Ribosome formation in HeLa cells in the absence of protein synthesis

J. Mol. BioI. (1966) 19, 373-382

Ribosome Formation in HeLa Cells in the Absence of Protein Synthesis J ONATHAN

R . W AR KER, M.

GIRARD ,

H.

LATHAM AN D J.

E.

DARKELL

Department of Biochemistry , Albert E instein College of 111edicine Y eshiva Uni versity , Ne w Y ork , N.Y., U.S.A. (R eceived 15 February 1966) R ibosomal RNA sy n t hesis and ribosome m aturation in H eL a cells can occ ur when protein synthesis has been complet ely stoppe d by cycloheximide. Thi s result implies the availability within the cell of eve ry protein necessar y to const ruct ribosomes. The ribosomes wh ich are newly formed in cyclohe x imide enter in t o associat ion wit h cha ins of mRNA.

1. Introduction Present evidence indi cates that ribo somes from all cells ar e composed of two rib onucleoprotein particles, a larger subunit (50 to 60 s in different cells) cont aining 23 or 28 s RNA and a smaller subunit (30 to 45 s in different cells) containing 16 to 18 s RNA (Kurland, 1960; Hiatt, 1962; Girard, Latham , P enman & Darnell, 1965). Th e protein of each ribo somal subunit apparently consists of a distinct set of at least 15 polypeptide chains (Leboy, Cox & Flaks, 1964; Waller, 1964). The present exp eriments utilize an inhibitor of protein synthesis, cycloheximide (Ennis & Lubin, 1964), to demonstrate that all of the prot eins necessary to allow the ini ti ation and completi on of rRNA t and the maturati on of ribosomes exist in finished form within HeLa cells, and that these proteins can be used in t he formation of rib osomes in the ab sence of further protein synt hesis. The amount of available ribo somal precursor proteins in HeLa cells has been shown in other experiments (Warner , 1966) t o be sufficient t o assemble rib osomes for one t o t wo hours (about one-tw elfth of a generation t ime ). In experiments on bacteria, most investigations of ribo some formation in the absence of continuing protein synthesis have been mainly concerned with the nature and fate of abnormal subribosomal particles during inhibition (Nomura & Watson, 1959; Nakada, Anderson & Magasanik, 1964). None of these studies with bacteria has explored the question of whether, imm ediatel y afte r prot ein synthesis has been inhibited , any new ribo somal RNA appears in complete d ribo somes or ribosomal subunits.

2. Materials and Methods The methods of cell cult ure, labeling of cell RNA with radioa cti ve uridine, prepara t ion of cytoplas mic extracts, release and sedime ntation analys is of cytoplas m ic RNA, and extra cti on of t otal cell RNA have all been des cribed in the precedi ng paper (Warner, Soeiro, Bi rnboim , Girard & Darnell, 1966), or elsewhere (Scherrer & Darnell, 1962; Penman, Scherrer, Becker & Darnell, 1963; Girard, Penman & Darnell, 1964). Cycloheximide

t Abbreviations used: rRNA, ribosomal RNA; mRNA, m essenger RNA. 373

374

J. R. WARNER, M. GIRARD, H. LATHAM AND J. E. DARNELL

(actidione) was obtained from Upjohn and actinomycin D was a gift of Merck, Sharp &

Dohme, Although previous reports clearly demonstrate that cycloheximide is an effective inhibitorof protein synthesis in animal cells (Ennis & Lubin, 1964) we wished to quantitate the speed and extent of inhibition for HeLa cells. Accordingly, amino acid incorporation was studied when cycloheximide (150 IJ-g/ml.) was added 1·5 min after (14C]amino acids. Figure l(a) shows that incorporation was halted by the cycloheximide within seconds. When the observation period was extended to 120 min (Fig. l(b)), i~ WaB clear that linear incorporation was continuing in both the control and drug-treated culture. The rate in cycloheximide WaB, however, reduced to less than 1 % of the control.

500

/

•

120

60 (b)

(al Time (minI

FIG. 1. Inhibition of protein synthesis by cycloheximide. Logarithmically growing HeLa cells were centrifuged and resuspended in fresh Eagle's medium containing one-fourth the normal concentration of valine. The culture was labeled at 0 time with 0·15 /-,c/m1. of (UC]valine (100 /-,cl /-'0101e). After 1·5 min, the culture was divided and cycloheximide (150 /-,g/ml.) added to one half. 1-011. samples were removed and acid-precipitable radioactive material determined after brief alkali treatment (Penman es al., 1963). The data are plotted so that the speed of action of the drug is seen in (a) while the extent of inhibition is seen in (b). Control; -0-0-, cycloheximide-treated.

-e-e-,

3. Results Previously published studies showed that almost no labeled ribosomal RNA enters the HeLa. cell cytoplasm during the first 20 minutes after exposure to [3H]uridine (Girard et al., 1964; Girard et al., 1965). By 30 minutes, new 16 s rRNA is detected and by 40 minutes new 28 s RNA has also appeared. The initial appearance of new ribosomal RNA occurs only in the form of ribosomal subunits. Considerable evidence indicates that these subunits then enter polyribosomes directly without first becoming 74 s particles (Girard et al., 1965; Joklik & Becker, 1965a,b; McConkey & Hopkins, 1965). The basic techniques used in these previous studies 011 the emergence of newly synthesized ribosomal RNA were: (I) the preparation of cytoplasmic extracts from cells swelled in hypotonic buffer, followed by {2} the sedimentation analysis of RNA released from various cytoplasmic structures by the action of sodium dodecyl sulfate.

RIBOSOME FORMATION IN HeLa CELLS

375

Ribosomal RNA is first synthesized in the nucleus as a 45 s molecule, which is a precursor to the 28 and 16 s RNA found in the ribosomes. This precursor molecule is never found in cytoplasmic extracts. Further, it was shown in the previous work that the ribosomal subunits which contained the first labeled cytoplasmic rRNA were indistinguishable in sedimentation behavior from ribosomal subunits derived by disaggregation of completed ribosomes. No evidence existed therefore for cytoplasmic pre-ribosomal particles other than completed subunits. The sites of both rRNA manufacture and ribosomal subunit assembly are effectively excluded in the preparation of a cytoplasmic extract. In the study of ribosome formation, this is a major advantage of animal cells as compared to bacteria, because it allows an assessment of ribosome synthesis by simply observing the appearance of labeled 28 and 16 s RNA in the cytoplasm. In the experiments which follow we will describe (1) the appearance of newly formed ribosomal RNA in cytoplasmic extracts from cells that have been exposed to the "pulse-chase" conditions detailed in the previous paper (Warner et al., 1966) and (2) the effect of various drugs on this process of ribosomal RNA appearance. The plan for the experiment of Fig. 2 is shown in the diagram in the legend. Figure 2(a) shows the pattern of appearance of labeled rRNA at one, two and four hours after a five-minute (3H]uridine pulse-chase with no drug treatment. As previously described, the initial appearance of 16 s rRNA is more rapid than that of 28 s rRNA (Girard et al., 1965). Figure 2(d) demonstrates another previously reported result, namely that, after blockage of further RNA synthesis by actinomycin, both 28 sand 16 s ribosomal RNA can pass into the cytoplasm, although in greatly reduced amounts compared to the pulse-chase (Girard et al., 1964). It is of interest that the actinomycin block in ribosome completion is more extensive for the 28 s RNA than for the 16 s RNA. Figure 2(b) demonstrates that even when protein synthesis had been completely halted by cycloheximide before exposure to (3H]uridine, the cells were still able to initiate and complete the synthesis of new ribosomal RNA which reached the cytoplasm. If the addition of cycloheximide was delayed until 20 minutes after chase, at which time very little or no rRNA would have entered the cell cytoplasm, the subsequent appearance of 16 and 28 s RNA was only slightly affected (compare Fig. 2(c) with Fig. 2(a)). The control cells (Fig. 2(a)) which were to be used for comparison to the drugtreated cultures in this experiment were exposed to a "pulse-chase"; but as described in the previous paper, the "chase" does not completely prevent (3H]uridine incorporation into RNA. Figure 3(a) gives a measurement of total labeled RNA (nuclear and cytoplasmic) in samples from the experiments shown in Fig. 2(a) and (c). It can be seen that in the control culture an 80% increase in total radioactive RNA occurred between one and six hours after the chase. Thus, there would still be a 15 to 20% per hour increase in cytoplasmic radioactive rRNA, even if all the ~uclear RNA precursors were converted to cytoplasmic ribosomes within an hour. Also, in the later points of the experiment more label would be expected in the cytoplasm of pulse-chased cells than in the cycloheximide-treated cells, in which there was no increase in total radioactivity after one hour. Therefore the curve of cytoplasmic 28 s rRNA accumulation in the "pulse-chase" culture (Fig. 2(a) ) was normalized in accord with the total amount of radioactive RNA in the culture at one hour. The rise in total 28 s rRNA in the corrected curve then falls

1000

1000

'E

.,

u

'"

a.

.c .:;

~ 0

c:

......

'" ::J

oJ

.,

L.

:;:;

'" ::J

U

L.

0

0

'0

3

0

ce::

I

0

FIG. 2. Effect of cycloheximide and actinomycin on appearance of cytoplasmic rRNA. Experimental plan: All cultures

1-[3R]uridine-1

(a)

I I - - - - - - - - - - - n o drug

(b)

I--unlabeled uridine---j

----------------cycloheximide

(c)

I I

l---cycloheximide--I

(d)

-5

o

5 Time (min)

J--actinomycin 25

--I 360

Logarithmically growing ReLa cells were centrifuged and resuspended at 4 X 105 cellsjml. in fresh medium at 37°C. Mter 30 min incubation, the culture was divided and cycloheximide (150 f'g/ml.) added to the smaller portion (b). 5 min later both portions were labeled with [3R]uridine (0,3 f'c/mI., 20 mC/flmole) and after 5 min further, unlabeled pyrimidine nucleosides were added (final concentrations, uridine, 0·1 mM; cytidine, 0·05 mM; and thymidine, 0·05 mM). After an additional 20 min of incubation, the larger untreated portion of the culture was divided into 3 parts to which was added, ~.a} nothing further, (c) 150 flg/mI. of cycloheximide, and (d) 5 f'g of actinoruycin/ml. All the cultures were continued at 37°C; samples were withdrawn at intervals, and a cytoplasmic extract prepared. RNA was released from the total cytoplasm by sodium dodecyl sulfate treatment and examined by zone sedimentation in sucrose gradients (Girard et al., 1964). The data presented in the Figure represent the acid-precipitable radioactive material in the regions of the resulting sucrose gradients containing the rRNA (only the 5 to 35 s regions are plotted). The numbers 28 and 16 on the abscissa indicate the peak tubes for the two species of rRNA. All data have been normalized to equal amounts of rRNA as determined by absorbancy at 260 txu»:

RIBOSOME FORMATIO~ I~ HeLa CELLS

377

(a)

1·8

1500

I

E

1·1

1·2 o

+ 0::::'--- -------,,--l>--

';;;

u I

0 0 .........

_

I

( (

(

. (b)

./

c:

-E

~u ::; 20,000 N

"0

~

.~

~ __-----_o

.~---~ ,__----A 0 ......

A""

e/"

/ .....0

A..........

.>

.".,.-, ..........

",-

3

6

Time (hrl

FIG. 3. Total radioactive RNA and 28 s cytoplasmic H~A from control and cycloheximidetreated cells. (a) From the cell samples of the experiment shown in Fig. 2 the following measurements were made. Total cytoplasmic radioactivity was determined by summing the counts from the gradients shown in Fig. 2. A portion of the nuclear fraction wall assayed for total acid. precipitable radioactive material, The com b ined nuclear and cytoplasmic totals from cultures (a) (- 0-0-, pulso-ehaee) and (c) (-/:::"-t:.-, cycloheximide) are plotted in Fig. 2. In Fig. 2 graphs were not presented for all the points taken in the experiment. The figures above the points on the curve for the pulse-chase culture represent the increase in total radioactivity relative to the 60·min point. (b} The a ccumulation in the cytoplasm of the total radioactive 28 s rRNA in the "pulsechaso" (-e-e-l and cycloheximide (--/:::,.--/:::,.--) cultures. The curve for the "pulse-chase" culture was then corrected (--0--0--) for continuing incorporation by dividing the total 28 s counts at each point by the figures above the points for the total incorporation seen in (a}.

on the dotted line shown in Fig. 3(b). It is clear from this corrected curve that most of the 28 s RNA synthesized in the first hour that is destined to appear as cytoplasmic ribosomal RNA, has done so within another two hours. Furthermore, in the culture treated with cycloheximide 20 minutes after pulsechase (culture (c), Fig. 2), almost as much ribosomal RNA appeared in the cytoplasm as in the control culture (Fig. 2(11». Thus the cell contains and can efficiently use a pool of ribosomal proteins to construct ribosomes (ribosomal subunits) in the absence of protein synthesis. The cycloheximide seems to have depressed the rate of rRNA appearance. A similar depression in the rate of appearance of preformed ribosomal proteins has also been observed (Warner, 1966). One final point emerges from a comparison of the pulse-chase culture (Fig. 2(a» with the cycloheximide-treated culture (Fig. 2(b» . The sedimentation profile of the RNA which has entered the cytoplasm within 60 minutes in culture (b) shows very little rRNA but an amount of heterogeneously sedimenting RNA (6 to 30 s) at least as great, if not greater, than in the control cells. This type of RNA has also been shown to enter the cytoplasm in puromycin-treated cells (Latham & Darnell, 1965) and probably represents mRNA.

J. R. WARNER, M. GIRARD, H. LATHAM AND J. E. DARNELL

378

A second type of experiment with cycloheximide-treated cells was then performed to determine whether the rRNA which entered the cytoplasm was in ribosomal particles, and, if so, to determine the distribution of radioactivity between polysomes, single ribosomes and ribosomal subunits. Cells were treated for five minutes with cycloheximide before a pulse-chase ofuridine. A companion culture not treated with cycloheximide served as a control. Cytoplasmic extracts were prepared at a time when the majority of the cytoplasmic radioactivity was in 28 and 16 s rRNA (i.e. thrce hours after pulse-chase, sec Fig. 2; this result was reconfirmed for the experiment shown in Fig. 4). A portion of the extract was sedimented to display polysomes (Fig. 4(a) and (a /)), and another portion centrifuged so as to obtain good resolution of the 74 s and smaller structures (Fig. 4(b) and (b ')). The polysomes were slightly smaller in cycloheximide but still contained as much material as the control, based on absorbancy at 260 mfL. The majority of the radioactive material had entered the polysomes in both cultures. ~o new ribosomes entered the 74 s pool in the treated cultures, however, (a)

{cl

(b)

400

0 0\

I,

; I I I

1000

400

o

~

000

J

c:

\~

J

·e

<,

~

..;:

I

J460 45

.

\

0'

SO 30\0,}'

Z;-

's

(a')

v

{c1

{b1

0

0

;:; 0 ex:: 400 o.

300· 200

/I

01 ,0

I

I

i\

J'" . . o ,

74-60 45

SO 30·

Relative sedimentation

FIG. 4. Cytoplasmic localization of new RNA appearing in presence of cycloheximide. A culture was divided and one part treated with cycloheximide as described in Fig. 2 «a'), (b'), (c')); the other portion was not treated «a), (b), (c)). Mter 5 min, the cells were labeled as in Fig. 1 with [3H]uridine and "chased" 5 min later; cytoplasm WlL8 then prepared 3 hr after the beginning of label. The distribution of acid-precipitable radioact.ive material within cytoplasmic structures was investigated by layering one-half the cytoplasmic extract on each of two 15 to 30% sucrose gradients (sucrose wfw in 0·01 M-Tris (pH 7'4), 0·01 M-NaCI and 0·0015 M-MgCI2) and centrifuging for two different times (Girard et al., 1965), a "short spin" to display polysomes «a), (a'), 100 min, 4°C, 25,000 rev.fmin) and a "long spin" to display 74 s and smaller structures «b), (b'), 16 hr, 20,000 rev·fmin, 4°C). The pellets of the "long spin" «b), (b')) were resuspended in a buffer containing 0·01 M-EDTA, 0·01 M-Tris (pH 7·4) and 0·1 M-NaCI and layered on a 5 to 15% sucrose gradient in the same buffer and centrifuged for 5 hr at 25,000 rev.froin «c), (c'». Ctsfmin on all graphs represent acid-precipitable radioactive material (-0' - 0-). Solid line ( - - ) represents absorb. anoy at 260 mIL.

RIBOSOME FORMATION IN HeLa CELLS

379

implying that although attachment to the messenger RNA can occur in the absence of protein synthesis, the equilibration with free 74 s ribosomes requires the completion of polypeptide chains. The polysome pellets from the bottom of the gradients shown in Fig. 4(b) and (b') were resuspended in an EDTA buffer to convert the ribosomes to subunits of 50 and 30 s (Girard et al., 1965). These were then examined on other gradients (Fig. 4(c) and (c')), which showed that the newly formed ribosomal subunits which had entered the polysomes in the cycloheximide-treated cells were normal with respect to sedimentation behavior and stability in EDTA. Effect of puromycin on ribosome synthesis Although the results with cycloheximide indicate that concomitant protein and RNA synthesis is unnecessary for ribosome formation, another inhibitor of protein synthesis was found to have a drastically different effect. It was previously shown (Latham & Darnell, 1965) that if cells are exposed to puromycin before [3H]uridine, no detectable labeled ribosomal RNA appears in the cytoplasm. Moreover, if puromycin is added after a five-minute label and 20-minute chase, there is a dramatic reduction in the emergence of new ribosomes into the cytoplasm compared to cells treated in the same way with cycloheximide (Fig. 5).

[J

1\

'I "19 I'

I I

c:

, I

E

<,

I

I

:Eu

, I

1 I

,

p

-0

I I

VI

~ ~

o .Q

o

0:::

10,000

[J

,

,,

,

[J

I I

I I

o

o

Relative s-value

FIG. 5. Comparative effect of puromycin and cycloheximide on ribosome maturation. A culture labeled with [3H]uridine as in Fig. 1 was divided 20 min after a "chase" and either not treated further (--0--0--), or treated with cycloheximide 150/Lg/ml. (-e-e-); or puromycin 150 iLg/ml. (-0-0-). Cytoplasm was prepared 2 hr after the addition of [3H]uridine and RNA analyzed as in Fig. 2.

380

J. R. WARNER, M. GIRARD, H. LATHAM AND J. E. DARNELL

In an effort to explain this difference between the two drugs, their relative effect on RNA synthesis was studied . Both drugs almost immediately depress RNA synthesis at least twofold (Fig. 6(a)); however, the sedimentation pattern of tho total rapidly labeled RNA formed in the presence of eit her inhibitor is not detectably changed from normal. Furthermore, if puromycin is added to cells which have been labeled for five minutes followed by a 15-minute cha se, the conversion of the~larger nuclear RNA precursor (45 s) to smaller rRNA molecules is not affected (Fig. 6(b)). Thus, the lesion in ribosome formation caused by puromycin is not in the synthesis or maturation of rRNA, but possibly in the assembly process of rRNA and ribosomal protein, or in the transport of new particles to the cytoplasm.

1<'10.6. Effect of puromycin and cycloheximide on formation of ribo somal precursor RNA. (a) A control culture and two cultures treated for 5 min with 150 ,.,.g/ml. of eit her puromycin or cycle hexirnide were labeled with [3H]uridine for 5 min and chased as in Fig. 2. 15 min after the chase, total cellular RNA WaB extracted and analyzed on sucrose gradients (-0-0-, control culture; -e-e-, cycloheximide; -6.-6.-, puromycin). (h) A portion of the control culture from (a) was treated with puromycin (150 ,.,.g/ml.) at the time the samples for (a) were collected. This puromycin-treated culture and another portion of the control culture were then incubated for 30 additional minutes. Total cellular RNA WaB extracted and analyzed at that time (-0-0-. control; --/::"--/::"--. puromycin-treated).

4. Discussion The main conclusion to be drawn from the experiment describ ed in this paper has already been stated: ribosomal RNA synthesis can be initiated and ribosomes completed in the presence of cycloheximide, which blocks at least 99% of protein synthesis. Since ribosomes contain at least 5 % of the total cellular protein (Warner, 1966), a functional pool of ribosomal protein must therefore exist. If, as ha s been suggested (Otaka, Osawa & Sibatani, 1964; Nakada, 1965), rRXA functions in some way as a messenger in the synthesis of ribo somal protein, it is clear that a ribosomal RNA

RIBOSOME FORMATION IN HeLa CELLS

381

molecule (or precursor molecule) need not necessarily perform such a function in order to appear ultimately in a ribosome. In another paper a study of the appearance of ribosomal proteins in the HeLa cell cytoplasm and detailed considerations of the mechanisms of assembly of rRNA and ribosomal proteins will be presented (Warner, 1966). Several additional points emerge from the present experiments with cycloheximidetreated cells. (1) The synthesis of mRNA appears relatively unaffected for at least an hour (see Fig. 2(b)). (2) Ribosomal synthesis not only continues in the presence of the drug bat the subunits which enter the cytoplasm become associated with mRNA (whether only at the beginning or throughout the chain is not. known) since they occur in polyribosomes of virtually normal size. (3) It is clear from Fig. 5 that the ribosomes which are newly formed in cycloheximide do not equilibrate with the pool of 74 s ribosomes. The precise mode of action of cycloheximide is unknown, but nascent protein chains remain attached to ribosomes (Ennis & Lubin, 1964; Wettstein, Noll & Penman, 1964) and most of the ribosomes are apparently "frozen" in polysomes. Apparently the new subunits can begin an association with mRNA chains but cannot complete the entire translation, or can do so at such a slow rate that equilibration does not occur within three hours. Maximum effective protein synthesis is therefore not a prerequisite for the continued synthesis and entrance into the cytoplasm ofmRNA. If ribosomes have a vital role (Byrne, Levin, Bladdin & Nirenberg, 1964; Stent, 1965) in the continuing transcription and movement into the cytoplasm of mRNA, it is probably sufficient that a ribosome (ribosomal subunit) make an initial interaction with the chain of mRNA. In the course of this work, it was found that ribosome completion was effectively interrupted by puromycin. This result resembles the effect of actinomycin D in that only a small proportion of RNA formed prior to drug addition ever appears in the cell cytoplasm (compare Fig. 2(d) with Fig. 5). Whether there is a parallel in the action of the two drugs in halting ribosome completion is far from clear at present. Because of the results of the cycloheximide experiments, however, it can be stated that some action other than simply the interruption of protein synthesis must be responsible for this particular action of both puromycin and actinomycin D. This work was supported by a National Institute of Health grant no. CA 07861-02 and a National Science Foundation grant no. GB 2477. One of the authors (M. G.) is a Fellow of the Comite de Biologie Moleeulaire, Delegation Generale a la Recherche Scientifique et Technique, Paris, France, two others (J. R. W. and J. E. D.) are Career Scientists of the Health Research Council of the City of New York. REFERENCES Byrne, R., Levin, J. G., Bladdin, H. A. & Nirenberg, M. W. (1964). Pmc. Nat. Acad. Sci., Wa8h.52, 140. Ennis, H. L. & Lubin, M. (1964). Science, 146, 1474. Girard, M., Latham, H., Penman, S. & Darnell, J. E. (1965). J. Mol. Biol. 11, 187. Girard, M., Penman,S. & Darnell, J. E. (1964). Proc, Nat. Acad. Sci., Wa8h. 51, 205. Hiatt, H. H. (1962). J. Mol. Biol. 5, 217. Jokhk, W. K. & Becker, Y. (1965a). J. Mol. Biol. 13, 496. Joklik, W. K. & Becker, Y. (1965b). J. Mol. Biol. 13, 511. Kurland, C. G. (1960). J. Mol. Biol. 2, 83. Latham, H. & Darnell, J. E. (1965). J. Mol. Biol. 14, 13. Leboy, P. S., Cox, E. R. & Flaks, J. G. (1964). Proc, Nat. Acad. Sci., Wa8h. 52, 1367. McConkey, E. H. & Hopkins, J. (1965). J. Mol. Biol. 14, 257.

382

J. R. WARNER, M. GIRARD, H. LATHAM AND J. E. DARNELL

Nakada, D. (1965). J. Mol. Biol. 12, 695. Nakada, D., Anderson, I. A. C. & Magasanik, B. (1964). J. Mol. Biol. 9, 472. Nomura, M. & Watson, J. D. (1959). J. Mol. Biol. 1, 207. Otaka, E., Osawa, S. & Sibatani, A. (1964). Biochem, Biophys. Res. Comm. 15, 568. Penman, S., Becker, Y. & Darnell, J. E. (1964). J. Mol. Biol. 8, 541. Penman, S., Scherrer, K., Becker, Y. & Darnell, J. E. (1963). Proc. Nat. Acad.Sci., Wash. 49,654. Scherrer, K. & Darnell, J. E. (1962). Biochem, Biophys. Res. Comm, 7, 486. Stent, G. (1965). Mendel Centennial Symposium, Royal Soc., London. Waller, J. P. (1964). J. Mol. Biol. 10, 319. Warner, J. (1966). J. Mol. Biol. 19, 383. Warner, J., Soeiro, R., Birnboim, H. C., Girard, M. & Darnell, J. E. (1966).J. Mol. Biol.19, 349. Wettstein, F., Noll, H. & Penman, S. (1964). Biochim. biophys. Acta, 87, 525.