Transcription during cell differentiation in Naegleria gruberi. Preferential synthesis of messenger...

Biochimica et Biophysica Acta, 312 (1973) 52-71 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 97679

TRANSCRIPTION DURING CELL DIFFERENTIATION IN N A E G L E R I A

G R U B E R L PREFERENTIAL SYNTHESIS OF MESSENGER RNA

CHARLES WALSH and CHANDLER FULTON

Department of Biology, Brandeis University, Waltham, Mass. 02154 (U.S.A.)

(Received January 5th, 1973)

SUMMARY

Amebae of Naegleria gruberi undergo phenotypic transformation into flagellates when the aqueous environment is changed from axenic growth medium to non- nutrient buffer. This differentiation, which requires 80 min at 25 °C, is dependent on transcription during the first 40 min. The nature of the RNA synthesized during differentiation is the subject of this report. Net synthesis of RNA continues for the first 30 min after differentiation is initiated, but thereafter degradation exceeds synthesis leading to an overall decrease of 6 % in total RNA per cell by 90 min. [14C]- Uracil is incorporated into RNA during differentiation. Incorporation is linear until roughly 10 min before the appearance of flagella, at which time the rate of incorporation declines. Sucrose gradient analysis has been used to compare the RNA synthesized by cells differentiating in buffer versus amebae in growth medium. There is proportionally more synthesis of messenger-like RNA and less of rRNA in differentiating cells. The messenger-like RNA synthesized during differentiation is heterogeneous in size, but very large molecules (> 45 S) have not been found. This rapidly- labeled RNA is first present in the nucleoplasm, and moves rapidly to the cytoplasm, reaching equilibrium (nucleus/cytoplasm) within 10 min. In the cytoplasm it is found associated with polysomes, where it has the EDTA and ribonuclease sensitivity expected of mRNA. These observations support the interpretation that the rapidly- labeled RNA is mRNA. It is not yet known whether this mRNA includes the RNA essential for differentiation.

INTRODUCTION

Transcription and translation are required for the phenotypic transformation of Naegleria amebae into flagellates (Fulton, C. and Walsh, C., unpublished). Amebae grown axenically in a partially defined medium differentiate into flagellates when transferred to non-nutrient buffer. At 25 °C, flagellates are first seen about 65 min after the change in environment; 50 % of the cells have flagella at 80 min, and > 90 of the cells have differentiated by 100 min. Differentiation involves a complete re- structuring of the cell and synthesis of all the organelles of the flagellar apparatus,

RNA SYNTHESIS D U R I N G NAEGLERIA D I F F E R E N T I A T I O N 53

including flagella 1. RNA synthesis is required during the first 40 of the 80 min required for 50 ~ differentiation, and protein synthesis during the first 60 min (Fulton, C. and Walsh, C., unpublished).

The Naegleria system of rapid and synchronous differentiation is advantageous for studying the control of transcription. This paper provides a foundation, giving both a general description of RNA synthesis during differentiation and of methods for studying RNA in Naegleria. These studies are a necessary preliminary to any attempt to determine which RNA species are required for differentiation.

METHODS

Cell culture Naeoleria 9ruberi strain NEG-M was grown axenically at 28 °C in suspension

culture in Medium M (0.5 ~o (w/v) Difco yeast extract, I0 ~ (v/v) fetal calf serum dialyzed against water, 0.3 mM L-methionine, 30 mM dextrose, and 1 mM phosphate buffer (pH 6.6)) (Fulton, C., unpublished). Cultures were maintained in 125-ml DeLong flasks with 20 ml of medium, and were shaken at 80 oscillations per min (3.8-cm strokes). Under these conditions amebae grow with a doubling time of 8 h and reach a stationary phase density of 2 • 106-3 • 106 cells/ml.

Differentiation Conditions for ameba-to-flagellate transformation have been previously de-

scribed in detail 2. Suspensions of amebae, usually late growth or early stationary phase, at 1.0" 106-2 .5 " 106 cells/ml were harvested by centrifugation at room temperature in a clinical centrifuge (International Equipment Co. Model CL with No. 215 head) for 45 s at a rheostat setting of 6. The centrifuge was quickly brought to a stop with the aid of a mechanical brake, and the supernatant decanted. The pellet of amebae was suspended in Tris-KClbuffer (10mM KC1, 2 mM Tris-HC1, pH 7.2 at 25 °C). This initiates differentiation and all times are measured from this point. The resulting cell suspension was centrifuged again in the same way, the cells were resus- pended in Tris-KC1 buffer, and centrifugation and resuspension repeated a third time. The final cell suspension was incubated in Tris-KCl buffer, usually 10-15 ml in a 125-ml flask, at 25 °C in a shaking water bath at 100 oscillations per min (2.5-cm strokes). The time from first suspension in Tris-KC1 buffer, initiation, until the cell suspension was placed in the bath was typically 3.5 min.

The extent of differentiation was evaluated by fixing small aliquots of cell suspension in Lugol's iodine and counting the percentage of cells with flagella using phase-contrast optics 2. In all reported experiments involving differentiating cells, at least 80 ~ of the ceils formed flagella with kinetics similar to those in Fig. 2.

RNA extraction and purification The standard detergent and phenol methods of RNA extraction yielded only

RNA fragments of low molecular weight, presumably produced by high ribonuclease activity in Naegleria cell extracts. We have developed a method, based on that of Solymosy et al. 3 using diethyl pyrocarbonate as a ribonuclease inhibitor, that is ef- fective and convenient. Since diethyl pyrocarbonate has been found to cause cleavage of the purine ring of adenine when reacted with the pure base 4, there has been debate about its use to prepare RNA. It has also been reported to cause destruction of the

54 C. WALSH, C. FULTON

infectivity of purified virus RNA 5-7. Incubation of purified RNA with high concen- trations of diethyl pyrocarbonate at room temperature for 8 h or more results in changes in the ultraviolet spectrum and possibly some degradation s . On the other hand, diethyl pyrocarbonate has been used in the preparation of polysomes capable of continued protein synthesis 9, transfer RNA capable of activation in vitro ~°, and RNA which can serve as a template for protein synthesis in a cell-free system ~ ~. The ability to recover RNA with intact biological properties seems to be a function of the conditions of extraction and especially the presence of protein which may serve to protect RNA s. The recurrent observation 5-~ that the presence of diethyl pyrocarbonate during the extraction of RNA from cells and tissues does not lead to any changes in the sedimentation pattern or ultraviolet spectrum of the RNA extracted indicates its suitability for the work reported here.

Extraction solution contains 1 ~ (w/v) sodium dodecyl sulfate, 5 mM MgCI2, 50 mM Tris-HCl (pH 7.7). Aliquots of the solution are dispensed to test tubes on ice, and 3 ~ (v/v) diethyl pyrocarbonate is added less than 10 min before use. The sodium dodecyl sulfate forms a precipitate which is ignored. The extraction solution is usually added at a ratio of 3.0 ml of solution per 107 cells but one-half this amount has been used successfully. The solution, a weak emulsion, is thoroughly mixed with a vortex mixer just before addition to cells. Cells are pelleted using a clinical centrifuge, the supernatant is decanted, and the centrifuge tube is placed on ice. Immediately a pre- measured quantity of ice-cold extraction solution is poured onto the pellet of cells, and the tube is agitated for a few seconds on a vortex mixer and placed on ice. The cells lyse immediately on suspension in the extraction solution and this procedure al- lows breakage and thorough mixing within seconds of pelleting the cells.

Lysates are warmed to 37 °C for 5 min, and then NaCI is added at the ratio of 0.1 g per 1.0 ml of extraction solution. Lysates are mixed on a vortex mixer for ap- proximately 30 s and incubated at 37 °C for an additional 5 min. At the end of the second incubation lysates are cooled to 0 °C on ice and then centrifuged at 10 000 x g for 20 min at 4 °C.

The supernatant containing the RNA is poured into 2 vol. of ice-cold 95 ethanol and kept at -- 16 °C for 2 h or more. The precipitated RNA is collected by centrifugation at 10 000 x g for 10 min at 4 °C and dissolved in Buffer 1 (1 mM MgC12, 10 mM Tris-HC1, pH 7.2). The resulting solution is centrifuged at 20 000 x g for 20 min to remove any insoluble material. The final supernatant is stored at -16 °C.

RNA is extracted from cytoplasmic or soluble cytoplasmic fractionsl(see below) by adding sodium dodecyl sulfate (25 ~ (w/v) solution in 20 ~o (v/v) ethanol) to a concentration of 1 ~o (w/v) and then diethyl pyrocarbonate to a concentration of 3 (v/v). This suspension is mixed and then processed as described for lysates from cell pellets.

This extraction procedure typically gives 80 ~ recovery of RNA from amebae or flagellates. The ratio of absorbance at 260-280 nm of the final preparation usually is greater than 2.0 indicating very little protein contamination. RNA prepared in this way is free of ribonuclease activity as judged by the absence of hydrolysis when incubated at 37 °C for up to 3 h.

RNA extracted from cells labeled with [3H]adenosine during growth is treated with 10/~g/ml of electrophoretically purified deoxyribonuclease (EC 3.1.4.5, Worth- ington Biochemical Corporation) at 25 °C for 10 min.

RNA SYNTHESIS DURING NAEGLERIA DIFFERENTIATION 55

RNA determinations The concentration of RNA in solutions prepared as described above is esti-

mated by measuring the absorbance at 260 nm (assuming ~lr'l°/°cm = 200). RNA in cells is determined by a slight modification of the method of Munro

and Fleck x2, in which we minimize hydrolysis time in KOH to prevent protein contamination. Using purified Nae#leria RNA and whole cell extracts we found that 60 min heating in 0.3 M KOH is sufficient to solubilize all of the RNA (using 5 • 10 6

cells/ml KOH). This was determined by following the kinetics of the release of material absorbing at 260 and 232 nm and the release of ribose as determined by the orcinol method ~3. The ultraviolet spectrum of the RNA fraction of whole cells produced by this method is very similar to that obtained from hydrolysis of purified Naeoleria RNA.

Sucrose 9radients Linear 5-20 ~ (w/w) sucrose gradients of 4.5 ml in Buffer 1 are overlaid with

0.5-ml samples and centrifuged in the SW-39 head of a Spinco Model L ultracentrifuge at 37 500 rev./min for 210 rain at 20 °C. Sucrose solutions are treated with diethyl pyrocarbonate to destroy nuclease activity as described by Solymosy et al. 3.

Radioactivity is determined by precipitating each fraction at 0 °C with an equal volume of ice-cold 10 ~ (w/v) trichloroacetic acid in the presence of 20/~g of yeast nucleic acid (Schwartz Laboratories) as carrier. After more than 20 min at 0 °C the samples are collected on 24-mm filters (Millipore Corporation type HA or Schleicher and Schuell type B-6 membrane filters, or in later experiments Reeve Angel type 934AH glass-fiber filters). Filters are washed with ice-cold 5 ~ trichloroacetic acid and dried under an infrared lamp (membrane filters) or in a vacuum oven at 100-110 °C (glass-fiber filters). Filters are counted in 4.0 ml of scintillation fluid (6.0g/l 2,5-diphenyloxazole plus 0.1 g/1 1,4-bis-2-(5-phenyloxazolyl)-benzene in toluene) in 1 dram shell vials placed inside standard scintillation vials.

In order to facilitate comparison of RNA patterns between gradients, patterns of fractions are plotted as a function of their volume from the bottom of the gradient. This is calculated by dividing the total volume of the gradient by the total number of drops, and assuming the drop size within a gradient to be constant. Drop size varied between gradients, and presenting the data as a function of volume rather than drop number increases the reproducibility of the patterns.

Cell fractionation The observed fragility of Naeoleria nuclei, the suspected presence of high

nuclease activity, and the qualitative nature of most nuclear isolation methods prompt- ed the development of the method described below, based on that of O'Brien 14.

Amebae washed with Tris-KC1 buffer or differentiating cells in Tris-KCI buffer are pelleted in a clinical centrifuge for 45 s at a setting of 6, the supernatant is decanted and the tube is drained briefly and placed on ice. Ice-cold Buffer 2 (0.075 (v/v) Nonidet P.40 (Shell Chemical Ltd), 6 mM MgCI2, 25 mM KCI, 50 ktg/ml poly- vinylsulfate, 10 mM Tris-HC1 pH 7.4 at 25 °C) containing 1 M sucrose is added at a ratio of 0.5-1.0 ml per 10 7 cells and the tube gently shaken, just enough to suspend the ceils. After 3 min on ice, during which time the cells lyse, the lysate is mixed on a vortex mixer for 1-2 s to separate the nuclei from loosely attached membranous


material. Up to 1.0 ml of lysate is layered over 1.0 ml of Buffer 2 containing 1.62 M sucrose in a 3.5-mi (10.9 mm x 77.2 mm) polycarbonate centrifuge tube (Ivan Sorvall No. 270). This tube is centrifuged in the HB-4 head of a Servall RC-2 centrifuge at 4 °C for 12 min (timed from reaching speed) at 10 000 ×9 and braked to a stop. The upper and lower sucrose layers are carefully removed together, using a chilled capil- lary pipette, paying particular attention to remove any material at the interface of the two layers of sucrose. This supernatant is designated the cytoplasmic fraction, while the pellet consists of nuclei (Fig. 1B).

Fig. 1. Nuclear and nucleolar fractions. Amebae were lysed and fractionated as described in Methods. A, ameba with two nuclei in the upper right cytoplasm; B, isolated nuclei; C, nucleoli. All at 1000 ×.

The efficiency of this procedure was determined by hemocytometer counts with phase contrast optics of fractions stained with an equal volume of 0.5 ~ toluidine blue in Buffer 2. Cell lysis under these conditions was found to be complete, with no intact cells remaining after 3 min of incubation. Axenically grown amebae were found to have an average of 1.3 nuclei per cell by counting stained cells. In five separate cell fractionations, the average recovery of nuclei was 105 ~ with less than 1 ~o of the nuclei remaining in the cytoplasmic fraction.

The cytoplasmic fraction is subdivided essentially as described for slime molds by Cocucci and Sussman is. The fraction is diluted with 3 vol. of Buffer 2 and centrifuged at 10 000x9 for 10 min at 4 °C. The supernatant is designated the soluble cytoplasmic fraction while the pellet is designated the particulate cytoplasmic fraction.

Nucleoli are prepared from the nuclear fraction by sonication 16. The nuclear pellet is suspended in Buffer 2 containing 1 M sucrose and 1.0-ml aliquots in 15-ml conical plastic centrifuge tubes are treated at a setting of 2 (approx. 2.3 A) with a small conical probe on a Branson Sonifier (Model LS75) for 10 s while the tube is immersed in an ice-water bath. Up to 1.0 ml of the resulting sonicate is then layered over 1.0 ml of Buffer 2 containing 1.62 M sucrose and centrifuged as described for nuclei. The supernatant is designated the nucleoplasmic fraction while the pellet consists of nucleoli (Fig. IC).


Quantitation of this procedure with toluidine blue staining and hemocytometer counts was facilitated by the large size of the nucleoli and their intense orange-red staining. Nuclei proved very sensitive to sonication while nucleoli were very resistant. Doubling the length of sonication did not reduce the yield of nucleoli. The conditions described provided preparations where intact nuclei could not be found and the average recovery of nucleoli was 99 ~o (for 6 preparations). Some small pieces of material which stain like nucleoplasm were occasionally seen on nucleoli prepared in this way.

A polysomal fraction is prepared as suggested by Cocucci and Sussman 15 using the non-ionic detergent Cemusol NPT-12 (Soci6t6 des Produits Chimiques de Syn- th~se). Cells are pelleted by centrifugation in a clinical centrifuge at a setting of 6 for 45 s, the supernatant is decanted and the cells lysed by the addition of ice-cold Buf- fer 3 (20 mM KCI, 40 mM MgCI 2, 50 mM Tris-HCl, pH 7.4 at 25 °C) containing 4 ~o (v/v) NPT-12 and 5 ~ (w/v) sucrose. The lysate is mixed briefly and then centrifuged at 10 000 ×# for 105 s at 4 °C. The resulting supernatant is immediately layered over linear ice-cold 7 4 2 ~ (w/v) sucrose gradients in Buffer 3 and centrifuged in the SW-39 head of a Spinco Model-L ultracentrifuge at 4 °C for 120 min at 26 000 rev./ min. Fractions were collected into ice-cold test tubes by puncturing the centrifuge tubes as described above.

In order to extract RNA from the polysome region of sucrose gradients without loss or degradation, the fractions are collected into 2 vol. of ice-cold 95 ~ ethanol 17. Desired fractions are pooled, held at -16 °C for at least 2 h, and the RNA pelleted by centrifugation at 10 000 ×# for 20 min. The RNA is extracted by addition of extraction solution to the pellets as described above.

Radioactive compounds [2 -14C]Uracil (56.3 mCi/mole), [2,8-3H2 ]adenosine (7.15 Ci/mmole) and [G-

3H]lysine (3 Ci/mmole) were obtained from New England Nuclear Corporation.

RESULTS

RNA synthesis durin# differentiation When amebae differentiate into flagellates under the conditions used in this

study, there is no change in cell number and no net synthesis of RNA. It is not sour- prising that there is no increase in total RNA, since the cells have no external source of nutrients and therefore any synthesis during differentiation is dependent on endo- genous resources. Careful measurements indicated that the amount of RNA per cell increased about 5 ~ during the first 30 min and then decreased about 11 ~o during the next hour (Fig. 2B).

Isotopic precursors of RNA are incorporated during differentiation. For example, the incorporation of [14C]uracil into acid insoluble material accelerates for about 10 min and then is linear until about the time flagella begin to appear (Fig. 2A). The incorporation of uracil is restricted to RNA as shown by sensitivity (> 96 ~ ) to mild-alkali hydrolysis. Uracil incorporation indicates RNA synthesis but does not give an estimate of the magnitude of the synthesis. The decrease in total RNA after 30 min indicates that degradation exceeds synthesis, but in the absence of data on the labeling of the immediate precursor pools no measure of the absolute rate of RNA synthesis is possible 18,19

58 C. WALSH, C. F U L T O N

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Fig. 2. R N A synthesis during differentiation. (A) Incorporation of uracil. Differentiation of amebae from late growth phase cultures (1.3 • 106 per ml) was initiated at 0 rain as described in Methods. After the final suspension (6.5 min after initiation), [2-14C]uracil was added to give 0.15/~Ci/ml and 1.2 • 106 cells/ml. Samples (1.0 ml) were taken to 2.0 ml of ice-cold Tris-KCl buffer, cells were pelleted by centrifugation for 1 min in a clinical centrifuge, and then suspended in 2.0 ml of ice-cold 5 ~ (w/v) trichloroacetic acid. The precipitates were collected on membrane filters, washed with 5 ~ trichloroacetic acid, and the radioactivity measured. Flagellates were counted in Lugol's iodine fixed samples• (B) Net synthesis o f R N A . Differentiation of amebae from late growth phase cultures (8.0 • l0 s per ml) was initiated, and at intervals samples (0.5 ml) of the cell suspension were taken to 0.25 ml of ice-cold 0.6 M HC104 and the R N A content determined. Cell number was determined using a Coulter Counter. Percent flagellates, A; 14 C cpm, 0 ; #g RNA, O; cells/ml, V].

Classes of RNA synthesized RNA was isolated from cells at successive intervals during differentiation and

fractionated on sucrose gradients. No change in the distribution of total RNA in cells was seen by this method. To determine the kinds of RNA synthesized, cells were labeled with uracil for short intervals and the RNA examined on sucrose gradients.

To have a basis for comparison with differentiating cells, RNA synthesis in growing cells was examined by adding [14C]uracil to late log phase amebae suspended in growth medium at 28 °C, and after brief intervals isolating the RNA for analysis. Even after only 5 or 10 min of labeling most of the label is in ribosomal RNA (Figs 3A and B). What we know of the patterns of RNA synthesis found in growing cells is consistent with what is known from studies of other eucaryotic cells2°: rapid labeling of heterogeneously sedimenting RNA, processing of ribosomal R N A (rRNA) from higher molecular weight precursors, and synthesis of soluble RNA. The rapidly labeled heterogeneous RNA sedimenting with a broad peak at roughly 16-17 S is referred to as messenger-like RNA, since as is shown below much of this RNA has the characteristics of mRNA. The processing of rRNA, and the synthesis of soluble RNA, are not further considered here. Within about 20 min of labeling of growing

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Fig. 3. Sucrose gradient analysis o f R N A labeled dur ing growth and differentiation. (A and B) Growth . A late growth phase culture o f amebae (1.6 • 106 per ml) was adjusted to 1.0 ffCi/ml [2-14C]- uracil by addit ion o f a sterile 25 ffCi/ml solut ion in T r i s - K C l buffer. Incubat ion at 28 °C was con- t inued under growth condit ions. After 5 min (A) and 10 min (B) an al iquot o f cells (8.8 • 106 per ml) was removed, diluted with 4 vol. o f ice-cold Tr i s -KC1 buffer, and centrifuged in a clinical centrifuge for 1 rain. The superna tan t was decanted, and the R N A extracted f rom the pelleted cells. ( C - H ) Differentiation. Washed amebae f rom late growth phase cul tures (1.6 • 106/ml) were incubated in T r i s - K C l buffer at 25 °C and 1.5 • 107 cells/ml. Al iquots (0.6 ml) were removed to centrifuge tubes at 25 °C conta in ing 0.1 ml o f [~4Cluracil solut ion (25 ffCi/ml). Al iquots were taken at 5 min (C), 16 min (D), 27 min (E), 38 min (F), 49 rain (G), and 60 min (H) after initiation. Incubat ion with isotope was cont inued for l0 min , then 6 vol. o f ice-cold T r i s - K C l buffer were added and the suspension centrifuged in a clinical centrifuge for 1 min. The superna tan t was decanted and the R N A was extracted f rom the pelleted cells. R N A (equivalent to 8 .8 ' l06 cells in A and B and 6.4. l06 cells in C - H ) was analyzed on linear 5-20 % (w/w) sucrose gradients.

cells (not shown), the distribution of label resembles the absorbance at 260 nm (total RNA), though there is still higher specific activity in the region of the gradient containing RNA slightly smaller than the 17-S* rRNA. The progressive shift of pattern in growing cells toward equilibrium between labeling and total RNA indicates that in these short intervals messenger-like RNA is turning over as rRNA accumulates.

* The size o f r R N A was es t imated by coelectrophoresis with E. coli R N A on polyacrylamide gels 21. The two r R N A peaks are 17-S and 25-S, similar in size to the r R N A o f m a n y eucaryotic protists 22.


Differentiating cells were labeled for successive 10-min intervals. The labeling pattern is different from that in growing cells. Ribosomal RNA is much less evident and messenger-like RNA more evident (cf Fig. 3B with Figs 3C-3H). There are slight variations but the basic labeling patterns are similar throughout differentiation. The change from the growth pattern is already evident by the 5-15-min labeling interval, and remains similar through the 60-70-min labeling interval, which is after RNA synthesis is no longer required for differentiation (40 min). The change in pattern indicates that differentiating cells have shifted the relative proportions of synthesis of messenger-like RNA and rRNA. As compared to growing cells, differentiating cells show preferential synthesis of messenger-like RNA and proportionally limited synthesis of rRNA.

If differentiating cells are exposed to label for long intervals, most of the radioactivity is found in rRNA. For example, if these cells are labeled for 60 min the radioactivity pattern is similar to growing cells labeled for 10 min. The fact that label accumulates in rRNA on longer labeling indicates that even though synthesis of rRNA is proportionally reduced, the rRNA synthesized is more stable than the messenger- like RNA.

The labeling of growing cells was carried out with undisturbed cultures at 28 °C, whereas differentiating cells were disturbed mechanically (by repeated centri- fugations and resuspensions in transferring them to buffer) and were at 25 °C. Grow- ing cells can be labeled by suspending amebae in fresh growth medium, with or without washing in Tris-KC1 buffer, either at 25 or 28 °C. These various procedures affect the amount of isotope incorporated, but the pattern of incorporation is similar to that illustrated for the minimally disturbed cultures in growth medium (Figs 3A and B).

The marked change in the pattern of RNA synthesis in going from growth to differentiation conditions is the result of the shift-down to a non-nutrient environment and is independent of the differentiation 23. Although the observed pattern is not unique to differentiating cells, the pattern is obtained under the conditions re- gularly used to study the differentiation, and the RNA synthesized must include the RNA required for differentiation. The rest of this paper continues the description of RNA synthesis in cells differentiating in non-nutrient buffer.

lntracelhdar distribution of RNA Any attempt to assign a functional role to different classes of RNA requires

knowledge of their position in the cell. For example if the heterogeneous RNA synthesized during differentiation is in fact mRNA, then one would expect that much of it would be synthesized in the nucleoplasm and move to the cytoplasm where it would be found on polysomes.

The intracellular distribution of RNA was examined in cells labeled for 15 min during differentiation. Amebae were grown for 48 h in the presence of [3H]adenosine to label total cell RNA. This permitted calculation of the recovery of RNA in fractions, and examination of the distribution of RNA in fractions containing insufficient RNA to examine by absorbance. Differentiation was initiated by washing the cells into buffer. 15 min after initiation [1 ~C]uracil was added and the incubation continued for an additional 15 min. At this time the cells were fractionated into subcellular fractions, and the RNA from these fractions was analysed on sucrose gradients. The data are presented in Fig. 4.


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Fig. 4. Intracellular distribution of stable and rapidly labeled RNA. Amebae were grown for 48 h in the presence of 3.0/~Ci/ml of [2,8-3H2]adenosine. Differentiation was initiated, and 15 min after initiation [2-14C]uracil was added to give 2.5/~Ci/ml and 1.4. 107 cells/ml. After 15 min with isotope, an aliquot of cells (1.3 • 108) was diluted with 1.5 vol. of ice-cold Tris-KCI buffer and pelleted by centrifugation for 1 min in a clinical centrifuge. (A-G) The cells were lysed by suspension at 2.7 • 107 per ml in ice-cold Buffer 2 containing 1 M sucrose. The lysate was fractionated into subcellular components, and the RNA extracted from these fractions was analysed on linear 5 to 20 % (w/w) sucrose gradients. (H) An additional aliquot of the cells was taken at the same time for the preparation of a polysomai fraction (equivalent to 1.3 • 107 cells) which was analysed on a linear 7 to 42 % (w/v) sucrose gradient.

The distribution o f total R N A in the various subcellular fractions as revealed by 3H radioactivity is essentially the same as has been described in other eucaryotes 2°. This tends to corroborate the fractionation method and indicates that there has not been extensive degradation o f R N A during the cell fractionation. Where the fractions contained enough R N A to measure the absorbance pattern, this pattern and that for 31-t were superimposible. The proport ions o f R N A in various subcellular fractions are similar whether determined by 3H radioactivity or chemically (Table I). The results o f this inventory (Fig. 4 and Table I) indicate that the nucleus contains about 5 % o f the cell's R N A . There is a predominance o f 25-S R N A in the nucleus, and this is most ly localized in the nucleolus. The 25-S and 17-S R N A in the nucleoplasm, account ing for 1% o f the cell R N A , could be due to r ibosomes on the nuclear envel- ope 24. The cytoplasm contains the majority o f the cell R N A , with 4 % in the particulate cytoplasm. About 83 % of the R N A in the soluble cytoplasm is r R N A , with 25-S

62 C. W A L S H , C. F U L T O N

T A B L E I

S U B C E L L U L A R D I S T R I B U T I O N OF R N A S Y N T H E S I Z E D D U R I N G G R O W T H AND, D I F F E R E N T I A T I O N

Cells were labeled dur ing growth with [3H]adenosine and dur ing differentiation with [ '4C]uraci l a s described in Fig. 4. Siibcellular fract ions are as described in Methods .

Subcellular 14 C 3 H Total** fract ion cpm/lO a cells °~*/o cpm/lO a cells %* %*

Total 50 208 92 975 766 90 98 Nuclear 15 906 29 47 883 4 6 Cytoplasmic 38 916 71 1036 072 96 94 Nucleolar 8 038 15 23 722 2 2 Nucleoplasmic 4 695 9 16 830 2 1 Soluble

cytoplasmic 23 867 44 757 929 70 73 Particulate

cytoplasmic 3 279 6 40 008 4 6 Polysome 9 570 17 254 193 23 --

* Percentage o f nuclear plus cytoplasmic fractions. ** Tota l R N A content as determined chemically. Average o f two experiments .

and 17-S in a 2 : 1 ratio. Some 73 % of the RNAin the particulate cytoplasm is rRNA: this constitutes 3 % of the total rRNA in the cell and is presumed to be from mito- chondrial ribosomes.

The RNA synthesized during differentiation (14C radioactivity) obviously has different sedimentation characteristics than the bulk of the RNA (3H radioactivity) (Fig. 4) and the proportions are quite different (Table I). In the nucleus the newly synthesized ribosomal and preribosomal RNA species are confined to the nucleoli, while the 14C-labeled RNA in the nucleoplasm is smaller and heterogeneous. Most of the newly synthesized cytoplasmic RNA is in the soluble cytoplasm; this RNA is slowly sedimenting with a broad peak near the soluble region of the gradient. Thc new RNA in the particulate cytoplasm, about 12 % of that in the total cytoplasm, contains a greater proportion o f rRNA than the soluble cytoplasm.

The pattern of total RNA in the various subcellular fractions made extensive RNA degradation during the fractionation unlikely, and the RNA patterns obtained in other organisms with similar fractionation procedures have usually been accepted as representing the intracellular distribution of RNA. However, the indications of high nuclease activity in Nae#leria (see Methods) suggested caution in interpreting the patterns. Degradation or losses could be of two types, non-selective as to the RNA species involved leading to low recoveries without influencing the proportions, or preferential losses or degradations leading to a distorted RNA pattern. Preferential losses have been seen before, and in some cases traced to a greater sensitivity of mRNA than rRNA to nucleases 25. Some RNA is lost during cell fractionation, and roughly similar amounts are lost from total RNA and differentiation-labeled RNA (Table II). There is little loss during the initial fractionation of nucleus and cytoplasm, but losses occur during further subfractionation.

If all RNA losses were nonselective, the gradient patterns of fractions, corrected for losses, should sum to give back the pattern of the whole. The patterns as


TABLE II

RECOVERY OF RNA SYNTHESIZED DURING GROWTH AND DIFFERENTIATION

The data are from Table I.

14 C a H

(Nuclear + cytoplasmic)/total 1.09 1. I 1 (Nucleoplasmic + nucleolar)/nuclear 0.80 0.85 (Soluble cytoplasmic+particulate cytoplasmic)/cytoplasmic 0.70 0.77

usually presented (Fig. 4) cannot be added because the fraction volume is not precisely reproduced from gradient to gradient. In order to make the patterns additive it is necessary to correct for differences in the volume of fractions between gradients. This is done by plotting concentration in each fraction (cpm/ml) v e r s u s position of the fraction in the gradient. This method of presentation makes it possible to compare the amount of radioactivity in restricted portions of a gradient or between gradients by comparing the areas under the curves. Areas in this case are equal to the radioactivity. In order to allow patterns to be quantitatively compared, they were made additive by adjusting for losses using the data in Table II. This involves multiplying the value for each fraction from a gradient by a constant factor. It does not change the shape of the pattern but simply shifts the position of the curves along the ordinate (radioactivity scale). The results of this analysis of the data are shown in Fig. 5. Using these patterns it is possible to determine graphically whether fractions are additive.

All losses of total R N A (3H radioactivity) appear to be nonselective, in that in all cases the 3H patterns of fractions are additive. For example, in separation of the nuclear fraction into nucleolus and nucleoplasm only 86 ~o of the RNA was re- covered (Table II) but these two subfractions add up to give the same gradient pattern as the nuclear fraction (Figs. 5C and D). The additivity of total RNA fractions provides a visual indication that the method of analysis is valid.

There is one clear indication of selective degradation or loss in the newly synthesized RNA, shown by the failure of nucleus and cytoplasm patterns to sum to the total pattern. The non-additivity, indicated by the shaded areas in Fig. 5A, is due to the cytoplasm having a lower proportion of rapidly sedimenting RNA. The most ob- vious explanation for this is selective degradation of the higher molecular weight RNA species, presumably messenger-like RNA. This is the only discrepancy in the recovery of newly synthesized RNA. The nucleolar and nucleoplasmic patterns sum to that for the whole nucleus. The patterns of the soluble cytoplasm plus the particulate cytoplasm equal that for the cytoplasmic fraction, indicating that no further selective degradation of the cytoplasmic RNA occurred during subsequent fractionations.

The selective loss of some of the newly synthesized cytoplasmic RNA of higher molecular weight is most easily explained as a degradation of some RNA molecules during the lysis of the cells or during separation of nuclei from cytoplasm. To test this possibility cells were labeled as in the previous experiment but the fractionation procedure was modified. At the end of 15 min of incubation with [14C]uracil, a sample of cells was diluted with ice-cold buffer, sedimented, and the RNA extracted. This material provided a sample for total RNA without previous lysis of the cells. An aliquot of the same cells was lysed as in the previous experiment but instead of separa-

64 C. W A L S H , C. F U L T O N

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Fig. 5. Evaluat ion o f selective loss or degradat ion o f R N A dur ing cell f ract ionat ion. The pa t t e rns o f R N A distr ibut ion in Figs 4 and 6 were corrected for non-specific losses or degradat ion as explained in the text us ing the recovery data in Table I. In order to make possible the addi t ion o f the pa t te rns f rom var ious cell fractions, radioactivity is expressed as concentra t ion (cpm per 0.1 ml) rather than cpm per drop or fraction. This adjusts for smal l variat ions in drop size between gradients. All frac- t ions have been adjusted to 108 cell equivalents per gradient. The pat tern for the sum o f two curves was obta ined by addit ion o f cor responding points on each curve. The series o f curves on the left hand side o f the figure present the da ta for R N A labeled with [ '4C]uraci l dur ing differentiation. The right hand set o f curves is the da ta f rom the same fractions for R N A labeled with Ja i l ]adenosine dur ing growth. (A and B) The R N A pat tern o f nuclei ( O - O ) is added to that o f the total cy top lasm ( . - .) to give a curve ( . . . . . ) for compar i son with tha t seen in the unfrac t ionated lysate ( A - A ) . (C and D) The R N A pat tern o f nuc leoplasm ( O - O ) is added to the pat tern o f nucleoli (. -- .) to give a curve ( . . . . . ) for compar i son with that seen in unfrac t ionated nuclei ( A - A ) . (E and F) The R N A curve o f the part iculate cy top lasm ( O - O ) is added to that o f the soluble cy top lasm (. -- .) to give a curve ( . . . . . ) for compar i son with that o f the unfrac t ionated cy top lasm ( A - A ) . (G and H) The R N A profile observed in the nuclear plus part iculate cytoplasmic fraction ( O - O } is added to tha t o f the "qu ick cy top lasm" fraction (. -- .) to give a curve ( . . . . ) for comparisor~ with that o f unfract ionated cells ( A - A ) .

ing the nuclei by 12 min centrifugation through sucrose, the lysate was centrifuged for 105 s at 10 000 xg and the supernatant and pellet immediately extracted for RNA. This supernatant fraction is designated the "quick cytoplasm" fraction; the pellet contained nuclei and particulate cytoplasm. When RNA is extracted under these conditions the total sucrose gradient pattern (Fig. 6A) resembles that seen in the 10-min pulses (Figs 3C-3H). The newly synthesized cytoplasmic RNA (Fig. 6B) is larger

RNA SYNTHESIS DURING NAEGLER1A DIFFERENTIATION 65

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Fig. 6. Distr ibut ion on sucrose gradients o f R N A from cell fractions prepared rapidly. Amebae f rom stationary phase cultures (3.1 • l0 6 per ml), labeled with [al l ]adenosine as described in Fig. 4, were transferred to buffer. [2-14C]Uracil was added 15 min after initiation to give final concentra- tions of 3 ffCi/ml and 2.0' 107 cells/ml. After 15 min additional incubation a sample of cells was diluted with 10 vol. of ice-cold Tris-KC1 buffer and pelleted by centrifugation in a clinical centrifuge for 1 min. RNA was extracted from the pelleted cells. A second sample of cells was taken at the same time and pelleted. This cell pellet was lysed with ice-cold Buffer 2 containing 1 M sucrose and fractionated into a "quick cytoplasm" fraction and a nuclear plus particulate cytoplasmic fraction as discussed in the text. RNA extracted from these fractions was analysed on sucrose gradients as in Fig. 4. (A) RNA from 7.1 • 106 unfractionated cells. (B) RNA from the "quick cytoplasm" fraction of 1.4 • 1 0 7 cells. (C) RNA from the nuclear plus particulate cytoplasmic fraction of 1.4- 107 cells.

than observed in the previous fractionation (Fig. 4E). The sum o f the "quick cyto- p lasm" fraction and the nuclear plus particulate cytoplasm closely resembles that o f the total (Fig. 5G) indicating that selective degradat ion o f R N A was minimal under these fract ionation conditions.

In conclusion, the intracellular distribution o f R N A obtained by the stepwise fract ionation procedure (Fig. 4) appears reliable for all fractions except for rapidly labeled cytoplasmic R N A . For that class o f R N A the "quick cytoplasm" procedure (Fig. 6) is necessary to minimize degradation.

lntraeellular movement o f RNA The distribution o f newly synthesized R N A in cells provides a static picture.

These same fractionation methods can be used to examine the movement o f R N A within cells. The kinetics o f labeling o f various subcellular fractions is shown in Fig. 7. After a lag, all f r a c t i o n s - nucleus, soluble cytoplasm, and particulate c y t o p l a s m - incorporate [14C]uracil into R N A at a constant rate th roughout the experiment. After an initial interval, the p ropor t ion o f label in the three fractions remains constant even though all fractions continue to accumulate radioactivity in R N A (Figs 7 and 8). In growing amebae the nucleus contains 5 ~ o f the cell R N A (Table I) but in differentiating cells 25 ~ o f the accumulated label remains associated with the nucleus (Fig. 8). The nucleus continues to accumulate radioactive R N A in a constant propor t ion much larger than the steady state level. This suggests that a certain fraction o f all R N A being made in the nucleus remains there or that different classes o f R N A move to the cytoplasm at different rates with degradat ion in the cytoplasm just ba-


"°[/ / ; ° o.O.Or. r y /

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Fig. 7. The kinetics of labeling of cell fractions during differentiation. Amebae from late growth phase cultures (2.5 • 106 per ml) were transferred to buffer. 15 min after initiation [2-14C]uracil was added to 6/~Ci/ml and 9.3 - 104 cells/ml. 0.5-ml samples of cell suspension were taken to 4.5 ml of ice-cold Tris-KCl buffer and the cells pelleted by centrifugation for 45 s in a clinical centrifuge. Pelleted cells were lysed by suspension in 0.5 ml of ice-cold Buffer 2 containing 1 M sucrose, and fractionated into nuclei, soluble cytoplasm and particulate cytoplasm. Radioactivity was determined by precipitating each fraction with HC104 (0.2 M final concn) and collection of the precipitates on filters for liquid scintillation counting. Whole cells, ~,; soluble cytoplasm, (3; nuclei, ~; and particulate cytoplasm, F].

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Fig. 8. The proportion of radioactivity incorporated into cell fractions. The data from Fig. 7 are expressed as the per cent of the total incorporation found in nuclei, soluble cytoplasm, and particulate cytoplasm. Isotope was added at 15 rain.

lancing accumula t ion that would otherwise occur. Many interpretat ions of this

equi l ibr ium are possible. Of greatest interest are the results of labeling dur ing the first few minutes

(Fig. 7 insert; Fig. 8). Initially incorpora t ion is more rapid in the nucleus; after one min of labeling 50 % of the labeled R N A is in the nucleus. This propor t ion decreases as the propor t ion in the cytoplasm increases. Equi l ibr ium is reached within 10 min. These kinetics suggest initial synthesis in the nucleus and movement to the cytoplasm.

RNA associated with polysomes Most of the rapidly synthesized heterogeneous R N A is localized in the cyto-

plasm. If that R N A is messenger R N A it would be expected to be on polysomes. In order to prepare polysomes, cells were rapidly lysed, the nuclei and mi tochondr ia sedimented, and the superna tant layered on sucrose gradients and centrifuged. An a l iquot of the double-labeled cells used for the subcellular f ract ionat ion experiment

R N A S Y N T H E S I S D U R I N G NAEGLERIA D I F F E R E N T I A T I O N 67

of Fig. 4 was treated in this manner, with the results shown in Fig. 4H. The distribution of total 3H-labeled RNA is typical of this kind of preparation in other eucaryotic cells, with a sharp peak of monosomes and a broad peak of polysomes 2°'26. The newly synthesized t4C-labeled RNA is restricted to the polysome region and to the top of the gradient, and conspicuously absent from the area under the monosomes. This pattern indicates that little degradation of polysomes to monosomes occurred during the fractionation 27. The absence of any 14C label in the monosome region is not surprising, since there is little if any 14C-labeled ribosomal RNA in the soluble cytoplasm of these cells (Fig. 4F).

To determine whether the rapidly labeled RNA sedimenting with the polysomes is in fact part of the polysomes, this fraction was further examined using several procedures others have used to characterize polysomes 26. In one series of tests, the control gradient (Fig. 9A) is similar to that of the preceding experiment (Fig. 4H} except for the presence of some newly synthesized RNA (containing 14C) in the monosome region. This is presumed due to some degradation of polysomes to monosomes during the fractionation, which took longer in this experiment. In some other

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Fig. 9. Character iza t ion o f the po lysomal fraction. Amebae f rom late growth-phase cultures (7.4 - 105-9.6 • 105 per ml) , labeled dur ing growth as described in Fig. 4, were t ransferred to buffer. 4 mir~ after init iat ion [2-1¢C]uracil was added to give 3 #Ci /ml and 1.6 • 106 cells/ml. Incuba t ion was con- t inued at 25 °C for 16 min at which t ime the cell suspens ion was diluted with 15 ml ice-cold T r i s - K C l buffer and pelleted by centr i fugat ion for 1 min in the clinical centrifuge. The pelleted cells were lysed by the addi t ion o f 1.5 ml ice-cold Buffer 3 conta in ing NPT-12 as described in Methods . The resul t ing lysate was centr ifuged for 105 s (after reaching speed) at 10 000 × # at 4 °C and the superna tan t decanted. 0.4-ml al iquots o f this superna tan t were mixed with 0.1 ml o f ice-cold solution: A, 40 m M MgClz , 2 0 m M KC1, 50 m M T r i s - H C l (pH 7.4); B, 0.15 M E D T A (pH 7.4); D, buffer as in A with 10 #g o f pancreat ic r ibonuclease (EC 2.7.7.16, Sigma type X-A heated at 80 °C for 10 min in 0.15 M NaCI-0 .015 M sod i um citrate). Sample D was warmed to 25 °C for 2 rain and immedia te ly chilled in ice. Samples A and D were layered over ice-cold 4.5-ml linear 7-42 % (w/v) sucrose gradients prepared in the buffer used in A. Sample B was layered over a similar gradient prepared in the same buffer wi thout magnes ium. After the sucrose gradients were centrifuged, fract ions f rom gradients B and D were prepared for radioactivity measurement• Fract ions f rom gradient A kept at 4 °C were divided in half. A n equal vo lume o f a solut ion conta in ing 2.6 ktg/ml o f r ibonuclease (as above) was added to one set o f half-fractions. An equal vo lume o f the same buffer wi thout r ibonuclease was added to the second set. Both sets were incubated in an ice-water slurry for 60 rain and then prepared for radioactivity measurements . A, half-fract ions treated with buffer; C, half- fract ions treated with r ibonuclease.

68 c. WALSH, C. FULTON

organisms dissociation of polysomes to ribosomal subunits with EDTA has revealed an EDTA-insensitive RNA cosedimenting with polysomes but not associated with them 2s. In Naegleria, dissociation of ribosomes to subunits with EDTA prior to sedimentation leads to a loss of rapidly labeled RNA from the polysome region (Fig. 9B) suggesting that this RNA is in fact associated with the polysomal structure. There is no EDTA-insensitive residual radioactivity in the polysome region of the gradient. Treatment of the lysate with ribonuclease before sedimentation results in a loss of polysomes and proportional increase in monosomes (Fig. 9D). The shift of RNA from the polysome region to the monosome region is consistent with the view that the structure is held together by RNA and that small pieces of rapidly labeled RNA are retained with the monosomes 29. In the next test, half of each fraction of a gradient was subjected to mild ribonuclease digestion. The preferential sensitivity of the rapidly labeled RNA in the polysome region (Fig. 9C, cf. Fig. 9A) is consistent with the idea that the rapidly labeled RNA is relatively unprotected, as others have found polysome-associated mRNA to be 3°. These data show polysome associated rapidly labeled RNA has the characteristics usually associated with messenger RNA holding together ribosomes in a polysome structure 3°.

The rapidly labeled RNA associated with polysomes was examined. RNA extracted from the polysome region of gradients comparable to Figs 4H and 9A was examined on sucrose gradients comparable to Figs 4E and 6B. The RNA in the polysome region is predominately rRNA but the newly synthesized RNA is almost entire- ly messenger-like RNA (Fig. 10).

The polysomes of differentiating cells are actively engaged in protein synthesis, as indicated by their rapid incorporation of [3H]lysine (Fig. 11). The absence of radioactivity coincident with the monosome peak supports the conclusion that this polysome fraction was undegraded 29. Ribonuclease treatment of a sample of this polysome fraction degraded the polysomes to monosomes (as in Fig. 9C). The radioactivity was found in the monosome peak, indicating that it was associated with the polysomes rather than simply cosedimenting with them. Treatment with EDTA led to degradation of the polysome structure to ribosomal subunits (as in Fig. 9B). In this case the lysine radioactivity was released to the top of the gradient, indicating that while the radioactivity is associated with ribosomes it is not incorporated in them. All these observations support the conclusion that the radioactivity is in nascent polypeptides in the polysomes.

DISCUSSION

RNA synthesis and processing in Naegleria are basically similar to those described in other eucaryotic cells, especially HeLa 2°. They occur faster in Naegleria, on a time-scale more closely resembling that of procaryotes. For example, rapidly- labeled RNA is found on Naegleria polysomes within 5 min (at 25 °C) after addition of label (unpublished observation), whereas this process requires at least 15 min (at 37 ~C) in HeLa 28. Another difference is the apparent absence in Naegleria of rapidly labeled RNA with sedimentation values greater than about 45 S, i.e. heterogeneous nuclear RNA 2°' 31. This class of RNA has also been reported to be absent from the cellular slime mold, Dictyostelium discoideum 32. The conclusion that these organisms lack heterogeneous nuclear RNA must be considered tentative, however, since it has


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1.01 2.(31 3,01 4.01 5,o' o ~ n l ~ - t " I ~ I ~ ' o ~0 1.0 2.0 3.0 4.0 ~ . v

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Fig. 10. Sucrose gradient analysis of RNA extracted from polysomes. Differentiation of amebae from late growth phase cultures (1.3" 106-1.8 • 106 per ml) was initiated. Ten min after initiation 12-t 4C]uracil was added to give 5 #Ci/ml and 9.6 • 106 cells/ml. At 30 min after initiation, 3 ml cell suspension were diluted with 5 ml ice-cold Tris-KCl buffer. The cells were pelleted, lysed, and a polysomal fraction prepared and centrifuged on 3 sucrose gradients as described for Sample A in Fig. 9. Gradients were fractionated by alternately collecting 4-drop fractions into 0.6 ml ice-cold 95 % ethanol and 1-drop fractions into empty tubes. The 1-drop fractions were diluted with 0.7 ml of 20 mM KC1, 50 mM Tris-HC1 (pH 7.6) and the absorbance at 260 nm determined. The absorbance pattern was used to pool all the 4-drop fractions from the region of the gradients sedimenting faster than the monosome peak. RNA from the pooled fractions was analysed on a linear 5-20 % (w/v) sucrose gradient.

Fig. 11. Incorporation of [3H]lysine into polysomes. Differentiation of amebae from a late growth phase culture (2.0- 106 per ml) was initiated. Fifteen min after initiation [G-3H]lysine was added to give 100yCi/ml and 1.2. 107 cells/ml. After an additional 10 min of incubation at 25 °C, an aliquot of cells (1.6 • 107) was diluted with an equal volume of ice-cold Tris-KC1 buffer and pelleted by centrifugation for 1 min in a clinical centrifuge. A polysomal fraction was prepared from the pelleted cells and analysed on a sucrose gradient as in Fig. 9A. C)-O, absorbance at 260 nm;• - - . , SH cpm.

not yet been possible to rule out selective degradat ion or loss during isolation. Differentiating cells make proport ional ly more m R N A and less r R N A than

growing cells. This major shift is caused by the shift to a non-nutr ient environment 23. It could be due to faster m R N A synthesis or slower m R N A turnover, or to slower r R N A synthesis or faster r R N A turnover. We propose it is due to slower r R N A synthesis, but cannot support this rigorously in the absence o f measurements o f absolute rates o f synthesis and turnover. Our interpretation is consistent with many studies in procaryotes and eucaryotes, where decreases in r R N A synthesis occur as a result o f nutritional shift-downs 33-37.

Most o f the R N A synthesized during differentiation is messenger-like R N A . This R N A has the characteristics o f m R N A 3°. It is rapidly labeled, heterogeneous, first appears in the nucleoplasm and then moves to the cytoplasm. There it is associated with polysomes in a form more susceptible to nuclease attack than r R N A . These results support the conclusion that this R N A is m R N A . The only unequivocal cri- terion for any messenger R N A , which we have not yet achieved, is its translation in

vi tro to give a recognizable peptide. R N A synthesis is required for differentiation (Fulton, C. and Walsh, C., un-

published). The major R N A synthesized is m R N A , and it is tempting to propose that this is the required R N A . The recent finding that at least 70 % of the flagellar tubulin is synthesized de novo at a particular time during differentiation 38 makes it reasonable

70 c. WALSH, C. FULTON

to fOCUS on the m R N A for tubulin. We are a t tempt ing to determine if flagellar tubulin

m R N A is synthesized de novo during differentiation.

ACKNOWLEDGMENTS

The research was supported by grant GB-16334 f rom the Nat iona l Science

Foundat ion . C. W. was supported by a Nat iona l Institutes o f Heal th fellowship

1FO2Ca32627 f rom the Nat iona l Cancer Insti tute and a traineeship f rom training

grant T1-HD-22 of the Insti tute o f Child Heal th and H u m a n Deve lopment o f the

Nat iona l Institutes o f Health. The dedicated assistance o f Carol Rob inson is grate-

fully acknowledged.

REFERENCES

1 Dingle, A. D. and Fulton, C. (1966) J. Cell Biol. 31, 43-54 2 Fulton, C. (1970) in Methods in Cell Physiology (Prescott, D. M., ed.), Vol. 4, pp. 341-476,

Academic Press, New York 3 Solymosy, F., Fedorcs~ik, I., Guly~s, A., Farkas, G. L. and Ehrenberg, L. (1968) Eur. J. Biochem.

5, 520-527 4 Leonard, N. J., McDonald, J. J. and Reichmann, M. E. (1970) Proc. Natl. Acad. Sci. U.S. 67,

93-98 5 Guly~is, A. and Solymosy, F. (1970) Acta Biochim. Biophys. Acad. Sci. Hun#. 5,235-238 60xelfelt, P. and Arstrand, K. (1970) Biochim. Biophys. Acta 217,544-547 70berg, B. (1970) Biochim. Biophys. Acta 204, 430-440 8 Solymosy, F., HiirOs, P., Guly~is, A., Kapovits, L, Gaal, 0., Bagi, G. and Farkas, G. L. (1971)

Biochim. Biophys. Acta 238,406-416 9 Means, A. R., Comstock, J. P. and O'Malley, B. W. (1971) Biochem. Biophys. Res. Commun.

45, 759-766 10 Abadom, P. N. and Elson, D. (1970) Biochim. Biophys. Acta 199, 528-531 11 Fedorcs~ik, I., Natarajan, A. and Ehrenberg, L. (1969) Eur. J. Biochem. 10, 450-458 12 Munro, H. N. and Fleck, A. (1966) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. XIV,

pp. 133-136, Interscience, New York 13 Dische, Z. (1955) in The Nucleic Acids (Chargaff, E. and Davidson, J. N., eds), Vol. 1, pp.

300-301, Academic Press, New York 14 O'Brien, B. R. A. (1964) J. Cell Biol. 20, 521-525 15 Cocucci, S. M. and Sussman, M. (1970) J. Cell Biol. 45, 399-407 16 Muramatsu, M., Smetana, K. and Busch, H. (1963) Cancer Res. 23, 510-518 17 Pine, K. and Klein, A. O. (1972) Dev. Biol. 28, 280-289 18 Plagemann, P. G. W. (1971) J. Cell. Physiol. 77, 241-258 19 Weber, M. J. and Rubin, H. (1971) J. Cell. Physiol. 77, 157-167 20 Darnell, J. E. (1968) Bacteriol. Rev. 32, 262-290 21 Bishop, D. H. L., Claybrook, J. R. and Spiegelman, S. (1967) J. MoL BioL 26, 373-387 22 Attardi, G. and Amaldi, F. (1970) Annu. Rev. Biochem. 39, 183-226 23 Walsh, C. and Fulton, C. (1971) Abstr. 11th Annu. Meet. Am. Soc. CellBiol., New Orleans, p.317. 24 Blobel, G. and Potter, V. R. (1966) Science 154, 1662-1665 25 Hadjiolov, A. A. (1967) Pro#r. Nucleic Acid Res. Mol. Biol. 7, 195-242 26 Warner, J. R., Knopf, P. M. and Rich, A. (1963) Proc. Natl. Acad. Sci. U.S. 49, 122-129 27 Noll, H. (1969) in Techniques in Protein Biosynthesis (Campbell, P. N. and Sargent, J. R., eds),

Vol. 2, pp. 107-110, Academic Press, London 28 Penman, S., Vesco, C. and Penman, M. (1968) J. Mol. Biol. 34, 49-69 29 Arnstein, H. R. V. (1969) in Subcellular Components (Birnie, G. D. and Fox, S. M., eds), pp.

110-113, Plenum Press, New York 30 Kuechler, E. and Rich, A. (1969) Proc. Natl. Acad. Sci. U.S. 63,520-527 31 Attardi, G., Parnas, H., Hwang, M.-I. H. and Attardi, B. (1966) J. Mol. Biol. 20, 145-182

RNA SYNTHESIS D U R I N G NAEGLERIA DIFFERENTIATION 71

32 Kessin, R. H. (1971) Aspects of RNA Metabolism in Dictyostelium discoideum, Doctoral dissertation, Brandeis University

33 Byfield, J. E. and Scherbaum, O. H. (1968) Exp. Cell Res. 49, 202-206 34 Loening, U. E. (1965) Biochem. J. 97, 125-133 35 Mitchison, J. M., Cummins, J. E., Gross, P. R. and Creanor, J. (1969) Exp. Cell Res. 57, 411-422 36 Mizukami, Y. and Iwabuchi, M. (1970) J. Biochem. Tokyo 67, 501-504 37 Watson, J. D. and Ralph, R. K. (1966) Cancer Res. 26, 2362-2367 38 Kowit, J. D. (1972) Programmed Tubulin Synthesis Duriny Differentiation of the Amebo-flayellate

Naeyleria yruberi, Doctoral dissertation, Brandeis University

Transcription during cell differentiation in Naegleria gruberi. Preferential synthesis of messenger...

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