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IN TER A C TIO N OF D N A WIT H RIBOSOMES
321
Interaction of D N A with ribosomes in cell-free protein
synthetizing systems of Chlorella pyrenoidosa
G. G a l l in g
Pflanzenphysiologisches Institut der Universität Göttingen
(Z. Naturforschg. 24 b , 321— 327 [1969] ; ein g eg a n g en am 4. Septem ber 1968)
1. In cell-free protein synthetizing systems of the green alga Chlorella pyrenoidosa, DNA from
various sources enhances the amino acid incorporation.
2. This stimulation is neither inhibited by actinomycin D nor by chloramphenicol or cycloheximide (actidione).
3. In the presence of ribonuclease, some precipitable polypeptide is formed with DNA, although
the endogenous incorporation is completely inhibited by ribonuclease.
4. After sucrose density gradient centrifugation, polysomal aggregates of ribosomes with DNA
are found. Electron micrographs of such polysomes show a direct association of the DNA molecule
with several ribosomes.
5. In Chlorella, direct translation of DNA can be obtained also in the presence of neomycin.
The kinetics of this reaction are different from those of endogenous m-RNA mediated and of DNA
stimulated polypeptide synthesis.
Cell-free protein synthetizing systems are shown
to decode usually messenger RNA during the for­
mation of polypeptides. From the green alga C h lo­
rella, cell-free extracts were shown to incorporate
amino acids into polypeptides by an energy depen­
dent, GTP stimulated, ribonuclease sensitive reac­
tion *. This endogenous incorporation can be sti­
mulated by the addition of RNA from several cell
fractions 2> 3.
Recently, a direct interaction of DNA with ribo­
somes was described in cell-free systems obtained
from rat liver nuclei 4. This stimulation was inter­
preted as direct translation of DNA. It could not be
obtained with extracts from bacterial cells, in which
DNA exhibits an inhibitory effect5. With contrast to
this, bacterial cell-free systems are able to translate
directly DNA of various sources in the presence of
n eom ycin6-8. This effect of neomycin could not be
observed in cell-free systems of rat liver nuclei 5. In
C hlorella , DNA enhances the amino acid incorpora­
tion in vitro 9 while systems of the blue-green alga
A n a cystis nidulans are not stimulated by the addi­
tion of DNA 2.
The results of experiments reported here indi­
cate, that DNA of various sources enhances the
Z. Naturforschg. 2 1 b , 993 [1966],
Z. Naturforschg. 2 2 b , 687 [1967].
3 G . G a l l i n g , Planta 79, 44 [1968].
4 H. N a o r a , Biochim. biophysica Acta [Amsterdam] 123,
151 [1966].
5 H. N a o r a and K . K o d a i r a , Biochim. biophysica Acta
[Amsterdam] 1 2 3 ,4 2 5 [1966].
1 G. G a llin g ,
2 G . G a llin g ,
amino acid incorporation in cell-free systems from
Chlorella. This stimulation also takes place in the
presence of various antibiotics like actinomycin D
and chloramphenicol. Ribonuclease does not com ple­
tely inhibit the formation of polypeptides in the pre­
sence of DNA. Electron micrographs show that the
ribosomes form complexes with single DNA m ole­
cules in vitro. These complexes are also formed in
the presence of actinomycin D. Furthermore, ribo­
som es of Chlorella are also able to translate directly
the DNA in the presence of neomycin.
M aterials and Methods
Materials
Chlorella pyrenoidosa, strain 211/8 b was obtained
from the Algensammlung des Pflanzenphysiologischen
Instituts der Universität Göttingen. Salmon sperm
DNA, calf thymus DNA and neomycin sulphate were
obtained from Serva, Heidelberg. (2-14C) thymine came
from The Radiochemical Centre, Amersham. The
amino acid mixture, uniformly 14C-labelled, was from
New England Nuclear, Inc. Transfer RNA (yeast), the
nucleoside triphosphates, and chloramphenicol (paraxin) were obtained from Boehringer, Mannheim. Cycloheximide (actidione) came from The Upjohn Comp.,
Kalamazoo. Actinomycin D was a gift from Merck,
Sharp and Dohme, New York.
8 J. J. H o l l a n d and B. J. M c C a r t h y , Proc. nat. Acad. Sei.
USA 52, 880 [1965].
7 B . J. M c C a r t h y , J. J. H o l l a n d , and C . A. B u c k , Cold
Spring Harbor Sympos. quantitat. B i o l . vol. XXXI, 683
[1967].
8 A. R. M o r g a n , R. D. W e l l s , and H. G. K h o r a n a , J.
molecular Biol. 26, 477 [1967].
9 G . G a l l i n g , Z. Naturforschg. 22 b, 348 [1967].
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322
G. GALLING
Methods
Electron microscopy
Preparation of cell-free systems
Aliquots of the polysomal fractions were plated on
carbon coated formvar films. After drying, the pre­
parations were stained with uranyl acetate overnight,
washed several times in bidist. water, and dried. Elec­
tron microscopy was performed in a Philips EM 200
electron microscope equipped with a cooling device.
Chlorella pyrenoidosa was growrn in sterile, axenic
culture in inorganic salt medium 10 under continuous
illumination at 30 °C. The single steps of the prepara­
tion of ribosomes and supernatant enzymes have been
previously described *. After disruption of the cells
with glass beads, the homogenate was precentrifuged
30 min at 30 000 g. From this supernatant, the ribo­
somes were pelleted after 90 min at 105 000 g 0 . The
protein of the supernatant was precipitated with am­
monium sulphate at full saturation in the cold and
dialyzed overnight against buffer. The ribosome pellet
was resuspended and centrifuged 180 min at 105 000
g 0 . From this pellet, the upper layer consisting of
small chloroplast particles was discarded and the clear
opalescent ribosome pellet was resuspendet and cleared
by centrifugation in 20 min at 20 000 g 0 . All steps
were performed near 0 °C.
R e su lts
In cell-free amino acid incorporating systems of
consisting of ribosomes and enzymes,
D NA of various sources enhances the incorporation
of amino acids into peptides (table 1 ). The require­
ments of this reaction are the same as in endo­
genous mRNA mediated protein synthesis. Trans­
fer RNA, nucleoside triphosphates, and an energy
regenerating system as phosphoenolpyruvate and
Chlorella,
Preparation of labelled and unlabelled DNA from
Chlorella
Expt.
To obtain radioactive DNA from Chlorella, the al­
gae were grown 5 days in the presence of 0.2 /^C/ml
of (2-14C) thymine. After preparation of the nucleic
acids with the phenol method n , DNA was purified by
chromatography on MAK-columns. The DNA-containing fractions were collected, and the nucleic acids
precipitated with ethanol. After dialysis, the volume
was decreased by dialysis against a 30 percent dextrane solution in buffer 12. Unlabelled DNA from Chlo­
rella and also from the blue-green alga Anacystis nidulans were prepared and purified by direct isolation of
macromolecular DNA after deproteinization with isopropanol and chloroform 13.
1
2
Incubation and analysis of polysomal fractions
The incubation mixture of the cell-free system of
Chlorella has been previously described 1. For precipi­
tation and purification of the polypeptides formed in
vitro, the paper filter disc method was used 14. In some
experiments, aliquots of the samples were layered after
10 min of incubation on top of a 10 through 40 per­
cent linear sucrose gradient in tris buffer, supplied with
Mg20 and K®, and centrifuged 60 min in the SW 651
rotor of the Spinco ultracentrifuge writh 65 000 rev/min
at 0 °C. After puncturing the bottom of the tubes, the
optical density of the effluent at 260 m/t was recorded
automatically in a Zeiss PM Q II spectrophotometer
equipped with continuous flow quartz cell.
10 A. K u h l , Beitr. Physiol, u. Morphol. d. Algen, G. Fischer,
Stuttgart, 1962, p. 157.
11 G . G a l l i n g and G. R i c h t e r , Biochim. biophysica Acta
[Amsterdam] 123, 613 [1966].
3
conditions
counts/min.mg
ribosomes
basic system
1,530
t= 0
320
510
500
390
4,860
5.260
3.260
2,970
— transfer RNA
— enzymes
+ ribonuclease (5 /ug)
+ 100 fig thymus DNA
+ 100 fig salmon sperm DNA
+ 100 fig Anacystis DNA
+ 100 fig Chlorella DNA
basic system + 100 fig salmon
sperm DNA
t= 0
— ribosomes, — enzymes
— transfer RNA
— ATP, — phosphoenolpyruvate,
— kinase
+ ribonuclease (5 fig)
basic system
- CTP, - UTP
+ 100 fig salmon sperm DNA
+ 100 fig salmon sperm DNA,
- CTP, - UTP
4.030
625
50
1,225
1,285
1,550
1,860
1,740
3,040
2,980
Table 1. Stimulation of amino acid incorporation by DNA in
cell-free systems of Chlorella pyrenoidosa. The incubation
mixture contained in a total volume of 0.4 ml (in /mmoles) :
tris 10, Mg-Acetate 4. KC1 12, CaCl2 0.1, Z nS04 0.02, ATP
0.4, GTP 0.2, CTP 0.1, UTP 0.1, phosphoenolpyruvate 1,
pyruvate kinase 20 u g, transfer RNA (yeast) 0.2 mg, ribo­
somes 1 mg, supernatant protein 1 mg, and 0.5 /uC of a r e ­
labelled amino acid mixture, specific activity 40 mC/milliatom C. Incubation: 30 min at 38 °C, pH 7,6.
12 J. C. D r a c h and J. B. L i n g r e l , Biochim. biophysica Acta
[Amsterdam] 123, 345 [1966].
13 J. M a r m u r , J. molecular Biol. 3, 208 [1961].
14 R. M a n s and D. G. N o v e l l i , Arch. Biochemistry 94, 48
[1961].
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IN TER A C TIO N OF D N A WIT H RIBOSOMES
pyruvate-kinase are needed. The stimulatory effect
of DNA on the system can be shown also when CTP
and UTP are ommited. In the presence of DNA, the
incorporation is not completely inhibited by the ad­
dition of ribonuclease. The same concentration of
ribonuclease completely inhibits the endogenous
mRNA mediated reaction.
To obtain more information on the role of DNA
in polypeptide formation in vitro, two concentrations
of actinomycin D were added to cell-free systems
(table 2 ). With 5 fig actinomycin, the endogenous
incorporation is not affected. In the presence of
DNA and the antibiotic, the same stimulation as in
untreated controls is observed. U sing 10 fig of acti­
nomycin D, the endogenous incorporation is strongly
affected, while in mixtures with DNA, the incorpora­
tion nearly reaches the value of the untreated con­
trol.
Other antibiotics were added to the D NA stimu­
lated reaction mixture to study the type of ribo­
somes involved in this reaction. Chloramphenicol
only slightly inhibits the endogenous mRNA mediated
amino acid incorporation in the cell-free system of
Chlorella. In the presence of DNA and chloramphe­
nicol, only a little more polypeptide is formed. The
stimulation is strongly reduced in the presence of
the antibiotic (table 2 ) . The other antibiotic used,
actidione, strongly affects the endogenous incorpora­
tion. Only thirty percent of the control without acti­
dione is reached. If DNA and actidione are present
in the reaction mixture, the incorporation comes up
E xpt.
conditions
to 77 percent as compared with the control in the
presence of DNA. Here, the DNA stimulated incor­
poration is affected comparatively little by actidione.
Ribosomes of liver and thymus glands have been
shown to form electrophoretically stable complexes
with DNA in vitro lo. This complex is stable during
centrifugation. Ribosomes of Chlorella were incu­
bated with labelled DNA of the same organism in
the presence of all cofactors required for cell-free
protein synthesis except labelled amino acids. After
incubation, the mixture was centrifuged under con­
ditions known to be sufficient only for sedimentation
of ribosomes but not of DNA. As shown in table 3,
an average of 4 0 percent of the labelled D NA can be
found in the fraction of the ribosomes.
ribosome fraction
supernatant fraction
total
counts/m in. ml
percent o f total
4,765
6,960
11,725
40,5
59.5
100,0
Table 3. Association of labelled DNA from Chlorella with
ribosomes after incubation with cofactors and centrifugationIncubation mixture as in table 1. Instead of 14C-labelled
amino acids, the mixture contained 4,4 OD260 of (2 —14C)
thymine —labelle d DNA from Chlorella, specific activity:
3000 counts min. OD26o • Öfter incubation, the ribosomes
were pelleted by centrifugation in 60 min at 105 000 g, 0 °C,
and aliquots of the suspended ribosome fraction and of the
supernatant were counted in liquid scintillation fluid.
counts/m in.m g
ribosomes
1
basic system
+ 5 f i g actinom ycin D
+ 100 f i g D N A
+ 100 fi g D N A + 5 fig
actinom ycin D
3.710
3,700
6.920
2
basic system
-j- 10 jug actinom ycin D
-j- 100 f i g D N A
+ 100 fi g D N A + 10 fig
actinom ycin D
3,875
710
5,000
3
basic
+ 10
+ 10
basic system +
+ 10
-j- 10
3,875
2,755
1,225
5,000
3,115
3,885
system
jug chloramphenicol
jug actidione
100 f i g D N A
jug chloramphenicol
f i g actidione
323
7,010
2,525
Table 2. Effect of various antibiotics on the stimulation of
amino acid incorporation by DNA in cell-free systems of
Chlorella. Experimental conditions: see table 1. DNA = sal­
mon sperm DNA.
bottom
dr° P n r
*■
top
Fig. 1. Sucrose density gradient pattern of freshly prepared
Chlorella ribosomes. The ribosomes were layered directly
after preparation on top of a 10 to 40 percent linear sucrose
gradient and centrifuged 60 min at 65 000 rev/min, 0 °C in
the SW 65 1 rotor of the spinco ultracentrifuge. The tubes
were punctured and the optical density of the effluent was
measured and recorded automatically. The arrow indicates
the monomer position (80 s ) .
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324
G. GALLING
To study the role of DNA — ribosome complexes
formed in vitro during the incubation with cofactors,
required for polypeptide synthesis, sucrose density
gradient centrifugation and electron microscopy
were performed. Ribosomes prepared from Chlorella
were centrifuged in density gradients before and
after incubation with DNA. Fig. 1 shows the den­
sity gradient pattern of unincubated ribosomes di­
rectly after the preparation from freshly harvested
cells. Most of the ribosomes from a single peak con­
sisting of monomers (fraction 80 to 8 8 ). Ahead of
this peak, several smaller peaks of polysome frac­
tions are seen. Part of the ribosomes are disinte­
grated forming two peaks of subunits, visible in frac­
tions 92 and 102. Possibly they come from chloroplasmic ribosom es wich easily disintegrate as was
recently shown for C h la m y d o m o n a s 16. In fig. 2 the
density gradient pattern of DNA alone and of ribo­
somes after incubation with DNA and all cofactors
are shown. The DNA used in these experiments is
unable to migrate through the sucrose gradient of
10 to 40 percent. The DNA therefore only can be
found on top of the gradient. The ribosom es, centri­
fuged after incubation with all cofactors required
for cell-free protein synthesis and with DNA, still
show some clear polysom e peaks as seen in fractions
52, 62, and 76. From such a gradient centrifugation,
samples of the different peaks were taken for elec­
tron m icroscopie studies. The arrow in fig. 2 indi­
cates the fraction from which the electron micro­
graph, shown in fig. 3 * was taken. In this figure, the
direct association of a DNA molecule with several
minutes — ►
Fig. 2.
Sucrose density gradient pattern of ribosomes of
Chlorella after incubation with DNA and cofactors, and of
DNA alone. The incubation mixture as in table 1 was layered
after 10 min of incubation on top of a 10 to 40 percent linear
sucrose gradient, and centrifuged and measured as in fig. 1.
One gradient tube contained only 100 jug of salmon sperm
DNA, dissolved in bidist. H20 . Solid line: gradient pattern
of the incubation mixture. The arrow indicates the fraction
from which the electron micrograph, fig. 3., is taken. Broken
line: salmon sperm DNA.
15 H. N a o r a and H. N a o r a , Biochim. biophysica Acta [Am­
sterdam] 134, 277 [1967].
Fig. 4. Kinetics of the amino acid incorporation by cell-free
systems of Chlorella. Two times the reaction mixture (see
table 1) was incubated a) without further additions O—O,
b) with 100 /ag salmon sperm DNA • — # . c) with 1 0 0 /<g
salmon sperm DNA and 50 /ug neomycin A - A- Aliquots
were taken to the given time and peptides were precipitated
and purified 14.
ribosomes is shown. On an unbranched fiber, which
is an average of 24 Ä in thickness, four ribosomes
are located directly. The ribosomes are 180 to 200 Ä
in diameter. Many pictures of such associations have
been obtained in different preparations of ribosomes
16 R. S a g e r and M. G. H a m i l t o n , Science [Washington]
157, 709 [1967].
* Fig. 3 s. Tafel S. 326 a.
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IN TERA CTION OF D N A WITH RIBOSOMES
after the incubation with DNA. After density gra­
dient centrifugation of unincubated ribosomes or
from incubation mictures without DNA, clusters of
ribosomes have been found in the position of poly­
somes. These clusters are very sim ilar to polysomes
seen in electron m icrographs from other organisms.
We have never seen interaction of ribosomes with
DNA fibers in fresh preparations of cell-free systems
incubated without D N A. The ribosomal fraction of
Chlorella itself contains no D N A. Also in the enzyme
preparation from the ribosome-free supernatant,
only trace amounts of DNA are found.
In bacterial cell-free extracts, a direct translation
of D NA by ribosomes resulting polypeptides has
been reported in the presence of neomycin and other
streptomycines. This was also tested with ribosomes
and supernatant enzymes of Chlorella. Table 4 shows,
that neom ycin stimulates the rate of polypeptide syn­
thesis in the presence of D NA to a high extent. The
325
reaction rate is linear for at last 40 min and only
decreases slightly during the 60 min incubation ex­
periment.
Discussion
endogenous mRNA mediated amino acid incorpora­
tion is not affected by neom ycin alone, while in the
presence of DNA and neom ycin up to 28-fold in­
crease is reached. The table shows also that the reac­
tion needs both, ribosomes and enzymes.
Attempts to obtain electron micrographs of the
direct interaction of DNA with ribosomes in the
presence of neomycin have not been successfull.
In fig. 4, the kinetics of the amino acid incorpo­
ration under the three conditions described above
are shown. The endogenous m RNA mediated incor­
poration levels off after little more than ten minutes.
In the presence of D N A, the reaction proceeds at a
linear rate for 10 min, then levels off slowly during
another 30 minutes. With neom ycin and DNA, the
The concept of protein synthesis of the living cell
has been formed and proven after experiments on
different types of cells, organs, and organelles. It
includes the transcription of the genetic message
from the DNA to a messenger RNA and the trans­
lation of the mRNA to polypeptides by the protein
synthetizing apparatus consisting of ribosomes,
amino acyl transfer RNA, and different enzymes.
The process of transcription and translation may
occur in vitro in a complex formed of D N A, newly
synthetized mRNA, ribosom e clusters, and enzymes
as found in cell-free extracts from Escherichia
coli 17>18.
However, recently another type of expression of
the genetic message has been proposed for some cellfree systems of higher organism s. In extracts from
liver nuclei, DNA effects a strong stimulation of the
amino acid incorporation in vitro 4’ 5. A lso in cellfree systems of Chlorella, such a stimulation by DNA
extracted from various organisms was sh ow n 9,
while in extracts from the blue-green alga A n a cystis
nidulans, which does not possess a true nucleus, no
stimulation was observed 2. An involvement of newly
synthetized mRNA in the stimulation by D NA in the
Chlorella system may be excluded on the basis of the
following arguments. The direct stimulation of DNA
is found also in the presence of actinomycin D. This
antibiotic is well known to inhibit the formation of
mRNA by the DNA dependent RNA polymerase re­
action. Furthermore, ribonuclease of the same con­
centration which inhibits the formation of poly­
peptides by endogenous mRNA, does not inhibit
completely the DNA stimulated amino acid incor­
poration. An interaction of preformed mRNA and
part of the DNA in the incubation mixture cannot
be completely excluded. However, a complex for­
mation of Chlorella m RNA with DNA from various
other organisms would be an unexpected result.
Studies with antibiotics other than actinomycin
gave no clear answer to the question as to wich type
of ribosomes is involved in the D NA stimulated
system. In the presence of DNA, more polypeptide
17 R.
18 H. A.
E xpt.
conditions
basic system
1
t = 0
+
2
+
+
-f-
+ 100 f i g D N A
+ 50 n g neom ycin
100 fi g D N A + 50 f i g neom ycin
basic system
+ 50 fi g neom ycin
100 fi g D N A + 50 f i g neom ycin
D N A , + neom ycin, — ribosomes
D N A , + neom ycin, — enzym es
counts/m in.m g
ribosomes
790
140
1,380
750
19,520
2,240
2,000
13,500
2,550
170
Table 4. Effect of neomycin on the amino incorporation in
cell-free systems of Chlorella. Experimental conditions: see
table 1. DNA = salmon sperm DNA.
J. G. L e v i n , H. A. B l a d e n and M. W.
Proc. nat. Acad. Sei. USA 52, 140 [1964],
B yrne,
berg
,
N ir e n -
R. B y r n e , J. G. L e v t n and M. W.
J. molecular Biol. 11, 78 [1965].
B laden,
berg,
N ir e n -
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326
G. GALLING
is formed with ribosom es treated with chlorampheni­
col and also with actidione, even though the effect of
chloramphenicol is clearly weaker. Chloramphenicol
in the concentration used here is known to inhibit
the cell-free protein synthesis only in bacterial and
chloroplasmic ribosom e systems 19. In Chlorella, the
endogenous mRNA mediated incorporation is only
slightly affected by chloramphenicol. In the presence
of chloramphenicol and DNA, the incorporation is a
little higher than without DNA, but the initial rate
of the control is not reached. In contrast to this,
actidione is strongly affecting the endogenous incor­
poration in the Chlorella system. Actidione is an in­
hibitor of protein synthesis in cell-free systems of
higher organisms. In plant cells, cytoplasmic ribo­
somes are strongly affected by actidione. With con­
trast to the endogenous incorporation, the stim ula­
tion of the system by DNA also takes place in the
presence of actidione, although the rate of the con­
trol with DNA is not reached. Higher concentrations
of both antibiotics, than used here, did not lower
more the amino acid incorporation in vitro.
With the electron microscope, a direct association
of D N A with ribosom es was observed after incuba­
tion of the protein synthetizing system with DNA.
This association is interpreted as direct interaction
of DNA molecules with ribosomes. The DNA m ole­
cules used here are not able to m igrate alone through
the sucrose gradient used. Therefore, they cannot be
together with ribosom es by chance in the polysome
region of the gradient from which the electron m icro­
scopic preparations were taken. Specific interaction
must have taken place. Ribosomes of thymus glands
and of liver have been shown to possess specific
binding sites for D N A , leading to electrophoretically
stable complexes after incubation 15. To this time it
is not possible to decide if the complexes shown here
are due to the involvement of the D N A in polypeptide
formation or to a binding to other than the messen­
ger site of the ribosome.
Many electron micrographs of the kind shown
have been obtained with several preparations of cellfree systems after incubation with DNA. The elec­
tron micrograph given here looks somewhat similar
to pictures of aggregations of D N A , mRNA, poly­
some clusters, and enzymes, shown in cell-free incu­
bation m ixtures from Escherichia coli 17’ 18. B ut there
are some striking differences. In the cell-free ex­
tracts of E. coli, several clusters of ribosom es are
aggregated to a DNA fiber which is branched by
molecules of newly synthetized m R N A . Such a
branching of the DNA fiber was never seen in p re ­
parations from the Chlorella system . F u rth e rm o re ,
in the latter system, only single ribosom es are a tta ­
ched to the DNA m olecule. T his com plex of DNA
and ribosom es is form ed also in the presence of ac ti­
nom ycin D, under conditions which should suppress
the form ation of mRNA by the polym erase reaction.
The DNA fiber show n in the electron m icro g rap h
does not exceed 24 Ä in thickness. T his value r e ­
presents the thickness of a double stran d ed DNA
molecule after uranyl acetate staining. The fiber
therefore cannot be an ag g reg atio n by chance of
several DNA molecules. Recently, a direct in te r­
action of double stranded RN A m olecules w ith rib o ­
somes form ing ribonuclease stable clusters of p o ly­
somes w ith 5 and 11 to 14 ribosom es has been re ­
ported after phage infection of b acterial cells 20, 21.
However, any mechanism of tran slatio n of double
stranded molecules of nucleic acids by ribosom es is
still unknow n.
The direct interaction of DNA w ith ribosom es
in the presence of neom ycin has yet been described
only in bacterial cell-free system s 6 > 8 : 22. In m ost
of the cases it is in terp reted as a direct tran slatio n
of the genetic m essage of the D N A, b ut som etim es,
neom ycin leads perhaps to unspecific m isread in g of
artificial m essenger polynucleotides, w hen DNA is
present in the reaction m ix tu re 22. O ur results in d i­
cate, th at ribosom es of Chlorella are also able to
translate DNA in the presence of neom ycin. It is p o s­
sible that only the b acterial type of ribosom es is
capable to this reaction, w hile cytoplasm ic ribosom es
of higher organism s are not. Chlorella seems to p o s­
ses two types of ribosom es, one of them com ing from
the chloroplast, the other fro m the cytoplasm , as des­
cribed for other algae and h ig h er p la n ts 16. T he
nuclear ribosom e system, obtain ed from ra t liver,
which is stim ulated directly by DNA, shows not a
fu rth er stim ulation by the ad d itio n of n e o m y c in 5.
T his system contains only ribosom es of the cytoplasm atic type.
19 J. M. E i s e n s t a d t a n d G. B r a w e r m a n , J. m o le c u la r
10. 392 [1964].
20 B. H o t h a m -I g l e w s k i and R. M. F r a n k l i n . P r o c .
Acad. Sei. USA 58. 743 [1967].
21 G .
B io l.
nat.
N. G o d s o n and R.
23. 495 [1967].
22 T. E.
L ik o v e r
and C.
L . S in s h e im e r ,
G. K u r l a n d ,
J. molecular Biol.
Proc. nat. Acad. Sei.
USA 58, 2385 [1967].
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G . G a l l i n g , I n te rac ti on of D N A
w it h r i b o s o m e s in c e l l-fr e e - p r o te in s y n t h e t i z i n g s y s t e m s of C h lo r e l la p y r e n o i d o s a (S . 3 2 1 )
Fig. 3. Electron micrograph of polysomes formed from ribo­
somes incubated with DNA and purified by sucrose density
gradient centrifugation. For preparation details see Materials
and Methodes. Magnification = 304 000 times. Electron
micrograph : Prof. Dr. F. A m e l u n x e n .
Zeitschrift für Xaturforschung 24 b. S e ite 326 a.
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C hr.
F.
B a rd e le .
U ltras truktur <lcr „Körnchen“ auf den A xo po dien von Raph id io p h rys (Cen trohelida, Hetiozoa) (S. 362 )
fr
Abb. 2. Raph idiophry s ambigua. Abkürzungen s. Abb. 1.
Die Pfeile weisen auf die kontrastarme Aussparung im Yorderstück des Organells, die Pfeilspitzen auf die helle Linie
unterhalb der Oberfläche desselben. Abbildungsmaßstab
2a 50000 : 1. 2b 75000 : 1, 2c 60000 : 1.
Zeitschrift für Xaturforschung 21 b. Seite 326 b.
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IN TERA CTIO N OF D N A WITH RIBOSOMES
The kinetics of the amino acid incorporation
with neomycin and DNA in the cell-free system of
Chlorella are similar to those of polypeptide syn­
thesis with artificial messenger polynucleotides like
poly (U) in bacterial systems. In this respect, the
reaction is completely different from the DNA sti­
mulated amino acid incorporation without neomycin
and from the endogenous mRNA mediated poly­
peptide synthesis.
The direct stimulation of polypeptide synthesis by
DNA may be discussed with regard to its possible
biological role in vivo. From the green alga C h lo ­
rella ellipsoidea and also from a number of higher
organisms, a rapidly labelled fraction of D N A has
been described which differs from the bulk D NA in
base composition and sedimentation behaviour 23-25.
23 T. I w a m u r a and S . K u w a s h i t a , Biochim. biophysica
[Amsterdam] 80, 678 [1964].
24 M. S a m p s o n , A. K a t o h , Y. H o t t a and H . S t e r n , Proc.
nat. Acad. S e i . USA 50, 459 [1963].
327
On behalf of the relatively large amount of DNA
needed for stimulation of the amino acid incorpora­
tion in vitro, it is possible, that only part of the
DNA is involved in the stimulatory effect. The direct
stimulation of protein synthesis in vitro by DNA has
been yet found only in systems from higher orga­
nisms. Therefore, the localization of the reaction in
the nucleus is believed. However, any quantitative
relation between mRNA mediated protein synthesis
and direct involvement of DNA as messenger cannot
yet be given.
I
wish to thank Professor Dr. F. A m e l u n x e n for
performing electron microscopy, and Dr. J. G r a e b e for
helpfull discussions. This work was supported by the
Deutsche Forschungsgemeinschaft.
25 V. H e m l e b e n - V i e l h a b e n ,
[1966],
Z.
Naturforschg.
21 b,
983
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