BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY
427
NOTES
BBA 21 144
On the occurrence of histones in yeast
In recent studies on the role of histones (reviewed b y BUSCH1) attention is
focussed on the possibility that histones function as gene suppressors in the process
of tissue differentiation. Experimental evidence is accumulating, however, which indicates that histones, and the very lysine-rich histone in particular, also function in
the structural organization of chromatin and chromosomes (e.g. refs. I, 2).
In view of these supposed functions we m a y expect histones to be absent in
unicellular organisms, particularly in those that do not show condensed metaphase
chromosomes. Good evidence for the presence of histones in bacteria is lacking 3, while
a very lysine-rich type of histone is found in unicellular organisms in which condensed
chromosomes do occur, such as Chlorella 4 and Tetrahymena 5.
As yeast is an organism that does not form condensed chromosomes during
cell division 6, but, in contrast to bacteria, contains a well defined nucleus e, we thought
t h a t an investigation on the presence of histones in yeast could be very useful in
the study of the function of histones.
The present communication reports the extraction of proteins resembling histones from yeast chromatin.
From pressed baker's yeast ("Koningsgist", Delft) chromatin can be obtained
by the following procedure. 20 g of yeast, suspended in 35 ml 0.05 M phosphate buffer
(pH 6.5), containing I mM MgSO 4 (Medium A), is disintegrated by shaking with 45-ml
glass beads (diameter o.25 mm) for 5 min in the apparatus of MERKENSCHLAGER7.
The glass beads are allowed to settle and the supernatant is then decanted and centrifuged for 40 min at 8000 × g. A sediment is obtained which consists of 2 layers.
The brown loosely packed upper layer is resuspended by shaking with fresh Medium A
and next centrifuged for 20 min at 8000 × g. The resulting pellet is washed twice
with Medium A and once with Medium B (0.05 M Tris buffer (pH 8.0) containing
I mM MgS04). The washed pellet is resuspended in Medium B to a concentration of
0.5 mg DNA/ml and homogenized in a Potter-Elvehjem type homogenizer with a
very close-fitting Teflon pestle. Of the homogenate, IO ml is layered on a sucrose
gradient consisting of IO ml 2 M sucrose and IO ml 1. 5 M sucrose, both in Medium B,
and centrifuged for 1-2 h in a SW-25 swinging-bucket head at 25 ooo rev./min. The
purified chromatin is then obtained as a pellet. All manipulations are performed in
the cold (0-5°). The isolated chromatin contains DNA, RNA and protein in a ratio
of 1:o.3:4.3.
A weakly basic protein, further called histone, can be extracted from the chromatin with cold 0.25 M HC1. The amino acid composition of the extracted protein is
given in Table I (first column).
Fractionation of this histone was achieved by chromatography on CM-cellulose s.
Two fractions could be eluted with o.17 M acetic acid-o.o5 M N a O H - o . 4 2 M NaCI
Biochim. Biophys. Acta, 124 (1966) 427-429
428
PRELIMINARY NOTES
( p H 4.2) a n d w i t h o.o2 M HC1. E a c h of t h e s e f r a c t i o n s c o m p r i s e d a b o u t h a l f of t h e
p r o t e i n a p p l i e d t o t h e c o l u m n . I n c o n t r a s t t o t h y m u s h i s t o n e s 8, y e a s t h i s t o n e s d o
n o t c o n t a i n a f r a c t i o n t h a t c a n b e e l u t e d w i t h o . o i M HC1.
B o t h f r a c t i o n s a r e c o m p l e t e l y s o l u b l e in t h e r e a g e n t of MIRSKY AXl) POLL~STER 9,
a s a r e all h i s t o n e s . E l e c t r o p h o r e s i s of t h e i s o l a t e d f r a c t i o n s o n p o l y a c r y l a m i d e gel
a t p H 4.2 s h o w e d t h a t t h e y d i f f e r e d g r e a t l y in e l e c t r o p h o r e t i c b e h a v i o u r a n d t h a t
TABLE 1
AMINO
ACID
COMPOSITION
OF Y E A S T
HISTONES
Values are expressed as moles %. A refers to acidic amino acids; B to basic amino acids.
A m i n o acids
Whole
Fraction •
yeast kistone
Fraction 2
Trichloroacetic acidextractable
Asp I A
Glu t
Gly
Ala
Val
Leu
Ile
Phe
Tyr
Trp*
Ser
Thr
Pro
Met
Cys
Arg /
His i B
7.4
lO. 3
7.4
lO. i
5.7
8. 5
5 .8
2.()
2. 7
o. 5
8.4
5.0
4" I
0.6
o
7.4
6. 7
Io.8
7.5
9.3
6.o
0.o
5 .8
3.2
2.2
o. 4
7.4
6.,
3 .0
0. 5
o
9.2
ii.o
12.3
17. I
8.o
3.7
4.4
2.7
r.7
~.7
2.I
2.0
2.I
2.
I~NS
IO.S
7 .0
i.I
I 1.()
8.5
1.o
IO.4
7.9
L2
0.5
I '5
2. I
1. I
2.S
NHa**
t3/A
l.ys/Arg
7 .8
lo. 7
6.5
i i .3
5-3
8.2
5-7
2.1
2. 7
o. 4
9. l
o. I
4.3
0. 7
o
5.5
13.2
5.2
5.9
0.5
o
2.8
7.8
* Tryptophan determined according to BEAVEN AND HOLIDAY11.
** These vaIues were consistent with those for amide ammonia estimated directly.
b o t h w e r e still r a t h e r h e t e r o g e n e o u s . T h e a m i n o a c i d c o m p o s i t i o n of t i m 2 f r a c t i o n s
( T a b l e I) s h o w s t h a t a v e r y l y s i n e - r i c h t y p e of h i s t o n e is a b s e n t i n y e a s t . B o t h
f r a c t i o n s , h o w e v e r , s h o w s o m e r e s e m b l a n c e w i t h t h e o t h e r f r a c t i o n s of t h y m u s hist o n e s , a l t h o u g h t h e y a r e less b a s i c in t e r m s of t h e r a t i o of b a s i c t o a c i d i c a m i n o a c i d s
( B / A , T a b l e I). E l e c t r o p h o r e s i s a t p H 9 i n s t a r c h gel c o n t a i n i n g 4 M u r e a s h o w e d
that both fractions moved to the cathode. Since ammonia was found in the amino
a c i d a n a l y s i s ( T a b l e I), t h e b a s i c i t y i n t h e s e e l e c t r o p h o r e s i s e x p e r i m e n t s m u s t b e
a s c r i b e d m a i n l y t o t h e p r e s e n c e of a c i d i c a m i n o a c i d s i n t h e a m i d e f o r m . T o e x c l u d e
the possibility that Fraction I does contain a very lysine-rich histone contaminated
w i t h a n a c i d i c p r o t e i n , we e x t r a c t e d w h o l e y e a s t h i s t o n e w i t h 5 % t r i c h l o r o a c e t i c
a c i d 1°. O n l y 5 % c o u l d t h u s b e e x t r a c t e d a n d a m i n o a c i d a n a l y s i s ( T a b l e I) r e v e a l e d
t h a t t h e e x t r a c t e d p r o t e i n h a d n o r e s e m b l a n c e a t all t o t h e v e r y l y s i n e - r i c h h i s t o n e
f o u n d in o t h e r o r g a n i s m s .
T h a t t h e a c i d - e x t r a c t a b l e p r o t e i n s w e r e p r e s e n t in t h e c h r o m a t i n a s s o c i a t e d
Bioch im. Biophys. Acta, 124 (1966) 427-429
PRELIMINARY NOTES
429
with the DNA, could be shown in further experiments. We found that extraction of
the purified chromatin with water yielded an extract in which all of the material
behaved as a single nucleoprotein complex during sucrose-gradient centrifugation,
and this complex contained essentially all of the acid-extractable proteins found in
the chromatin.
From the foregoing experiments we can conclude that the acid-extractable
proteins in yeast chromatin are histones, as they are basic proteins associated with
the DNA. These proteins, however, although having some resemblance with the histones of higher organisms, differ from them in several respects. Particularly striking
is the absence of a very lysine-rich type of histone, which is found in all higher
organisms and which is probably the predominant type of histone in unicellular
organisms like Chlorella* and Tetrahymena 5. This would support the view advanced
above that the very lysine-rich type of histone appears to be present only in those
organisms that show condensed chromosomes. We are therefore convinced that the
very lysine-rich histone plays an essential role in the structural organisation of metaphase chromosomes.
The very interesting question whether the yeast histones with their different
composition are also able to inhibit the priming function of DNA in the RNA-polymerase reaction is now being investigated.
The authors wish to thank Dr. C. H. MONFOORT for the amino acid analysis.
This work was supported in part by U.S. Public Health Service Research Grant
TW-5o-o3 and by the Netherlands Foundation for Chemical Research (S.O.N.) with
financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
Laboratory for Physiological Chemistry,
The State University,
Utrecht (The Netherlands)
I
2
3
4
5
6
7
8
9
io
II
G. J. M. TONINO
TH. H. RozIJN
H. BUSCH, Histones and other nuclear Proteins, Academic Press, New York, 1965, p. 91.
V. C. LITTAU, C. J. BURDICK, V. G. ALLFREY AND A. E. MIRSK¥, J. Cell. Biol., 27 (1965) I24A.
J. S. ROTH, Nature, 207 (1965) 599.
K. IWAI, in J. BONNER AND V. TS'O, The Nucleohistones, Holden-Day, San Francisco, 1964,
P-59.
K. IWAI, H. SHIOMI, T. ANI~O AND T. MITA, J. Bioehem. Tokyo, 58 (1965) 312.
TH. H. RozljN AND G. J. M. TONINO, Biochim. Biophys. Aeta, 91 (1964) lO5.
M. MERKENSCHLAGER, Biochem. Z., 329 (1957) 332.
E. W. JOHNS, D. M. P. PHILLIPS, P. SIMSON AND J. A. V. BUTLER, Bioehem. J., 77 (196o) 631.
A. E. MIRSKY AND A. W. POLLISTER, J. Gen. Physiol., 30 (1946) 117.
E. H. DE NooY AND H. G. K. WESTENBRINK, Biochim. Biophys. Acta, 62 (1962) 608.
G. H. BEAVEN AND E. R. HOLIDAY, Advan. Protein Chem., 7 (1952) 369 •
Received April 25th, 1966
Biochim. Biophys. Acta, 124 (1966) 427-429