Eur. J. Biochem. 29, 444-454 (1972)

An Analysis of DNA-Containing Particles Recovered from the Cytoplasm of Differentiating Chick-Muscle Cells

Eugene BELL, Charlotte MERRILL, and Charles B. LAWRENCE

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts

(Received March 23,1972)

Labeled DNA-containing particles from the cytoplasm of embryonic chick muscle can be dissociated in 0.5M KC1 to a basic particle of buoyant density 1.57 g/ml in CsC1. DNA- containing particles of density 1.43 g/ml are found in a large distribution of distinct sizes whose sedimentation values in sucrose range from 16s to greater than 100s. DNA-containing particles in a range of densities centering about a peak of 1.52 g/ml are found cosedimenting with polyribosomes in sucrose. The latter particles contain at least two subunits which are dissoci- able by treatment with EDTA; the DNA-containing subparticle has a density of 1.43 g/ml.

Newly made RNA is found in association with DNA-containing particles banding a t 1.43 g/ml which are about 40 S and larger but not with particles of density 1.57 g/ml.

Virtually all of the rapidly labeling cytoplasmic RNA taken from the polyribosomal region of a sucrose gradient is associated with particles of densities 1.43 g/ml and 1.55 g/ml; a minor portion may be associated with particles of density 1.52 g/ml which are found cosedimenting uniquely with polyribosomes.

Practically all nascent chain protein is associated with particles of density 1.55 glml, which is the density of ribosomes or polyribosomes.

Addition of labeled DNA to cytoplasmic extracts does not yield any of the foregoing particles, and no binding of label to polyribosomes or cosedimenting structures is observed. DNA- containing particles from the nucleus band at densities different from particles recovered from the cytoplasm.

We propose that the particles recovered from the cytoplasm are naturally occurring and not artifacts of fractionation and processing, and we present a model for their utilization in the transfer and use of genetic information.

Eyidence for the existence of a DNA entity (I-DNA) which is described hypothetically as a gene copy that passes from the nucleus to the cyto- plasm has been presented recently [l-31. Control experiments which deal with the possibility that I-DNA and I-somes may be artifacts of tissue fractionation or mitochondria1 DNA have been per- formed [2-51 and we continue to be alert to all interpretations of the significance of I-DNA.

I-DNA has been distinguished from nuclear DNA with inhibitors of DNA synthesis which depress nuclear and I-DNA synthesis to different degrees [3]. Dactinomycin has also been used to differentiate between nuclear and I-DNA synthesis [6]. At low concentrations it increases three-fold the amount of labeled I-DNA recoverable from cytoplasmic extracts without affecting the level of synthesis of nuclear DNA.

Abbreviation. Triton X-100, p-isocetylpolyoxyethylene- phenol polymer.

Membrane-associated non-mitochondria1 DNA which has been shown rigorously to belong in the cytoplasm has been isolated from the cytoplasm of cultured plasma cells by another group [7].

In the present paper we again show that I-DNA is not nuclear DNA randomly adsorbed to other cell structures or cell proteins since we can recover I-DNA- containing particulates of specific and unvarying buoyant density after glutaraldehyde-fixation of fractions isolated in a relatively high salt buffer which prevents the formation of unnatural complexes [S] between nucleic acids and proteins.

MATERIALS AND METHODS For the experiments, breast or thigh muscle

tissue from chick embryos of 11 - 12 days’ incubation was used. Details of the procedures for labeling tissue with radioactive compounds, dissociating the tissue into cells by means of detergent, and preparing

Vo1.29, N0.3,1972 E. BELL, C. MERRILL, and C. B. LAWRENCE 445

subcellular fractions without homogenization have been described previously [3,9]. Cell lysates were prepared in buffer M (0.25M KCl; 0.01 M Tris, pH 7.4; and 0.01 M MgC1,) [lo] and all "cytoplasmic extracts" were supernatants of a 10-min centrifuga- tion a t 12000 xg.

Standard methods were used for sedimentation in sucrose, and sucrose gradients were collected with a Gilford continuous recording flow-through spectrophotometer. All sucrose gradients were made up with buffer M. Fixation in glutaraldehyde and isopycnic banding of particulates in CsCl were done according to the procedure of Baltimore and Huang [lo] except that gradients were spun for somewhat longer periods (10-12 h) and a t higher centrifugal forces (220000~9 in the middle of the gradient). All CsCl gradients were made up in buffer M containing 0.8 O/, Brij-58 (polyoxyethylene cetyl- ether). Refractive indices were measured a t 25 "C with a Bausch and Lomb refractometer. Acid- insoluble material was precipitated by the addition of an equal volume of loo/, trichloroacetic acid plus approximately 50pg of bovine serum albumen to each fraction. The precipitates were collected on Millipore filters, type HAWG, and washed with 5O/, trichloroacetic acid. The filters were dried, placed in vials with a toluene-Omnifluor (New England Nuclear) scintillation fluid, and radioactivity was measured by a Packard scintillation spectrometer.

Purified embryonic chick nuclear DNA was prepared by a modification of the procedure develop- ed by Marmur [4,12].

Low speed centrifugations were carried out in a Sorvall SS34 rotor. Ultracentrifugation was per- formed using a Spinco SW 41 rotor.

[3H]Thymidine labeled in the methyl group and having a specific activity of 19Ci/mmol was ob- tained from New England Nuclear Corporation. [5-3H]Uridine having a specific activity of about 20 Ci/mmol and 14C-labeled reconstituted protein hydrolysate ( [14C]-labeled hydrolysate), having a specific activity of approximately 300 mCi/mmol, were purchased from SchwarzIMann. Dactinomycin was a gift of Merck, Sharp and Dohme, and 5-fluoro- deoxyuridine was kindly provided by Hoffman LaRoche Company.

RESULTS Density Gradient and Isopycnic Analysis

of I -DNA- Containing Particles which Sediment more Slowly than 35 S

Tissue was labeled for 4 h with [SHIthymidine, wahed, and incubated for 2 additional hours in Tyrode's solution a t 37.5 "C. The tissue was dissociat- ed, the cells were lysed, and the cytoplasmic frac- tion was layered onto a 15-300/, sucrose gradient. Sharp peaks of radioactivity sedimenting at 16, 22,

1 16s I 0.75

32s 27s 22s 1400 E

1200 > 1000 s m 0.50

s z1

m 800 ,z .p, .I-

n 600 .$

*

s N c .,d - rz

._ 0

U Q: 0.25

400

200

0 0

0 5 10 15 20 25 30 35 40 45 Fraction number

Fig. I . Sucrose gradient fractionation of DNA-containing particles lighter than 40 S. Tissue was labeled for 4 h with 75 pCi/ml [SH]thymidine, washed, and incubated 2 additional hours in Tyrodes solution at 37.5 "C. Cells obtained by dissociation of tissue with trypsin were filtered through gauze and then flannel and ruptured with 0.5O/, Triton X-100; the entire extract was spun for I0 min at 1200Oxg in a Sorvall centrifuge. The supernatant was layered onto a 15-30°/, gradient of sucrose made up in buffer M and centrifuged at 40000 rev./min in an SW 41 rotor for 18 h at 3 "C. Eight-drop fractions were collected. Acid-preci- pitable radioactivity contained in the fifth drop of each fraction was determined and is plotted; the remaining 7 drops of each fraction remained available for further processing. Fractions indicated by bars were fixed in glutar-

aldehyde for banding in CsCl

27 and 32 S (Fig. 1) were resolved when the extract was centrifuged for 18 h at 3 "C.

Material taken from the zones at 16, 22, 27 and 32 S was fixed in glutaraldehyde, layered onto pre- formed CsCl gradients, and banded. Each of the samples contained two types of labeled particles, one having a buoyant density of 1.57 g/ml and the other 1.43 g/ml. The examples shown in Fig.2 are from a single experiment while the values cited above represent averages of a t least six experiments.

Labeling of Particles Taken from the 40-50 S, and from the Polysomal Regions

of Sucrose Gradients Spun for 4.75 Hours Materials having a sedimentation value of about

4 0 8 also had two density peaks, one at about 1.43 g/ml and the other at about 1.57 g/ml, while that with a value of 50 S had essentially one peak a t 1.43 glml with only a suggestion of a high density peak (Fig.3A and C). Thus, the DNA-containing particles sedimenting at 40- 50 S are predominately

446 DNA Particles in the Cytoplasm Eur. J. Biochem.

Fraction number

- e .- E . In .. e 3 0 0 - 1200 4- .- > ._ 2 800 0 -0 ._

400

C

I 1.585

1.575 I

Fraction number

D

d.580

Fraction number Fraction number

I

Fraction number

Fig. 2. Density-gradient analysis of DNA-containing particles lighter than 40 S. The fractions indicated by bars in Fig. 1 were fixed in glutaraldehyde, layered onto preformed CsCl gradients and spun for 12 h a t 38000 rev./min in an SW 41 Spinco Rotor a t 5 "C. The acid-insoluble activity contained

in fractions collected from these gradients is plotted. One aliquot of 16-S material from a gradient was made 0.05 M with EDTA and allowed to remain for 0.5 h in the cold before fixation. (A) 16 S; (B) 22 S; (C) 27 S; (D) 32 S; (E) (6 S

treated with EDTA

Vol.29, N0.3, 1972 E. BELL, C. MERRILL, and C. B. LAWRENCE 447

2 2000 .- E

2 1600 . 3 0

u 2. 1200 c .- w .- L 2 800

' 400

0 D ._

0

1.7 h - E

1.6

1.5 f

1.4

ol

5.

u

c s

500

w

.; 200

f2 100 0 D

0

1.5m 4

1.435

I 1 I 1

. E I 11.7 0 - E

1.6 -- ol

x -

1.5 .: C

6 1,4

Fraction number Fraction number

Fig.3. Density-gradient analysis of DNA-containing particles sedirnenting in sucrose at 4 0 8 and 5 0 8 . A cytoplasmic extract like the one described in the legend to Fig. 1 was spun into a 15-30°/, sucrose layered onto a 2ml 60°/, sucrose cushion. After 4.75 h of centrifugation at 40000 rev./ min at 3 "C, cuts at 40 S and 50 S were taken and divided

into 2 aliquots each. One aliquot of each cut was made 0.05M with EDTA; after 30min these and the untreated aliquots were fixed in glutaraldehyde, layered onto preform- ed CsCl gradients, and spun for 12 h at 38000rev./min. Fractions were collected and acid-insoluble radioactivity was determined. (A) 40-S cut; (B) 40-S cut treated with

EDTA; (C) 50-5 cut; (D) 50-5 cut treatedwith EDTA

of low density in contrast with those a t 16 S which are mainly of high density.

The density distribution of radioactivity in the material cosedimenting with polyribosomes showed two peaks, one at 1.515 g/ml and one a t 1.43 g/ml (Fig.4A). Material of density 1.43 gfml banded sharply while the peak a t 1.515 lay in a broad band. Such a broad band could possibly be composed of polyribosomes and DNA with protein in varying molar ratios. A shoulder of the absorbance, which peaked a t 1.545, coincided with the 1.515g/ml peak of radioactivitv.

labeled cells had been sedimented. They were treated with 0.05 M EDTA, fixed and then banded in CsC1.

Under these conditions of fixation the particle of lower density (1.43 g/ml) from all sucrose frac- tions tested became slightly greater in density, banding a t about 1.445 g/ml. When either the 16-5 or the 40-5 particles were fixed in the presence of EDTA, the density of the heavier peak, 1.57 g/ml was unaltered (Fig.2E and 3B). I n addition, the radioactivity taken from the polyribosome fractions which had banded a t 1.515g/ml when fixed in the absence of EDTA was altered to band a t 1.58 to

in

1.60 g/ml when fixed after treatment with 0.05 M EDTA (Fig.4B).

I n order to determine whether the density shifts observed when fixation was performed in the pres- ence of EDTA should be attributed to chelation of divalent cations or merely to the increase in ionic strength caused by the addition of EDTA, experi- ments were performed in which particles were fixed

Effect of Ionic Strength and of Bivalent-Cation Deficiency on the Buoyant Density of

DNA- Containing Particles Taken from Sucrose Gradients

Fractions were selected from sucrose gradients which cytoplasmic extracts of [SHIthymidine-

448 DNA Particles in the Cytoplasm Eur. J. Biochem.

Fraction number

Fig.4. Density-gradient analysis of DNA-containing particles cosedimenting with polyribosomes. Polyribosomes from cells labeled with [aH]thymidine as described in legend to Fig. 1 were collected at the interface of a 600/, sucrose cushion and a 15-30°/, sucrose gradient. Four 10-drop fractions

were pooled and divided into 2 aliquots. One was made 0.05 M with EDTA and after 30 min at 4 "C both were fixed in glutaraldehyde and spun into a preformed CsCl gradient for 12 h at 5 "C. Fractions were collected and acid- insoluble radioactivity waa determined. ., Absorbance;

0, radioactivity; (B) EDTA-treated; (A) control

in the absence of EDTA but in the presence of varying concentrations of KC1. The typical pattern obtained when 40-5 particles were fixed in a buffer of ionic strength 0.20-0.24 (due primarily to KC1) is shown in Fig.5A in which the high and low density particles (1.57 and 1.43 g/ml) already de- scribed (Fig. 3) are again resolved ; the low density particle predominates. With an ionic strength of 0.29 (Fig.5B) the concentration of the high and low density particles is about equal. Further raising the ionic strength to 0.54 (Fig.5C) results in the disap- pearance of the low density particle. This suggests that the 1.43 g/ml particle is a complex of a protein moiety (density 1.3 g/ml) with a DNA-containing moiety of density 1.57 g/ml.

If the ionic strength is raised not with KC1 but with 0.05M EDTA to 0.55 (Fig.BD), the effect is similar to that due to KC1 of ionic strength 0.29. The DNA of the 1.43 g/ml particle appears tightly bound to protein and is released only when the ionic strength is made higher than that in the intact cell.

Polyribosome-containing fractions were made 0.24 or 0.49 ionic strength with KC1 or 0.20 ionic strength with 0.01 M EDTA to try to distinguish

between the effect of ionic strength and divalent cation deprivation on DNA radioactivity associated with the 1.515 g/ml and the 1.43 g/ml particles found cosedimenting with polyribosomes. With 0.24 ionic strength buffer the 1.515 and 1.43 g/ml particles remain intact as seen after fixation and banding (Fig. 6A). The high ionic strength buffer dissociates both the high and the low density particles so that the DNA contained in them is found banding in a denser part of the gradient (Fig.6B). This result is similar to that obtained when 40-5 particles are treated with high salt concentration ; however, whereas particles (I-somes) which band sharply at 1.57 g/ml are released from 1.43 g/ml particles sedimenting a t 40 S, they are not released so cleanly from particles cosedimenting with polyribosomes. Rather, a range of labeled particles of varying densities, mostly greater than 1.54 g/ml, is observed (Fig.6B). EDTA of low ionic strength neatly disso- ciates the entire 1.43 g/ml particle from the 1.515 g/ml particle (Fig. 6C), strongly suggesting that the 1.43 g/ml particle is bound to a poly- ribosome or some polyribosome-cosedimenting par- ticulate through a divalent cation.

Vo1.29, N0.3,1972 E. BELL, C. MERRILL, and C. B. LAWRENCE 449

240

200

160

120

5 80 2 40

- ._

UJ

c .,-

Y

.: 0

5 9

200 I ",. 160

120

80

-

40

0

1.6 2 m

1.5

1.4 0"

. c ._ (0 c

80

40

0

B

- 7.6

1.5 'z 1.4 6

-. m

c ._ UJ t

1.6 2 m

1.5 )r

1.4;

. .-- c c

Fig.5. Dissociation of the 40-S-sedimenting D N A particle by means of high salt concentration. A cytoplasmic extract was obtained as described in legend to Fig.1 from cells labeled with [3H]thymidine. Particles sedimenting a t 40 S in a 15-30°/,, sucrose gradient spun for 4.75 h a t 40000 rev./min in a Spinco SW 41 rotor were collected and divided into 4 aliquots which were treated as follows. The ionic strength

of the control aliquot was 0.20-0.24 due primarily to the KCl present in buffer M (A); one aliquot was made 0.29 ionic strength with KCl (B); another was made 0,54 ionic strength, also with KCl (C); a fourth was made 0.55 ionic strength with 0.05 M EDTA (D). The samples were then fixed in glutaraldehyde and banded in CsC1 gradients. Gradient fractions were collected and assayed for acid-

insoluble radioactivity

Isopycnic Banding of Particles after Labeling with ("1 Uridine and Separation in Sucrose

The cytoplasmic extract from tissue labeled for 30min with [3H]uridine was displayed in sucrose as described in Fig.1 and 3. The 16-S, 27-5, 40-5, and polysomal cuts from sucrose gradients were fixed in gluraraldehyde and banded in CsCl gradients (Fig.7). Material taken from 16-S and 27-5 zones showed no fHIuridine label associated with the 1.57 g/ml particles; a t 27 S there was only a sugges- tion, in one experiment of three, of labeling of particles of density 1.43 g / d . Polyribosome and 40-S cuts both showed radioactivity peaks having a density of 1.43 g/ml (Fig.7C, 8 and 9). The 40-5 cut yielded a peak of radioactivity a t 1.50 g/ml also (Fig.7C), while the polyribosome cut yielded a peak a t 1.55g/ml and a shoulder a t 1.515g/ml (Fig. 8 and 9).

Tissue was labeled for 10 min with [3H]uridine, then divided into two aliquots; one was processed without further incubation while the other was washed and subjected to a 30-min chase with 30 Ear. J. Biochem., Vo1.29

60 pg/ml dactinomycin. Polysomes were collected, fixed and banded in preformed CsCl gradients. A dense and a light peak of radioactivity are observed in the gradient in which particles from cells labeled for 10 min are displayed (Fig.8). The heavy peak a t 1.54 g/ml is coincident with the absorbance peak due to ribosomes and polysomes. The radioactivity peak has a distinct shoulder of density 1.515 g/ml which is coincident with a shoulder of the absorbance peak. The light peak, which from a num- ber of experiments was determined to have a mean density of 1.43 g/ml, is coincident with the light [3H]thymidine peak which also has a mean density of 1.43 g/ml. There is a suggestion that the shoulder at 1.515g/ml decays more rapidly than the peak radioactivity at I .54 g/ml (Fig. 8 B).

Treatment of 13H] Uridine-Labeled Particles with EDTA

Polyribosomes collected from cells labeled for 25min with [SH]uridine were divided into two

450 DNA Particles in the Cytoplasm Eur. J. Biochem.

30(

20(

lo(

C

-2 ._ 2oc E . Lo Y c 3 0

2 1oc % +J > Y

.-

.-

I

g c n

4 0 C

30C

200

100

0

1.6

1.5

1.4

1.6 - &

1.5: Ol Y

1.42

6 .- (I)

1.6

1.5

1.4

1.6

1.5

1.4 Fig. 7. Density-gradient analysis of 16-S, 27-3, and 40-5 particles containing rapidly labeling RhrA. Cytoplasmic extracts from tissue labeled for 30 min with [5-3H]uridine were spun into sucrose gradients. Fractions taken from the 16-S, 27-8, and 40-S regions were fixed with glutaraldehyde and banded in CsCI. Details may be found in legends of

Fig.l-3 Fraction number

Fig.6. Effects of high salt concentration and EDTA on the polysome-cosedimenting DNA particles. The polyribosome fraction collected from cells labeled with [3H]thymidine as described in legend to Fig.1 was divided into 3 parts. One part (A) was made 0.24 ionic strength with KCI; the second (B), 0.49 ionic strength with KCI; and the third (C) was made 0.20 ionic strength with 0.01 M EDTA. All were spun into preformed CsCl gradients for 12 h and fractions were processed to determine acid-precipitable radioactivity. 0, [3H]Thymidine; 0, absorbance a t 260 nm

aliquots. One aliquot was treated with EDTA; then both were fixed with glutaraldehyde and banded in CsCl gradients. In the gradient from the control aliquot two distinct peaks of radioactivity were observed, one coincident with a peak of absorbance a t 1.550 g/ml where ribosomes and polysomes band

and the other a t about 1 . 4 4 g / d (Fig.9A). A well defined shoulder occurs at a density of 1.515g/ml, the density a t which [3H]thymidine radioactivity peaks. On treatment with EDTA the shoulder a t 1.515 as well as the 1.55g/ml peak of radio- activity are displaced (Fig. 9B) ; the radioactivity peaks a t 1.50g/ml, coincident with the small ribosomal subunit, and there is a shoulder at 1.57 which might represent nascent RNA associated with a free I-some or template RNA bound to the large-ribosomal subunit, or both.

Isopycnic Sedimentation of Particles Containing Nascent Protein Chains

Tissue was labeled for 3 min with [14C]-labeled hydrolysate. The polyribosome-cosedimenting por-

Vol. 29, N O . 3, 1972 E. BELL, C. MERRILL, and C. B. LAWRENCE 451

tion of the cytoplasmic extract was isolated, fixed, and banded in CsCl as described in the legend to Fig. 4. The radioactivity was recovered banding sharply a t 1.54 g/ml, coincident with the polyribo- soma1 absorbance peak (Fig. 10).

Addition of Labeled DNA to Unlabeled Cell Extracts A8 a control experiment purified 3H-labeled

chick nuclear DNA was mixed with an unlabeled

..5

.4 h - E m -4. - x c .-

.6 E a

.5

.4

Fraction number

Fig. 8. Density-gradient analysis of particles containing rapidly-labeling RNA which cosediments with polyribosmes in sucrose. Tissue was labeled with lOO-pCi/rnl [ELaH]- uridine for 10 min and divided into 2 aliquots, one of which waa processed immediately (A) while the other was washed and reincubated for 30min with 60pg/ml dactinomycin without isotope (B). Polysomes were collected as described in legend to Fig.4, fixed and banded in preformed CsCl

gradients. 0, Radioactivity; 0, absorbance

whole cell extract. As in the other experiments, the ionic strength of the buffer used, buffer M, was 0.29. Nuclei were spun out and the cytoplasmic extract was layered onto a 15-30°/, sucrose gradient

1400 1200

1000 800

: 600

5 400 ._

2 200 $ 0 a,

U

3

.- 'Z 2000

F 1800 o, 1600 iB n '400 1200 1 i\ 400 0.100 200 0.050

0 5 10 15 20 25 40 Fraction number

Fig.9. The effect of EDTA on the density of polysome-cosedi- menting particles containing rapidly labeling RNA. Tissue was labeled for 25 min with 100 pCi/ml [5-aH]uridine; polyribosomes were collected as described in legend to Fig.4 and divided into 2 parts. One aliquot was made 0.05 M with EDTA and after 30 min a t 4 "C each was fixed and layered onto a preformed CsCl gradient. The control aliquot (A) was not exposed to EDTA, while the second aliquot (B) was brought to 0.05M EDTA. After 30min a t 0 ° C both were fixed and banded in preformed CsC1

gradients. 0, Radioactivity; 0, absorbance

E

8

0.1 $

0.2 z 2

D

n 4

Fraction number

Fig. 10. Density-gradient analysis of particles containing nascent protein chains. Cells were labeled for 3 min with 10 pCi/ml [W]-labeled hydrolysate. Polyribosomes were collected, fixed in glutaraldehyde and banded in CsCl as described in legend

to Fig.4. The acid-insoluble radioactivity in each fraction was counted for 60 min. 0, Radioactivity; 0, absorbance

30.

452 DNA Particles in the Cytoplasm Eur. J. Biochem.

1400 - 1200 - 1000 -

800

600 -

-

2001 I t

C

1.6

1.5

1.4

1.6 - E

1 . 5 2

- - A

1.4 ,z c W

0

- 1.6

- 1.5

- 1.4

- 1.3

I J k Id1

0 5 10 15 20 25 F 4 0 Fraction number

Fig. 11. Control to rule out nuclear leakage as the source of the observed cytoplasmic DNA-containing particles. Purified [3H]thymidine-labeled chick nuclear DNA (100000 counts/ min) was mixed with a whole cell extract fiom 11-day-old muscle cells. The ionic strength of the buffer was 0.29. Nuclei were spun out during a 10-min centrifugation a t I0000 rev./min in a Sorvall SS34 rotor and the cytoplasmic extract layered onto a 15-30°/, sucrose gradient formed on a 60°/, sucrose cushion. After a 4.75-h spin a t 40000 rev./min fractions were collected and one drop of each counted. The radioactivity was recovered as a sharp peak sedimenting at about 20s and virtually no radioactivity was seen at 40 S or wit,h polyribosomes. Ordinarily after labeling cells with thymidine, a peak of radioactivity accumulates at the cushion-gradient interface but in this experiment none did. Aliquots from the 20-5 peak were treated as follows: one was allowed to remain 0.29 ionic strength (A); a second was made 0.01 M EDTA and had a final ionic strength of 0.20 (B); and a third was brought to 0.54 ionic strength with KCI (C). The samples were then fixed and banded in CsCl

as described elsewhere

Table 1. Labeled particles from cytoplasmic extracts with which DNA is msociated

Each determination was made at least six times ~

Particle Buoyant density

s/ml

I-somes 1.57 -1.58 Transcription complex 1.43 -1.44 Transcription-translation complex 1.515-1.52 Ribosomes 1.54 -1.55 Polyribosomes 1.54 -1.55

Small ribosomal subunit 1.50 -1.51 Large ribosomal subunit 1.57

layered over a go0/, sucrose cushion. After centri- fugation for 4.75 h a t 40000 rev./min, fractions were collected and the radioactivity contained in one drop of each fraction was counted. No adventi- tious binding of radioactivity was observed in the polysomal or the 40-S region of the gradient, but a single peak of radioactivity sedimenting at about 20 S was seen.

Samples from this peak, which represented about 65O/, of the radioactivity loaded, were eit-her brought to 0.54 ionic strength with KC1, diluted and made 0.20 ionic strength with EDTA, or allowed to remain in 0.29 ionic strength buffer M. Each aliquot was then fixed in glutaraldehyde and banded in a pre- formed CsCl gradient. Low and high density com- plexes are observed when material was fixed in low ionic strength buffer (Fig. 11 B). But the low density (1.36 glml) complex, which did not form at ionic strengths of 0.29 or higher (Fig.11.A and C) may be contrasted with particles found in cytoplasmic extracts from labeled cells, all of which were stable at an ionic strength of 0.29. The high density com- plex seen banding at 1.63 glml under all conditions of fixation was never observed to occur naturally in a cytoplasmic extract derived from labeled cells.

Heasurement of Buoyant Density of Nuclear DNA Protein Complexes

After isolation and fixation in glutaraldehyde, buoyant densities of chromosomal and slow sedi- menting (sedimenting a t 35 S or less) nuclear DNA- containing particles were measured ; the former has a buoyant density of 1.40 g/ml and the latter 1.61 g1 ml, both different from the densities of particles found in the cytoplasm.

DISCUSSION

Labeled particles from cytoplasmic extracts with which we have found DNA associated are summarized in Table 1. We hypothesize that they constitute a class of organelles which function in transcription and possibly in translation as well.

Vol. 29, No. 3, 1972 E. BELL, C. MERRILL, and C. B. LAWRENCE 453

They do not appear to be artifacts of tissue prep- aration, having neither the characteristics of labeled nuclear DNA particles nor of particles formed by the association of protein from unlabeled cell extracts mixed with labeled DNA. They remain stable in buffers of relatively high ionic strength, while the low density artificially prepared particles do not, suggesting that the latter cannot exist in the ionic atmosphere of the cell. It is important to point out that labeled nuclear DNA added to cell extracts does not bind to polyribosomes or cosedimenting structures.

We have found that synthesis of nonmitochon- drial cytoplasmic DNA is relatively insensitive to the action of fluorodeoxyuridine, which greatly depresses synthesis of nuclear DNA, and that hy- droxyurea does the reverse, favoring synthesis of nuclear DNA over I-DNA [3]. These results provide further support for a fundamental difference be- tween chromosomal DNA and I-DNA.

The particles observed a t 16, 22, 27 and 32 S in sucrose gradients appear to be members of a related series of sizes and may be monomer (16 S) and multimers (the other sizes).

The particle of density 1.57 g/ml is stable a t ionic strengths as high as 0.54 and appears to be of a constitution to which other nonmitochondrial DNA-containing particles are reducible. Table 2 summarizes the relationships among the particles.

Particles having a buoyant density of 1.43 g/ml are of a gradation of sizes ranging from those found with 1.57 g/ml particles peaking at 16, 22, 27 and 32 S to those which cosediment with polyribosomes. The 1.43 g/ml particle appears to consist of il 1.57 g/ml moiety plus protein which a high salt buffer can remove. Even though the theoretical ionic strength of 0.05M EDTA is similar to the ionic strength of 0.5 M KC1 the effective ionic strength is clearly less (Fig.4 and 5) since 0.05 M EDTA does not dissociate the 1.43 g/ml particle. It may remove a small amount of protein since the buoyant density of the particle does increase slightly to 1.445 g/ml. The 1.515 g/ml particle found only cosedimenting with polyribosomes in sucrose can be dissociated by low ionic strength EDTA (0.01 M) yielding a DNA-containing entity which bands a t 1.43 g/ml. Thus, the 1.43 g/ml particle appears to be bound to the polyribosome or other particulate of similar size through a divalent cation. Low ionic strength EDTA does not alter the density of any particle other than the 1.515 g/ml particle. The breadth of the band which peaks at 1.515 g/ml suggests that it might be composed of polyribosomes and DNA with protein in varying molar ratios.

The free I-somes or 1.57 g/ml particle is found sedimenting in several size classes in sucrose; however, no free I-somes are found sedimenting more rapidly than 40 S. The free 1.57 g/ml particle

Table 2. Summary of effect of various treatments on DNA- containing particles

Treatment Effect ~ ~ _ _ _ _

glml

1.43 Buffer Little or no change

1.43 EDTA Little or no change

1.43 High salt concentra- 1.67 g/ml + protein,

1.616 Buffer No change

1.515 EDTA 1.43 g/ml + polysomes

(0.3 ionic strength)

(0.01 M or 0.05 M)

tion (0.5 M KCl)

(0.3 ionic strength)

(0.01 M) or cosedimenting par-

1.30 g/ml

ticle 1.515 High salt concentra-

tion (0.5 M KCl)

1.515 EDTA (0.05 M)

1.57 Buffer

1.57 EDTA

1.57 High salt concentra-

(0.3 ionic strength)

(0.01 or 0.05 M)

tion (0.5 M KCI)

1.54-1.60 g/ml + poly- somes or cosedimenting particle + protein 1.54-1.60 g/ml + poly- somes or cosedimentimg particle.:+ protein No change

No change

No change

is not functional; that is, newly made RNA is not found in association with it. Likewise, little if any newly made RNA is found in association with 1.43 g/ ml particles which are smaller than 40 S.

DNA associated with 40-5 and larger particles is functional. After a ten-minute label with [3H]uridine particles containing newly made RNA which cosediment with polyribosomes in sucrose are found to band in peaks a t densities of 1.43 and 1.54-1.55 g/ml and as a shoulder of radioactivity at 1.515g/ml. A similar result is obtained when the exposure to [SHIuridine lasts only for 3min. The rapidity of appearance of label with the poly- ribosomes leads us to suggest that the RNA is being synthesized in the cytoplasm. However, while DNA is found in polyribosome-cosedimenting particles of densities 1.43 and 1.515 g/ml, little if any labeled DNA is present in the 1.55 g/ml particles which are taken to be free polysomes or ribosomes. Therefore, if the labeled template RNA associated with poly- ribosomes is synthesized from I-DNA contained in the 1.515 g/ml particle it quickly dissociates to become part of a free polyribosome. After 3 min of labeling with [14C]labeled hydrolysate, nascent protein chains are also seen to band a t a density of 1.55 g/ml as a peak.

We would like to suggest that 1.43 g/ml particles which have become larger than 4 0 s accept ribo-

454 E. BELL, C. MERRILL, and C. B. LAWRENCE : DNA Particles in the Cytoplasm Eur. J. Biochem.

somes soon after initiation of template RNA syn- thesis.

The newly made RNA found with DNA-con- taining particles in the cytoplasm has not yet been characterized. We have found, though, that a-ama- nitin-sensitive DNA-dependent RNA-polymerase activity is found in association with polyribosomes and cosedimenting structures [13].

We propose the following scheme for synthesis, transfer, and utilization of I-DNA: I-DNA or gene copies synthesized in the nucleus and proteinized reach the cytoplasm in the form of I-somes of different sizes; these I-somes all have a buoyant density of 1.57-1.58g/ml. In the cytoplasm they grow by accretion of a “stabilizing” protein which is assumed to have a density of 1.3 g/ml. The I-some is saturated with protein when it reaches a buoyant density of 1.43 g/ml. Initially the 1.43 g/ml particles are inactive, but when one has grown in size by polymerization to greater than 40 S it is activated and RNA polymerase molecules attach to it. This particle, still of density 1.43 g/ml, may be thought of as a transcription complex in which the I-DNA serves as template for the synthesis of template RNA. Ribosomes can attach to the nascent strands of RNA with the resultant formation of a transcrip- tion-translation complex of a buoyant density of 1.515g/ml. A free polysome having a density of 1.55 g/ml dissociates from the complex as soon as synthesis of the strand of template RNA is com- pleted.

Alternately, the particle a t 1.515 g/ml might consist of template RNA in association not with a ribosome but with some yet unidentified particle. The template RNA might then be handed off to a ribosome from this entity. Further details have been described elsewhere [2]. The coupling of tran-

scription and translation has been “visualized” in a fragile strain of Escherichia coli [14].

A simpler model would suggest that the 16,22,27 and 34 S non-functional DNA containing particles are pieces rejected from larger particles in which only a portion of the DNA is retained for use.

We wish to acknowledge the help of John Brown for his critical reading of th manuscript and for making many useful suggestions. This work was supported by Grant GB-27326X from the National Science Foundation.

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E. Bell, C. Merrill, and C. B. Lawrence Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139, U.S.A.