JOURNAL OF BACTERIOLOGY, Apr. 1978, p. 76-830021-9193/78/0134-0076$02.00/78Copyright X 1978 American Society for Microbiology

Vol. 134, No. 1

Printed in U.S.A.

Regulation of Macromolecular Synthesis During Hyphal GermTube Emergence from Mucor racemosus Sporangiospores

MICHAEL ORLOWSKIt AND PAUL S. SYPHERD*Department ofMedical Microbiology, College ofMedicine, University of Califomia, Irvine, California 92717

Received for publication 31 October 1977

Protein and RNA syntheses were examined during hyphal germ tube emergencefrom sporangiospores of a dimorphic phycomycete, Mucor racemosus. Bothclasses of macromolecules were synthesized immediately upon introduction of thedormant sporangiospores into nutrient medium. The specific rates of synthesis ofboth protein and RNA accelerated during initial germ tube emergence andreached a maximum when the emergence of new germ tubes ended. The specificrates of synthesis later decreased during further hyphal elongation. The distri-bution of ribosomes between active polysomes and monosomes and inactivesubunits was determined by sucrose density gradient centrifugation, and the rateof amino acid addition to nascent polypeptide chains was calculated throughoutthe developmental sequence. The results showed that both the percentage ofribosomes active in protein synthesis and the velocity of ribosome movementalong the mRNA were continuously adjusted throughout hyphal germ tubedevelopment. The free intracellular amino acid pools were measured throughoutdevelopment. Alanine, glutamate, and aspartate were present at very high con-centrations in the dormant spores but were rapidly depleted during hyphal germtube emergence. The results of these studies are discussed in relation to hyphalgerm tube development from yeast cells of Mucor and dormant spores of otherfungal species.

Mucor racemosus is a dimorphic phycomy-cete that grows vegetatively either in the formof budding yeasts or branching hyphae, depend-ing upon the composition of the atmosphereunder which the cells are held. The cells grow asyeasts under a CO2 atmosphere if a hexose ispresent. Changing the atmosphere from C02 toair results in the emergence of hyphal germtubes from the yeast cells (5, 20). The organism,when grown on a solid medium under air, pro-duces aerial hyphae which yield many sporan-giospores. Upon introduction to liquid medium,the sporangiospores swell and, if under C02,develop into budding yeasts or, ifunder air, formhyphal germ tubes (5, 21).We have previously reported the occurrence

of a characteristic acceleration in the specificrate of protein synthesis, as measured by thekinetics of radioactive amino acid incorporation,during the initial emergence of germ tubes fromyeast cells after an atmospheric shift from C02to air (33). Once germ tube emergence was com-pleted and all further growth proceeded by hy-phal elongation, the specific rate of protein syn-thesis decreased (33). Investigations into molec-ular mechanisms that could account for the ob-

t Present address: Department of Biology, University ofCalifornia of San Diego, La Jolla, CA 92093.

served quantitative regulation of protein synthe-sis were undertaken. The percentage of ribo-somes active in protein synthesis was observedto decrease throughout the morphological tran-sition. The rate ofamino acid addition to nascentpolypeptide chains was calculated, and the tran-sit time of mRNA translation was measured.Both experimental methods revealed a signifi-cant increase in the velocity of ribosome move-ment along the message that was continuouslyadjusted throughout hyphal development (M.Orlowski and P. S. Sypherd, Biochemistry, inpress). The intracellular location of active pro-tein synthesis was examined by using autora-diography of whole cells that had been pulse-labeled with radioactive amino acids during theemergence of germ tubes from both yeast cellsand sporangiospores. It was determined thatprotein synthesis occurs in all regions of the cell(34), not preferentially at the growing tip as isthe case for cell wall synthesis (4).

In view of the morphological similarity of hy-phal germ tube development from yeast cellsand sporangiospores ofMucor, the present studywas performed to characterize the pattern ofprotein synthesis and its regulation during aero-bic germination of sporangiospores from M. ra-cemosus. The rates of RNA synthesis were also

76

SPORANGIOSPORE GERMINATION 77

measured. The findings are compared with thosepreviously reported for the yeast-to-hyphaetransition in Mucor (3; Orlowski and Sypherd,in press) and the germination of spores fromseveral other species of fungi.

MATERIALS AND METHODSOrganism and cultivation. M. racemosus (M.

lusitanicus) ATCC 1216B was used in all experiments.The growth medium (YPG) contained 0.3% (wt/vol)yeast extract, 1.0% (wt/vol) peptone (Difco), and 2.0%(wt/vol) glucose (pH 4.5). Sporangiospores were pro-duced on YPG agar plates kept at 220C for 3 to 5 days.Sporangiospores were scraped from the plates intodistilled water and inoculated into liquidYPG mediumat a final concentration of 5 x 105 spores per ml.Compressed air was bubbled through the culture atflow rates of approximately 10 volumes of gas pervolume of culture fluid per min.

Pulse-labeling with L-[14C]leucine and [3H]ur-acil. The procedure of Orlowski and Sypherd (32) wasused to pulse-label cells at stages throughout sporan-giospore germination with L-[U-'4C]leucine (290pCi/mmol; final concentration, 2.5 ,uCi/ml) or [5-3H]-uracil (17 Ci/mmol; final concentration, 12.5 uCi/ml).Measurement ofintracellular free amino acids.

The procedure of Orlowski and Sypherd (33) was usedto extract and measure the free amino acid pools ofMucor throughout sporangiospore germination.Polysome profiles. Cells at the appropriate stage

of development were incubated in the presence ofcycloheximide (200 ,ug/ml) for 5 min, rapidly collectedon a Teflon filter (Millipore Corp., type LC, 10.0-gmpore size), washed briefly with cold TMK buffer (50mM tris(hydroxymethyl)aminomethane-hydrochlo-ride [pH 7.25]-10 mM magnesium acetate-500 mMKCI) containing cycloheximide, quickly placed into amortar full of liquid N2, and vigorously ground with a

pestle for 2 min. Cell breakage with this method wasalways greater than 50% (as determined by the percentrelease of incorporated radioactive amino acids into a30,000 x g supernatant fraction) and microscopicallyappeared representative of all cell types present (i.e.,germ tubes and swollen spores broke with equal effi-ciency). The broken cells were resuspended in cold,sterile buffer (TMK) containing 200 jig of cyclohexi-mide per ml. Cold, sterile glassware and solutions wereused throughout these procedures. The suspensionwas centrifuged at 15,000 x g for 10 min at 40C. Thesupernatant fraction (S-15) was gently decanted awayfrom the pellet. A volume ofthe S-15 fraction sufficientto contain an absorbancy of 4.0 at 260 nm was layeredon top of a 10 to 40% (wt/wt) linear sucrose gradient(in TMK buffer, 11.0-ml volume) that rested on a 2 Msucrose cushion (0.8-ml volume). The gradients werecentrifuged in an SW41 rotor (Beckman) at 150,000x g for 60 min at 40C. The gradients were scanned at254 nm with an ISCO density gradient fractionator,model 180, equipped with a chart recorder. Additionof Triton X-100 to the breaking buffer did not increasethe recovery of polysomes from extracts.

Protein and RNA measurements. Total cellularprotein was determined by the method of Lowry et al.(26). Total cellular RNA was measured by the orcinol

procedure (11) or the method of Cheung et al. (8). Theresolution of total cellular RNA into the soluble andrRNA forms was done as follows. Cells were filteredand washed with and resuspended in coldTMK buffer.The suspension was passed through a chilled Frenchpress. Sodium dodecyl sulfate was immediately addedto the broken-cell suspension to a final concentrationof 1% (wt/vol). The preparation was extracted threetimes with cold, water-saturated redistilled phenolcontaining 0.1% (wt/vol) 8-hydroxyquinoline. RNAwas precipitated from the aqueous phase by additionof 2 volumes of absolute ethanol and 0.3M ammoniumformate (final concentration) at -20°C. The RNA wascollected by centrifugation, lyophilized, resuspendedin ice-cold TMK buffer, and treated with deoxyribo-nuclease (10 ,g/ml, ribonuclease-free) for 20 min. Thispreparation was again extracted with water-saturatedphenol and precipitated with ethanol and formate.The precipitate was lyophilized and then dissolved ina buffer composed of 10 mM sodium acetate-100 mMNaCl-1 mM ethylenediaminetetraacetic acid (pH 5.3).A volume of solution containing an absorbancy of 1.5at 260 nm was layered on top of a 5 to 20% (wt/wt)linear sucrose gradient (in ANE buffer, 11.0-ml vol-ume) that rested on a 2 M sucrose cushion (0.8 ml).The gradients were centrifuged in an SW41 (Beckman)rotor at 77,000 x g for 18 h at 40C. The gradients werescanned at 254 nm on an ISCO fractionator, and theareas under the peaks were integrated gravimetricallyto determine the percentage of each RNA species.

RESULTSGrowth, morphogenesis, and pulse-label-

ing kinetics. The exact time course of sporan-giospore germination varied by as much as 2 to3 h in individual experiments, but the followingdescription generally characterizes the process.Between 0 and 8 h following introduction toaerated YPG medium, the sporangiosporesswelled to many times their original size. Be-tween 6 and 8 h, germ tubes began to emergefrom the swollen spores. By 8 to 10 h, gern tubeemergence had ended in all cells and all furthergrowth occurred in the form of hyphal elonga-tion and branching. Growth, represented as totalcellular protein per milliliter of culture, occurredexponentially until approximately 1 mg of cel-lular protein per ml had accumulated (Fig. 1).Thereafter, growth occurred at a continuallyslower rate until about 2 mg of cellular proteinper ml had accumulated.When L-['4C]leucine or [3H]uracil was admin-

istered to cells in YPG medium and its incor-poration was measured at intervals over a shortperiod of time (20 min), a set of linear kineticswas obtained (not shown). An instantaneous rateof incorporation was determined from the slopeof the linear kinetics and expressed in terms ofcounts per minute incorporated per minute.Such rates were determined over the course ofhyphal germ tube emergence from sporangio-

VOL. 134, 1978

78 ORLOWSKI AND SYPHERD

12

10

a.' a

a..i6

a.EL

1000

6I0-.

100100 ya.M.

t00 2 4 6 8 10 12 14 16 18 a0

hoursFIG. 1. Accumulation of total cellularprotein and

specific rates ofprotein synthesis during hyphalgermtube emergence from sporangiospores of M. racemo-sus. The time course of the morphogenetic change isdepicted on the bottom of the figure. Symbols: (0)total accumulatedprotein measured by theprocedureof Lowry et al. (26); (0) specific rate ofprotein syn-thesis determined by. measuring the kinetics ofL-[4C]leucine incorporation into hot trichloroaceticacid-insoluble material (33).

spores and normalized to total cellular protein,in the case of L-[14C]leucine, or total cellularRNA, in the case of [3H]uracil. The calculatedvalues are referred to as the specific rate ofprotein or RNA synthesis.

Figure 1 shows the observed pattern of thespecific rate of protein synthesis throughout hy-phal germ tube development. In a previous pa-per we established that L-[14C]leucine is takenup by all morphological forms of Mucor, is notaltered from the form of L-leucine, and is incor-porated exclusively into protein. We have shownthat the intracellular pool of free L-leucine issmall, remains constant in size, and is rapidlyequilibrated with exogenous leucine. The sameinternal specific radioactivities were attainedshortly after addition of L-['4C]leucine in all celltypes examined. Other "4C-labeled L-amino acidswhen substituted for L-['4C]leucine showed thesame kinetics of incorporation (33). Therefore,if one assumes the absence of appreciable pro-tein turnover, the data collected in the pulse-labeling experiments represent the instanta-neous rates of protein synthesis. Protein synthe-sis commenced immediately upon introductionof the spores to nutrient medium (Fig. 1). Cyclo-

heximide immediately inhibited all L-['4C]leu-cine incorporation and completely preventedgermination of sporangiospores (the spores re-mained unswollen for at least 48 h). The specificrate of protein synthesis increased as the growthrate increased during late spore swelling andgerm tube emergence, reached a maximum inmidexponential growth after germ tube emer-gence had ended, and decreased in late exponen-tial growth when the hyphae were elongating(Fig. 1).The intracellular pools of free L-leucine re-

mained relatively constant throughout hyphalgerm tube emergence from sporangiospores, thesame as they did during the yeast-to-hyphaetransition (33). However, Table 1 shows thatseveral other amino acids fluctuated quite pro-foundly.

Figure 2 shows the specific rate of RNA syn-thesis and total cellular RNA accumulation dur-ing hyphal germ tube development. Experi-ments have indicated that [3H]uracil is taken upat rates that would not limit incorporation by allmorphological forms of Mucor. The incorpora-tion is immediately inhibited by actinomycin D,and the product of incorporation is completelyhydrolyzed by ribonuclease A (data not shown).It is of interest to note that the specific rate ofRNA synthesis followed a time course very sim-ilar to that for the specific rate of protein syn-thesis with respect to the morphogenetic se-

TABLE 1. Free intracellular amino acidpoolsduring hyphal germ tube emergence from

sporangiospores ofM. racemosus

Free amino acid concn (umol/g of cellular pro-Amino acid tein)

Toa T8 T,o T12 T14Asp 98.6 27.8 81.3 24.3 24.6Thr 45.0 8.8 13.3 15.0 12.2Ser 35.0 7.2 13.0 4.7 6.6Glu 211.6 88.3 201.1 99.9 101.9Pro 23.5 4.3 27.1 16.9 17.9Half-cys 5.5 NDb 4.2 ND NDGly 15.2 9.8 19.5 5.2 7.7Ala 623.7 19.8 64.3 65.2 45.2Val 9.7 8.6 12.4 5.5 7.0Met 2.8 0.5 0.5 0.6 0.2Ile 2.0 2.7 2.3 1.5 1.6Leu 1.7 1.9 2.0 1.5 1.6Tyr 13.7 6.9 15.0 5.5 4.8Phe 4.2 6.4 6.0 5.6 3.3His 7.8 2.7 5.9 2.1 2.2Lys 4.9 13.3 55.7 22.7 22.9Arg 7.6 27.4 105.0 35.8 37.6Try ND ND ND ND ND

a Time the sporangiospores had been incubated inaerated nutrient medium in hours. To samples werespores collected directly from agar plates.

b ND, Not detectable.

*oo ° , X2

0

-

8

4 .-

2 .

0

J. BACTERIOL.

VOL. 134, 1978

a

1

a.

a1.14

U 2 4 a 8 1U 13 14 16 18 50

hoursFIG. 2. Accumulation of total cellular RNA and

specific rates ofRNA synthesis during hyphal germtube emergence from sporangiospores ofM. racemo-sus. The time course of the morphogenetic changes isdepicted on the bottom of the figure. Symbols: (0)total accumulated RNA measured by the method ofCheung et al. (8); (0) specific rate ofRNA synthesisdetermined by measuring the kinetics of fH]uracilincorporation into cold trichloroacetic acid-insolublematerial (33).

quence. It is also worth noting, for purposes ofcomparison with other fungal species, that RNAwas synthesized, although at low rates, imme-diately upon introduction ofthe sporangiosporesinto liquid medium. Actinomycin D preventedthe formation of germ tubes but allowed thecells to complete the swelling stage.Polysome profiles. The conditions neces-

sary for producing the maximum polysomeyields from cells of Mucor were described in aprevious paper (Orlowski and Sypherd, in press).Figure 3 represents the distribution of ribosomesbetween polysomes, monosomes, and subunitsin cells throughout hyphal germ tube develop-ment from sporangiospores. The top of eachgradient is at the far right of each gradient scanshown. Low-molecular-weight material, whichnever entered the gradients, is the first peak onthe right, observed off-scale. Proceeding right toleft, one observes peaks identified as "40S sub-units," "60S subunits," "80S monosomes," andpolysomes of increasing size up to about 12 to 14monosome equivalents in the best-resolved gra-dients. The high salt concentration used in allbuffers and gradients should have caused anyrunoff 80S ribosomes (i.e., not attached to

SPORANGIOSPORE GERMINATION 79

A B 11000

FIG. 3. Ribosome distribution in cells sampled attimes throughout hyphalgerm tube development fromsporangiospores ofM. racemosus. Cell morphology isshown in Fig. 1. An absorbancy at 254 nm trace of S-15 preparations fractionated by centrifugation (150,-0XX x g for 60 min) on 10 to 40% sucrose densitygradients. (A) Dormant sporangiospores. (B) 3 h,swollen spores. (C) 7.5 h, swollen spores and a fewinitial germ tubes. (D) 9 h, germ tube emergence. (E)11 h, germ tubes. (F) 13 h, elongating hyphae. (G) 14.5h, elongating hyphae. (H) 18 h, elongating hyphae,decelerating growth rate. (a) 30 h, stationary-phasehyphae. Top of each gradient at the far right.

mRNA) to dissociate into 40S and 60S subunits(29, 30). Consequently, all 80S particles occur-ring in the sucrose gradients should representsingle, actively translating ribosomes or break-down products of polysomes. The procedure ofForchhammer and Lindahl (13) was used todetermine the percentage of 80S ribosomes ac-tive in protein synthesis. The results, whichshowed that the 80S peaks were labeled withthe same specific radioactivity as found in thepolysome regions of the gradients after pulse-labeling ofthe cells with radioactive amino acids,indicated that all 80S ribosomes are active (Or-lowski and Sypherd, in press). The percentageof all cellular ribosomes active in protein syn-thesis is therefore taken to be the sum of allpolysomes plus all 80S ribosomes. Ribosomesfrom ungerninated sporangiospores (Fig. 3A)were entirely in the form of dissociated 40S and

. O o o o0,Oj4. . ..T '.

80 ORLOWSKI AND SYPHERD

60S subunits. Early in germination, when pro-

tein synthesis was still accelerating (as indicatedin Fig. 1), the percentage of ribosomes in poly-somes was relatively high (Fig. 3B and C). Whenthe emerging germ tubes had reached their max-imum number, and the specific rate of proteinsynthesis was highest, the percentage of ribo-somes found in polysomes was at its lowestvalues (Fig. 3D and E). During hyphal elonga-tion, when the specific rate of protein synthesisdeclined, the percentage of ribosomes in poly-somes increased again (Fig. 3F through H). Poly-somes were present in very large amounts rela-tive to ribosomal subunits even in stationary-phase cells (Fig. 3I).Rate of nascent polypeptide chain elon-

gation. Calculation of the rate of polypeptidechain elongation was determined by using thebasic procedures and theory of Forchhammerand Lindahl (13). The parameters measuredwere those necessary to complete the followingrelationship:

protein120

.x y x ln2

rRNA

mol

x %AR x 3,600mol wt

= amino acids/second per ribosome

where ,u is the growth rate in doublings perhours, ln2 is from the growth rate equation, ARis active ribosomes, 3,600 is the number of sec-onds per hour, and 120 represents the averagemolecular weight of the 20 naturally occurringL-amino acids. Total cellular protein, rRNA, and

the percentage of ribosomes active in proteinsynthesis were measured as described above.The molecular weights ofthe rRNA species fromM. racemosus have been reported (25). Thepercentage of total cellular RNA contained inthe rRNA species was experimentally deter-mined to be 85.7 ± 2.2%. The calculated rates ofpolypeptide chain elongation in cells throughouthyphal germ tube development from sporan-giospores are presented in Table 2 (column 6).The values roughly correlated with the specificrates of protein synthesis calculated from thekinetics of L-['4C]leucine incorporation (Fig. 1).The polypeptide elongation rate was about 1.2amino acids added/s per ribosome during initialswelling of the sporangiospores after introduc-tion to YPG medium. This value acceleratedand reached a maximum of 5.1 amino acids/sper ribosome when the maximum number ofgerm tubes had emerged. The value afterwardsdropped to a relatively stable level of about 4amino acids/s per ribosome during hyphal elon-gation. When exponential growth had ceased,the value returned back to about 1 amino acid/sper ribosome (Table 2). Several other measuredparameters of interest are presented in Table 2.The growth rates (column 2) are similar to pub-lished values (31, 34). The total cellular RNA-protein ratio (column 3) and number of ribo-somes per milligram of cellular protein (column4) increased about 25% between 0 to 11 h, an

amount that could not alone account for theobserved fourfold increase in rate of proteinsynthesis. The percentage of ribosomes active inprotein synthesis is shown in column 5 of Table2.

TABLE 2. Polypeptide chain elongation rate and other measuredparameters during hyphal germ tubeemergence from sporangiospores ofM. racemosusa

Ratio of Ribosomes/mg of pro- % Active ribo- Polypeptide chainTime (h) u (doublings/h) RNA/protein tein somes elongation rate (amino

acids/s per ribosome)

0 0 0.373 9.4 X 1013 0 03 0.082 0.379 9.6 x 1013 71.5 1.157.5 0.278 0.447 11.3 x 1013 76.3 3.249 0.357 0.468 11.8 x 1013 57.4 5.0711 0.357 0.500 12.6 x 1013 62.2 4.3713 0.357 0.499 12.6 x 1013 69.1 3.9614.5 0.357 0.460 11.6 x 1013 75.2 3.9418 0.102 0.455 11.5 x 1013 82.4 1.0430 0 NDb ND 78.2 0

a Sporangiospores of M. racemosus were inoculated into nutrient medium and vigorously aerated (seeMaterials and Methods). The cells were sampled at the times indicated in column 1. Zero time samples weresporangiospores coliected directly from agar plates and not introduced into nutrient medium. The growth rate(column 2) was measured on the basis of total protein accumulation in the culture with time. The number ofribosomes per milligram of protein (column 4) was calculated by using the combined molecular weights of M.racemosus rRNA reported by Lovett and Haselby (25), i.e., 2.04 X 106. Dividing this value by Avogadro'snumber yields the weight of rRNA per 80S ribosome, i.e., 3.38 x 10-18 g/ribosome. The data in column 3 and thepercentage ofrRNA (85.7%) permit the final calculation of this value (shown in column 4). All other values weredetermined as described in the text.

' ND, Not determined.

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SPORANGIOSPORE GERMINATION 81

DISCUSSION

The specific rates of protein and RNAsyntheses were measured throughout hyphalgerm tube development from sporangiospores ofMucor. Synthesis of both classes of macromole-cules commenced immediately upon exposure ofspores to nutrient medium, and the measuredrates attained a maximum when the emergenceof new germ tubes ended. A correlation of peakprotein synthetic activity with germ tube emer-gence from Mucor yeast cells has previouslybeen reported (33). Perhaps these observationsrepresent a general phenomenon of acceleratedprotein synthesis that has an integral role in theformation ofhyphal germ tubes in this organism.The function of the acceleration is not known,but presumably it could regulate qualitativechanges in the protein composition of the orga-nism. Protein synthesis is generally required forgermination of fungal spores and occurs imme-diately upon introduction to nutrient medium orbreaking of dormancy (7, 16, 31, 39, 40). Therates of protein synthesis are regulated in pat-terns specific to the organism, and generally theyincrease during germination (17, 24, 39, 40, 42).Although fungal spores can immediately syn-

thesize protein in vivo, cell-free extracts pre-pared before stimulation with germinants oftenhave lesions in the protein synthetic apparatus.The lesions are quickly repaired during germi-nation (1, 17, 41). Autoinhibitors represent an-other mechanism reported to prevent proteinsynthesis in dormant spores (3, 23). All ribo-somes from dormant sporangiospores of Mucorexist as 60S and 40S subunits. Therefore, activeprotein synthesis does not occur in these cells.Whether protein synthesis is blocked by one ofthe above mechanisms or merely a lack ofmRNA is not known, but should prove to be aninteresting area of future study.Ribosomes recovered from dormant fungal

spores have been reported to exist either asinactive 80S particles (15, 17, 24) or in the formof polysomes (7, 9, 38), depending upon thespecies. It is claimed that the polysomes containstable mRNA that will be translated duringgermination. Three additional forms of evidencehave been presented to suggest the presence ofpreformed mRNA in dormant fungal spores: (i)inability of actinomycin D to inhibit germina-tion, (ii) failure to detect incorporation of radio-active RNA precursors during germination (7,16, 19, 42), and (iii) isolation of polyadenylicacid-containing RNA from dormant spores (18).The first two results, of course, could be due tothe failure of the added compounds to penetratethe spores. In addition, actinomycin D may onlyincompletely inhibit mRNA synthesis (12). In

Mucor, RNA was synthesized as soon as thesporangiospores were introduced into nutrientmedium. Actinomycin D prevented germ tubeemergence but not the initial swelling of spores.A definitive statement cannot be made at thistime about the existence of preformed mRNA inMucor sporangiospores. The hypothesis does,however, provide a plausible explanation for therapid appearance of polysomes and immediatesynthesis of protein after exposure of the sporesto nutrient medium.The nascent polypeptide chain elongation rate

was calculated at stages throughout hyphal germtube development from sporangiospores of Mu-cor. The rate increased during swelling of thespores and emergence of germ tubes. A maxi-mum value was attained when the emergence ofnew germ tubes had ended. The values de-creased gradually during further hyphal elon-gation. The temporal pattem of the polypeptidechain elongation rate correlates well with thepattern of the rate of protein synthesis measuredby radioactive amino acid incorporation. Adjust-ment of the rate of amino acid polymerizationper ribosome is therefore involved in regulationof the overall rate of protein synthesis. A similarobservation was made during gern tube emer-gence from yeast cells of Mucor .(Orlowski andSypherd, in press). Adjustment of the percent-age of cellular ribosomes active in protein syn-thesis is another mechanism involved in regulat-ing the overall rate of protein synthesis duringhyphal germ tube development from both sporesand yeast cells ofMucor (Orlowski and Sypherd,in press). The percentage of ribosomes active inprotein synthesis was lowest when protein syn-thesis occurred at the fastest rates. This couldbe due to a failure of the rate of polypeptidechain initiation to increase commensurate withthe accelerated rate of polypeptide chain elon-gation. We have no direct data concerning chaininitiation. The polypeptide chain elongation ratehas been frequently studied in other organisms(2, 6, 14, 28, 37, 43), but rarely in connectionwith morphogenesis (22; Orlowski and Sypherd,in press).The composition of free amino acid pools dur-

ing development has recently become an objectof intense investigation (10, 20, 27, 36). Charac-terization of the pools might yield informationon possible readily metabolizable storage com-pounds or changes in intermediary metabolicpathways. The largest pools in dormant spor-angiospores of Mucor were alanine, glutamate,aspartate, serine, and threonine. These were allgreatly reduced during germination and hyphalgrowth. Enzymes of glutamate metabolism havehigh activity in developing hyphae (J. Peters,unpublished data), and terminal oxidation of the

VOL. 134, 1978

82 ORLOWSKI AND SYPHERD

compound has been shown to occur at this time(C. Inderlied, unpublished data). Glutamate, al-anine, and aspartate may play a central role incarbon, energy, and nitrogen metabolism duringgermination ofMucor sporangiospores. Arginineincreased profoundly during germination. Therelationship of this compound to polyamine con-

centrations in this organism during hyphal de-velopment is presently being studied (J. Peters,unpublished data).

ACKNOWLEDGMENISThese studies were supported by Public Health Service

grant GM 23999 from the National Institute of General Med-ical Sciences.We thank Alf Larsen for helpful discussions about the

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15. Henney, IL R., and R. Storck. 1964. Polyribosomes andmorphology in Neurospora crassa. Proc. Natl. Acad.Sci. U.S.A. 51:1050-1055.

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