RNA composition of the planarian, Dugesia dorotocephala

RNA Composition of the Planarian, Dugesicr dorotocephala '

KATHLEEN STEWART, ALAN B. GOODMAN, AND JAY 3. BEST Depaement of Physiology and Biophysics, Cotorado State University, Fort Collins, Colorado

ABSTRACT The RNA of anterior and posterior regions of normal and regenerating planarians was extracted by means of a modification of the phenol procedure, and further purified. The nucleotide composition of this RNA was determined by subjecting it to alkaline hydrolysis and resolving the constituent nucleotides by thin layer electrophoresis. Contrary to older reports there was no difference in either amount or base composition of the total RNA from anterior and posterior regions or between regenerate and control worms. Resolution of the various molecular weight species of the planarian RNA by sucrose density centrifugation and gel filtration revealed a typical distribution between ribosomal and transfer RNA as well as the explanation of the negative result. Constancy of base composition of the predominate ribosomal RNA would mask any anticipated changes in base composition.

In spite of the use of the fresh water planarians for a number of interesting experiments on the mechanisms of morpho- genesis (cf. Child, '41 ), regeneration (cf. reviews by Bronsted, '55; Wolff, '62) and learning (cf. reviews by Jacobson, '63; Mc- Connell, '66), the basic anatomical phy- siological, and biochemical information has been insufficient for elaboration of these mechanisms. Recently we have made some progress in this direction (Best, Rosenvold, Souders and Wade, '64; Morita and Best, '65, '66; Best and Elshtain, '66; Best, Elshtain and Wilson, '67; Morita, '65; Best, '67; Best, Hand and Rosenvold, '68). In view of the demonstrated role of ribonucleic acid (RNA) in the information transcrip- tion from DNA and translation in protein synthesis, it might be anticipated that the axial gradient in morphogenetic potential which has been demonstrated in planarians, as well as the cellular differentiation changes which must accompany regeneration, would find expression in differences of the RNA which must control these processes. In keeping with such concepts, Clement ('47) and Lindh ('57) reported alterations in the amount and base composition of RNA in regenerating planarians. Henderson and Eakin ('61) found irre- versible alterations of their differentiated tissues to be produced by purine analogs. Coming and John ('61) found that ribonuclease treatment of planarians regenerating

J. EXP. ZOOL., 169: 9-14.

a new head abolished the effects of previous conditioning in the regenerate worm.

The studies to be described in this paper originally addressed themselves to the ques- tion of whether there is, in fact, any difference in the base composition of the RNA from head and tail regions and whether these undergo any alteration during regeneration of the worm. In order to account for the results which we obtained from these experiments we, as a sequel, per- formed further studies directed toward as- certaining the proportions of the various molecular weight species of RNA present in the planarian.

METHODS

General procedure We extracted the RNA from the anterior

and posterior halves of uncut control and regenerating planarians by a modification of the phenol extraction procedure. This RNA was further extracted to rid it of protein, then purified by precipitation and repetitive washing with cold ethanol. To determine the nucleotide composition we subjected it to alkaline hydrolysis and resolved the nucleotides by thin layer electrophoresis. To determine the proportions of the various molecular weight species of RNA present we used gel filtration chro-

1 This work was supported in part by grant NsG-625 from the National Aeronautics and Space Administra- tion and grant MH 07603 from the National Institute of Mental Health.

9

10 K. STEWART, A. B. GOODMAN AND J. B. BEST

matography and fractionation on a sucrose density gradient in an ultracentrifuge. De- tails of these various procedures are given in sections b-f below.

Experimental animals The planarians used in these experi-

ments were Dugesia dorotocephala approximately 15 mm long that had been maintained in colonies of approximately 500 in white enamel dishpans in the dark, or dim illumination, at a temperature of 70 2 1" F. Prior to their use as experimental subjects they were fed twice weekly on raw beef liver. Following each feeding all food particulates were removed and the water completely changed. The water in which the planarians were maintained was tap- ped from the upper regions of a large aquarium tank with a substantial growth of green algae on its walls and a layer of crushed limestone through which the water of the tank circulated. This water, hence- forth referred to as M-water, yielded the analysis given in table 1 and had been found to maintain colonies of these planarians in a state of good health and growth for periods of a year or more. The analysis given in table 1 provided no information regarding trace elements and it is not known which of these are present or essential.

The worms to be used as experimental subjects were fasted for five to seven days prior to the beginning of the experiment. One hundred worms were used for each experimental replication. Fifty of these were sectioned by a transverse cut at the midpharyngeal level and placed in the M-water. Fifty uncut worms of the same size were also placed in the M-water at the same time.

At the end of 24 hours the regenerating pieces, as well as the uncut worms, were rinsed in fresh M-water and the uncut worms sectioned by a transverse midpharyngeal cut. All four groups of pieces (normal-anterior, normal-posterior, regen- er ating-anterior , and regenerating posterior) were transferred to pieces of parafilm for determination of the wet weight of tissue. The complete experiment was rep- licated, as described, three times. An ad- ditional replication was carried out on regenerating heads and tails so that 350 planarians were used in the experiments dealing with nucleotide composition.

R N A extraction RNA was extracted by a modification of

the procedure of Kirby ( '62) . A 0.5% solution of napthalene disulfonate (NDS) was adjusted to pH 7 with 0.01N NaOH. A phenol solution (P) was prepared by addition of 10 ml of distilled water and 0.1 gm of 8-hydroxyquinoline to 90 gm of distilled liquid phenol. Each group of worm pieces was homogenized in a glass tissue grinder with 2 ml of solution P and 4 ml of NDS solution. The homogenates were centrifuged at 15,000 g for ten minutes. The upper aqueous phase was removed and re- extracted with 2 ml of solution P. This process was repeated until the interfacial region between the two phases was clear. Two volumes of cold 95% ethanol were added to the aqueous phase and the RNA allowed to precipitate overnight at 0" C . The precipitate was then centrifuged for ten minutes at 15,000 g. The pellet was washed five times with large volumes of cold 70% ethanol, resuspended in 70% ethanol, and resedimented. The pellet was dissolved in 0.2 ml of 0.3N KOH and

TABLE 1 Results of analysis of M water

hydrolyzed overnight at 37" C. After com- pletion of the hydrolysis slightly over 0.01 ml of 28% perchloric acid was added to the hydrolysate to yield a pH of 2-3. The hydroIysate was then centrifuged at 15,-

Amount in ppm Characteristic

Total alkalinity Total solids Organic matter Calcium Magnesium Iron

16 64 6 32 1 0.01

000 g for ten minutes to remove potassium perchlorate and any DNA which may have been present.

Nucleotide composition determination Nucleotides in the supernatant solution

were resolved by a modification of the thin 7.1 PH

RNA COMPOSITION OF THE PLANARIAN, DUGESIA DOROTOCEPHALA 11

layer electrophoresis method of DeFilippes ('64). Aliquots of the supernatant solution were spotted with a microliter syringe onto several thin layer strips (Brinkman MN polygram, Cel 300/UV) 4 cm from one end. The strips were sprayed with citric acid-sodium citrate buffer (pH 3.3), placed into an electrophoresis tray (Gelman model 51170) with more of the same buffer and subjected to voltage gradients of 20 volts/ cm for 4-5 hours. The positions of the spots corresponding to the four nucleotides were located and marked on the dried strips by examination under ultraviolet light.

The cellulose of each of these regions was then quantitatively removed and eluted in a centifuge tube with 2 ml of 0.2N HCl for 24-48 hours. Blank control strips were treated in the same way (in- cluding electrophoresis) and corresponding regions removed and eluted in the same manner. The quantity of nucleotide was determined by measurement of the 0.D at 260 m!.i in a Zeiss spectrophotometer using the speciiic molar absorbancy values for each nucleotide from the literature (Beaven et al., '55).

Gel filtration chromatography Two kinds of columns were used for gel

filtration fractionation, a K 15/90 column (Pharmacia Fine) packed with Sephadex G-100 (1.5 X 90 cm, 154 ml bed volume) and one packed with Sephadex G-25 (1.5 X 42 cm, 74 ml bed volume). Both columns were equilibrated with 0.05 M sodium phosphate buffer, pH 6.7, prior to use. Flow rates were 9 and 45 ml per hour respectively. U.V. absorbancy of the outflow was monitored with a Uvicord I (LKB Instru- ments) monitor recorder.

Sucrose density centrifugation The RNA extract was also fractionated

by means of a 5-20% sucrose gradient (5 ml total volume) made up in 0.05 M sodium phosphate buffer, pH 6.7. The sucrose gradients were prepared by layer- ing 20, 15, 10 and 5% sucrose solutions (1.1, 1.2, 1.3, and 1.3 ml respectively) and allowing interdiffusion at 5" C overnight. Samples, layered on top of the gradient, were centrifuged for five and one-half hours at 37,000 rpm (Beckman SW50 rotor) at the same temperature. After centrifugation the gradients were fractionated by means of an ISCO Model D Density Gradient Fractionater and the U.V. absorbancy of the outflow recorded with a Uvicord I (LKB Instruments) monitor recorder.

RESULTS The average ODzso/ODzao for six nonhy-

drolyzed samples was 0.55 while the OD~SO/ ODsso was 0.48. The measures of spectrum profile indicate a good RNA preparation that is relatively free of protein and ex- tractant solutions, The average yield of material obtained was 1.19 mg/gm wet weight of planarian.

Table 2 shows the mole percent of each of the four nucleotides obtained from the hydrolysates of the extracts from the head and tail regions of normal and regenerating planarians. It is evident from a com- parison of the columns of table 2 that there is no significant difference in the base compositions between the RNA extracts of head and tail regions or between normal and regenerating planarians. Standard Deviations of the Means have been calculated according to the method shown in the appendix.

Figure 1 is a fractionation profile from the unhydrolyzed RNA preparation that is

TABLE 2 Mole percent nucleotide composition of the RNA in the anterior and posterior regions of

regenerating Dugesia dorotocephala

Normal Normal Regenerating Regenerating anterior posterior anterior posterior

Cytidylic A. 17.4 f 0.7 18.01 0.7 17.8 f 0.4 18.4 -C 0.4

Adenylic A. 26.8 2 0.7 26.0f 0.7 27.0 f 0.4 26.7 .C 0.4

Guanylic A. 32.1 2 0 . 7 31.5 2 0.7 30.6 2 0.4 30.8 -C 0.4

Uridylic A. 23.6 f 0.7 24.5 & 0.7 24.5 f 0.4 24.2 f 0.4


Fig. 1 Resolution of planarian RNA by sucrose density centrifugation.

typical of those obtained by the sucrose density centrifugation.

Figure 2 is a G-100 gel filtration profile of the non-hydrolyzed RNA. It is interesting to compare the profiles of figures 1 and 2. Peaks I1 and I11 of figure 1 would, by their relative sizes and positions in the gradient, correspond approximately to those anticipated for the 18s and 28s ribosomal RNA components. Since Peak A of figure 2 is eluted in the void volume of the column and hence is comprised of material of molecular weight greater than 100,000, it would be comprised of the same material giving rise to Peaks I1 and I11 or figure 1. In agreement with this, the area under Peak A, as well as the summed areas under I1 and 111, both make up 75% of the area under the sample profile.

Peaks B and C of figure 2 are of molecular weights somewhat less than 100,000, Their elution positions suggest that they are most likely 5s ribosomal and 4s transfer RNAs (Watson and Ralph, '67; Knight and Darnell, '67; Galibert et al., '66). Peak D which accounts for approximately 12% of the total area under the sample profile is low molecular weight material. From its elution position it would appear to be of molecular weight of 1000 or less. Prelimin- ary inquiry into the identity of this peak indicates that it is made up primarily of ATP and ADP (Wade et al., '68) and is not the result of a degradation of the RNA. Peak I of figure 1 is comprised of the material giving rise to Peaks B, C and D of figure 2. The finding of the low molecular weight Peak D was unexpected and means that the values given in table 2 are high in adenylic acid, which does not, however, invalidate the finding that there was no difference in RNA base composition between the four treatment groups. In order to correct for the non RNA adenylic acid contribution to the values of table 2 we extracted three groups of head pieces and three groups of tail pieces, using the same procedure as before, and separated the low molecular weight fraction on a Sephadex G-25 column. In these six replications we found that an average of 10.3% of the sample, by weight, was in this low molecular weight fraction. There was no signifi-

Fig. 2 Resolution of planarian RNA by elution through a Sephadex G-100 column.

RNA COMPOSITION OF THE PLANARIAN, DUGESIA DOROTOCEPHALA 13

cant difference between heads and tails in this regard. The standard deviation of the individual measurements was 2.2%.

Averaging the values in each row of table 2 and correcting for the contribution of the non RNA adenylic acid of Peak D one obtains 20.0, 18.0, 35.0 and 27.0 for the respective mole percents of the cytidylic acid, adenylic acid, guanylic acid and uridylic acid in the RNA of the planarians. Excluding Peak D, Peak A accounts for 85% of the total RNA, Peak B 3%, and Peak C 12%.

DISCUSSION

It is clear that neither the amount nor the overdl base composition of the RNA of the planarians employed in these experiments shows any difference between the anterior and posterior halves. Nor is there any difference in this base composition of the RNA extracted from either half between nonregenerating planarians and ones that have regenerated for 24 hours. These results are contrary to those reported by Lindh ('57). An explanation for this negative result is evident from resolution of the various molecular weight species of the RNA by gel filtration and sucrose density gradient centrifugation.

The various molecular weight species of RNA which we resolved by these methods, as well as their relative amounts, were en- tirely typical of those which have been found in other species. Peaks 111, I1 and B probably correspond to the 28S, 18s and 5s ribosomal RNA subunits. Peak C probably corresponds to the 4s transfer RNA. Our methods do not resolve a separate peak for messenger RNA, however, one might expect it to occur as a polydisperse RNA through the general region in which the ribosomal RNA is eluted.

One can thus identify about 85% of the RNA as ribosomal and about 12% as transfer RNA. In other organisms, e.g. bacteria, approximately 80 to 85% of the total RNA is ribosomal, about 10 to 15% is transfer, and only about 3 to 5% is messenger. Since the relative amounts of ribosomal and transfer RNA which we found for planarians accord well with these typical proportions, it would be reasonable to suppose that the proportion of messenger RNA would also be about the same.

The constancy of overall RNA base composition which we found is therefore not surprising. Changes in base composition might be anticipated in messenger but not in ribosomal RNA, but, even large changes in the base composition of the messenger would be obscured by the relatively large amount of ribosomal RNA present. In view of such considerations, the differences in base composition reported by Lindh ('57) for the total RNA are surprising and need to be confirmed.

The contamination of the RNA extracts with the low molecular weight peak de- serves mention as a cautionary note for other investigators. especially those meas- uring RNA turnover by 32P incorporation into RNA purified by the present method. To our knowledge this contaminant has not been previously reported. With this same procedure for extraction and purification we found that rat liver preparations yielded a similar low molecular weight contaminant but log phase E. CoZi did not. This peak, in planarians, labels relatively rapidly with 32P when it is supplied in the form of inorganic phosphate. Two other facts, in addition, show it is not a degradation product of high molecular weight RNA. First is that addition of Bentonite (which inhibits RNAase) does not reduce this peak. Second is that thin layer chromatography shows the peak to be mostly comprised of ATP and ADP rather than the oligonucleo- tides, monophosphates, ribosides, or free bases which would result from RNA degradation.

We have, in the present experiments, only sampled regenerating worms 24 hours after transection. Other experiments (Morita and Best, '68; Best, Hand and Rosenvold, 6'8; Goodman, Best and Riegel, '68) indicate an increase in cell division and RNA synthesis beginning around ~ 4 0 hours after transection. However, in view of our findings, this limitation should not alter the results or conclusions of the present study.

APPENDIX The standard deviations of table 2 were

calculated in the following way. Let XU denote an individual determination of the value to be entered in the ith row and jth column of the table. Let denote the


mean of these for this ~th cell. xi, is the value actually entered in the table. Since there is no reason, from either the data or the method of measurement, to expect any difference in the precisions with which these various Xi, are determined, the best estimate of the variance of Xi, will be the pooled estimate given by the expression

2 (Xii-%u)a . - -.

Var (Xu) = all 66 - 16

where 66 is the total number of individual measurements and 16 is the number of cells in the table. The standard deviations of the values in the first two columns wil l thus be

StdDevyij = VVar (Xu)/3 and for the second two columns will be

Std Devxij = \Nar (X3)/4.

LITERATURE CITED Beaven, G. H., E. R. Holiday and E. A. Johnson 1955 Optical properties of nucleic acids and their components. In: The Nucleic Acids, Vol. 1 E. Chargaff and J. N. Davidson, eds. Academic Press.

Best, J. B. 1967 The neuroanatomy of the planarian brain and some functional implications. In: The Chemistry of Learning: A n Evaluation of planarian research and other invertebrated preparatives. W. Corning and S. Ratner, eds.

Best, J. B., and E. Elshtain 1966 Biophysics of unconditioned response elicitation in planaria by electric shock. Worm Runner’s Digest, 8:

1966 Unconditioned response to electric shock: Mechanism in planarians. Science, 151:

Best, J. B., E. Elshtain and D. Wilson 1967 Single monophasic square-wave electric pulse excitation of the planarian Dugesia doroto- sephala. Jour. Comp. and Physiol Psychol., 63:

Best, J. B., S. Hand and R. Rosenvold 1968 Mitosis in normal and regenerating planarians. J. Exp. Zool., (in press).

Best, J. B., M. Morita and J. Noel 1968 Fine structure and function of planarian goblet cells. Jour. Ultrastructure Res., (in press).

Best, J. B., R. Rosenvold, J. Souders and C. Wade 1965 Studies on the incorporation of isotopic- ally labeled nucleotides and amino acids in planaria. J. Exp. Zool., 159: 397-404.

Best, J. B., and I. Rubinstein 1962 Maze Learn- ing and associated behavior in planaria. J. Comp. and Physiol. Psychol., 55: 560-566.

8-24.

707-709.

198-207.

- 1962 Effect of environmental familia- rity on feeding in planaria. Science, 135: 916- 917.

Brgnsted, H. V, 1955 Planarian regeneration. Biol. Rev., 30: 65-126.

Clement, N. 1944 Les Acides pentose nucleiques et la regeneration. Ann. SOC. rov. Zool Belg., 75: 25-33.

Corning, W., and E. R. John 1961 Effect of ribonuclease on retention of conditioned re- sponses in regenerated planarians. Science,

DeFilippes, F. M. 1964 Nucleotides: Separa- tion from an alkaline hydrolysate of RNA by thin layer electrophoregis. Science, 144: 1350- 1356.

Galibert, F., J. C. Ldong, C. J. Larsen and M. Boiron 1966 Comparative kinetics of synthesis of transfer RNA and 5s RNA in bacteria and mammalian cells. J. Col. Biol., 12: 385-390.

Goodman, A. B., J. B. Best and V. Riegel 1968 (in preparation).

Henderson, T. R., and R. E. Eakin 1961 11- reversible alteration of differentiated tissue in planaria by purine analogues. J. Exp. Zood.,

Jacobson, A. L. 1963 Learning in flatworms and annelids. Psychol. Bull., 66: 74-94.

Kirby, K. S. 1962 Ribonucleic acids 11. Im- proved preparation of rat-liver ribonucleic acid. Biochem. Biophys. Acta, 55: 545-546.

Knight, J. E., and J. E. Damell 1967 Distribu- tion of 55 RNA in HeLa cells. J. Mol. Biol., 28:

Lindh, N.O. 1957 The nucleic acid composition and nucleotide content during regeneration in the flatworm Euplanaria polychroa. Arkiv f.

McConnell, J. V. 1966 Comparative physiology: Learning in invertebrates. Ann. Rev. Physiol.,

Morita, M. 1965 Electron microscopic studies on planaria. I. Fine structure of the muscle fiber in the head of the planarian. Dugesin dototocephala. Jour. Ultrastructure Res., 13:

Morita, M., and J. B. Best 1965 Electron microscopic studies on planaria 11. Fine structure of the neuronsecretory system in the planarian Dugesia dorotocephala. J. Ultrastructure Res.,

134: 1363-1365.

146: 253-263.

491-502.

ZOO^., 11: 153-166.

28: 107-136.

383-395.

13: 396-408. - 1967 Electron microscopic studies on planaria 111. Some observations on the fine structure of planarian nervous tissue. J. Exp. Zool., 161: 391412.

Wade, C., V. R. Riegel and A. B. Goodman 1968 ( in preparation).

Watson, J. D., and R. K. Ralph 1967 Stability of 5s Ribonucleic acid in mammalian cells. J. Mol. Biol., 26: 541-544.

Wolff, E. 1962 Recent research on the regeneration of planaria. In: Regeneration. D. Rudnick, ed. The Ronald Press, New York.

RNA composition of the planarian, Dugesia dorotocephala

Documents

Transcript of RNA composition of the planarian, Dugesia dorotocephala