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A choice of morphogenetic pathways in Dictyostelium discoideum induced by the adenosine analog formycin B
J. Mol. Biol. (1974) 99, 529-539
A Choice of Morphogenetic Pathways in Dictyostelium discoideum induced by the Adenosine Analog Formycin B ROBERT
W.
BRACKENBURP,
JOEL SWINDLER,
AND MAVRICE
STEPHEN ALEXANDER
SUSSM~LN
Section of Developmental and Molecular Biology, Institute of Life Sciences Hebrew University, Jerusalem, Israel, and Department of Biology Brundeis University, Waltham, Mass., U.S.A. (Received 26 April 1974, a& in recised form 23 September 1974) Depending on several external parameters, a newly formed multioellular aggregate of Dictyostelium diecoideuna can elect either to construct a fruiting body directly at the site of aggregation or to transform into a migrating slug and then to construct a fruiting body hours or days later when external conditions permit. When aggregates were incubated in the presence of Formycin B under conditions that ordinarily induce fruiting body construction, they followed the normal program of fruit construction but stopped at a late stage and transformed into structures resembling migrating slugs. Shortly thereafter they resumed the fruiting program. The choices of morphogenetic pathways dictated early by onvironmental parameters and later by Formycin B produce characteristic, dramatic changes in the patterns of enzyme accumulation and disappearance. The control of these patterns appears to act at the level of transcription.
1. Introduction In Dictyostclium discoideum, vegetative cells that have entered the stationary growth phase collect together into organized multicellular aggregates. Influenced by several environmental parameters (see legend to Fig.* l), each aggregate elects either to construct a fruiting body directly at the site of aggregation or to transform into a migrating slug and move away. (Figure 1 shows drawings of these morphogenetic events.) The slug can migrate for many days (Slifkin & Bonner, 1952) but if exposed to omnidirectional light and/or shifted to conditions favoring fruit construction, it immediately stops migrating and constructs a fruiting body over a seven-hour period (Newell et d., 1969). The patterns of accumulation and disappearance of several developmentally regulated enzymes have been shown to depend crucially on whether the aggregate develops into a fruit or a slug and, if the latter, whether it subsequently elects to stop migrating and start fruiting (Newell t Sussman, 1970 ; Ellingson et al., 1971). These patterns and their moditications appear to be convenient and useful objects with which to elucidate the regulatory system that controls gene expression in these organisms (Sussman & Newell, 1972). The present communication describes the capacity of the adenosine analog, Formycin
B, 3-@D-ribofuranosyl)pyrazolo(4,3-d)6(H)7-pyrimidone 629
(Koyama
et al., 1966)
R. W. BRACKENBURY
530
ET
AL.
to influence the choice of morphogenetio and biochemical pathways as above. Formyoin B can induce aggregates at a late stage of fruiting body tion to suspend their activities and to transform into structures resembling slugs. These slugs subsequently resume the program of fruit construction. chemical consequences of these shifts are described.
described construomigrating The bio-
Condition A
Omnidirectional light, plus shift to condition t3
I
c
16 \
/
Add Formycin
18
L1
18
20
1 ’
20
22
1 ”
22
24
’
24
1 ’
26
(1 28
Fro. 1. Alternative morphogenetic pathways in D. discoidcu‘eumand the effect of Formycin B. The important parameters for this choice (pH, salt concentration, humidity, accumulation of metabolites and light) were controlled by the following experimental procedures. Cells were harvested from growth plates, washed and dispensed in portions of lo* cells on 2 in. Millipore or Whatman No. 50 filter circles (Sussman, 1966). To induce aggregates to develop directly into fruiting bodies (condition B) the fllters were incubated on absorbent pads saturated with LPS inside 60 mm Petri dishes. Pads cemented to the dish covers were saturated with 1 Mphosphate solution (pH 6.0) in order to absorb volatile, alkaline materials. To induoe aggregates to develop into migrating slugs (condition A), filters were incubated on pads saturated with water or weak (10 mM) phosphate (pH 6.5) solution. Disk covers without absorbent pads were used and the plates were incubated in the dark or in a weak, horizontal light gradient. Alternatively, washed cells were dispensed directly on 2% agar in water and incubated in the dark or in a horizontal light gradient (Ellingson et d., 1971). If at any time aggregates that have developed into migrating slugs (condition A) are shifted to oondition B, and/or exposed to omnidirectional light, the slugs are rapidly transformed into structures resembling the 16 h stage of fruit construction and these follow the normal pathway to mature fruiting bodies over a 7-h period. If at any time prior to the 17-h stage of fruit construction, filters are shifted from condition B to condition A, the aggregates stop fruiting body construction and are transformed into migrating slugs. However, between 16 and 17 h of fruit
MORPHOGENETIC
PATHWAYS
IN
D.
DlSCOIDEUM
631
2. Materials and Methods (a) Organism
ati
culture methods
D. &coideum (haploid) strain DdB was grown in association with Aerobaoter awogenes as previously described (Sussman, 1966). Cells were harvested from the growth plates in cold water and washed free of exogenous bacteria by three centrifugations at 1200 g for 5 min. Finally they were suspended in a solution (LPS) containing 40 mm-phosphate (pH 6.5), 20 mM-HCl, 2.5 man-Mg& and O-7 maa-streptomycin sulfate. To permit fruiting body construction or the formation of migrating slugs, the cells were incubated as described in the legends to Figs 1 and 3. (b) Enzyme (i) UDPglucose
assays
pyrophosphoryluse
Cells were harvested in 0.1 a6-Tricine (pH 75) solution and stored at -20°C. The cells were lysed by addition of Cemulsol NTPl2 (see section (d) below) to a fuml concentration of 0.15°h and the extracts were assayed immediately as described previously (Newell & Sussman, 1969). 1 unit of activity is defined as 1 nmol product formed/mm at 37°C. (ii) UDPGal-C-epimeraae Cells were harvested in 0.1 M-N-Tris(hydroxymethyl)methylglycine (pH 7.5) solution containing 20% glycerol, lysed as described above and assayed immediately as described previously (Telser & Sussman, 1971). 1 unit of activity is defmed as 1 nmol product formed/mm at 37°C. (iii)
synthetase
Trehalose-6-phosphate
Cells were harvested in 0.01 a6-2(iV-morpholino)ethane sulfonic acid (pH containing 7.3 w-sodium thioglycollate, sonicated at 2 A intensity for Branson sonifier, and mayed immediately as described (Roth & Sussman, et al., 1972). 1 unit of activity is defined as 1 nmol of product formed/min at (iv) Alanine
6.5) solution 30 s/ml in a 1968; Newell 37°C.
transaminase
Cells were harvested in a 15% glycerol solution containing 0.2 mMdithiothreito1 and frozen. They were thawed, sonicated and assayed as described previously (Firtel & Brackenbury, 1972). 1 unit of activity is defined as 1 nmol product formed/mm at 28°C.
(v) N-AcetyZglucosaminidaae Cells were harvested previously described formed/min at 37°C.
in water (Loomis,
and frozen. They were thawed, sonicated and assayed as 1969). 1 unit of activity is defined as 1 nmol product (0) Protein
These were performed
by the method
estimation et al. (1951).
of Lowry
(d) Chemicals Formycin B, a Meiji product, was purchased from Calbiochem. N-Tris-(hydroxymethyl) methylglycine and 2(N-morpholino)ethane sulfonic acid were purchased from Calbiochem. Cemulsol NPTlP was obtained from Melle Bezons Inc., France. Actinomycin D was a kind gift of Dr Harlyn Halvorson, Rosenstiel Basic Medical Sciences Research Centre, Waltham, Mass. Cycloheximide was purchased from Upjohn. Alanine, cr-ketoglutarate and p-nitrophenyl-N-acetyl-fi-n-gluoosaminide were purchased from Sigma. All other chemicals were reagent grade. construction, the aggregates become committed to that pathway and continue to construct fruits despite the shift to condition A. To produce the Formycin effect (condition C), the filters were incubated in condition B until the 17-h stage w&s reached and then shifted to pads containing 1 mpn-Formycin in LPS. Pads cemented to the Petri dish covers were saturated with 1 M-phosphate, pH 6.0.
532
R. W. BRACKENBURY
ET’ AL.
3. Results (8) Morphogenesis in the presence of Formycin Figure 1 (conditions A and B) show the alternative morphogenetic pathways of D. dkwideum. As previously described (Newell et al., 1969) it is possible to control experimentally the entrance of cell aggregates into or their progress along either pathway (see legend to Fig. 1 for details). Figure 1 (condition C) shows the morphogenetic pathway followed by aggregates that are allowed to develop in condition B but then are exposed to Formycin starting from 17 hours. Instead of assuming the normal Mexican hat shape at 18 hours the aggregate assumes a deviant form with a greatly expanded apex and reduced basal periphery. The apex progressively assumes the shape of a slug, while the basal portion continues to regress until the whole aggregate is transformed into a migrating slug. This condition is very transitory. Almost immediately the slug is transformed into a shape resembling an early Mexican hat and thereafter follows the normal sequence of fruiting body construction. In summary, the pathway followed corresponds to that induced by condition A except that the decision of the cell aggregate to transform into a migrating slug is made at a late stage of fruit construction and in environmental conditions that ordinarily induce fruiting body construction directly. Additional conclusions regarding the effect of Formycin together with the pertinent experimental results are given below. (1) The response illustrated in Figure 1C is qualitatively unaltered when cells are exposed to Formycin B prior to 17 hours. Thus cells exposed to the agent from zero time did begin aggregation one to two hours early but otherwise developed normally to the Mexican hat stage and then diverged along pathway C. (2) Other nucleosides (adenosine, inosine, hypoxanthine) at similar concentrations were without effect on morphology or accumulation of UDPglucose pyrophosphorylase (data not shown). (3) The late decision to transform into a migrating slug induced by Formycin B is separate and distinct from the same decision induced earlier by pH, ionic strength, etc. (see legend to Fig. 1). Thus, cell aggregates induced to develop along pathway A to the migrating slug stage were exposed to Formycin. The slugs remained unaltered and continued to migrate. However, if shifted to condition B and exposed to omnidirectional light while in the presence of Formycin, the slugs stopped migrating, entered the program of fruit construction, reached the Mexican hat stage and the?L reverted to migrating slugs once again before ultimately producing normal fruiting bodies. (4) The re-entrance of the Formycin-induced slugs to the fruiting program is not due to the disappearance of Formycin B. Thus cells were exposed to Formycin B from zero time. At 20 hours the aggregates had diverged along pathway C and were developing into slugs. The filters were then switched to fresh pads saturated with 1 mlrr-Formycin B in LPS. The slugs reverted to the fruiting program at the same time as slugs that had not been switched to fresh Formycin B. (b) Effect of Formycin on the patterns of enzyme accumulation and disappearance (i) UDPglucose pyrophosphorylase In aggregates that are constructing fruiting bodies directly (condition B) UDPglucose pyrophosphorylase activity accumulates and disappears in the manner shown
MOBPHOGENETIC
PATHWAYS
/
/’
IO
IN
D.
DISCOIDEUM
Light plus shift to condition B
5 .-
OO
17
Lightplusshift to condition 8
, 1 1 I 1 i
I :
c
\
:
-----:--,-e-3 20
533
30
40
-50
Time(h) FIQ. 2. Solid lines: Cells were dispensed on filter circles as described in the legend to Fig. 1, and in Materials and Methods. The filters were incubated in condition B: (-O-O-), exposed to 1 mrd-Formycin starting from zero time; -O-O--, exposed to Formycin starting at 17 h, A-, untreated aontrols. Measurements of specific by shifting the filters to fresh pads; -A-enzyme aotivity were made as described in Materials and Methods. Broken lines. These reference curves are taken from a previous publication (Newell & Sussman, 1970). They show UDPglucose pyrophosphorylase and UDPGal-4-epimerase specific aotivities in aggregates that formed migrating slugs and which, subsequently exposed to omnidirectional light and shifted to condition B, constructed fruiting bodies.
in Figure 2(a) (--A--A-) and elsewhere (Ashworth & Sussman, 1967). In migrating slugs (condition A) the activity accumulates very slowly but reaches the se,me peak level and does not disappear (broken line in Fig. 2(a); Newell & Sussman, 1970). If after this plateau is reached, the slugs are induced to stop migrating and construct fruiting bodies, they initiate a complete second round of UDPglucose pyrophosphorylase synthesis at a rapid rate (broken line in Fig. 2(a); Newell %, Sussman, 1970). As seen in Figure 2(a) (--O-O--) aggregates exposed to Formycin starting at 17 hours completed a normal first round of enzyme accumulation and then after s, short lag initiated a second round. The lag coincided with the transformation of the Mexican hats into slugs and the initiation of the second round coincided with the reversion to fruiting body construction. Cells exposed to Formycin from zero time, Figure 2(a) (-O-O-) performed similarly except that the program was advanced one to two hours, in step with the earlier onset of aggregation. When migrating slugs are induced to construct fruiting bodies before they have finished the first round of UDPglucose pyrophosphorylase accumulation, they simply complete the first round at an accelerated rate snd do not initiate a second round (broken line in Fig. 3; Newell & Sussman, 1970). Cells were allowed to develop into
634
R.
W. BRACKENBURY
IO
ET
AL.
& Time(h)
FIU. 3. Solid linea. Cells were harvested and washed as described in Materials and Methods and suspended in cold water at a density of 8 x lo* cells/ml. A sample of 60 ~1 was deposited in a thin line at one edge of 2% agar/water substratum in a rectangular plastic container. The container was covered with aluminum foil. In order to produce a horizontal light gradient a small hole was made opposite the line of cells. Before covering the containers, Millipore filters had been placed on the agar so that they would lie between the cells and the light source. The oontainers were incubated for a period of 12 h (to permit slug formation) plus enough time to allow the slugs to migrate on to the Alters (at a rate of 2 mm/h). At the time designated by the arrow the boxes were opened and the titers were shifted to pads saturated with LPS with (-a-e--) and without (-O-O-) 1 mad-Formycin and exposed to omnidirectional light (fluorescent ceiling fixtures). Measurements of UDPglucose pyrophosphorylase speoiflc activity were made as described in Materials and Methods. Broken lines. These reference curves were taken from a previous publication (Newell & Sussman, 1970). They show the accumulation and disappearance of UDPglucose pyrophosphorylase by aggregates that had constructed fruiting bodies directly or had transformed into migrating slugs.
migrating slugs. Before they had finished the fist round of pyrophosphorylase accumulation they were shifted to condition B and illuminated with omnidirectional light in the presence of Formyoin. These slugs shifted to the program of fruit construction, reached the Mexican hat stage, reverted to the slug stage very briefly, and then fruited. As indicated in Figure 3 (-O-e-) they did not stop synthesizing the pyrophosphorylase when they reached the usual level but continued on to a peak equivalent to two rounds of synthesis. (ii) UDPGal-d-epimerase
In aggregates that are constructing fruiting bodies directly, UDPGal-4-epimerase accumulates and disappears in the manner shown in Figure 2(b) (-AA-) and elsewhere (Telser & Sussman, 1971). In migrating slugs the epimerase does not accumulate at all. But whenever these slugs are induced to stop migrating and to construct fruiting bodies they initiate a complete round of epimerase accumulation (broken line in Fig. 2(b); Newell & Sussman, 1970). As seen in Figure 2(b) (-O-(-J--) cells exposed to Formycin from zero time did not accumulate epimerase activity initially but only after the Formyoin-induced slugs had reverted to the program of fruiting body construction. The peak value of epimerase activity in the Formycin-treated cells was somewhat lower then in the controls but their development was somewhat asynchronous and
MORPHOGENETIC
PATHWAYS
IN
D. DISCOIDEUM
636
this may account for the difference. In past experiments (Telser & Sussman, 1971) untreated populations, which were correspondingly asynchronous, also attained peaks of 10 units/mg or slightly less. (iii) Alanine transaminase and N-acetyl gEucoaaminicEase These enzyme activities begin to accumulate at the very beginning of the morphogenetic sequence, even before the overt formation of cell aggregates. One might expect that their developmental programs would be unaffected by the morphogenetic pathway elected by the aggregate at later stages of development and therefore be insensitive to the presence of Formycin. As shown in Figure 4, this expectation is realized. (a)
1
G OOW
Time (h) FIG. 4. Cells were tiestad md washed as desaribed in Materiels and Methods and dispensed on filter oiroles. The filters were incubated in oondition B (as desoribed in the legend to Fig. 1): (0) 1 mlrr-Formycin present from zero time; (A) untreated controls. Enzyme-specifia activities were meesured 8s described in Materials and Methods.
Trehdoee-6-phosphate synthetwe Figure 5(a) (-A-A-) shows the accumulation and disappearance of this activity during fruiting body construction. Sister cells incubated in the presence of Formycin from zero time were, as usual, morphogenetically advanced by about two hours and as seen in Figure 5(a) (-O-O-) the accumulation of the enzyme activity was correspondingly advanced. They attained the same peak as did the controls and did not embark upon a second round of accumulation as was the case for UDPglucose pyrophosphorylase. However, the subsequent disappearance of activity was much slower than in the controls. If the Formycin-induced behavior is closely related to (iv)
636
R. W. BRACKENBURY
ET
AL.
(b i2(b) E
IO-
t
FIG. 6. (a) Cells were dispensed on filter circles as described and incubated in condition B: controls. Measurements of --O-O-, exposed to 1 mM-Formycin from zero time; -A--&-, enzyme activity were made as described in Materials and Methods. (b) Cells were prepared and incubated &8 described in the legend to Fig. 3. The arrow refers to the time at which filters with migrating slugs were shifted to condition B and illuminated with omnidirectional light.
that of aggregates that have elected to transform into migrating shgs, one would predict that the latter would accumulate trehalose-6-phosphate q&hetase activity to normal levels and retain it until induced to stop migrating and construct fruits. Thereafter the cells would not initiate a second round of accumulation but would lose the activity accumulated during the first round. This enzyme activity in migrating slugs had not previously been examined. Since the above prediction represented a specific test of the hypothesis the experiment was performed and, as seen in Figure 5(b), the result was precisely in accord with the prediction. (c) Sensitivity of the Fowsycin e#ect to actinomycin D and cycloheximide Cells were allowed to construct fruiting bodies in the presence of Formycin B from zero time. At 22 hours the filters were shifted to fresh pads, saturated with LPS containing Formycin B and cycloheximide, shown previously to inhibit protein synthesis in D. discoideum. Cycloheximide stopped further morphogenesis and as seen in Figure 6 (-m---D--) stopped further accumulation of UDPglucose pyrophosphorylase.
BIORPHOGENETIC
PATHWAYS
IN
D.
Cycloheximide
DISCOIDEUM
637
[\ I
I
: 0
k+I
O
I’ /I / Ii
Actinomycin
(&-----10
’
1
I
20
3c
Time (h)
FIG. 6. Solid lines. Cells were incubated as described in the legend to Fig. Z(a): ( l ) at 14.6 h and (0) at 16.6 h shifted to fresh pads saturated with LPS aontaining 126 pg actinomycin D/ml; (0) at 22 h shifted to fresh pads saturated with LPS containing 500 pg cycloheximide/ml. Specific enzyme activities were measured as described in Materials and Methods. Broken lines. Enzyme accumulation and disappearance in untreated controls. The points for this curve have already been shown in Fig. 2(a) (these experiments were performed simultaneously).
During fruit construction, cells exposed to actinomycin D at any time prior to six hours fail to accumulate pyrophosphorylase activity. Those exposed after 15 to 16 hours accumulate the full complement. Those exposed at times between these, accumulate progressively increased levels. Cells exposed to actinomycin D at 14.5 and at 16.5 hours continued to develop normally for one to two hours and then stopped. They did not display the morphogenetic deviations normally induced by Formycin, and as seen in Figure 5(b) they failed to initiate an additional round of UDPglucose pyrophosphorylase accumulation.
4. Discussion (a) Effect of Formycin B on morphogenesis Previous results (Newell et al., 1969) have shown that if a newly formed D. discoideum cell aggregate is exposed to high pH, low ionic strength, high humidity and the local accumulation of a metabolite it transforms into a migrating slug and moves away from the site of aggregation. If exposed to the opposite set of conditions, it develops directly into a fruiting body. At any time up to the 16-hour stage of fruit construction, the aggregate can be caused to abandon that program in favor of slug migration by an appropriate change of external conditions. The reverse shift from slug migration to fruit construction can be effected at any time by an appropriate change of external conditions, as noted above, and/or by exposure of the slug to omnidirectional light. At present the metabolic bases for these choices of morphogenetic pathways are completely unknown. The results reported here indicate that at a still later stage of fruit construction, the aggregate can by exposure to Formycin B be induced (and only then induced)
638
R. W. BRACKENBURY
E’T
AL.
to transform into a migrating slug under conditions which otherwise favor fruit construction. Presumably because of the latter condition, this reversion is transitory and the Formycin-induced slug then resumes the program of fruit construction. These two decisions in favor of slug migration appear to be separate and distinct since the same cell aggregate can be induced to abandon (and continue) the program of fruit construction on two separate occasions by sequential shifts in the external conditions and exposure to Formycin B. Since Formycin B is an analog of inosine and adenosine, the possibility emerges that changes in the nucleoside and/or nucleoticle economy of the cells provide the metabolic signals that trigger the choice of morphogenetic pathways. A systematic examination of nucleoside and nucleoticle profiles of aggregates embarked on the alternative pathways, whether in the presence of Formycin B or otherwise, is being carried out at present in the hope of testing this possibility. (b) Morphogelaetic feedback The patterns of accumulation and disappearance of four developmentally regulated enzymes? are dramatically inlluenced by the choice of morphogenetio pathways (Newell 6 Sussman, 1970; Ellingson et al., 1971). The shifts of pattern observed in aggregates that have abandoned and re-entered the program of fruit construction as a result of exposure to Formycin B are entirely consistent with these earlier data, given the times at which the program is abandoned and re-entered. This gives further indication that the program of gene expression of a cell within a developing aggregate is not only influenced by its relative position within the assembly but also by the flow of morphogenetic events occurring to the assembly as a whole. Thus cells having corresponding relative positions appear to be doing decidedly different things depending upon the progression of the cell assembly along one morphogenetio pathway or the other. (c) Quanta2 control The patterns of enzyme accumulation and disappearance have also been studied in cells dispersed from developing aggregates and allowed to reaggregate, to recapitulate their previous morphogenesis, and to develop further (Newell et al., 1972). The pattern of regulation observed here and in aggregates developing along alternative morphogenetic pathways has been termed “quanta1 control” (Sussman t Newell, 1972). Depending on the morphogenetic circumstance, a particular enzyme may accumulate rapidly or slowly, now or later, or not at all. There can be one or two, or even three, successive rounds of accumulation. But in each such round, whether fast or slow, immediate or delayed, initial or additional, a quanta1 amount of activity characteristic of each enzyme accumulates. Each such round requires a separate prior period of transcription. The pattern of control displayed by the Formycininduced slugs appears to be consistent with the quanta1 control model. t The fourth This work
enzyme is UDPGal: was performed
with
polysaocharide
transferam.
the aid of a grant
from
the Authority
for Researoh
and
Development, Hebrew University, Jerusalem, Israel and from the National Institute of Health (GM 18689). One of the authors (R. W. B.) is a Prado&oral Fellow of the National Science Foundation (U.S.A.). Another of the authors (8. A.) is a Predoctoral Trainee, National Institutes of Health Graduate Training Program T.HD22, administered by Brandeis University.
MORPHOGENETIC
PATHWAYS
IN
D.
DISCOIDEUM
539
REFERENCES Ashworth, J. M. t Sussman, M. (1967). J. Bid. Chem. 242, 16961700. Biophye. Acta, 244, 388-395. Ellingson, J. S., Telser, A. & Sussman, M. (1971). B&him. Firtel, R. & Brackenbury, R. W. (1972). Dew. Bid. 27, 307-321. Koyama, G., Maeda, K., Umezawa, H. t Iitaka, Y. (1966). Tetruhedron Letters, 6, 597602. Loomis, W. F., Jr (1969). J. Bacterial. 97, 1149-1154. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Bid. Chem. 193,
265-275. Newell, P. C. & Sussman, M. (1969). J. Biol. Chem. 244, 2990-2995. Newell, P. C. & Sussman, M. (1970). J. Mol. BioZ. 49, 627-637. Newell, P. C., Telser, A. & Sussman, M. (1969). J. Bacterial. 100, 763-768. Newell, P. C., Franke, J. & Sussman, M. (1972). J. Mol. BioZ. 63, 373-382. Roth, R. & Sussman, M. (1968). J. BioZ. Chem. 243, 5081-6087. Slifkin, M. K. & Bonner, J. T. (1952). BioZ. Bull. (Woo& Hole), 102, 273-277. Sussman, M. (1966). In Methods in Cell Phyeiology (Prescott, D., ed.), vol. 2, pp. 387-409, Academic Press, New York. Genetics and Developmental Biology Sussman, M. & Newell, P. C. (1972). In Molecular (Sussman, M., ed.), pp. 275-302, Prentice Hall, New Jersey. Telser, A. & Sussman, M. (1971). J. BioZ. Chem. 246, 2252-2257.
A Choice of Morphogenetic Pathways in Dictyostelium discoideum induced by the Adenosine Analog Formycin B ROBERT
W.
BRACKENBURP,
JOEL SWINDLER,
AND MAVRICE
STEPHEN ALEXANDER
SUSSM~LN
Section of Developmental and Molecular Biology, Institute of Life Sciences Hebrew University, Jerusalem, Israel, and Department of Biology Brundeis University, Waltham, Mass., U.S.A. (Received 26 April 1974, a& in recised form 23 September 1974) Depending on several external parameters, a newly formed multioellular aggregate of Dictyostelium diecoideuna can elect either to construct a fruiting body directly at the site of aggregation or to transform into a migrating slug and then to construct a fruiting body hours or days later when external conditions permit. When aggregates were incubated in the presence of Formycin B under conditions that ordinarily induce fruiting body construction, they followed the normal program of fruit construction but stopped at a late stage and transformed into structures resembling migrating slugs. Shortly thereafter they resumed the fruiting program. The choices of morphogenetic pathways dictated early by onvironmental parameters and later by Formycin B produce characteristic, dramatic changes in the patterns of enzyme accumulation and disappearance. The control of these patterns appears to act at the level of transcription.
1. Introduction In Dictyostclium discoideum, vegetative cells that have entered the stationary growth phase collect together into organized multicellular aggregates. Influenced by several environmental parameters (see legend to Fig.* l), each aggregate elects either to construct a fruiting body directly at the site of aggregation or to transform into a migrating slug and move away. (Figure 1 shows drawings of these morphogenetic events.) The slug can migrate for many days (Slifkin & Bonner, 1952) but if exposed to omnidirectional light and/or shifted to conditions favoring fruit construction, it immediately stops migrating and constructs a fruiting body over a seven-hour period (Newell et d., 1969). The patterns of accumulation and disappearance of several developmentally regulated enzymes have been shown to depend crucially on whether the aggregate develops into a fruit or a slug and, if the latter, whether it subsequently elects to stop migrating and start fruiting (Newell t Sussman, 1970 ; Ellingson et al., 1971). These patterns and their moditications appear to be convenient and useful objects with which to elucidate the regulatory system that controls gene expression in these organisms (Sussman & Newell, 1972). The present communication describes the capacity of the adenosine analog, Formycin
B, 3-@D-ribofuranosyl)pyrazolo(4,3-d)6(H)7-pyrimidone 629
(Koyama
et al., 1966)
R. W. BRACKENBURY
530
ET
AL.
to influence the choice of morphogenetio and biochemical pathways as above. Formyoin B can induce aggregates at a late stage of fruiting body tion to suspend their activities and to transform into structures resembling slugs. These slugs subsequently resume the program of fruit construction. chemical consequences of these shifts are described.
described construomigrating The bio-
Condition A
Omnidirectional light, plus shift to condition t3
I
c
16 \
/
Add Formycin
18
L1
18
20
1 ’
20
22
1 ”
22
24
’
24
1 ’
26
(1 28
Fro. 1. Alternative morphogenetic pathways in D. discoidcu‘eumand the effect of Formycin B. The important parameters for this choice (pH, salt concentration, humidity, accumulation of metabolites and light) were controlled by the following experimental procedures. Cells were harvested from growth plates, washed and dispensed in portions of lo* cells on 2 in. Millipore or Whatman No. 50 filter circles (Sussman, 1966). To induce aggregates to develop directly into fruiting bodies (condition B) the fllters were incubated on absorbent pads saturated with LPS inside 60 mm Petri dishes. Pads cemented to the dish covers were saturated with 1 Mphosphate solution (pH 6.0) in order to absorb volatile, alkaline materials. To induoe aggregates to develop into migrating slugs (condition A), filters were incubated on pads saturated with water or weak (10 mM) phosphate (pH 6.5) solution. Disk covers without absorbent pads were used and the plates were incubated in the dark or in a weak, horizontal light gradient. Alternatively, washed cells were dispensed directly on 2% agar in water and incubated in the dark or in a horizontal light gradient (Ellingson et d., 1971). If at any time aggregates that have developed into migrating slugs (condition A) are shifted to oondition B, and/or exposed to omnidirectional light, the slugs are rapidly transformed into structures resembling the 16 h stage of fruit construction and these follow the normal pathway to mature fruiting bodies over a 7-h period. If at any time prior to the 17-h stage of fruit construction, filters are shifted from condition B to condition A, the aggregates stop fruiting body construction and are transformed into migrating slugs. However, between 16 and 17 h of fruit
MORPHOGENETIC
PATHWAYS
IN
D.
DlSCOIDEUM
631
2. Materials and Methods (a) Organism
ati
culture methods
D. &coideum (haploid) strain DdB was grown in association with Aerobaoter awogenes as previously described (Sussman, 1966). Cells were harvested from the growth plates in cold water and washed free of exogenous bacteria by three centrifugations at 1200 g for 5 min. Finally they were suspended in a solution (LPS) containing 40 mm-phosphate (pH 6.5), 20 mM-HCl, 2.5 man-Mg& and O-7 maa-streptomycin sulfate. To permit fruiting body construction or the formation of migrating slugs, the cells were incubated as described in the legends to Figs 1 and 3. (b) Enzyme (i) UDPglucose
assays
pyrophosphoryluse
Cells were harvested in 0.1 a6-Tricine (pH 75) solution and stored at -20°C. The cells were lysed by addition of Cemulsol NTPl2 (see section (d) below) to a fuml concentration of 0.15°h and the extracts were assayed immediately as described previously (Newell & Sussman, 1969). 1 unit of activity is defined as 1 nmol product formed/mm at 37°C. (ii) UDPGal-C-epimeraae Cells were harvested in 0.1 M-N-Tris(hydroxymethyl)methylglycine (pH 7.5) solution containing 20% glycerol, lysed as described above and assayed immediately as described previously (Telser & Sussman, 1971). 1 unit of activity is defmed as 1 nmol product formed/mm at 37°C. (iii)
synthetase
Trehalose-6-phosphate
Cells were harvested in 0.01 a6-2(iV-morpholino)ethane sulfonic acid (pH containing 7.3 w-sodium thioglycollate, sonicated at 2 A intensity for Branson sonifier, and mayed immediately as described (Roth & Sussman, et al., 1972). 1 unit of activity is defined as 1 nmol of product formed/min at (iv) Alanine
6.5) solution 30 s/ml in a 1968; Newell 37°C.
transaminase
Cells were harvested in a 15% glycerol solution containing 0.2 mMdithiothreito1 and frozen. They were thawed, sonicated and assayed as described previously (Firtel & Brackenbury, 1972). 1 unit of activity is defined as 1 nmol product formed/mm at 28°C.
(v) N-AcetyZglucosaminidaae Cells were harvested previously described formed/min at 37°C.
in water (Loomis,
and frozen. They were thawed, sonicated and assayed as 1969). 1 unit of activity is defined as 1 nmol product (0) Protein
These were performed
by the method
estimation et al. (1951).
of Lowry
(d) Chemicals Formycin B, a Meiji product, was purchased from Calbiochem. N-Tris-(hydroxymethyl) methylglycine and 2(N-morpholino)ethane sulfonic acid were purchased from Calbiochem. Cemulsol NPTlP was obtained from Melle Bezons Inc., France. Actinomycin D was a kind gift of Dr Harlyn Halvorson, Rosenstiel Basic Medical Sciences Research Centre, Waltham, Mass. Cycloheximide was purchased from Upjohn. Alanine, cr-ketoglutarate and p-nitrophenyl-N-acetyl-fi-n-gluoosaminide were purchased from Sigma. All other chemicals were reagent grade. construction, the aggregates become committed to that pathway and continue to construct fruits despite the shift to condition A. To produce the Formycin effect (condition C), the filters were incubated in condition B until the 17-h stage w&s reached and then shifted to pads containing 1 mpn-Formycin in LPS. Pads cemented to the Petri dish covers were saturated with 1 M-phosphate, pH 6.0.
532
R. W. BRACKENBURY
ET’ AL.
3. Results (8) Morphogenesis in the presence of Formycin Figure 1 (conditions A and B) show the alternative morphogenetic pathways of D. dkwideum. As previously described (Newell et al., 1969) it is possible to control experimentally the entrance of cell aggregates into or their progress along either pathway (see legend to Fig. 1 for details). Figure 1 (condition C) shows the morphogenetic pathway followed by aggregates that are allowed to develop in condition B but then are exposed to Formycin starting from 17 hours. Instead of assuming the normal Mexican hat shape at 18 hours the aggregate assumes a deviant form with a greatly expanded apex and reduced basal periphery. The apex progressively assumes the shape of a slug, while the basal portion continues to regress until the whole aggregate is transformed into a migrating slug. This condition is very transitory. Almost immediately the slug is transformed into a shape resembling an early Mexican hat and thereafter follows the normal sequence of fruiting body construction. In summary, the pathway followed corresponds to that induced by condition A except that the decision of the cell aggregate to transform into a migrating slug is made at a late stage of fruit construction and in environmental conditions that ordinarily induce fruiting body construction directly. Additional conclusions regarding the effect of Formycin together with the pertinent experimental results are given below. (1) The response illustrated in Figure 1C is qualitatively unaltered when cells are exposed to Formycin B prior to 17 hours. Thus cells exposed to the agent from zero time did begin aggregation one to two hours early but otherwise developed normally to the Mexican hat stage and then diverged along pathway C. (2) Other nucleosides (adenosine, inosine, hypoxanthine) at similar concentrations were without effect on morphology or accumulation of UDPglucose pyrophosphorylase (data not shown). (3) The late decision to transform into a migrating slug induced by Formycin B is separate and distinct from the same decision induced earlier by pH, ionic strength, etc. (see legend to Fig. 1). Thus, cell aggregates induced to develop along pathway A to the migrating slug stage were exposed to Formycin. The slugs remained unaltered and continued to migrate. However, if shifted to condition B and exposed to omnidirectional light while in the presence of Formycin, the slugs stopped migrating, entered the program of fruit construction, reached the Mexican hat stage and the?L reverted to migrating slugs once again before ultimately producing normal fruiting bodies. (4) The re-entrance of the Formycin-induced slugs to the fruiting program is not due to the disappearance of Formycin B. Thus cells were exposed to Formycin B from zero time. At 20 hours the aggregates had diverged along pathway C and were developing into slugs. The filters were then switched to fresh pads saturated with 1 mlrr-Formycin B in LPS. The slugs reverted to the fruiting program at the same time as slugs that had not been switched to fresh Formycin B. (b) Effect of Formycin on the patterns of enzyme accumulation and disappearance (i) UDPglucose pyrophosphorylase In aggregates that are constructing fruiting bodies directly (condition B) UDPglucose pyrophosphorylase activity accumulates and disappears in the manner shown
MOBPHOGENETIC
PATHWAYS
/
/’
IO
IN
D.
DISCOIDEUM
Light plus shift to condition B
5 .-
OO
17
Lightplusshift to condition 8
, 1 1 I 1 i
I :
c
\
:
-----:--,-e-3 20
533
30
40
-50
Time(h) FIQ. 2. Solid lines: Cells were dispensed on filter circles as described in the legend to Fig. 1, and in Materials and Methods. The filters were incubated in condition B: (-O-O-), exposed to 1 mrd-Formycin starting from zero time; -O-O--, exposed to Formycin starting at 17 h, A-, untreated aontrols. Measurements of specific by shifting the filters to fresh pads; -A-enzyme aotivity were made as described in Materials and Methods. Broken lines. These reference curves are taken from a previous publication (Newell & Sussman, 1970). They show UDPglucose pyrophosphorylase and UDPGal-4-epimerase specific aotivities in aggregates that formed migrating slugs and which, subsequently exposed to omnidirectional light and shifted to condition B, constructed fruiting bodies.
in Figure 2(a) (--A--A-) and elsewhere (Ashworth & Sussman, 1967). In migrating slugs (condition A) the activity accumulates very slowly but reaches the se,me peak level and does not disappear (broken line in Fig. 2(a); Newell & Sussman, 1970). If after this plateau is reached, the slugs are induced to stop migrating and construct fruiting bodies, they initiate a complete second round of UDPglucose pyrophosphorylase synthesis at a rapid rate (broken line in Fig. 2(a); Newell %, Sussman, 1970). As seen in Figure 2(a) (--O-O--) aggregates exposed to Formycin starting at 17 hours completed a normal first round of enzyme accumulation and then after s, short lag initiated a second round. The lag coincided with the transformation of the Mexican hats into slugs and the initiation of the second round coincided with the reversion to fruiting body construction. Cells exposed to Formycin from zero time, Figure 2(a) (-O-O-) performed similarly except that the program was advanced one to two hours, in step with the earlier onset of aggregation. When migrating slugs are induced to construct fruiting bodies before they have finished the first round of UDPglucose pyrophosphorylase accumulation, they simply complete the first round at an accelerated rate snd do not initiate a second round (broken line in Fig. 3; Newell & Sussman, 1970). Cells were allowed to develop into
634
R.
W. BRACKENBURY
IO
ET
AL.
& Time(h)
FIU. 3. Solid linea. Cells were harvested and washed as described in Materials and Methods and suspended in cold water at a density of 8 x lo* cells/ml. A sample of 60 ~1 was deposited in a thin line at one edge of 2% agar/water substratum in a rectangular plastic container. The container was covered with aluminum foil. In order to produce a horizontal light gradient a small hole was made opposite the line of cells. Before covering the containers, Millipore filters had been placed on the agar so that they would lie between the cells and the light source. The oontainers were incubated for a period of 12 h (to permit slug formation) plus enough time to allow the slugs to migrate on to the Alters (at a rate of 2 mm/h). At the time designated by the arrow the boxes were opened and the titers were shifted to pads saturated with LPS with (-a-e--) and without (-O-O-) 1 mad-Formycin and exposed to omnidirectional light (fluorescent ceiling fixtures). Measurements of UDPglucose pyrophosphorylase speoiflc activity were made as described in Materials and Methods. Broken lines. These reference curves were taken from a previous publication (Newell & Sussman, 1970). They show the accumulation and disappearance of UDPglucose pyrophosphorylase by aggregates that had constructed fruiting bodies directly or had transformed into migrating slugs.
migrating slugs. Before they had finished the fist round of pyrophosphorylase accumulation they were shifted to condition B and illuminated with omnidirectional light in the presence of Formyoin. These slugs shifted to the program of fruit construction, reached the Mexican hat stage, reverted to the slug stage very briefly, and then fruited. As indicated in Figure 3 (-O-e-) they did not stop synthesizing the pyrophosphorylase when they reached the usual level but continued on to a peak equivalent to two rounds of synthesis. (ii) UDPGal-d-epimerase
In aggregates that are constructing fruiting bodies directly, UDPGal-4-epimerase accumulates and disappears in the manner shown in Figure 2(b) (-AA-) and elsewhere (Telser & Sussman, 1971). In migrating slugs the epimerase does not accumulate at all. But whenever these slugs are induced to stop migrating and to construct fruiting bodies they initiate a complete round of epimerase accumulation (broken line in Fig. 2(b); Newell & Sussman, 1970). As seen in Figure 2(b) (-O-(-J--) cells exposed to Formycin from zero time did not accumulate epimerase activity initially but only after the Formyoin-induced slugs had reverted to the program of fruiting body construction. The peak value of epimerase activity in the Formycin-treated cells was somewhat lower then in the controls but their development was somewhat asynchronous and
MORPHOGENETIC
PATHWAYS
IN
D. DISCOIDEUM
636
this may account for the difference. In past experiments (Telser & Sussman, 1971) untreated populations, which were correspondingly asynchronous, also attained peaks of 10 units/mg or slightly less. (iii) Alanine transaminase and N-acetyl gEucoaaminicEase These enzyme activities begin to accumulate at the very beginning of the morphogenetic sequence, even before the overt formation of cell aggregates. One might expect that their developmental programs would be unaffected by the morphogenetic pathway elected by the aggregate at later stages of development and therefore be insensitive to the presence of Formycin. As shown in Figure 4, this expectation is realized. (a)
1
G OOW
Time (h) FIG. 4. Cells were tiestad md washed as desaribed in Materiels and Methods and dispensed on filter oiroles. The filters were incubated in oondition B (as desoribed in the legend to Fig. 1): (0) 1 mlrr-Formycin present from zero time; (A) untreated controls. Enzyme-specifia activities were meesured 8s described in Materials and Methods.
Trehdoee-6-phosphate synthetwe Figure 5(a) (-A-A-) shows the accumulation and disappearance of this activity during fruiting body construction. Sister cells incubated in the presence of Formycin from zero time were, as usual, morphogenetically advanced by about two hours and as seen in Figure 5(a) (-O-O-) the accumulation of the enzyme activity was correspondingly advanced. They attained the same peak as did the controls and did not embark upon a second round of accumulation as was the case for UDPglucose pyrophosphorylase. However, the subsequent disappearance of activity was much slower than in the controls. If the Formycin-induced behavior is closely related to (iv)
636
R. W. BRACKENBURY
ET
AL.
(b i2(b) E
IO-
t
FIG. 6. (a) Cells were dispensed on filter circles as described and incubated in condition B: controls. Measurements of --O-O-, exposed to 1 mM-Formycin from zero time; -A--&-, enzyme activity were made as described in Materials and Methods. (b) Cells were prepared and incubated &8 described in the legend to Fig. 3. The arrow refers to the time at which filters with migrating slugs were shifted to condition B and illuminated with omnidirectional light.
that of aggregates that have elected to transform into migrating shgs, one would predict that the latter would accumulate trehalose-6-phosphate q&hetase activity to normal levels and retain it until induced to stop migrating and construct fruits. Thereafter the cells would not initiate a second round of accumulation but would lose the activity accumulated during the first round. This enzyme activity in migrating slugs had not previously been examined. Since the above prediction represented a specific test of the hypothesis the experiment was performed and, as seen in Figure 5(b), the result was precisely in accord with the prediction. (c) Sensitivity of the Fowsycin e#ect to actinomycin D and cycloheximide Cells were allowed to construct fruiting bodies in the presence of Formycin B from zero time. At 22 hours the filters were shifted to fresh pads, saturated with LPS containing Formycin B and cycloheximide, shown previously to inhibit protein synthesis in D. discoideum. Cycloheximide stopped further morphogenesis and as seen in Figure 6 (-m---D--) stopped further accumulation of UDPglucose pyrophosphorylase.
BIORPHOGENETIC
PATHWAYS
IN
D.
Cycloheximide
DISCOIDEUM
637
[\ I
I
: 0
k+I
O
I’ /I / Ii
Actinomycin
(&-----10
’
1
I
20
3c
Time (h)
FIG. 6. Solid lines. Cells were incubated as described in the legend to Fig. Z(a): ( l ) at 14.6 h and (0) at 16.6 h shifted to fresh pads saturated with LPS aontaining 126 pg actinomycin D/ml; (0) at 22 h shifted to fresh pads saturated with LPS containing 500 pg cycloheximide/ml. Specific enzyme activities were measured as described in Materials and Methods. Broken lines. Enzyme accumulation and disappearance in untreated controls. The points for this curve have already been shown in Fig. 2(a) (these experiments were performed simultaneously).
During fruit construction, cells exposed to actinomycin D at any time prior to six hours fail to accumulate pyrophosphorylase activity. Those exposed after 15 to 16 hours accumulate the full complement. Those exposed at times between these, accumulate progressively increased levels. Cells exposed to actinomycin D at 14.5 and at 16.5 hours continued to develop normally for one to two hours and then stopped. They did not display the morphogenetic deviations normally induced by Formycin, and as seen in Figure 5(b) they failed to initiate an additional round of UDPglucose pyrophosphorylase accumulation.
4. Discussion (a) Effect of Formycin B on morphogenesis Previous results (Newell et al., 1969) have shown that if a newly formed D. discoideum cell aggregate is exposed to high pH, low ionic strength, high humidity and the local accumulation of a metabolite it transforms into a migrating slug and moves away from the site of aggregation. If exposed to the opposite set of conditions, it develops directly into a fruiting body. At any time up to the 16-hour stage of fruit construction, the aggregate can be caused to abandon that program in favor of slug migration by an appropriate change of external conditions. The reverse shift from slug migration to fruit construction can be effected at any time by an appropriate change of external conditions, as noted above, and/or by exposure of the slug to omnidirectional light. At present the metabolic bases for these choices of morphogenetic pathways are completely unknown. The results reported here indicate that at a still later stage of fruit construction, the aggregate can by exposure to Formycin B be induced (and only then induced)
638
R. W. BRACKENBURY
E’T
AL.
to transform into a migrating slug under conditions which otherwise favor fruit construction. Presumably because of the latter condition, this reversion is transitory and the Formycin-induced slug then resumes the program of fruit construction. These two decisions in favor of slug migration appear to be separate and distinct since the same cell aggregate can be induced to abandon (and continue) the program of fruit construction on two separate occasions by sequential shifts in the external conditions and exposure to Formycin B. Since Formycin B is an analog of inosine and adenosine, the possibility emerges that changes in the nucleoside and/or nucleoticle economy of the cells provide the metabolic signals that trigger the choice of morphogenetic pathways. A systematic examination of nucleoside and nucleoticle profiles of aggregates embarked on the alternative pathways, whether in the presence of Formycin B or otherwise, is being carried out at present in the hope of testing this possibility. (b) Morphogelaetic feedback The patterns of accumulation and disappearance of four developmentally regulated enzymes? are dramatically inlluenced by the choice of morphogenetio pathways (Newell 6 Sussman, 1970; Ellingson et al., 1971). The shifts of pattern observed in aggregates that have abandoned and re-entered the program of fruit construction as a result of exposure to Formycin B are entirely consistent with these earlier data, given the times at which the program is abandoned and re-entered. This gives further indication that the program of gene expression of a cell within a developing aggregate is not only influenced by its relative position within the assembly but also by the flow of morphogenetic events occurring to the assembly as a whole. Thus cells having corresponding relative positions appear to be doing decidedly different things depending upon the progression of the cell assembly along one morphogenetio pathway or the other. (c) Quanta2 control The patterns of enzyme accumulation and disappearance have also been studied in cells dispersed from developing aggregates and allowed to reaggregate, to recapitulate their previous morphogenesis, and to develop further (Newell et al., 1972). The pattern of regulation observed here and in aggregates developing along alternative morphogenetic pathways has been termed “quanta1 control” (Sussman t Newell, 1972). Depending on the morphogenetic circumstance, a particular enzyme may accumulate rapidly or slowly, now or later, or not at all. There can be one or two, or even three, successive rounds of accumulation. But in each such round, whether fast or slow, immediate or delayed, initial or additional, a quanta1 amount of activity characteristic of each enzyme accumulates. Each such round requires a separate prior period of transcription. The pattern of control displayed by the Formycininduced slugs appears to be consistent with the quanta1 control model. t The fourth This work
enzyme is UDPGal: was performed
with
polysaocharide
transferam.
the aid of a grant
from
the Authority
for Researoh
and
Development, Hebrew University, Jerusalem, Israel and from the National Institute of Health (GM 18689). One of the authors (R. W. B.) is a Prado&oral Fellow of the National Science Foundation (U.S.A.). Another of the authors (8. A.) is a Predoctoral Trainee, National Institutes of Health Graduate Training Program T.HD22, administered by Brandeis University.
MORPHOGENETIC
PATHWAYS
IN
D.
DISCOIDEUM
539
REFERENCES Ashworth, J. M. t Sussman, M. (1967). J. Bid. Chem. 242, 16961700. Biophye. Acta, 244, 388-395. Ellingson, J. S., Telser, A. & Sussman, M. (1971). B&him. Firtel, R. & Brackenbury, R. W. (1972). Dew. Bid. 27, 307-321. Koyama, G., Maeda, K., Umezawa, H. t Iitaka, Y. (1966). Tetruhedron Letters, 6, 597602. Loomis, W. F., Jr (1969). J. Bacterial. 97, 1149-1154. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Bid. Chem. 193,
265-275. Newell, P. C. & Sussman, M. (1969). J. Biol. Chem. 244, 2990-2995. Newell, P. C. & Sussman, M. (1970). J. Mol. BioZ. 49, 627-637. Newell, P. C., Telser, A. & Sussman, M. (1969). J. Bacterial. 100, 763-768. Newell, P. C., Franke, J. & Sussman, M. (1972). J. Mol. BioZ. 63, 373-382. Roth, R. & Sussman, M. (1968). J. BioZ. Chem. 243, 5081-6087. Slifkin, M. K. & Bonner, J. T. (1952). BioZ. Bull. (Woo& Hole), 102, 273-277. Sussman, M. (1966). In Methods in Cell Phyeiology (Prescott, D., ed.), vol. 2, pp. 387-409, Academic Press, New York. Genetics and Developmental Biology Sussman, M. & Newell, P. C. (1972). In Molecular (Sussman, M., ed.), pp. 275-302, Prentice Hall, New Jersey. Telser, A. & Sussman, M. (1971). J. BioZ. Chem. 246, 2252-2257.