Chapter 3 Some Genetic and Biochemical Aspects of the Regulatory Program for Slime Mold Development*

CHAPTER 3

SOME GENETIC AND BIOCHEMICAL ASPECTS OF THE REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT* Maurice Sussmn DEPARTMENT OF BIOLOGY, BRANDEIS UNIVERSITY, WALTHAM, MASSACHUSETTS

I. Introduction

.....................................

11. The Succession of Developmental Events and the Regu-

lated Appearance of Specific Biochemical Products .... 111. Alteration of the Overall Developmental Program in Mutant Strains ...................................... IV. FR-17, a Temporally Deranged Mutant .............. V. The Regulatory Program for UDP-Gal Polysaccharide Transferase ...................................... VI. Specific Requirements for RNA and Protein Synthesis in the Transferase Program ........................... VII. The Role of Protein Synthesis during Genetic Transcription ............................................ References ......................................

61

62 65 66

67 70 76 82

1. Introduction

The developmental cycle of a cell or multicellular organism comprises a complex progression of phenomic alterations which themselves are expressions of underlying changes in macromolecular constitution. This progression is under rather precise temporal, spacial, and quantitative The work reported here was performed with the aid of grants from the National Science Foundation (GB-1310) and National Institutes of Health (C-4057). 61

62

MAURICE SUSSMAN

control, i.e., developmental events occur in fixed chronological order, at specific intracellular sites and/or within specific cells of a multicellular assembly, and to fixed amounts or extents. Such precision dictates the operation of an overall regulatory program whose molecular basis represents one of the main current mysteries of developmental biology. Previously, it was difficult even to ask meaningful questions about the genetic and biochemical properties of such regulatory programs let alone to answer them. But recent conceptual and technological advances in molecular genetics and RNA and protein biosynthesis, as well as insights into specific metabolic controls which operate during bacterial growth, now make both the asking and the answering feasible. The intent of this essay is to describe the beginnings of that kind of experimental approach to slime mold development. II. The Succession of Developmental Events and the Regulated Appearance of Specific Biochemical Products

Figure 1 is a schematic summary of the developmental cycle carried out by Dictyostelium discoideum. Thick-walled dormant spores germinate into amoebae which feed on live or dead bacteria and increase exponentially in number. Upon entering the stationary growth phase, the amoebae prepare to form multicellular aggregates and then do so. Each aggregate becomes further organized into a conical, finger-like aerial projection (pseudoplasmodium or slug) which lies on its side and migrates over the substratum. This is followed by a complex series of morphogenetic movements which result in the construction of a fruiting body (or fruit) consisting of a lemon-shaped mass of spores at the top surmounting a cellulose-ensheathed stalk, made up of tightly packed, vacuolated cells, which rests upon a basal disk. The developmental fate of a given cell appears to be determined by its position within the aggregate and pseudoplasmodium and thus may be the result of a matrix of cell interactions (Raper, 1940; Bonner, 1944, 1950). In general, two experimental systems are available for biochemical and serological analyses. Vegetative amoebae are harvested, washed, and dispensed on a solid substratum. The latter can be either non-nutrient agar, or a 2-inch Millipore filter resting on an absorbent pad saturated with inorganic salts-streptomycin solution inside a 60-mm petri dish ( M . Sussman and Lovgren, 1965). Morphogenetic synchrony is good on agar, even better on millipore filters. Alternatively, single aggregates,

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

63

slugs, and fruits may be employed for histochemical study (Gregg and Bronsweig, 1956). A variety of specific products, detectable by serological and biochemical means, appear and in some cases disappear at particular stages of this sequence. The patterns of their appearance are generally deranged

0

f

-0 \ Spor.e

8,

Amoeba

Growth,and

rnultiplicotion

I

19

‘Qz

A

-

Aggregation

Pseudo plasmodium

FIG.1. The developmental cycle of Dictyostelium discoideum.

in mutants with altered morphogenetic capacities. Thus, a considerable number of new antigenic determinants are acquired. One of these appears and accumulates just prior to and during aggregation (Sonnebom et at., 1964). Species which can coaggregate with cells of D. discoideum also accumulate a serologically detectable material which cross-reacts with this antigen; but a member of another genus, which cannot coaggregate, does not. In vivo, the antiserum (prepared against homogenates of aggregating cells and absorbed out with homogenates of vegetative amoebae) specifically inhibits D. discoideum aggregation

64

MAURICE SWSSMAN

without affecting cell viability. Preliminary fractionation suggests that this antigen is a lipoprotein associated with the cell membrane. The act of slime mold cell aggregation may be closely allied to analogous phenomena in animal cells (Moscona and Moscona, 1952; Moscona, 1957; Trinkaus and Groves, 1955), i.e., it may be mediated by the formation of divalent cationic bridges (DeHaan, 1959) and directed by specific macromolecular components of the cell surface (Moscona, 1963; Humphreys, 1963) of which the “aggregation” antigen (Sonnebom et al., 1964) may be one. At high cell density, as on growth plates or in shaken liquid suspension ( Gerisch, 1960), random collisions are sufficiently frequent to ensure rapid aggregation. At lower initial cell densities, however, the appearance of locally high cell concentrations is assured by a chemotactic mechanism ( Runyon, 1942; Bonner, 1947; Shaffer, 1953). Although investigation of this process is impeded by the lack of a quantitative assay system, it is already clear that chemotaxis is promulgated by the controlled release of a specific chemotactic factor which is unstable as the result of extracellular enzymic destruction (M. Sussman et al., 1956; Shaffer, 1956). Several steroids have some chemotactic activity, among them A-22-stigmastene, which is present in the amoebae in considerable amounts ( Heftmann et al., 1959). Production of the chemotactic agent apparently continues in migrating slugs and may play a role in later morphogenetic movements ( Bonner, 1949). Other antigenic determinants successively appear at later developmental stages. Some are concentrated in the spore contingent of the mature fruit, others in the stalk (Gregg, 1961; Takeuchi, 1963; Sonneborn, 1962). In addition, at least one antigen carried by vegetative cells is preferentially lost by the spores but not by stalk cells (Sonneborn et al., 1965). The synthesis of at least three polysaccharides and one disaccharide accompanies the developmental sequence. One of the former is a starchlike polymer which accumulates to a level of approximately 2% of the dry weight during cell aggregation and slug migration and disappears rapidly from the trichloroacetic acid ( TCA )-soluble fraction during fruit construction (White and Sussman, 1963a,b). An analogous glucose polysaccharide is subsequently found in the fruiting stalk (C. Ward and Wright, 1966), but the relationship between the two is not yet clear. A second polysaccharide fraction, a glucose polymer, almost certainly cellulose, insoluble in TCA and alkali, appears during fruit construction and ultimately makes up about 4% of the dry

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

65

weight of the fruiting body (White and Sussman, 1963a; C. Ward and Wright, 1966). Histological studies ( Raper and Fennell, 1952; Muhlethaler, 1956; Gezelius and Ranby, 1958) have shown the fruiting body stalk to be sheathed in a cellulose wall. The third fraction is an acid mucopolysaccharide (White and Sussman, 1963b), composed of galactose (55% ), galactosamine (25% ), and galacturonic acid ( 15% ) . It reacts serologically with sera against slime mold spores and with pneumococcic capsular type VII antiserum. It first appears in early pseudoplasmodia, accumulates to about 2% of the dry weight, and is found only in the spore mass of the mature fruit. The disaccharide is trehalose (glucose l,l-aD-glucoside), present in low concentration in vegetative amoebae but comprising 5 7 % of the spore dry weight. It is lost during germination, presumably serving as a carbon and energy source (Clegg and Filosa, 1961) . A variety of enzyme activities have been followed during development. These include several DPN- and TPN-linked dehydrogenases (Wright and Anderson, 1959; Wright, 1960), succinic dehydrogenase and cytochrome oxidase ( Takeuchi, 1960), alkaline phosphatases ( Gezelius and Wright, 1965) and esterases (Solomon et al., 1964), UDPG synthetase (Wright, 1960), UDP-galactose polysaccharide transferase ( M. Sussman and Osborn, 1964), UDPG cellulose transferase (C. Ward and Wright, 1966), UDPG trehalose transferase (Roth and Sussman, 1966), and trehalose (Ceccarini, 1966). The specific activities of at least some of these enzymes change drastically during morphogenesis. The detailed program for one of them ( UDP-Gal polysaccharide transferase) is described in Section V. Finally, the appearance of the mature fruiting body is followed closely by the fabrication of a yellow pigment concentrated in the spore mass. The chemical composition is unknown, but the pigment may be a carotenoid possibly related to phytol ( Ennis, personal communication). 111. Alteration of the Overall Developmental Program in Mutant Strains

Mutants can be isolated which, taken as a group, run a very wide gamut of morphogenetic aberrations (R. R. Sussman and Sussman, 1953; Rafael, 1962; Kahn, 1964; Sonneborn et al., 1963). Some strains construct morphologically altered fruiting bodies ( e.g., “fruity, bushy, forked, curly, glassy”), Others lack pigment or produce altered pigments. Still

66

MAURICE SUSSMAN

others are morphogenetically deficient, i.e., their development stops at a stage prior to the construction of a mature fruiting body and prior to the appearance of stalk cells and spores. These include stocks which either completely fail to aggregate or form loose, amorphous clumps (“aggregateless”). Still others are morphogenetically deficient. Of the latter, some mutants cannot aggregate at all while others can begin the morphogenetic sequence but stop at a stage short of the appearance of mature fruiting bodies with spores and stalk cells. Such mutants are incapable of accumulating the specific molecular products described above, since these normally arise at developmental stages subsequent to the morphogenetic block (White and Sussman, 1981, 1963a,b; Sonneborn et a,?., 1963; M. Sussman and Osborn, 1964). Thus, the effects of these mutations to morphogenetic deficiency are not confined each to a single metabolic activity but ramify into a wide variety of biosynthetic processes. It is particularly interesting to note that many of the deficient stocks, when mixed with one another or with the wild-type, can develop synergistically to the terminal morphogenetic stage ( M. Sussman, 1954; M. Sussman and Lee, 1955). IV. FR-17, a Temporally Deranged Mutant

Strain FR-17 (Sonneborn et al., 1963) grows normally but constructs amorphous flattened, papillated aggregates which develop no further in gross aspect but which are composed of terminally differentiated spores and stalk cells intermixed chaotically. In addition, all products characteristic of mature, wild-type fruits appear to be present in FR-17. Those whose presence has already been demonstrated include the aggregation antigen, a stalk-specific antigen, a spore-specific antigen, the starch-like polysaccharide, ,the mucopolysaccharide, and the corresponding UDPGal polysaccharide transferase, cellulose, pigment. The morphogenetic anomalies encountered in FR-17 are accompanied (and perhaps caused) by a drastic temporal derangement of the developmental program. In the mutant, most developmental events, including the accumulation of products listed above, start sooner and are accomplished faster than in the wild-type under comparable conditions, so that the mutant attains its terminal morphogenetic state (the papillated, flattened mass of spores and stalk cells) in about half the time required by the wild-type to construct a mature fruit. However the morphogenetic aberrations ultimately expressed by the mutant make it

3.

REGULATORY PROGRAM

FOR SLIME MOLD DEVELOPMENT

67

likely that at least some parts of the program are not correspondingly accelerated. The growth rate of the mutant vegetative amoebae is approximately equal to that of the wild-type, and this, too, argues against a general acceleration of all metabolic activity. Thus, in contrast to the variants discussed above, FR-17 provides an example of a mutation whose regulatory consequences accelerate the flow of developmental events without significantly changing the kinds of products fabricated or the terminal states of cytodifferentiation. The topographical relationships among the developing cells, however, both along the way and terminally, are affected drastically indeed. V. The Regulatory Program for UDP-Gal Polysaccharide Transferase

Galactose is incorporated into the mucopolysaccharide by the following transfer reaction: UDP-Gal

+ acceptor

-

galactosyl-acceptor

+ UDP

The reaction can be followed in crude pressates or sonicates by measur~ C UDP-Gal into the ethanol-insoluble ing the transfer of g a l a c t o ~ e - ~from fraction (M. Sussman and Osborn, 1964). The completed mucopolysaccharide or a smalIer-molecular-weight precursor can serve as the acceptor, and enzyme activity is assayed in the presence of a standard concentration of the latter, Under assay conditions, the incorporation is linear with time, directly proportional to enzyme concentration and the concentration of acceptor. Incorporated counts are recovered quantitatively as galactose after acid hydrolysis. Figure 2 shows the developmental kinetics of the transferase in D. rliscoideum wild-type. The activity first appears in early pseudoplasmodia, about 1 hour before the mucopolysacchande itself can be detected. The enzyme accumulates rapidly to a peak of specific activity which is attained shortly before the cessation of mucopolysaccharide synthesis and then it rapidly disappears. Mixed extracts from active and inactive stages show no evidence of inhibitors or destroyers of enzyme activity that might mask the presence of transferase in the latter stages. During the period of its accumulation all of the activity remains associated with the cells when they are concentrated by centrifugation at low speed. However, within a 2 hour period after the peak of activity is reached the bulk of the enzyme is released by the cells and up to 80% is found in the super-

68

MAURICE SUSSMAN

natant of centrifuged cells (Table I ) , This release is preferential, since the specific activity of the released enzyme is about thirtyfold greater than that of the small residuum still associated with the cells. As Fig. 3 indicates, most of the mucopolysaccharide is synthesized before the re-

FIG.2. The developmental kinetics of UDP-Gal polysaccharide transferase activity in D. discoideum. Dotted line: the accumulation of the mucopolysaccharide as measured by its bound, nondialyzable galactose content (White and Sussman, 1963b ) . TABLE I TIMECOURSEOF ENZYMERELEASE Enzyme BSSOC. with cells

Enzyme released in supernatant

Time, hrs.

% of Total act.

Spec. act.

% of Total act.

Spec. act.

10.5, 14.5 17 19 21.5 22.5

100 98 98 90 42 24

121 407 1390 1385 530 304

0 2 2 10 58 76

1750 9200 11,200

-

lease of enzyme, and the former remains bound to the cells long after the extrusion of the latter (M. Sussman and Lovgren, 1965). That the activity of the enzyme is under control of the over-all developmental program is indicated by the study of five morphogenetically

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

69

FIG. 3. Relation between the release of enzyme and the synthesis of polysaccharide. The data for the former are taken from Table I. The same samples were assayed for mucopolysaccharide content by quantitative complement fixation ( White and Sussman, 1963b).

deficient mutants. Two of these, Agg-204 and FR-2, do not reach the morphogenetic stage at which the mucopolysaccharide normally appears. They fail to synthesize it and, as Fig. 4 shows, do not accumulate any

t

I

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4

FIRST MATURE FRUITS

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I

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--WILD-TYPE o FR-17 6 FR-2 A Agg-204

t \ t 1

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FIG.4. The developmental kinetics of transferase activity in three mutant strains of D. discoideum.

70

MAURICE SUSSMAN

transferase activity. Two other mutants, KY-3 and KY-12 (Yanagisawa and Sussman, 1966), yield normal migrating slugs which fail to develop into mature fruits. The transferase does appear to accumulate in normal fashion but is not released or destroyed, events which in the wild-type accompany the last stages of fruit construction. The peak of specific enzyme activity attained by KY-3 is somewhat less than that of the wildtype and KY-12 accumulates about half as much. In FR-17, the synthesis of mucopolysaccharide is accelerated in approximate accord with the overall temporal derangement. As seen in Fig. 4, the developmental kinetics of transferase activity in the mutant are like that of the wild-type except that it appears sooner and accumulates and disappears faster. In summary then, the activity and location of UDP-Gal polysaccharide transferase seems to be entrained by the overall developmental program, and the control pattern for this enzyme includes the following steps: ( a ) initial appearance shortly after cell aggregates are transformed into slugs; ( b ) rapid linear accumulation to a peak of specific activity which is attained at a late stage of fruiting body construction; ( c ) preferential release by the cells of the bulk of the enzyme followed by its rapid destruction; ( d ) coincident disappearance of the small residuum still associated with the cells (possibly also preceded by release). VI. Specific Requirements for RNA and Protein Synthesis in the Transferase Program

The following experiments (M. Sussman, 1965; M. Sussman and R. R. Sussman, 1965) demonstrate that both the accumulation and subsequent disappearance of the transferase can be halted by coincident inhibition of protein synthesis (by cycloheximide) and the prior inhibition of RNA synthesis ( by actinomycin). Figure 5 shows the cycloheximide reversibly inhibited amino acid incorporation into TCA-insoluble material in strain FR-17. Under the same conditions, uridine incorporation was not altered. Figure 6 summarizes the effect of cycloheximide on transferase accumulation and disappearance. The cells were harvested, washed, dispensed on Millipore filters, preincubated for 16 hours at 15"C, and then switched to the standard temperature of 22°C. The preincubation at 15°C for 16 hours is equivalent to 2 hours of development at 22°C. Thus all of the morphogenetic and biochemical events ( including appearance and disappearance of transferase) occur 2 hours sooner in cells preincubated in this way, and

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

71

n W

G 2000c 0

a K 0

u

z 2"

m

g - 1000-

0

a 0

z E

a E n u

I

3

.

v

4

5

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6

7

I

8

FIG.5. 14C-Amino acid incorporation in the presence and absence of cycloheximide. I, control; 11, cycloheximide added at 4 hours; 111, cycloheximide added at 4 hours, removed at 6 hours.

4

HOURS

FIG.6. Effect of cycloheximide on the accumulation and disappearance of the transferase. I, control (open and closed circles are from different experiments); 11, cycloheximide added at 8 hours; 111, added at 6 hours; IV, added at 5 hours and removed at 7 hours.

72

MAURICE SUSSMAN

the remainder of the developmental sequence is subsequently completed in 12 hours instead of the 14 hours for cells not preincubated. Since this writer preferred a 12-hour to a 14-hour working day, all subsequent experiments involving strain FR-17 include the period of preincubation. As Fig. 6 indicates, the transferase activity appeared in the control cells 6.5 hours after the temperature switch, reached a peak at 10.5 hours, 2000

a a

-

0

xa

HOURS

FIG.7. Uridine-SH incorporation in the presence and absence of actinomycin.

and thereafter disappeared. Addition of cycloheximide at 6 hours prevented the elaboration of any transferase activity. When the agent was added at 8 hours (by which time the cells had accumulated about 30% of the peak activity), further increase in transferase activity stopped immediately and also its subsequent disappearance was prevented. When cycloheximide was added at 5 hours and removed at 7 hours, the rise in enzyme activity commenced after a lag period and proceeded at a rate usually equal to that of the control but sometimes slower. Both the

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

73

duration of the lag and the subsequent rate of accumulation depended upon the time at which cycloheximide was added and removed. Actinomycin D was found ( M . Sussman and R. R. Sussman, 1965) to reduce uridine incorporation to about 10-15% of the control (Fig. 7 ) . Sucrose density gradient centrifugation revealed that this residue was confined to the 4 S region and presumably reflected terminal labeling of transfer RNA by derived cytosine (Franklin, 1963). In contrast, amino acid incorporation was slightly accelerated. The effect of actinomycin on accumulation and disappearance of transferase is illustrated in Fig. 8.

FIG.8. Effect of actinomycin on transferase accumulation and disappearance in FR-17. The time values next to the curves represent the times at which actinomycin was added.

When the agent was added 2 hours after the temperature switch, transferase accumulation was completely inhibited. When actinomycin was added at 3, 4, and 5 hours, the activity appeared at the usual time but accumulated to levels that were 20, 55, and 80% of the control peak value; furthermore, in the first two activity did not disappear subsequently, and in the third it disappeared at a slower rate. When actinomycin was added at 6 hours or later, transferase activity at the normal time accumulated to 100-1200/0 of the control peak and thereafter declined, although at a rate usualIy slower than in the control. Figure 9 summarizes the relation between the time of actinomycin addition and the amount of enzyme activity ultimately detected. Figures 10 and 11 illustrate similar experiments using the wild-type.

74

MAURICE SUSSMAN

50-

00 -

-

v

/

I

8

4

TIME OF ACTINOMYCIN ADDITION (HOURS)

12

FIG. 9. Relation between the time of actinomycin addition and the amount of enzyme activity that subsequently accumulated in FR-17. Dotted line: the time course of actual enzyme accumulation taken from the control curve of Fig. 8.

TIME (hours) DEVELOPMENTAL STAGES IN CONTROL:

*9 f f

FIG. 10. Effect of actinomycin on accumulation and disappearance of transferase in wild-type D. discoideum.

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

75

Here the transferase activity appeared 12.5 hours after deposition of cells on the Millipore filters, accumulated to a peak at 21-22 hours, and thereafter declined. Exposure to actinomycin prior to 8 hours prevented the rise in transferase activity; exposure after 15 hours did not prevent the full rise. When added between these periods, the drug did not affect the time of the enzymic increase but did restrict the amount of activity accumulated, in a manner similar to that observed with FR-17. In the wildtype, however, actinomycin did not at any time dissociate enzyme accumulation from disappearance as it did in the mutant.

I

/

/

/

/D

5 10 15 20 TIME OF ACTINOMYCIN ADDITION (hours)

FIG. 11. Relation between the time of actinomycin addition and the amount of enzyme activity that subsequently accumulated in the wild-type ( C ) . Curve D, the time course of actual enzyme accumulation in the wild type taken from the control curve of Fig. 10. Curves A and B, the corresponding curves for FR-17 taken from

Fig. 9.

In summary, then (Fig. 12), treatment of FR-17 with actinomycin permits the delineation of a sensitive period between 2 and 6 hours after the temperature switch (i.e., between 4 and 8 hours after the start of the morphogenetic period ) during which specific RNA is synthesized, the presence of which is required for the subsequent accumulation of the UDP-Gal transferase. Within this period, a simple linear relation exists between the time of actinomycin addition and the amount of enzymic activity that can be elaborated. A corresponding period of actinomycin sensitivity is detected in the wild-type except that it extends between 7.5 and 15.5 hours after the start of morphogenesis. Thus, in the latter, the period begins later ( 7.5 versus 4 hours) and lasts longer ( 8 versus 4 hours) than in the former but there is approximate correspondence with the general acceleration of development observed in the mutant ( including

76

MAURICE SUSSMAN

the time of appearance of the enzyme itself and the rate at which its activity increased). It would appear, therefore, that the synthesis of at least some developmentally significant RNA is entrained by the overall regulatory program. Furthermore, the finding that transcription of one particular segment of the genome does not continue throughout the developmental sequence but in fact occupies less than a third of the total time is of great significance. It should also be noted that in both strains 7-

WILD TYPE

PERIOD OF ACTINOMYCIN SENSITIVITY FOR

-.I

ACCUMULATION-'

I -n

DISAPPEARANCE ENZYME ACCUMULATES ENZYME D i s m w m

I

10

-

ACCUMULATION

, 20

25

DISAPPEARS 30

35

TIME

FIG. 12. Schematic outline of the developmental program for UDP-Gal polysaccharide transferase in D. discoideum wild-type and strain FR-17.

there is a lag of 4-5 hours between the beginning of the actinomycinsensitive period and the appearance of transferase activity. This raises the possibility that temporal controls may operate at the level of translation of mRNA into protein. VII. The Role of Protein Synthesis during Genetic Transcription

Assuming that the actinomycin-sensitive period represents the time of transcription of the structural cistron( s ) for the UDP-Gal polysaccharide transferase, the lag of 4-5 hours mentioned above implies that athe mRNA has a correspondingly long functional life span. The question arises

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

77

whether this functional capacity is affected by the inhibition of protein synthesis coincident with or subsequent to transcription. The data presented below (M. Sussman, 1966) indicate that the nascent message is indeed rendered unusable by such interference but that after transcription it is no longer sensitive. It will be recalled that, in FR-17, the actinomycin-sensitive period extends between 2 and 6 hours after the temperature switch. When cycloheximide was added at 3 hours and removed at 5 hours (Fig. lk),

6oool

z c Y

6-8 hourr

4b-6h hourr

CYCLOHEXIMIDE AT; 3 - 5 hOUrl

w

ti 3000 W

0 k

u a

in

0

o

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8 1 HOURS CONTROL

0

-

A

0

0

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0

-

a ACTINOM

0

u -

0

CYU.

o

0

a ACTINOM

CONTROL

-

0

L ACTINOM.

CYCL.

FIG. 13. Effect of cycloheximide treatment during and after the period of transcription. Ordinate: cpm galactose incorporated per hour per milligram protein ( 918 cpm = 1 mpmole). Abscissa: time of incubation of the cells after the temperature switch from 12" to 22°C.

transferase activity did not appear at the usual time (6.5 hours) but instead required 3 more hours ( a total of 4.5 hours after removal of cycloheximide) and then accumulated to a level 8590% of that reached by the control. If, after cycloheximide was removed, actinomycin was added to prevent subsequent RNA synthesis, no enzyme appeared.* In agreement with previous results, addition of actinomycin at 5 hours to previousIy untreated celIs permitted the enzyme to appear at the usual time and to acoumulate to 75% of the peak level in the controls. Figure 13b illustrates the result of adding cycloheximide at 4.5 hours and removing

* In repeat experiments, cells treated in this fashion accumulated up to 10% of activity attained by the controls.

78

MAURICE SUSSMAN

it at 6.5 hours. Transferase activity appeared at 9.5 hours ( 3 hours after the controls and 3 hours after removal of cycloheximide) and accumulated to the same level as in the controls. However, when actinomycin was added immediately after removal of the cycloheximide, the transferase accumulated at a lower rate and to a level only 40% of the control, this despite the fact that actinomycin added at 6.5 hours to untreated cells permitted full accumulation. Figure 13c illustrates the result of addb

W

I

z

w

40001

I

2000

6

4

0 0 0

€

HOURS

0

-

A

0

A

0

CYCL.

-

ACTINOM

FIG. 14. Dependence of enzyme accumulation on RNA synthesis after the removal of cycloheximide. Ordinate and abscissa as in Fig. 13.

ing cycloheximide at 6 hours (i.e., at the end of the transcription period) and removing it at 8 hours. When actinomycin was added immediately after removal of cycloheximide, the cells could still accumulate enzyme to a level approximately the same as in the controls. Nevertheless, the appearance of activity was delayed 1.5 hours after removal despite the fact that overall protein synthesis as reflected by 14C-amino acid incorporation resumed immediately. When actinomycin was not added immediately after removal of cycloheximide at 8 hours, the cells failed to accumulate the full complement of enzyme. This paradox stems from the fact that the 6-8 hour exposure to cycloheximide delayed the appearance

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

79

of spores by only 1.5 hours (whereas the 2 4 or 4%-6$ hour exposures delayed it by 3 hours). Thus, in the former, the accumulation of enzyme was stopped by the premature entrance into dormancy. Figure 14 shows the results of an experiment in which actinomycin was added at different times after the cells had been exposed to cycloheximide between 3 and 5 hours. When added immediately after removal of cycloheximide, actinomycin permitted the enzyme to accumulate only to a level of about 5% of the control peak; when added 2 hours after removal k LA

I-0

2E 100-

0

of cycloheximide, it permitted accumulation of about 50% of the control peak; when added after a 4-hour lapse, it permitted normal accumulation. Figure 15 is a summary of two experiments of this kind and it shows in more detail the relation between the amount of enzyme activity accumulated and the time at which actinomycin was added during this second period of transcription. The combined results of these experiments are schematically summarized in Fig. 16. They suggest that, when the cells were exposed to cycloheximide during the normal period of transcription, the RNA synthesized during the 2-hour exposure plus that fraction made during the previous 30-60 minutes did not subsequently give rise to active UDP-Gal transferase. Instead, a second round of RNA synthesis followed after removal

80

MAURICE SUSSMAN

of cycloheximide and this second round accounted for part or all of the delayed accumulation of transferase activity depending on whether part or all of the first round had been made nontranslatable by treatment with the drug. Thus in FR-17, the transcription of a specific region of the genome is not automatically restricted to a particular period of time but may be considerably extended. It is noteworthy that the net amount of enzyme activity elaborated as a result of RNA synthesis before and after cycloheximide exposure together approximated the total which accumulated in the undisturbed system. This may reflect the operation of a feedback system which may conceivably regulate the amount of transcription

0

4 8 1 2 0 ' ' " '

HOURS I ' PERIOD OF RNA SYNTHESIS FOR ENZYME ACCUMULATION \ YO OF TOTAL ENZYME ACTUALLY ---a ACCUMULATED 0

=

CYCLO.

C 0 NT ROL

n

4

"

4

8

I

\

100

CYCLO

0

"

I

L=l

=

12

8 "

-

0

100

CYCLO.

1 2 0

4

8

12

0

1 1 1 m. D 0 100 0 100

FIG. 16. 13-15.

A schematic summary and interpretation of the data presented in Figs.

or translation or both. The data also indicate that the translation, normally lagging 4-5 hours behind transcription, can be made to lag at least 7-8 hours without affecting the amount of enzyme which accumulates. The mechanism by which cycloheximide renders the RNA nontranslatable is not yet clear. It certainly does not interfere with net incorporaH Sussman, 1965). But the RNA made in its presence tion of ~ r i d i n e - ~(M. is considerably more sensitive to RNase in neutralized TCA precipitates than in control preparations, a result to be expected if the former were not bound to protein. Furthermore RNA, pulse labeled with 32P in the presence of cycloheximide is distributed differently after sucrose gradient. centrifugation (Fig. 17), being skewed toward lower molecular weights

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

81

than is R N A synthesized by control cells ( M . Sussman, 1966). Corresponding studies in yeast have revealed essentially the same result (Fukuhara, 1965). However, the data cannot distinguish between the possibility that cycloheximide has a differential effect on the kind of R N A synthesized or that it renders the nascent R N A less stable. The main import of these experiments bears upon the control of the transcription process during development. The most naive picture would be one in which, given an initial triggering, the R N A polymerase would I-

1-19.700

H3.1001 cpm

cpm

3000

k

2000

I000

10

20

30

20

30

4000

3000

k 2000

I000

10

TUBE

NO.

FIG. 17. Sucrose density centrifugation of RNA synthesized during a 2-hour period in the presence (upper graph) ancl the absence (lower graph) of cycloheximide. The cells were harvested and then centrifuged at 5000 g, and the pellets were frozen and then thawed in the presence of 1% sodium dodecyl sulfate (SDS). The clear extracts were then layered over a 1540% sucrose-SDS gradient and spun 17 hours at 18°C. in an SW-25 Spincorotor. The tubes were emptied from below, passed through a Gilford recording spectrophotomer to measure OD,,,, and collected in 1-ml aliquots. The TCA-precipitable radioactivity of each fraction was then measured.

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contribute a sequential reading of the genome in an automatic fashion uninfluenced by further signals from the overall program. The appearance of messages specific for early phage proteins in T-2 infected E. coli seem to occur in this manner (Cohen et al., 1963). The results described in this section fail to support that view and suggest that transcription at least of the transferase-specific RNA is continually open to external control and may be extended if need be to guarantee the subsequent appearance of enzyme. REFERENCES Bonner, J. T. (1944). Am. J. Botany 31, 175. Bonner, J. T. (1947). J. Exptl. Zool. 106, 1. Bonner, J. T. (1949). J. Exptl. Zool. 110, 259. Bonner, J. T. (1950). Biol. Bull. 99, 143. Ceccarini, A. (1966) Science 151, 454. Clegg, V. S . , and Filosa, M. (1961). Nolure 192, 1077. Cohen, S. S., Sekiguchi, M., Stern, J. L., and Barner, H. D. (1963). Proc. Null. Acad. Sci. U S . 49, 699. DeHaan, R. L. (1959). J . Embryol. Exptl. Morphol. 7, 335. Franklin, R. M. (1963). Biochim. Biophys. Acta 72, 555. Fukuhara, H. (1965). Biochenz. Biophys. Res. Commun. 18, 297. Gerisch, G. (1960). Arch. Entroicklungsmeclz. Organ. 152, 632. Gezelius, K., and Ranby, B. G. ( 1958). Exptl. Cell Res. 12, 265. Gezelius, K., and Wright, B. E. (1965). J. Gen. Micr(iliol. 38, 309. Gregg, J. H. (1961). Develop. Biol. 3, 757. Gregg, J. H., and Bronsweig, R. (1936). J. Cellular Comp. Physiol. 47, 483. Heftmann, E., Wright, B. E.. and Liddel, C . U. (1959). J . Am. Chem. SOC. 81, 6525. Humphreys, T. (1963). Deoelop. Riol. 8, 27. Kahn, A. J. (1964). Develop. Biol. 9, 1. Moscona, A. (1957). PTOC.Nntl. Acad. Sci. US.43, 184. Moscona, A. (1963). Proc. Natl. Acad. Sci. U . S . 49, 742. Moscona, A., and Moscona, H. (1952). J. Anat. 86, 287. Muhlethaler, K. (1956). Am. J. Botany 43, 673. Rafael, D. E. (1962). Bull. Torrey Botan. Club 89, 312. Raper, K. B. (1940). J . Elisha Mitchell Sci. SOC. 56, 241. Raper, K. B., and Fennell, D. ( 1952). Bull. Torrey Botan. Club 79, 25. Roth, R., and Sussman, M. (1966). To be published. Runyon, E. H. (1942). Collecting Net 17, 88. Shaffer, B. M. (1953). Nature 171, 957. Shaffer, B. M. (1956). Science 123, 1171. Solomon, E., Johnson, E. M., and Gregg, J. H. (1964). Deoelop. Biol. 9, 314. Sonnebom, D. R. (1962). Ph.D Thesis, Brandeis University. Sonnebom, D. R., White, G. J., and Sussman, M. (1963). Deoelop. Biol. 7, 79. Sonnebom, D. R., Sussman, M., and Levine, L. (1964). J. Bacteriol. 87, 1321.

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REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

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