Regulation of four functionally related enzymes during shifts in the developmental program of Dictyostelium discoideum

J. Mol.

Biol.

(1972) 68, 373-382

Regulation of Four Functionally Related Enzymes during Shifts in the Developmental Program of Dictyostelium discoideum PETERC.

NEWELL,JAKOBFRANKEANDMAURICE STJSSMAN Microbiology University

Unit, Department of Biochemistry of Oxford, OX1 3&U, England and Department of Biology, Brandeis University Waltham, Mass. 02154, U.S.A. (Received 26 July

1971)

Partially developed cell aggregates of Dictyostelium discoideum engaged in fruiting body construction can be disaggregated to single cells and redeposited on solid substratum. The cells almost immediately reaggregate, recapitulate within two to three hours a previous course of morphogenesis that might have taken as much as 18 to 20 hours the first time, and then complete the construction of fruiting bodies with approximately normal timing. During particular stages of fruit construction, four functionally related enzymes accumulate, reach characteristic levels of specific activity, and then disappear partly or completely. They are: UDP-glucose pyrophosphorylase, trehalose-6-phosphate synthetase, UDP-galactose-4-epimerase and UDP galactose : polysaccharide galactosyl transferase. The disaggregated cells discontinued the initial rounds of enzyme synthesis, but when permitted to reaggregate and develop further, they accomplished complete second rounds of enzyme accumulation. If disaggregated and redeposited a second time, they accomplished complete third rounds of enzyme synthesis as well. All such additional rounds required new periods of RNA synthesis. A characteristic level of each enzyme activity accumulated during each round of synthesis regardless of the amount previously accumulated. In these additional rounds the temporal relations between transcription and enzyme synthesis were drastically altered,

1. Introduction After growth has stopped, the previously independent cellsof Dictyostelium discoideum collect together into an organized multicellular aggregate and begin a complex morphogenetic sequencethat culminates in the construction of a fruiting body with two differentiated components, spores and stalk cells. The sequenceis accompanied by the turnover and replacement of the bulk of the pre-existing cell RNA and protein, including the accumulation and disappearanceof a variety of enzymes (Sussman & Sussman, 1969). The entire successionof morphogenetic and biochemical events and, ultimately, the developmental fates of the individual cells (i.e. whether they are to become spores or stalk cells) depend upon a complex matrix of cell interactions which appear to operate partly through exchange of diffusible metabolites and partly 373

374

P.

C. NEWELL,

J.

FRANKE

AND

M.

SUSSMAN

through direct cell surface contacts (Raper, 1940,196O; Bonner, 1949; Sussman & Lee, 1955). Partially developed cell aggregates can be mechanically disrupted to yield suspensions of separated cells. When redeposited on a solid substratum, these cells immediately reaggregate and rapidly recapitulate the normal morphogenetic sequence. Reattaining the stage at which they were disaggregated, they then go on to complete the construction of the fruiting body with approximately normal timing (Newell, Longlands & Sussman, 1971). This interference with the hierarchy of cell associations was shown to have a profound effect on the accumulation of at least one developmentally regulated enzyme, UDP-glucose pyrophosphorylase (EC 2.7.7.9) (Ashworth & Sussman, 1967). The initially present at an activity of 50 units/mg/prot’ein, acpyrophosphorylase, cumulates to a peak of about 400 to 450 units/mg during fruit construction. Disaggregated cells, permitted to reaggregate and resume development, repeated t,he program of pyrophosphorylase synthesis, forming an additional 300 units of enzyme regardless of the level they had already accumulated prior to disaggregation. This second period of accumulation required an additional period of RNA synthesis which appeared to be triggered by the act of reaggregation (Newell et al., 1971). Three other developmentally regulated enzymes have been characterized which bear a close functional relationship with UDP-glucose pyrophosphorylase (I). These include (II) trehalose-6-P synthet,ase (EC 2.3.1.15) (Roth & Sussman, 1968), (III) UDP-galactose-4-epimerase (EC 5.1.3.2) (Telser & Sussman, 1971) and (IV) UDP galactose : polysaccharide galactosyl transferase (Sussman & Osborn, 1964). The metabolic relations are shown below:

+Gx

G-I-P’IUDPG

Trcholose-6

UDPGol

-P

F

Treholosc

Mucopolysocchoride

Ip_

Both products are uniquely associated with the spores of the mature fruiting body. It was of interest to determine if the quantitative nature of gene expression observed in the case of the pyrophosphorylase is common to all four enzymes. The results to be described indicate that it is indeed a general phenomenon.

2. Materials (a)

Organism

and

and Methods experimental

conditions

D. discoideum strain NC-4 (haploid) was cultivated as previously described (Newell, Telser & Sussman, 1969) with Aerobueter aerogenes strain 1033 as a food source. The amoebae were harvested from the late logarithmic phase of growth and freed from the remaining bacteria by repeated centrifugation at approx. 150 g. Samples of 0.5 ml. containing 10s cells were deposited on 42*5-mm Whatman no. 50 filters. These rested inside 60-mm Petri dishes on support pads saturated with lower pad solution containing in g/l.: KCl, 1.5; MgCls.6Hs0, 0.5; streptomycin sulphate, 0.5; NasHP04/KH2P04, 40 rnM at pH 6.4. Cemented to the dish cover was another support pad saturated with 1 M-phosphate buffer, pH 6.0 (Newell et al., 1969). Under these conditions lo* cells form about 1000 aggregates and construct fruiting bodies synchronously over a 24-hr period.

ENZYME

REGULATION

IN

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DISCOIDEUM

375

(b ) Disuggregation. Aggregates were harvested from about 10 filters at 22°C into 20 ml. lower pad solution. Disaggregation was accomplished by repeatedly sucking up and blowing out the suspension from a fine-tipped lo-ml. pipette. The operation was monitored by microscopic examination. About 70% of the cells were present singly with about 20% doublets and 1Oy; triplets. The suspension was centrifuged and the cells resuspended in lower pad solution at a density of 2 x 10s cells/ml. Samples of 0.5 ml. were then deposited on Whatman no. 50 filter circles as described above. The entire operation of disaggregation and redeposition took about 30 min. (c)

UDPG-pyrophosphorylase

and

UDPGal

epimeraae

assays

Cells were harvested from single filters in 1.5 ml. of 0.1 M-Tricine . NaOH (pH 7.5) containing 20% v/v glycerol. The cells were lysed at room temperature by addition of 0.15 ml. of Cemulsol NPT-12 detergent 1.5% (v/v). Th e extracts were immediately assayed in a Gilford recording spectrophotometer for UDP-glucose pyrophosphorylase activity (Newell & Sussman, 1969) and for UDP-galactose-4-epimerase activity (Telser & Sussman, 1971). One unit of pyrophosphorylase activity is defined as the amount capable of converting 1 nmole of UDPG to G-1-P per min at 37°C. One unit of epimerase activity is defined as the amount capable of converting 1 nmole of UDPGal to UDPG per min at 37’C. (d)

UDP-galactose

:polysaccharide

galactosyl

transferme

assay

Cells were harvested from single filters in 3 ml. of 0.1 M-Tricine 9 NaOH (pH 7.5) and frozen. They were broken by treatment with a Branson sonifier for 60 set at 2 A intensity. Extracts were assayed immediately by a procedure described elsewhere (Loomis & Sussman, 1966). Since the rate of the reaction depends on the concentration of mucopolysaccharide acceptor as well as of the enzyme, activity is expressed as cts/min of galactose transferred from UDP [l*C]galactose to a standard concentration of acceptor. (e)

Trehalose-6-P

synthetase

assay

Cells from 1 to 3 filters were harvested in 1 ml. of 0.01 M-Tris * HCl, pH 7.5, containing 0.5 mM-sodium thioglycollate and frozen. They were broken by treatment with a Branson sonifler for 30 set/ml. of extract at 2.0 A intensity. Extracts were then assayed immediately by a modification of the previous method (Roth & Sussman, 1968). The reaction mixture contained 0.64 ml. of 0.063 M-UDPGlucose, 0.4 ml. of 0.4 M-G-~-P, 0.76 ml. of 2.5 M-KC& 0.2 ml. of 1.5 M-MgCl, +0.05 M-EDTA. The final pH was 7.0. 0.1 ml. of this mixture was added to 50 to 150 ~1. of extract and the volume adjusted to 0.25 ml. with 0.01 m-Tris * HCl (pH 7.5) buffer. The final concentrations were : 0.008 M-UDPGlucose; 0.032 M-G-~-P ; 0.38 M-KCl; 0.060 M-MgCl,; O-002 M-EDTA; 0*004 M-Tris * HCl. Tubes were incubated for 30 min at 37°C. The reaction was stopped by 3 min incubation in a boiling water bath. The boiling period is critical since some UDPG is converted to UDP. The UDP produced by the catalytic activity of the synthetase was assayed as follows. 0.2 ml. of the boiled sample was mixed in a l-ml. cuvette with 0.8 ml. of solution containing: 5 units pyruvate kinase (Boehringer); 0.25 pmole NADH; 5 units lactate dehydroKCl; 80 pmole Tricine * NaOH pH 7.6. genase (Worthington) ; 10 pmole MgCl, ; 10 pmole The decrease in O.D.Q~~~~ after addition of 0.15 pmole phosphoenolpyruvate is stoichiometrically related to the amount of UDP initially present. One unit of synthetase activity is defined as the amount capable of converting 1 nmole of UDPG to UDP per min at 37%. (f) This

was

performed

N-Acetyl

as described

by (g)

Protein was measured 1951) using bovine serum

by the albumin

glucosaminidase Loomis

Protein

and

phosphoenol and G-6-P from

determinations

Folin procedure as a standard. (h)

NADH UDPGlucose

pyruvate Sigma

assay

(1969).

(Lowry,

Rosebrough,

Farr

& Randall,

Chemicals

were Chemical

purchased Co.; Tricine

from Boehringer-Mannheim (Tris-hydroxymethyl-methyl

;

376

P.

C. NEWELI,,

J.

FRANKE

AND

glycine A grade) from Calbiochem. Cemulsol detergent was Bezons, Bezons, France. Reagents for the pyrophosphorylase, assays are described elsewhere (Newell et al., 1969; Telser Sussman, 1966).

M.

SUSSMAN

generously donated epimerase, and & Sussman, 1971;

by Melletranaferase Loomis &

3. Results (a) The time-course

of reaggregationand subsequent development

Figure 1 shows the time-course of morphogenetic events in cell populations that were (i) allowed to develop undisturbed on filters and (ii) disaggregated after 16.5 hours development, redeposited on fresh filters at the original cell density and incubated further. Subsequent morphogenesisfollowed precisely the same time-course Disaggregated c

FIG. 1. Cell aggregates were harvested after 16.5 hr on the filters and disaggregated (see ;\;Iaterials and Methods). They were redeposited on fresh filters at the original cell density and allowed to develop further. The drawings show the morphogenetic stages attained by these and by control aggregates left undisturbed. The numbers refer to time in hours after the initial deposition on 6lters.

as has been described elsewhere (Newell et al., 1971). That is, the cells reaggregated almost immediately and within two to three hours reattained the stage at which they had been d&aggregated. They then went on to construct fruiting bodies with approximately normal timing. This general pattern was encountered with cells disaggregated even after as much as 20 hours development on the filters. However, after 18 hours, those cells that have already begun construction of the stalk cannot be recovered. (b) Enzyme

accumulation

after reaggregation

Cell sampleswere taken at intervals during the course of reaggregation and subsequent development and extracts were prepared (see Materials and Methods) in order to measure the specific enzyme activities. The results are summarized in Figure 2 for UDPG pyrophosphorylaseT and UDPGal-4-epimerase, and in Figure 3 for trehalose-6-P synthetase. After reaggregation, the cells promptly resumedenzyme synthesis and accumulated an additional quantum of each of the enzymes, apparently independently of the stage at which disaggregation occurred or of the level of activity previously accumulated. t The data for UDPG pyrophosphorylase in Fig. (Newell et al., 1971) and are included for comparison. Fig. 4(a).

2 were taken from A repetition of that

an earlier experiment

publication is shown

in

ENZYME

REGULATION

IN

D.

DISCOIDEUM

377

Disaggregated 3 --

O-

o>’ I

I

I

I

16

I

I

20

12 Time

(a) FIG. 2. (a) Cells were disaggregated on the filters and redeposited on fresh were harvested and lysed in order to (see Materials and Methods). -O-O-, (b) Cells were d&aggregated after the titers and redeposited as above. activity of UDP-Galactose-4-epimerase Undisturbed controls.

/O

O-

I

24

I

16

I

20

(hr)

I‘00

I

24

(b)

after 15 hr (--@---a-) or after 18 hr (--A----A--) filters at the original cell density. At intervals, cell samples measure the specific activity of UDPG pyrophosphorylase Undisturbed controls. 17 hr (-A----A--) or after 18.5 hr (--n--O--) on Cell samples were harvested at intervals and the specific wasdetermined (see MaterialsandMethods). -o--O--,,

In the case of the epimerase, the previously accumulated activity rapidly disappeared during disaggregation and reaggregation prior to accumulation of the additional quantum. Invariably these augmented delayed peaks of enzyme activity occurred at the same morphogenetic stages as did the normal undelayed peaks in the undisturbed controls.

20,

z

5-

2

Disaggregated

z

4

8

12 ‘ime

FIG.

(-•----a---)

3. The

I

I

I

16

20

24

(hr)

accumulation of trehalose-6-P synthetase or 19 hr (-A-b-). -O-O--,

activity after disaggregation Undisturbed controls.

at

15.5

hr

378

P.

C. NEWELL,

J.

FRANKE

AND

M.

SUSSMAN

800-

Dlsoggregoted

2 u g

600-

T

IO

1

/

1

16 (0)

1

22

I

28 Time

IO

16

t hr)

I

!

I 22

I 28

tb)

FIQ. 4. Cells were disaggregated after 19 hr on the filters, redeposited on fresh filters at the original density and reincubated so that they could recapitulate their previous morphogenesis and develop further. At 23 hr, 4 hr after their redeposition, the cells were disaggregated a second time and redeposited on fresh filters at the original cell density. Cell samples were harvested at intervals and assayed for enzyme activity. (a) UDPG pyrophosphorylase; (b) UDPGal-4-epimerase.

(c) Enzyme accumulation during repeated disaggregations and reaggregations Cell populations were allowed to develop on filters, were disaggregated and redeposited on fresh filters at the original density, and incubated so that they might recapitulate their previous morphogenesis and develop further. The same cells were then disaggregated a second time and redeposited on fresh filters. The course of recapitulation and further development of the twice disaggregated cells was precisely the same as of the once disaggregated cells, and normal mature fruiting bodies eventually appeared.

Time

Fro. 5. Successive disaggregations were performed described in the legend to Fig. 4. After harvesting, polyseccharide transferase activity.

(hr)

at the times noted and the oells treated samples were assayed for UDP-galactose:

as

ENZYME

REGULATION

IN

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DISCOIDEUM

379

Figures 4 and 5 show that subsequent to the second disaggregation, the cells once again accumulated an additional quantum of each of three of the enzymes. (Trehalose-6-P synthetase was not tested.) Each of the activity peaks was attained at characteristic morphogenetic stages. (d) Requirement for RNA

synthesis subsequent to disaggregation

In the earlier study (Newell et al., 1971) it was shown that when the disaggregated cells were redeposited on filters in the presence of actinomycin D, the specific activity of UDPG pyrophosphorylase did not increase thereafter to a significant extent. The poisoned cells could reaggregate, however, and recapitulate the prior morphogenetic sequence and could incorporate labelled amino acids for several hours at a progressively diminishing rate. Figure 6 shows the results for the epimerase. Under the conditions used, RNA synthesis, though it begins to slow down almost immediately, is not halted until about 30 minutes after the filters are transferred to fresh pads containing the drug. Cells exposed to actinomycin, starting 30 minutes before disaggregation formed no additional epimerase when allowed to reaggregate and develop further in the presence of the drug. Yet sister cells exposed to actinomycin at the same time, but not disaggregated afterward, continued to accumulate epimerase activity to a level more than half that in the control, even though RNA synthesis had been stopped. Cells exposed to actinomycin, commencing 15 to 30 minutes after disaggregation and redeposition on fresh filters, accumulated less than half the normal level of epimerase activity, whereas undisturbed cell aggregates exposed to actinomycin

(b)

0 Untreated control

I

sI :e .-E w”

-

v I

14

16

I

I

I

I

IO

22 Time

I

14

I

I

IS

I

I

I

22

(hr)

FIG. 6. Cells were deposited on filters at zero-time (a) -O--O-, Undisturbed controls; -n-n-, not fresh pads containing actinomycin D in lower pad solution actinomycin D at 16 hr, disaggregated at 16.5 hr, redeposited mycin D. (b) -aA-, D&aggregated at 16.5 hr and redeposited redeposited, and exposed to actinomycin D at 16.75 hr; exposed to actinomycin D at 16.75 hr; dotted line, tracing

and treated as described below. disaggregated but at 16 hr shifted to (125 pg/ml.); -m-u---, exposed to and incubated further with actino; - AA-, disaggregated at 16.5 hr, -- l -- l --, not disaggregated but of the control curve in (a).

380

P.

C.

NEWELL,

J.

FRANKE

a o-s&i-

AND

M.

SUSSMAN

24

Time thr)

FIG. 7. Disaggregations were performed at 15 hr or at 18 hr and the cells were redeposited on fresh iilters and reincubated. At intervals, N-acetyl glucosaminidase activity was measured (Loomis, 1969). -O-O-, Undisturbed oontrols; --e-O,disaggregated at 16 hr; (A), disaggregated at 18 hr.

at the same time accumulated substantially as much activity as did the controls. Similar results were obtained for trehalose-6-P synthetase and UDPGal polysaccharide transfer&se. (e) Requirement

for cell contact after disaggregation

In the earlier study (Newell et al., 1971) it was shown that when the disaggregated cells were redeposited at a cell density low enough to prevent reaggregation, further accumulation of the UDPG pyrophosphorylase was prevented. Similar experiments demonstrated that this is also true of UDPGal :polysaccharide transferase. The other enzymes were not t’ested. (f) Effect of disaggregation

and reaggregation (EC 3.2.1.30)

on N-acetyl

glucosaminidase

Loomis (1969) characterized an enzyme in D. discoideum which hydrolyses the chromogenic substrate p-nitrophenyl-N-acetyl-glucosaminide. The specific activity of this enzyme begins to rise as soon as the cells leave the exponential phase and enter into the stationary phase, hours before aggregation has begun. The synthesis of this enzyme appears to be neither triggered nor modulated by the matrix of cell interactions that follows aggregation. In addition, it appears to have no functional relation with the four enzymes previously described. Hence it was of interest to examine the accumulation of N-acetyl-glucosaminidase activity in cells disaggregated and then permitted to develop further. Figure 7 summarizesthe data. As reported by Loomis (1969), the enzyme increased in specific activity from about 60 units/mg protein to a plateau value of about 250 units/mg after approximately 16 hours. Some cells were disaggregatedat 15 hours, others at 18 hours, and deposited on fresh filters at the original densities. In contrast to the results obtained with the other four enzymes, no further accumulation of glucosaminidaseactivity was observed.

4. Discussion In D. discoideum,UDPG pyrophosphorylase, trehalose-6-P synthetase, UPDGal 4epimerase, and UDPGal :polysaccharide transferase accumulate dramatically and

ENZYME

REGULATION

IN

D.

DISCOIDEUM

381

reach peaks of specific activity during particular stages of fruiting body construction. They then disappear partly or completely. Two enzymes, the epimerase and transferase, are preferentially released into the extracellular space prior to their disappearance. These events are altered in a consistent. fashion in morphogenetically incompetent or deranged mutant strains (Sussman & Sussman, 1969). Accumulation of all four enzymes has been shown to cease immediately after exposure of the cells to cycloheximide (Sussman & Sussman, 1969), indicating that the increases in specific activities require concurrent protein synthesis. More direct evidence is available in the case of UDPG pyrophosphorylase. This enzyme has been purified to a state of apparent physical homogeneity. The rate and extent of the increase in enzyme activity during fruiting body construction was shown to be correlated with a proportionate accumulation of a single antigenic component all of which was accountable as active enzyme. Pulse-labelling of the cells with [35S]methionine during the period of enzyme accumulation demonstrated that some and perhaps all of the enzyme that does accumulate is composed of newly synthesized polypeptide monomers (Franke & Sussman, 1971). The accumulation of each of the four enzymes requires a prior period of RNA synthesis, as delineated by studies involving actinomycin D (Roth, Ashworth &r, Sussman, 1968; Telser & Sussman, 1971). These transcriptive periods are staggered throughout the morphogenetic sequence and each precedes the actual accumulation of the corresponding enzyme by a significant amount of time: 1.5 hours for the synthetase, 3 to 4 hours for the epimerase, 4 to 5 hours for the pyrophosphorylase and transferase. This program of transcription and translation is seen to be profoundly altered when developing aggregates are disrupted and the cells are forced to reaggregate and to recapitulate earlier morphogenetic events. The resumption of cell contacts brought about through reaggregation appears to trigger a re-run of the sequence of gene expressions delineated above. This biochemical recapitulation displays the following properties. (1) The sequence of transcription and translation already under-way is abandoned. Thus when cells were exposed to actinomycin D just before disaggregation and redeposited on fresh filters in the presence of the drug, additional enzyme accumulation ceased abruptly, though the cells could reaggregate normally and recapitulate the prior morphogenetic sequence. In contrast, when developing cell aggregates at corresponding stages were exposed to actinomycin D without being disrupted, they continued to accumulate substantial amounts of all four enzymes even though RNA synthesis had stopped completely within 15 to 30 minutes after exposure. While disaggregation appears to be the signal to jettison these preprogrammed enzyme syntheses, the initiation of new rounds of transcription and translation appears to require the reaggregation of the separated cells. The reinitiations appear to be specific; whereas the reaggregated cells accumulated additional amounts of the pyrophosphorylase, synthetase, epimerase and transferase, they did not accumulate additional amounts of N-acetyl-glucosaminidase. (2) The second rounds of transcription and translation (or even third rounds in the case of the twice disaggregated cell populations) result in the synthesis of approximately the same quantities of the enzymes as accumulate during the normal course of morphogenesis. This quantitative aspect has been observed during other shifts in the developmental

382

P.

C.

NEWELL,

J.

FRANKE

AND

M.

SUSSMAN

program (Newell & Sussman, 1970; Ellingson, Telser & Sussman, 1971). Depending on certain environmental parameters, a cell aggregate of D. discoideum can either construct a fruiting body directly at the site of aggregation or transform into a migrating slug and move to a new location. At any time thereafter the slug can, if given the appropriate environmental signal, be induced to stop migrating and construct a fruiting body. Such slugs, if allowed to migrate for many hours, accumulate the usual levels of pyrophosphorylase and transferase activities but no epimerase activity at all. (The synthetase was not examined.) Given the signal to fruit, these cells then proceed through the usual first round of epimerase synthesis and complete second rounds of pyrophosphorylase and transferase synthesis. Here, too, the basic control is at the level of transcription. (3) Certain temporal relations are significantly altered. As mentioned previously, in undisturbed cell aggregates engaged in fruit construction, time-lags of as much as four to five hours exist between the start of a transcriptive period and the appearance of the corresponding enzyme. But in the second and third synthetic rounds shown in Figures 2 to 6, these lags were uniformly cut to one hour or less. Furthermore, the transcriptive and translative periods for the four enzymes are normally staggered in time but were virtually synchronous during the second or third synthetic rounds. Since there is normally a ten-hour time-interval between the start of accumulation of trehalose-6-P synthetase, the earliest enzyme, and UDPGal-4-epimerase, the lat,cst (see Figs 2 and 3), this reflects a temporal shift of considerable magnitude. This research was supported

by grants from the Oxford Cancer Research Cancer Research Campaign and from the National Science Foundation One of us (M.S.) is the recipient of a Kational Institutes of Health Career Award (K3-GM-1313). The major portion of this work was carried out at the Marine Biological Woods Hole, Massachusetts. the

Committee (GB-5976X). Development

of

Laboratory,

REFERENCES Ashworth, J. M. & Sussman, M. (1967). J. Biol. Chcm. 242, 1696. Bonner, J. T. (1949). J. E.rp. ZooZ. 110, 259. Ellingson, J. S., Telser, A. & Sussman, M. (1971). Biochim. biophys. Actu, 244, 388. Franke, J. & Sussman, M. (1971). J. Biol. Chem. 246, 6381. Loomis, W. F., Jr. (1969). J. Bact. 97, 1149. Loomis, W. F., Jr. $ Sussman, M. (1966). J-. &foZ. Biol. 22, 401. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Cht~m. 193, 265. Newell, P. C., Longlands, M. & Sussman, M. (1971). J. Mol. BioZ. 58, 541. Newell, P. C. & Sussman, M. (1970). J. Mol. BioZ. 49, 627. Newell, P. C., Telser, A. & Sussman, M. (1969). J. Bad. 100, 763. Raper, K. B. (1940). J. Elisha MitcheZZ Sci. Sot. 56, 241. Raper, K. B. (1960). Proc. Amer. Phil. Sot. 104, 579. Roth, R., Ashworth, J. M. & Sussman, M. (1968). Proc. Nut. Acad. Sci., Wash. 59, 1235. Roth, R. &. Sussman, M. (1968). J. BioZ. Chem. 243, 5081. Sussman, M. & Lee, F. (1955). Proc. Nat. Acad. Xci., Wash. 41, 70. Sussman, M. & Osborn, M. J. (1964). Proc. Nut. Acad. Sci., Wash. 52, 81. Sussman, M. & Sussman, R. (1969). Syrnp. Sot. Ben. Microbial. 19, 403. Telser, A. & Sussman, M. (1971). J. BioZ. Chem. 246, 2252.