 ° decreases while the Tm of the remainder shifts to that of pure
DNA. After dissociation with i M NaC1 the melting curve was identical to that of
40
:~
z<
a
o
,
0
40
80
%
Protein removed
~
20
7<
~
o
I/
,
0
,
10
20
,
30
$2o~
Fig. 5. Percentage of D N A melting a t 83 ° as a function of the a m o u n t of protein removed from
deoxyribonucleoprotein.
Fig. 6. Changes in s e d i m e n t a t i o n a n d melting behavior of y e a s t deoxyribonucleoprotein upon
stepwise r e m o v a l of protein. Percentage of D N A melting a t 83 ° as a function of t h e s°,0,w value.
Biochim. Biophys. Acta, 195 (1969) 473-483
479
DEOXYRIBONUCLEOPROTEIN COMPLEX FROM YEAST
DNA. As can be seen in Fig. 6, there is a linear relationship between the changes in
sedimentation behavior and the changes in melting profile of yeast deoxyribonucleoprotein upon stepwise removal of protein.
Template activity o/ deoxyribonucleoprotein
The rate of RNA synthesis as a function of the template concentration is given
in Fig. 7. At all concentrations tested, the incorporation rate with deoxyribonucleoprotein as template is lower than with DNA as template. The template activity at
each concentration is independent of the time of incubation up to at least 60 miD.
From Fig. 7 it can be readily seen that the template activity of deoxyribonucleoprotein increases as the template concentration is decreased. At low template concentrations, however, a maximum value of 5o-60 % was reached. This value does not change,
when at low template concentration the enzyme concentration is increased (Figs. 8
and io), from which it can be concluded that a template activity of 50-60 % is indeed
the maximum.
LL
4[
12.0
_...._--o
100
&O
t~
60
<
4
3
4.0
c
20
2
1
0
I
I
I
I
8
16
24
32
I
I
I
0
40
48
56
D N A (JJg/Q25ml)
0
160
320
RNA polyrnerase ( u n i t s / Q 2 5 r n l )
Fig. 7. T h e r a t e of R N A s y n t h e s i s as a f u n c t i o n of t h e t e m p l a t e c o n c e n t r a t i o n w i t h y e a s t d e o x y r i b o n u c l e o p r o t e i n a n d DI%A as t e m p l a t e . I n c u b a t i o n w i t h 80 u n i t s of e n z y m e . F o r r e a c t i o n m i x t u r e
a n d f u r t h e r d e t a i l s see MATERIALS AND METHODS. O - - O , D N A ; /X-/X, d e o x y r i b o n u c l e o p r o t e i n .
Fig. 8. T h e e f f e c t of e n z y m e c o n c e n t r a t i o n on t h e t e m p l a t e a c t i v i t y a t low t e m p l a t e c o n c e n t r a tions. O - O , i.o/~g DN'A p e r 0.25 ml; O - Q , I.o/~g d e o x y r i b o n u c l e o p r o t e i n p e r 0.25 ml.
At these low template concentrations reproducible results are obtained, as can
be seen from incorporation data with five different deoxyribonucleoprotein preparations, at three different concentrations (Table II).
There are a number of possible explanations for the decrease in template
activity at increasing deoxyribonucleoprotein concentration, a phenomenon also
observed by other authors with bacterial as well as mammalian nucleoprotein 15-1~.
One explanation is that the deoxyribonucleoprotein is contaminated with ribonuclease activity. There is a detectable ribonuclease activity in yeast deoxyribonucleoprotein. Attempts to free the deoxyribonucleoprotein from ribonuclease by sucrose
gradient centrifugation according to RAAF AND DONNERis were unsuccessful. It is not
Bio6him. Biophys. Acta, 195 (1969) 473-483
P. CH, VAN DER VLIET el al.
480
TABLE II
TEMPLATE
PROPERTIES
OF FIVE DIFFERENT
DEOXYRIBONUCLEOPEOTEIN
PREPARATIONS
I n c u b a t i o n , i o m i n a t 37°; R N A p o l y m e r a s e , 80 u n i t s .
Preparation No. of
deoxyribonucleoprotein
A3/I P incorporated (nmoles)
5 fig DNA per
o.25 ml
z k*g DNA per
o.25 ml
o.25 #g DNA per
o.25 ml
I
2
3
4
5
3.4 °
3.73
3.52
3.5 o
3 .18
1.57
2.06
1.8o
1.82
1-65
0.67
0.72
o.72
o.6o
0-56
Average
3.48±0.20
1.78Jzo. I 9
o.64~ o.o 7
likely, however, that this contamination is responsible for the concentration dependence of the template activity for the following two reasons.
First the incorporation data at high deoxyribonucleoprotein concentration are
reproducible although the ribonuclease activity varies from preparation to preparation.
Secondly, when a mixture of DNA and ribonuclease with the same ribonuclease
activity as deoxyribonucleoprotein is used as template, the incorporation data with
different template concentrations closely resemble those obtained with ribonucleasefree DNA and are quite different from those obtained with deoxyribonucleoprotein
as a template (Fig. 9). The evidence remains circumstantial, however, because we do
not know the exact nature of the ribonuclease activity in deoxyribonucleoprotein.
10
T,
4
-~4o
2
~920
E
i
0
i
4
i
i
8
i
i
12
i
i
i
i
16
20
~gDNA/0.25ml
0
"
01
I
. . . . . .
O4
'
1.0
~
. . . .
4.0
' ' "
'
I
'
'"
10
50
~g DNA /025ml
F i g . 9. E f f e c t of r i b o n u c l e a s e on t h e r a t e of D N A - d i r e c t e d R N A s y n t h e s i s a t d i f f e r e n t t e m p l a t e
c o n c e n t r a t i o n s . 80 u n i t s R N A p o l y m e r a s e w e r e used. Q - O , y e a s t D N A ; V 3-~ , y e a s t D N A t o
w h i c h 3.6- lO -5 u n i t s p a n c r e a s r i b o n u e l e a s e a n d o. 7. lO -5 u n i t s T I r i b o n u c l e a s e p e r / z g D N A w a s
a d d e d ; t h i s m i x t u r e g i v e s t h e s a m e a m o u n t of a c i d - s o l u b l e c o u n t s / r a i n a f t e r i n c u b a t i o n w i t h
[14C]RNA as I / ~ g d e o x y r i b o n u c l e o p r o t e i n ; A - A , d e o x y r i b o n u c l e o p r o t e i n .
Fig. IO. T h e p e r c e n t a g e of t e m p l a t e a c t i v i t y of d e o x y r i b o n u c l e o p r o t e i n as a f u n c t i o n of D N A
c o n c e n t r a t i o n a t d i f f e r e n t R N A - p o l y m e r a s e c o n c e n t r a t i o n s . E]-[~, 24 ° u n i t s p e r 0.25 ml ; O - O ,
8o u n i t s p e r 0.25 m l ; / ' , - ZX, 15 u n i t s p e r o.25 ml. F o r f u r t h e r d e t a i l s see MATERIALS AND METHODS.
Biochim. Biophys. Acta, 195 (1969) 473-483
481
DEOXYRIBONUCLEOPROTEIN COMPLEX FROM YEAST
Another explanation could be that aggregation occurs at higher deoxyribonucleoprotein concentration, as has been suggested for deoxyribonucleoprotein of calf
thymus 17. To test this possibility, we centrifuged deoxyribonucleoprotein in the
template medium for 15 rain at 25 ooo ×g (ref. 18) and analyzed it for DNA. The
results (see Table III) show that there is only a slight concentration dependence for
the sedimentation of deoxyribonucleoprotein in this medium, which makes aggregation, as reason for the diminished template activity at higher deoxyribonucleoprotein
concentrations, not very likely.
TABLE III
SOLUBILITY OF YEAST DEOXYRIBONUCDEOPROTEIN IN THE TEMPLATE MEDIUM AT DIFFERENT CONCENTRATIONS
4 m l of t h e v a r i o u s d e o x y r i b o n u c l e o p r o t e i n s o l u t i o n s w e r e c e n t r i f u g e d for 15 r a i n a t 25 ooo × g.
A f t e r c e n t r i f u g a t i o n t h e D N A c o n c e n t r a t i o n in t h e u p p e r 2 m l w a s m e a s u r e d . The p e r c e n t a g e of
s o l u b l e D N A w a s e x p r e s s e d as ( c o n c e n t r a t i o n u p p e r h a l f ) / ( i n i t i a l c o n c e n t r a t i o n ) × i o o %.
Deoxyribonucleoprotein
conch. (tzg per o.25 ml)
Medium
Soluble D N A
5
io
2o
D N A (20/zg)
20
Template medium"
Template medium"
Template medium*
Template medium"
o.2 m M E D T A (pH 8.o)
86
84
78
95
95
(%)
* 2 - M e r c a p t o e t h a n o l is o m i t t e d .
Finally it is possible that upon incubation with deoxyribonucleoprotein the
effective enzyme concentration is lower than in the case of incubation with an equivalent amount of DNA (possibly by inactivation of enzyme).
Evidence for this view is given in Fig. IO where the template activity is given
as a function of DNA concentration at different enzyme concentrations. Deviation
of the maximum template activity occurs at low deoxyribonucleoprotein concentration when a low enzyme concentration is used and at higher deoxyribonucleoprotein
concentrations with higher enzyme concentration. The figures suggest that the observed concentration dependence of the template activity is caused by a change in
the enzyme/template ratio rather than by the template concentration itself.
At an enzyme/DNA ratio of about IO units/#g DNA, the maximum value for
the template activity is reached.
DISCUSSION
Yeast is generally regarded as an organism which occupies an intermediate
position between the prokaryotic organisms and the metazoa. Combined chemical
and electron microscopic studies of isolated nuclei which were extracted with salt
solutions, or treated with ribonuclease, deoxyribonuclease or pronase 19, revealed
that as in higher eukaryotes the nuclear chromatin of yeast is composed of DNA
complexed with proteins. The present paper describes the properties of a soluble
Biochim. Biophys. Acta, 195 !1969) 4 7 3 - 4 8 3
482
P. CH. VAN DER VLIET et al.
chromosomal DNA-protein complex (deoxyribonucleoprotein). The yeast deoxyribonueleoprotein resembles similar preparations from animal and plant cells in several
ways.
About 75 % of the associated proteins are acid-extractable basic proteins,
which show some resemblance to the histones of higher eukaryotes, although they
are definitely less basic 1,2.
The yeast deoxyribonucleoprotein can be dissociated by solutions of high ionic
strength. About 9° °/o of the histone fraction is dissociated by 0.8 M NaC1. This figure
indicates that the histones of yeast deoxyribonucleoprotein dissociate more easily
than the histone fractions of pea and mammalian nucleohistone. This may be correlated with a lower positive charge density of yeast histones as compared with mammalian histones.
As with the nucleohistones of higher eukaryotes, the associated proteins
stabilize the DNA in yeast deoxyribonucleoprotein against thermal denaturation.
Yeast deoxyribonucleoprotein shows a biphasie melting profile. About 5 ° 9/0 of the
DNA melts at 71° and about 50 % melts at 83 °, whereas the T m of pure DNA is 68 °.
The interpretation of these melting curves is difficult. Mitochondrial DNA cannot be
responsible for this phenomenon, as this melts at a temperature below the T m of
nuclear DNA and is only present in a minor quantity (5_+_2 %). Experiments in
which deoxyribonucleoprotein is extracted with NaC1 of increasing concentrations
show a remarkable correlation between the sedimentation coefficient and the percentage of DNA melting at 83 °. This suggests that the DNA melting at 83 ° represents
stretches of deoxyribonucleoprotein that are linked together by the associated protein,
thus forming a rather compact organization of the individual deoxyribonucleoprotein
molecules.
Like the histones of the higher eukaryotes, the proteins associated with DNA
in the yeast deoxyribonucleoprotein depress the template activity of the DNA for
RNA synthesis. The template activity of the yeast deoxyribonucleoprotein depends
on the enzyme/template concentration ratio but reaches a maximum of about 60 °/o
at high ratios. This could mean that a part of the DNA in yeast deoxyribonucleoprotein is not available for transcription.
Isolated nucleohistones of higher eukaryotes have in general a very low template
activity as compared to pure DNA, which suggests that histones are physiological
repressors of transcription. Some authors, however, reached the conclusion that a firm
aggregation or extensive crosslinkage in the isolated nucleohistone is partly responsible
for the low template activity 17,~°.
The studies of CHALKLEY AND JENSENlv on isolated calf-thymus chromatin
show clearly that when intermolecular crosslinks are destroyed the template activity
of the nucleohistone is approx. 50 % at low template concentrations. It is generally
accepted that in thymocytes most of the DNA is not available for transcription,
while this is not likely to be so in a non-differentiating unicellular eukaryote like yeast.
It is thus very surprising to find that the template activity of yeast deoxyribonucleoprotein is quantitatively very similar to that of comparable preparations of calf
thymus.
To decide to what extent the low template activity of yeast deoxyribonucleoprotein really means that a large part of the DNA in deoxyribonucleoprotein is not
available for transcription needs further studies. Such studies, including characterizaBiochirn. Biophys. Acta, 195 (1969) 473-483
DEOXYRIBONUCLEOPROTEIN COMPLEX FROM YEAST
483
t i o n of t h e R N A m a d e i n vitro o n y e a s t D N A a n d o n y e a s t d e o x y r i b o n u c l e o p r o t e i n
a n d c o m p a r i s o n oi t h e s e R N A ' s w i t h R N A m a d e i n vivo, e v i d e n t l y a r e n e c e s s a r y t o
gain more insight and are now in progress.
ACKNOWLEDGMENTS
The present investigations were supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organizat i o n for t h e A d v a n c e m e n t of P u r e R e s e a r c h ( Z . W . O . ) a n d b y a U n i t e d S t a t e s P u b l i c
Health Service Research Grant. We wish to thank the Koninklijke Nederlandse
G i s t - e n S p i r i t u s f a b r i e k N . V . , D e l f t , f o r s u p p l y i n g f r o z e n E . coli. T h e a u t h o r s a r e
g r a t e f u l t o P r o f e s s o r s E . P. H u l s t - S t e y n P a r v 6 a n d H . S. J a n s z for t h e i r c o n t i n u o u s
i n t e r e s t , a n d t o G. M. Z a n d v l i e t f o r s t i m u l a t i n g d i s c u s s i o n s .
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