JOURNAL OF BACTERIOLOGY, June 1972, p. 1065-1072 Vol. 110, No. 3 Copyright 0 1972 American Society for Microbiology Printed in U.S.A. Protein and Ribonucleic Acid Synthesis During the Diploid Life Cycle of arbuscula DANIEL J. BURKE,I THOMAS W. SEALE,2 AND BRIAN J. McCARTHY Departments of Biochemistry and Genetics, University of Washington, Seattle, Washington 98105 Received for publication 22 December 1971

The diploid life cycle of Allomyces arbuscula may be divided into four parts: spore induction, germination, vegetative growth, and mitosporangium forma- tion. Spore induction, germination, and mitosporangium formation are insensi- tive to inhibition of actinomycin D, probably indicating that stable, pre-ex- isting messenger ribonucleic acid (RNA) is responsible for these developmental events. Protein synthesis is necessary during the entire life cycle except for cyst formation. A system for obtaining synchronous germination of mitospores is described. During germination there is a characteristic increase in the rate of synthesis of RNA and protein although none of the other morphogenetic changes occurring during the life cycle are necessarily accompanied by an ap- preciable change in the rate of macromolecular synthesis.

Several investigators have emphasized the invaluable background for the present bio- potential usefulness of aquatic fungi for studies chemical studies (2, 5, 9, 12, 19, 21, 25, 28, 30). of the mechanisms of cellular differentiation Our initial studies with this organism were and morphogenesis (6, 14). Among the phyco- designed to determine the characteristics of mycetes, Allomyces arbuscula is an especially macromolecular synthesis associated with de- appropriate model system for the correlation velopment and differentiation and to begin to of cytological and biochemical changes occur- identify those points during the life cycle ring during differentiation. Allomyces pos- which are the most critical for the initiation of sesses stable alternation of haploid and diploid these processes. life cycles (Fig. 1), a property common to The studies to be reported were concerned members of the family Blastocladiaceae (5). with the temporal variation of the rate of ribo- The haploid and diploid stages seem to be nucleic acid (RNA) and protein synthesis identical in nutritional requirements and phys- during the diploid stage. In particular, this iology; they apparently differ only morphologi- communication examines the point in the life cally, the haploid producing paired gametan- cycle at which the genome is activated or at gia, and the diploid producing mito- and least genome activity is dramatically increased meiosporangia (20). Both the diploid and the and the several times during the life cycle haploid stages produce several differentiated when it is probable that development is con- cell types: mitospores (2N) and meiospores trolled by pre-existing stable messenger RNA (1N) in the diploid, male and female gametes (mRNA). In many of these instances differen- in the haploid, and an encysted single cell tiation occurs without profound changes in the stage, rhizoidal growth, hyphal growth, and rate of synthesis of RNA or protein. reproductive structures in both. The life cycle can be conveniently reproduced on defined MATERIALS AND METHODS medium, and large amounts of the various cell Organisms and media. All experiments were types can be obtained after proper manipula- performed with a stock of Allomyces arbuscula tion of growth conditions (8, 20, 21). Further- strain Brazil 2 G derived from a single meiospore more, the numerous cytological and physio- (1N) isolate of the original Brazil 2 G strain that was logical studies already published provide an kindly provided by Ralph Emerson. The gameto- phyte plant developed from this meiospore was in- ' Present address: Department of Microbiology, Univer- duced to release gametes at maturity, and the zy- sity of Illinois, Urbana, Ill. 61801. gotes obtained from this were used as the initial 2Present address: Department of Biological Sciences, source of diploid material. This strain has been des- University of Illinois at Chicago Circle, Chicago, Ill. 60680. ignated B2G-15-D. The preparation of YpSs me- 1065 1066 BURKE, SEALE, AND McCARTHY J. BACTERIOL. incubating them with label for either 5 or 10 min. Samples were precipitated with cold 10% trichloroa- cetic acid containing 200 gg of base or amino acids, or both, per ml as carrier, filtered on Whatman GFC filters, and counted in a Packard Tri-Carb scintilla- tion counter. Staining. Mitospores and rhizoid stage plants were stained by mixing a volume of culture with an equal volume of 2% toluidine blue in water. Chemicals. Actinomycin D and actidione (cyclo- heximide) were purchased from Calbiochem, Los Angeles, Calif. 3H-adenine, "4C-phenylalanine, 3H- phenylalanine, and l4C-glutamic acid were pur- chased from New England Nuclear Corp., Boston, Mass. The specific activities of the isotopes are given with each experiment. RESULTS Induction of mitospores. The diploid life (r) cycle can be conveniently divided into four FIG. 1. Life cycle of Allomyces arbuscula. (mt) parts: induction of the mitospore, spore en- Mitospores, (c) cyst, (r) rhizoid growth, (h) hyphae growth, (mts) mitosporangium, (mis) meiosporan- cystment and germination, rhizoidal and hy- geum, (mi) meiospore, (mg) male gametangium, (fg) phal growth (vegetative), and the formation of female gametangium, and (z) zygote. mitosporangia. On flooding of an agar plate culture with DS, the multinucleate cytoplasm in the sporangium cleaves into individual uni- dium, Machlis B medium (MB), and dilute salt solu- nucleate motile spores, and within 2 hr the tion (DS) has been described elsewhere (7, 17). MB are medium was modified by the addition of 15 g of sol- motile spores released through discharge uble starch per liter in place of glucose, 400 gg of L- papillae. The uninucleate nature of the spores leucine/ml, and 400 jig of L-/ml. In some cases, was confirmed by microscopic examination of 1.0 to 4.0 mg of an equal mixture of Casamino Acids stained material. (Difco) and tryptone (Difco) was added per ml to the To determine whether the induction is MB medium. merely a rearrangement of existing elements or Growth conditions. Mitospores were obtained in represents new synthetic events, the uptake of the following manner. YpSs agar plates of 5- to 10- RNA and protein precursors into acid-precipi- day-old plants were flooded with 10 to 20 ml of table material was measured in the presence sterile DS. Motile mitospores were released within 2 hr. For germination and growth experiments, mito- and absence of inhibitors (Table 1). RNA syn- spores in DS were inoculated to a final concentration thesis appeared to be unnecessary for induc- of between 5 x 104 to 2 x 10S spores/ml into YpSs tion: the number of spores induced in the pres- broth or MB broth plus additions and incubated at ence of actinomycin D was the same as that 28 C. The inoculum was never more than 10% of the induced in its absence. The RNA synthesized final volume. The growth vessel was either a 125-ml during induction in the absence of actino- Erlenmeyer flask with a stirring bar for agitation or mycin D might result from some residual vege- a 500-ml Bellco spinner flask. tative synthesis of some RNA species which Spore formation was also studied by growing are subsequently incorporated into the spore plants (this refers to either the rhizoidal or hyphal growth stage) (Fig. 1) for various lengths of time in during cleavage. In any case, it is not neces- liquid YpSs or MB broth. The mycelium was har- sary for further growth since spores induced in vested by centrifugation and resuspended in a the presence of actinomycin D developed nor- volume of DS equal to that of the original culture mally when compared to spores induced in the and incubated until mitosporangia were formed (20). absence of actinomycin D. Labeling. Mitospores were labeled by the addi- Protein synthesis does, however, seem to be tion of 3H-adenine and "4C-Phenylalanine along necessary for spore induction. Cycloheximide with the DS when plates were flooded. Germinated at concentrations as low as 0.1 ug/ml inhibited spores and the plants were labeled in two ways. (i) mitospore induction to a level of 0.01% or less Total uptake into trichloroacetic acid-precipitable of material, was determined by adding a labeled base than that control cultures. The protein syn- or amino acid, or both, to the medium and removing thesis taking place appeared to be directed by samples at various times. (ii) Determinations of the stable mRNA since the amount of synthesis rate of macromolecule synthesis were made by re- was the same in the presence or absence of ac- moving samples from a spinner flask culture and tinomycin D. The time at which this synthesis VOL. 110, 1972 DIPLOID LIFE CYCLE OF ALLOMYCES ARBUSCULA 1067 TABLE 1. Effect of inhibitors on mitospore cysts, and germlings was present for a 7-hr inductiona period after inoculation of the medium. The rate of synthesis of RNA and protein Incorporation was measured by means of the total uptake .No. of Additios (pmoles/ml) into trichloroacetic acid-precipitable material Additions spores/mispr/m Phenyl- Adenine of precursor by germinating spores (Fig. 3a). alanine Zero time was taken as the inoculation of of None 105 19 20 spores into medium capable supporting Actinomycin D, 105 <0.3 16 growth. Synthesis of RNA and protein com- 20 ug/ml menced at about 20 min, at which time encyst- Cycloheximide, 103 <0.3 <0.2 ment was complete. The rate of incorporation 20 Ag/ml of adenine, as determined in a similar experi- ment, revealed some interesting characteristics (Fig. 3b): it increased with time to a maximum a Ten milliliters of DS solution containing 20 gCi value, decreased, and then reached a constant of 3H-adenine (505 gCi/,umole) and 1 gCi of 14C- rate of followed phenylalanine (16.5 qCi/,qmole) was added to each value. The protein synthesis plate and incubation was carried out for 120 min precisely the same pattern after a 40-min lag, before measurement of isotope incorporation. and the nature of both incorporation curves was quite reproducible. Such rate increase coupled with the apparent lack of synthesis of occurred was determined by flooding the RNA by the spore is suggestive of activation of plates and then adding cycloheximide (20 the genome upon germination. It will be neces- ,gg/ml) after different times of incubation. sary to examine the type and amount of RNA Addition during the first 15 min completely made during germination to be able to rule out inhibited induction, addition from 15 to 30 mechanisms other than genome activation to min resulted in an increased percentage of explain the results obtained. Further analysis spores relative to a control incubated for 2 hr is necessary also to determine the significance without cycloheximide, and addition after 30 of the decrease in rate of synthesis that seems min was without effect. Addition at 30 min to take place. resulted in about 10% binucleate, double-sized The effects of actinomycin D and cyclohexi- spores. This suggests that the processes of mide on germination and outgrowth were also cleavage of individual spores and that of the examined (Table 2). Growth proceeded in acti- other events of induction may be separate. nomycin D to the rhizoid stage, whereas cyclo- Germination and outgrowth. The next heximide stopped growth after encystment stages of the life cycle are encystment, germi- (see Fig. 1). Thus, it appears that protein syn- nation, and the initial outgrowth of the rhizoid thesis, but not RNA synthesis, is necessary to from the germling. In order to enumerate indi- vidual events occurring during this time, a method for the synchronous germination and outgrowth of the mitospores was developed fol- iRn lowing the observation of Machlis (18). This was achieved by adding 400 ug of L-leucine I and 400 gg of L-lysine per ml to MB medium and incubating at 28 C rather than at room temperature as has previously been the case 20460

(Fig. 2). Under these conditions the spores re- VW14 Z" tracted their flagella within 20 min and formed iIz cysts. Germination began by 30 min and was essentially complete by 80 min as shown in Fig. 2. The actual germination time may be slightly less since the kinetics were monitored by the appearance of a germ tube, which may not be the earliest event in germination. The timing of these events was very reproducible Minutes and represented a considerable acceleration of FIG. 2. Synchronous germination of mitospores. germination when compared to incubation at Spores at a final concentration of 105/ml were inocu- 25 C in unmodified MB medium. Under the lated into modified MB medium (see Materials and latter conditions a mixed population of spores, Methods) and incubated at 28 C. 1068 BURKE, SEALE, AND McCARTHY J. BACTERIOL.

a) 0 0. o 0 vt 0 #o a' v - 0 to so so0 00 va E 0 T~? .Es a- E - in ~0 Q E c 0 O i a ) E -o 0)

Om 0o 0)4) 0

40 80 120 160 Minutes Minutes FIG. 3. Uptake of glutamic acid and adenine by germinating mitospores. Mitospores inoculated into modi- fied MB medium containing 2 UCi of 3H-adenine per ml (135 jiCi/limole) and 0.1 giCi of "4C-glutamic acid per ml (147 jiCi/jmole). (a) Total uptake; (b) slopes of total uptake during 20-min intervals. support further growth after encystment. Pro- TABLE 2. Effect of actinomycin D and tein and RNA synthesis were measured during cycloheximide on growtha germination in the presence of actinomycin D Actino- Cyclo- (Fig. 4a and b). Protein as measured by amino mycin D heximide acid incorporation into acid-precipitable mate- rial appeared to accumulate at 50% of the con- Spore induction _b +c trol rate for the first 80 min of germination Encystment _ and continued at about 25% of the control rate Germination _ + until 120 min before ceasing; RNA synthesis Hyphal growth + + was almost completely inhibited. It appears Mitosporangium formation - + that some in mRNA is made the absence of a Both inhibitors were present at 20 jig/ml. actinomycin D because of the higher rate of bIndicates no inhibition. protein synthesis in the control without actino- c Indicates inhibition. mycin D, but this RNA is not necessary for germination. Any mRNA necessary for further growth may apparently be synthesized later. thesizing at the same rate as the single nuclei When actinomycin D was removed from the prior to mitosis (Fig. 5). medium at 3 hr, the plants developed normally When an estimate of the hot water-extract- after a lag. able pool of adenine and glutamic acid was Post germination. Synthesis beyond the made in material from the synchronous spore initial 3-hr period was investigated by adding germination system, little change in pool sizes precursor to 3-hr cultures and measuring up- was observed over a 4-hr interval beginning take into acid-precipitable material from this from the onset of germination. It should be point. RNA and protein synthesis continued at emphasized that the kinetic picture is influ- a linear rate until 4.5 to 5 hr. At that time the enced by inoculum size and particular growth rate of RNA synthesis doubled and, approxi- conditions. Obviously the developmental mately 30 min later, the rate of protein syn- changes themselves are triggered by processes thesis doubled (Fig. 5). Nuclear staining indi- too subtle to be revealed by the overall kinetic cated that this is approximately the time of picture. the first nuclear division. The increase in rate Formation of mitosporangia. The forma- is consistent with the two daughter nuclei syn- tion of mitosporangia completes the diploid VOL. 110, 1972 DIPLOID LIFE CYCLE OF ALLOMYCES ARBUSCULA 1069

9.., U) 9) 9, Q)

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9t, 9, 9)z U) 9) 9,

Minutes Hours FIG. 4. Uptake of phenylalanine and adenine during germination in the presence of actinomycin D. Mito- spores were inoculated into modified MB medium containing 20 ug of actinomycin D per ml and either phen- ylalanine or adenine. (a) 1 gCi of 3H-phenylalanine per ml (165 MuCi/Mmole); (b) 1 gjCi of 3H-adenine per ml (135 1ACi/Mmole). life cycle. This process can occur in actively was sufficient to allow plants to form mito- growing plants, since on YpSs plates one of sporangia when they were transferred to DS. the two branches formed by the growing plant The effect of inhibitors upon mitosporangium often develops into a mitosporangium while formation was investigated with this regime the other hyphal branch continues to grow and (Table 3). Young plants in the rhizoid stage divide. However, this does not seem to be the did not form mitosporangia when transferred case in liquid medium. Normally, when spor- in the presence of actinomycin D or cyclohexi- angia do form, they appear at the tips of the mide; thus, both RNA synthesis and protein hyphae. The process of sporangium formation synthesis are necessary. In very young plants, in liquid medium seems to be initiated by at least, there is some RNA made during changes in cultural conditions which we have growth which is necessary and cannot be re- yet to understand. For example, sporangia placed after transfer since spores germinated often appeared in one culture but not in an- in the presence of actinomycin D will not form other parallel culture grown simultaneously. mitosporangia when transferred to DS either Also, the time at which mitosporangia were in the presence or absence of actinomycin D. formed and rate of RNA synthesis of the cul- Plants transferred after they had reached the tures forming mitosporangia varied greatly. hyphal growth stage had the ability to form Sometimes sporangia appeared in vigorously mitosporangia and release spores in actino- growing cultures and at other times in senes- mycin D but not in cycloheximide. Thus, RNA cent ones. There was a marked variability in synthesis is not necessary and the plant pre- the response of these cultures to actinomycin sumably contains stable mRNA set aside for D. In many cases sporangium formation was the synthesis of mitosporangia. actually more pronounced in the presence of We were unable to measure any synthesis of actinomycin D. either RNA or protein which was specific for It is possible to study mitosporangium for- mitosporangia formation. This was due to the mation by transferring plants from YpSs me- large amount of turnover of these macromole- dium to DS (20). We initially determined that cules as determined by an initial increase and growth in YpSs or MB for as little as 60 min then decrease in trichloroacetic acid-precipi- 1070 BURKE, SEALE, AND MCCARTHY J. BACTERIOL. reduction in the rate of protein synthesis ef- fected by actinomycin D. This synthesis of mRNA while development is being directed by other stable messengers is also reminiscent of early embryonic development (11, 26, 27). However, this mRNA synthesized during early germination is either dispensable for further development, or can be replaced after actino- mycin D is removed. Studies with Blastocla- diella emersonii, an organism closely related to Allomyces, have also implicated the presence of a stable mRNA that directs protein syn- thesis during germination, suggesting that this mechanism is common to the aquatic Phyco- mycetes (15). The presence of stable mRNA has also been demonstrated in other fungal Minutes systems (13, 23). In the slime mold (23) the FIG. 5. Uptake of phenylalanine and adenine mRNA which directs the synthesis of an en- during rhizoidal growth. The culture growing in zyme necessary for differentiation is made modified MB medium was labeled by the addition of several hours before its expression. In fact, this 2 1uCi of 3H-adenine per ml (135 ACi/,mole) and 0.1 1iCi of '4C-phenylalanine per ml (16.5 MCi/lmole) at same mechanism is also to be found in some 180 min. prokaryotes, namely in spore formation in Ba- cillus cereus (1) and microcyst germination in table counts of precursor for either RNA or Myxococcus xanthus (24). protein taking place in plants transferred to The involvement of stable mRNA in differ- DS. The kinetics of incorporation were domi- entiation is apparent in mitosporangium for- nated by this turnover rather than by events mation as well as in induction and germina- directly related to mitosporangium formation. tion of the mitospore. In each case this mecha- The lack of incorporation of adenine in the nism may be important for survival. For exam- presence of actinomycin D contrasted to the ple, the mRNA necessary for mitosporangium observed incorporation in its absence indicates formation accumulates early in hyphal growth that the drug is actually inhibiting RNA syn- and its translation is induced by starvation thesis. The results then are consistent with the conditions. This enables the organism to dif- gradual accumulation of a pool of stable ferentiate rapidly into a motile form which is mRNA capable of directing mitosporangium able to seek out more favorable conditions and formation which renders the process increas- reinstate the growth cycle. The ability to com- ingly refractory to actinomycin D as the plants pletely eliminate hyphal growth, a normal stage in its life cycle, and form mitosporangia age. is reminiscent of the observation in Myxo- coccus xanthus that fruiting body formation DISCUSSION A number of interesting speculations con- cerning the mechanisms responsible for differ- TABLE 3. Formation of mitosporangiaa entiation the life can be during cycle made Relative no. of from these experiments, the major one being mitosporangia formed (%) that at particular stages Allomyces contains Growth stage stable mRNA which directs specific develop- Minus actino- Plus actino- mycin D mycin D mental stages in a manner analogous to other eukaryotic cells. The stable RNA postulated to Rhizoid 10-30 1 direct germination and outgrowth of the mito- Young hyphae 40-50 0-20 spore seems to be analogous to stored maternal First branching 50-60 30-40 messenger vital to early development of higher Old plants 100 60-100 organisms (3, 11, 26, 27, 29, 31). Initial em- a All percentages are relative to the number of bryonic development in both amphibian and sporangia formed per plant in rhizoid and young sea urchin embryos has been shown to be hyphae cultures, and per hyphal tip in the other under the control of stable mRNA species ex- cases. Growth stage refers to the time in the diploid isting in the egg; Allomyces also synthesized life cycle at which plant material was transferred mRNA during germination, as shown by the from YpSs to DS (see Fig. 1). VOL. 110, 1972 DIPLOID LIFE CYCLE OF.ALLOMYCES ARBUSCULA 1071

can be omitted and microcysts formed directly pears to be equilibrated by this time. It might from vegetative organisms. represent the cessation of synthesis of the When conditions are manipulated to elimi- mRNA molecules necessary for further growth nate hyphal growth, Allomyces greatly resem- after the initial direction of translation by the bles Blastocladiella emersonii, whose life cycle preformed stable mRNA. To help substantiate is somewhat similar. Sporangium formation in this hypothesis, it will be necessary to demon- B. emersonii is also quite resistant to actino- strate that these same molecules are made in mycin D, again indicating the possibility of the lag period after removal of actinomycin D stable mRNA. This mechanism may not, how- from spores germinated in its presence. The ever, operate in all the Phycomycetes since it decrease might also represent some type of has been shown that in Achlya sporangium damping phenomenon regulating the rate of formation is inhibited in actinomycin D after rRNA synthesis. Clarification of this point induction although it does gradually become awaits good measurements of the relative pro- refractory to it (10). Further study of other portion of rRNA and mRNA present and being Phycomycetes will determine how widespread synthesized during germination. is the use of stable mRNA to direct sporangial formation. ACKNOWLEDGMENTS Several other explanations of these results This work was supported by Public Health Service post- doctoral fellowships 5 F02-CA-38, 197 (D.D.B.) from the can be tentatively ruled out. The differentia- National Cancer Institute, 1 F02 GM 42834-01 (T.W.S.) tions which take place in actinomycin D from the National Institute of General Medical Sciences, cannot be explained simply by a lack of and Public Health Service research grant GM 12449 (B.J.M.) permeability to actinomycin D because we from the National Institute of General Medical Sciences. an of the We wish especially to thank Ralph Emerson for the ad- have demonstrated inhibition uptake vice, the technical assistance, and the several Allomyces of adenine into trichloroacetic acid-precipi- strains that he provided us during the initiation of our table material in the presence of actinomycin studies on Allomyces. D during induction (Table 1), germination (Fig. 4), and formation of the mitosporangia LITERATURE CITED It is possible, however, (unpublished data). 1. Aronson, A. I., and M. Rosas Del Valle. 1964. RNA and that only ribosomal RNA (rRNA) synthesis protein synthesis required for bacterial spore forma- was inhibited and not mRNA. At least in one tion. Biochim. Biophys. Acta 87:267-276. case mRNA synthesis was shown to be par- 2. Blondel, B., and G. Turian. 1960. Relation between ba- sophilia and fine structure of cytoplasm in the tially inhibited; during germination in actino- Em. J. Biophys. Biochem. mycin D the amount of protein synthesis is Cytol. 7:127-134. less than the control, indicating that at least 3. Brachet, J., H. Denis, and F. DeVitry. 1964. The effects some mRNA synthesis is blocked. of actinomycin D and puromycin on morphogenesis in amphibian eggs and Acetabularia mediterranes. De- Further, the conclusion that macromolecular velop. Biol. 9:398-434. synthesis is blocked in the presence of the in- 4. Cochrane, J. C., T. A. Rado, and V. W. Cochrane. 1971. hibitors does not seem to be due to a change in Synthesis of macromolecules and polyribosome forma- the uptake of the precursor. Preliminary esti- tion in early stages of spore germination in Fusarium mates of the size of the precursors have solani. J. Gen. Microbiol. 65:44-55. pool 5. Emerson, R. 1941. An experimental study of life cycles indicated that there is no significant difference and of Allomyces. Lloydia 4:77-144. in the pool size of either RNA or protein pre- 6. Emerson, R. 1954. The biology of water molds. Aspects cursor in the presence of either actinomycin D of synthesis and order in growth, p. 171-708. In D. Rudnick (ed.), 13th Growth symposium, vol. 13. or cycloheximide as compared to a control cul- Princeton Press, Princeton, N.J. ture in which the inhibitors were not present. 7. Emerson, R. 1958. Mycological organization. Mycologia Thus, it is most probable that stable mRNA 50:589-621. does, in fact, program several aspects of differ- 8. Emerson, R., and C. Wilson. 1954. Interspecific hybrids entiation the diploid life cycle. and the cytogenetics of cytotaxonomy of Euallomyces. during Mycologia 46:393-434. Determination of the rate of RNA and pro- 9. Fuller, M. S., and L. W. Olson. 1971. The zoospore of tein synthesis during short intervals through Allomyces. J. Gen. Microbiol. 66:171-183. the germination process suggested that rather 10. Griffin, D. H., and C. Breuker. 1969. Ribonucleic acid in occur. This may result from synthesis during the differentiation of sporangia abrupt changes the water mold Achlya. J. Bacteriol. 98:689-696. activation of parts of the genome resulting in 11. Gross, P. R., and G. H. Cousineau. 1964. Macromolecule the synthesis of mRNA, rRNA, or both. How- synthesis and the influence of actinomycin on early ever, the brief decrease in the rate of synthesis development. Exp. Cell Res. 33:368-395. P. of is more to 12. Hill, E. 1969. The fine structure the zoospores and during germination difficult under- cysts of Allomyces macrogynus. J. Gen. Microbiol. 56: stand. It is probably not a pool phenomenon 125-130. since we found that the nucleotide pool ap- 13. Holloman, D. W. 1970. Ribonucleic acid synthesis 1072 BURKE, SEALE, AND McCARTHY J. BACTERIOL.

during fungal spore germination. J. Gen. Microbiol. enzyme synthesis by slime mold assemblies embarked 62:75-87. upon alternative developmental programs. J. Mol. 14. Lovett, J. 1967. p. 341-358. In F. H. Wilt and N. K. Biol. 49:627-637. Wessels (ed.), Methods in developmental biology. 24. Ramsey, W. S., and M. Dworkin. 1970. Stable mes- Thomas Y. Crowell Co., New York. senger ribonucleic acid and germination of Myxo- 15. Lovett, J. S. 1968. Reactivation of ribonucleic acid and coccus xanthus microcysts. J. Bacteriol. 101:531-540. protein synthesis during germination of Blastocla- 25. Renaud, F. L., and H. Swift. 1964. The development of diella zoospores and the role of the nuclear cap. J. basal bodies and flagella in Allomyces arbuscula. J. Bacteriol. 96:962-969. Cell Biol. 23:339-354. 16. Maalde, O., and N. 0. Kjeldgaard. 1961. Control of mac- 26. Smith, K. D. 1967. Genetic control of macromolecular romolecular synthesis. W. A. Benjamin, Inc., New synthesis during development of an ascidian: Ascidia York. nigra. J. Exp. Zool. 164:393-405. 17. Machlis, L. 1953. Growth and nutrition of water molds 27. Stavy, L., and P. Gross. 1969. Availability of messenger in the subgenus Euallomyces. II. Optimal composition RNA for translation during normal and transcription- of the minimal medium. Amer. J. Bot. 40:450-460. blocked development. Biochim. Biophys. Acta 182: 18. Machlis, L. 1968. Zoospore chemotaxis in the water 203-213. mold Allomyces. Physiol. Plant. 22:126-139. 28. Turian, G. 1962. Cytoplasmic differentiation and dedif- 19. Machlis, L. 1969. Zoospore chemotaxis in the water ferentiation in the fungus Allomyces. Protoplasma 54: mold Allomyces. Physiol. Plant. 22:126-139. 323-326. 20. Machlis, L., and J. Crasemann. 1956. Physiological vari- 29. Tyler, A. 1967. Masked messenger RNA and cyto- ation between the generations and among strains of plasmic DNA in relation to protein synthesis and water molds in the subgenus Euallomyces. Amer. J. processes of fertilization and determination in em- Bot. 43:601-611. bryonic development. Develop. Biol. Suppl. 1 16:170- 21. Machlis, L., W. H. Nutting, M. W. Williams, and H. 226. Rapport. 1966. Production, isolation and characteri- 30. Wilson, C. 1952. in Allomyces. Bull. Torrey Bot. zation of sirenin. Biochemistry 5:2147-2152. Club 79:139-160. 22. Murphy, M. N., Sr., and J. S. Lovett. 1966. RNA and 31. Whiteley, J. R., B. J. McCarthy, and A. H. Whiteley. protein synthesis during zoospore differentiation in 1970. Conservatism of base sequences in RNA from synchronized cultures of Blastocladiella. Develop. early development of echinoderms. Develop. Biol. 21: Biol. 14:68-95. 216-242. 23. Newell, P. C., and M. Sussman. 1970. Regulation of