Regulatory mechanisms in cellulase synthesis during hyphal morphogenesis in the water mold Achla

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Regulatory mechanisms in cellulase synthesis during hyphal morphogenesis in the water mold Achla
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Kane, Bernard Evan, 1935-
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Morphogenesis   ( lcsh )
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Thesis (Ph. D.)--University of Florida, 1971.
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Includes bibliographical references (leaves 90-94).
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by Bernard Evan Kane.
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Typescript.
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Vita.

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Regulatory Mechanisms in Cellulase Synthesis
During IIyphal Morphogenesis
in the Water Mold Achlya














By

BERNARD EVAN KANE, JR.


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
1971













ACKNOWLEDGMENTS


The writer wishes to express his thanks first of all to

Dr. J. T. Mullins, Chairman of the Supervisory Committee, who provided

liberally of his facilities, time, and assistance for this research.

Appreciation is also extended to Dr. M. M. Griffith, Dr. Daniel

B. Ward, Professor T. deS. Furman, and Dr. Chesley B. Hall for serving

on the Supervisory Committee.

Dr. Joe L. Key and his associates at the University of Georgia

are due a special debt of gratitude for their guidance in learning

special techniques for the isolation of ribosomes.

Use of the ultracentrifuge was generously provided by

Professor James F. Preston. Dr. Paul Smith allowed the writer the free

use of the scintillation counter, and Mr. Francis M. Bordeaux assisted

greatly with scintillation counting procedures. The writer expresses

..' ~his-thariks-to all' f them.

This study was supported by a National Defense Education Act

Title IV Fellowship.


















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... .... ...... .......... ii

LIST OF TABLES .. ... ... .. ... .. .. iv

LIST OF FIGURES . . .. v

LIST OF ABBREVIATIONS ....................... vii

ABSTRACT ......... ..... .......... .viii


INTRODUCTION ............. ............. 1

LITERATURE REVIEW ....................... 2

PLANT MATERIALS AND CULTURE METHODS . ... 13

EXPERIMENTS AND RESULTS .. . .. 19

SUMMARY AND CONCLUSIONS . . ... .. 78

APPENDICES
--A.--DATA-FROM-CULTURES- USED IN ISOLATION OF. RIBOSOMES 84

B. EFFECT OF INHIBITORS ON BRANCHING . .. 85

C. SCINTILLATION COUNTING OF 1C-URIDINE ... 87
14
D. EXTRACTION OF RNA CPM OF 1C IN EXTRACTS FROM
FOUR REPRESENTATIVE SAMPLES . ... 89


BIBLIOGRAPHY .......... ............. ........ 90

BIOGRAPHICAL SKETCH ...................... 95


















LIST OF TABLES


Table Page

1. Complete Medium ..................... 14


2. Enriched Medium .......... ...... .... 16


3. E87 d Sporulation Medium . . 17


4. Response of E87 c to Vegetative and Sexual Induction 21


5. Effect of Inhibitors on Branching .. 63


6. Effect of Inhibitors and Antheridiol on RNA Synthesis 70

















LIST OF FIGURES


Figure Page

1. Sucrose gradient profile of Achlya ribosomes from vege-
tative cultures homogenized with DEP. Experiment#1,
Sample A-. .. .. .. .. ..... 27

2. Sucrose gradient profile of Achlya ribosomes from vege-
tative cultures homogenized without DEP. Experiment #1,
Sample A-2 ......................... 28

3. Sucrose gradient profile of Achlya ribosomes from
antheridiol-induced cultures homogenized with DEP.
Experiment #2, Sample B-. . ... 32

4. Sucrose gradient profile of Achlya ribosomes from Casein
Hydrolysate-induced cultures homogenized with DEP.
Experiment #2, Sample B-2. ... . .. 33

5. Sucrose gradient profile of Achlya ribosomes from vegeta-
tive cultures homogenized with DEP. Experiment #2,
Sample B-3 ........ ;................. 34

6. Sucrose gradient profiles of mungbean ribosomes from
hypocotyls homogenized with DEP. Experiment #3,
-Samples C=l and C-2 (replicates). . ... 39

7. Sucrose gradient profiles of Achlya ribosomes from
antheridiol-induced cultures homogenized with DEP.
Experiment #3, Samples C-3 and C-4 (replicates). ... 40

8. Sucrose gradient profiles of Achlya ribosomes from
vegetative cultures homogenized with DEP. Experiment #3,
Samples C-5 and C-6 (replicates). . ... 41

9. Profile of sucrose concentration and viscosity of
a test gradient. ....... ......... 44

10. Sucrose gradient profile of mungbean ribosomes from
hypocotyls homogenized with DEP. Experiment #4,
Sample D-l. ... . . 46

11. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium and homogenized with 20X
DEP. Experiment #4, Sample D-3. . ... 47









LIST OF FIGURES (Continued)
Figure Page

'12. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium and homogenized with
100 X DEP. Experiment #4, Sample D-5. . ... 48

13. Sucrose gradient profile of mungbean ribosomes from
hypocotyls homogenized with DEP. Experiment #4,
Sample D-2. . . ... ...... 49

14. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium and homogenized with 20 X
DEP. Experiment #4, Sample D-4. . 50

15. Sucrose gradient profile of Achlya ribosomes from cultures
grown on enriched medium and homogenized with 100 X DEP.
Experiment #4, Sample D-6. . ... 51

16. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium homogenized without
DOC or Mg++. Experiment #5, Sample E-. ... 54

17. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium homogenized with DOC
but without Mg++. Experiment #5, Sample E-2. ... 55

18. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium homogenized without
DOC but with 10 mM Mg++. Experiment #5, Sample E-3. 57

19. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium homogenized with
DOC and 10 mM Mg+. Experiment #5, Sample E-4. ... 58

20. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium homogenized without
DOC but with 30 m Mg++ and 30 mM K+. Experiment #5,
Sample E-5 . . . 59

21. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium homogenized with DOC,
with 30 ml Mg++ and with 30 mM K+. Experiment #5,
Sample E-6. ....................... 60

22. Flow sheet for RNA extraction. . ... 68

23. Typical absorbance profile of RNA extract. ... 73

24. Effect of AD and antheridiol on cellulase activity
released in the medium by E87 d6 .. .......... 76

25. Effect of AD and antheridiol on cellulase activity in
the mycelium of E87 ... ............... 77
vi

















LIST OF ABBREVIATIONS


AD Actinomycin-D

A.U. Absorbance Unit

BU Barksdale Unit

CM Cushioning Medium

DEP Diethyl Pyrocarbonate

DOC Deoxycholate

EM Enriched Medium

FU 5-fluorouracil

HA Antheridiol (Hormone A)

HM Homogenizing Medium

S Svedberg Unit

SM Standard Medium

SM+T Suspension Medium









Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy


Regulatory Mechanisms in Cellulase Synthesis
During Hyphal Morphogenesis
in the Water Mold Achlya

By

Bernard Evan Kane, Jr.

March, 1971


Chairman: Dr. John Thomas Mullins
Major Department: Botany


Achlya ambisexualis Raper is a heterothallic Oomycete, Male

strains produce antheridia in response to exposure to antheridiol, a

steroid hormone secreted by female strains. The antheridia are formed

by branching of the male hyphae.

Increases in protein synthesis, cellulase activity, and respir-

ation precede or accompany the branching response. Certain metabolic

substrates such as casein hydrolysate can also induce branching. The

induction of cellulase is thought to be necessary for softening the

cellulosic hyphal wall to facilitate branching.

The hormonal control mechanisms were investigated in several

ways. Male cultures were treated sequentially with hormone and casein

hydrolysate to determine whether two control sites (one responding to

hormone and another responding to metabolic substrates) exist for the

branching response. Failure to demonstrate the presence of two sites

was taken as evidence that there may only be one.

Ribosomes were isolated on sucrose gradients with the goal of

determining whether shifts in the proportion of ribosomes present as

viii










polysomes could be detected following exposure to hormone. Variations

in procedures, including changes in magnesium concentration, use of

deoxycholate to release polysomes from membranes, use of diethyl pyro-

carbonate in various concentrations to inhibit ribonucleases, were

tried but only monosomes were obtained.

The techniques used were tested by isolating ribosomes from

mungbean hypocotyls and excellent polysomes were demonstrated.

Actinomycin-D and 5-fluorouracil were used to study the effect

of these inhibitors on RNA synthesis, cellulase synthesis and branching.

Actinomycin-D, at a concentration of 15 pg/ml, inhibited tran-

scription by more than 50%, and prevented branching in cultures exposed

to antheridiol. It did not affect uptake of the hormone or endogenous

cellulase activity. When cultures inhibited with actinomycin-D and

exposed to antheridiol were rinsed in medium free of the inhibitor,

a delayed branching response was observed.

Five-fluorouracil, at a concentration of 15 pg/ml, did not

inhibit branching and its effect on transcription was not clear.

Continued exposure to this inhibitor killed the mycelium.

Antheridiol was found to increase the rate of transcription by

"14% above vegetative controls. The increase was observed 15 minutes

after hormone treatment (when first measurements were taken) and con-

tinued for at least 1 hour (when the last measurement was taken).

The results suggest that antheridiol may control cellulase

synthesis and subsequent hyphal morphogenesis by a direct effect on

transcription. There is no evidence to indicate that it does not

also affect translation.



















INTRODUCTION


Achlya ambisexualis Raper is a fungus whose sexual morphogenesis

is controlled by a sequence of hormones [49]. The first hormone in the

sequence initiates, in male strains of the organism, a branching

response that'leads to the formation of the male gametangium or

antheridium. Studies have revealed that these morphological changes

are accompanied by changes in respiration [70], in protein synthesis

[54], and in the activity of specific enzymes [64].

The purpose of the present study was to examine possible

mechanisms by which the hormone might exert its control. Because of

the previous studies on this system and an accumulating body of evi-

dence which indicates that plant hormones cause changes in nucleic

acid metabolism or transcription and in the assembly of polysomes for

protein synthesis or translation, special emphasis was placed on

examining these two aspects of hormonal control.
















LITERATURE REVIEW


The Organism


Achlya is a water mold in the class Oomycetes, order Saproleg-

niales, family Saprolegniaceae. The vegetative thallus consists of

filamentous, coenocytic, colorless hyphae that aremacroscopically

visible. It may be found in nature growing saprophytically in aquatic

habitats. This heterotrophic, obligate aerobe can be cultured in the

laboratory on hemp seeds or other natural substrates in water. It may

also be grown on a chemically defined medium consisting of a carbon

source and an organic supply of both sulfur and nitrogen [4]. The

respiratory metabolism has been studied [70] and shown to consist of

enzymes of the hexose monophosphate pathway, the Embden-Meyerhof-Parnas

-----eheme, and-the tricarboxylic-acid-cycle with a terminal cytochrome

system, as well as glycerol phosphate dehydrogenase and lactic dehydro-

genase activities.

SVegetative reproduction is usually accomplished by means of

zoospores produced in a zoosporangium, or occasionally by means of

gemmae. Both of these structures are delimited from the parent hypha

by means of a septum [20].

Heterogamous sexual reproduction occurs in this genus, and

some species are homothallic while others are heterothallic. The

species used in this study, A. ambisexualis Raper, is heterothallic

but exhibits an ambivalent sexual response, as the name indicates.

2










Various strains may be arranged in a linear series from pure males

through self-fertile intersexes all the way to pure females [46].

The sexual progression in Achlya may be divided into five

distinct morphological stages: (1) production of antheridial initials

by the male; (2) production of oogonial initials by the female;

(3) directional growth of antheridial hyphae to the oogonial initials

followed, after contact, by the delimitation of the antheridium by

means of a septum; (4) delimitation of the oogonium by a septum, fol-

lowed by the formation of eggs or oospheres; (5) transfer of male

nuclei from the antheridium through fertilization tubes which penetrate

the oogonial wall to the oospheres [503.

These stages have been demonstrated to be initiated and coor-

dinated by a series of hormones. In several classic papers Raper [42, 43,

44,45,47,48,51,52] has described the details of this system.

'In Raper's scheme a complex of four hormones controls the

initiation of antheridia. This A-complex consists of A and A2 which
1 3
are secreted by the vegetative female thallus and A and A by the

vegetative male thallus. The two hormones secreted by the vegetative

plants of each sex are distinguished by different solubilities.

A and A2 from the female both initiate antheridial branching in the

male, while A1 augments this response and A3 inhibits it.

Further morphological differentiation is dependent upon hormone

B, which is secreted from males that have been sexually induced by the

A-complex. Hormone B sexually induces the vegetative female to produce

oogonial initials. This induced female then secretes hormone C, which

attracts the antheridial initials and causes septum formation, which

results in delimiting a distinct anthoridial cell. These antheridia










then produce the final hormone D, which causes the formation of a

septum that delimits a distinct oogonial cell.

Barksdale [6] has provided evidence that hormone A may not

only initiate branching in male strains, but can also perform the

functions attributed to Raper's hormone C. She has also questioned

whether hormone A which Raper described as augmenting the response

to hormone A, might not be more simply explained as a nutritional

effect [7,8].

There is no debate, however, that hormone A, secreted by the

vegetative female, does, under proper conditions, induce branching in

the male strains.


Hormone A or Antheridiol


Hormone A can be isolated from the medium in which vegetative

females were grown by extraction with acetone or chloroform and the

extract concentrated by evaporating the solvent. The activity of the

extract can be measured by the bioassay technique of Raper [44] or of

Barksdale [5]. In this work, the Barksdale unit (BU) will be used.

It is defined as the least amount of hormone in one ml which will

cause the production of antheridial branches in male strains, such as

E87 r, within 2 hr at 30 C.

Raper and Haagen-Smit [52] were the first to produce a con-

centrated extract of hormone A. They characterized some of the chemical

and physical properties of the hormone but did not determine its struc-

ture. Later McMorris and Barksdale [34] were able to obtain a highly

purified crystalline extract of the formula C29 H4205 which could induce

branding in concentrations as low as 2 X 10-8 mg/ml. They named
branching in concentrations as low as 2 X 10 mg/ml. They named











this compound antheridiol, and this name will be used in the rest of

this text.

The chemical structure of antheridiol has been described [2]

and two possible isomers have been synthesized in the laboratory.[14].

One of the isomers has the same range of activity and the same phys-

ical properties as the natural extract. It is composed of a normal

tetracyclic steroid nucleus with a hydroxyl at C-3 and a carbonyl

at C-7 and is cited as the first and only example of a steroidal sex

hormone in the plant kingdom. It differs from mammalian sex hormones

particularly in that it has a much longer side chain attached at C-17.

This is a 10-carbon chain composing an a, -unsaturated y-lactone ring

and contains 3 oxygen atoms.



The Male Response


Antheridiol is rapidly taken up by male strains of Achlya.

In 1 hr an uptake of 6 X 10 BU/g of fresh weight of mycelium is pos-

sible from a solution with an initial concentration of 105 BU/ml [5].

-Certain metabolic and morphological changes have been shown to

occur rapidly following this uptake. The respiratory rate increases

to 115%, as compared with vegetative controls, during the first 40 min

and continues to increase to a peak of 411% of the controls in 80 to

120 min when males are exposed to 100 BU/ml in an appropriate medium

[70]. Antheridiol also elicits a rise in activity of the enzyme cel-

lulase, which peaks within 2 hr, paralleling the increase in appear-

ance of antheridial branches [64]. Mullins [35] has proposed that the

induction of increased cellulase activity is a prerequisite to branch












initiation. This was supported with several kinds of data. First, as

already mentioned, there is a parallel increase in cellulase activity

and branch induction. Secondly, treatment with casein hydrolysate

(0.1%) or other similar metabolites induces a vegetative branching

response, and this too is paralleled by an increase in cellulase activ-

ity. Thirdly, the cellulase in Achlya is an endocellulase, i.e., not

a nutritional cellulase, but possibly a weakening agent for restructur-

ing the cellulose cell wall. Furthermore, the cell wall of Achlya has

been examined by Parker, Preston, and Fogg [40], who determined that it

is composed of 15% microfibrillar cellulose and 85% 1 an amorphous, non-

cellulosic, polysaccharide complex.



Mechanism of Hormone Action


Hormones regulate a wide variety of responses in plants and

animals. The hormones themselves are a heterogeneous group of com-

pounds. A single hormone applied to a single tissue may elicit numer-

ous physiological changes. These changes may be expressed in membrane

permeability, enzyme activity, nucleic acid metabolism or intracellu-

lar ionic concentrations. In a recent review article, Grant [17] dis-

cusses more than a dozen possible mechanisms by which steroid hormones

may exert their control. There is some experimental evidence for all

of the mechanisms he describes, and yet he concludes that it has not

been possible to prove the primary site of action for any hormone.

Rather, so many hormonal effects have been shown that it may be that

a single hormone acts at several sites within the cell.











The Karlson hypothesis [22], i.e., that hormones act at the

level of the gene as derepressors, thus exposing or activating repressed

genes that in turn code for mRNA, has been proposed as an extension of

the Jacob and Monod hypothesis [19] to eucaryotic organisms.

Considering the Karlson hypothesis, Grant [17] warns that,

"Despite the tendency to be hypnotized into believing that all forms

of control, including protein synthesis, are at the genetic level .

this is not the case." Thus he points out that there is good evidence

for membrane effects and direct allosteric effects upon key enzymes as

control mechanisms.

Tata, in another review of hormonal control [62], also con-

sidered the Karlson hypothesis to be inadequate to account for the

existing evidence. He calls attention to the sensitivity of rRNA

cistrons to hormones as a possible control mechanism. Transport of

mRNA from the nucleus to the cytoplasm is facilitated by the produc-

tion of ribosomal precursor particles. The accumulation of sufficient

-numbers of polysomes preceded with a hormone specific mRNA could

account for some of the control effects. Tata also cites evidence

that hormones influence membrane-ribosome associations, which, besides

stabilizing mRNA, may restrict free exchange between different popula-

tions of ribosomes and the mRNA formed before and after the cell is

exposed to a hormone, thereby serving as a control mechanism.

In cases where enzyme activity is altered by a hormone, it is

not usually clear whether enzyme activation or de novo synthesis is

responsible for the change. In a notable exception Varner [67] has

shown the de novo synthesis of ac-amylase in barley endosperm following

treatment with gibberellic acid. This is perhaps the most definitive










example of the mode of action of a plant hormone. Yet even here it is not

known if a new m-amylase specific message is being transcribed.

Varner [68] has noted that no mRNA has ever been purified or charac-

terized, nor has its rate of synthesis or stability been determined

in higher plants.

Hormones do affect nucleic acid synthesis, as was first shown

by Silberger and Skoog [57]. They found that in auxin-treated tobacco

pith culture, nucleic acid synthesis increased prior to the increase

in tissue weight. Since that report, numerous investigations have

shown that growth-promoting hormones enhance nucleic acid synthesis.

Key [23] has reviewed the relationship between nucleic acids and

hormones; he concludes that although hormones affect both the kind as

well as the amount of nucleic acids, there is no definitive evidence

that this is the primary effect.

Part of the central dogma of molecular biology is that infor-

mation can flow from DNA to RNA, and from RNA to protein [12]. This

flow of information, which defines the structure and function of the

cell, is dependent upon two fundamental processes: transcription and

translation. The first is the mechanism by which ribonucleic acids

are assembled at the level of the gene; the second is the process by

which the ribonucleic acids assemble proteins.

In view of the many sites that may be available for hormone

action, the question of primary site is generally considered in terms

of which of these two levels of control is affected.

The mechanism of hormonal control of Achlya is also not known,

but Achlya combines several features which provide a better system for

studying hormonal control than do other organisms. (1) It exhibits










de novo production and differentiation of organs under hormonal control.

(2) It is easily cultured aseptically on a chemically defined medium.

(3) It has a steroid hormone whose structure is known and which can be

administered quickly to the entire organism via its natural route.

(4) It is analogous to higher organisms because it is eucaryotic and

has cell walls composed of cellulose.



Ribosomes


The term ribosomee" was proposed in 1958 at the First Symposium

of the Biophysical Society to designate certain ribonucleoprotein

particles that seemed to be involved in protein synthesis [55).

These particles are isolated and analyzed by differential

centrifugation. They are usually characterized in terms of their

sedimentation rate under standard conditions, expressed in Svedberg

units (S). A Svedberg unit is the velocity of sedimentation per unit
-13
of gravitational field, or 1 x 10- cm/sec/dyne/g [11].

Special techniques for analytical ultracentrifugation used to

determine the sedimentation rates require special equipment and are

technically exacting. It is common practice therefore simply to com-

pare sedimentation of an unknown particle to that of a known particle.

Ribosomes isolated from E. coli have been found to sediment precisely

at 70S, using the analytical ultracentrifuge, and may be used as stan-

-dards for comparison with ribosomes from some unknown organism [72].

Ribosomes from a wide variety of organisms and subeellular

organelles seem to be of two types based on their sedimentation

velocities: (1) those from procaryotic organisms or mitochondria and

chloroplasts which have sedimentation velocities at or near 70S,











and (2) those from the cytoplasm of eucaryotic organisms which sediment

at or near 80S [73].

Ribosomes provide the structural base upon which mRNA is

translated to form proteins. The ribosome moves along the long mRNA

molecule, reading the code and assembling protein. Because of the

length of the mRNA, more than one ribosome may translate the mRNA at

one time; it is common for 2, 3, or more ribosomes to become attached

to one messenger. This complex of ribosomes and mRNA is called a

polysome [16]. There is little reason to believe that protein syn-

thesis ever occurs in the absence of ribosomes.

Williamson and Schweet [73,74] presented evidence supporting

the view of dynamic equilibrium of attachment and detachment of ribo-

somes from polysomes during protein synthesis; further, that the rate

of attachment of ribosomes to mRNA may be a rate-limiting step in

protein synthesis. Others [69] have emphasized that these mechanisms

of attachment may have a decisive role in metabolic control.

The effect of hormones on polysome formation in higher plants

is being explored. Trewavas [66] reported that auxin causes a shift

in the proportion of ribosomes present as polysomes in excised pea

internodes. Key and Ingle [25,26] have shown a similar shift in

soybean hypocotyl ribosomes that was related to a requirement for

synthesis of AMP-rich RNAs (DNA-like RNA and therefore possibly mRNA).

Reports of studies of ribosomes in fungi are limited and none

has been concerned with hormonal control mechanisms. The few reports

that have been published show that the fungal ribosome has sedimenta-

tion coefficients varying from 77S to 81.5S, which is indicative of













normal eucaryotic ribosomes [3]. These reports were on ascomycetes;

none was found from phycomycetes.



Inhibitors


One of the methods for examining control mechanisms is the use

of selective metabolic inhibitors. An array of such compounds is

available to the biologist. The advantage these offer is that they

may permit selective inhibition of specific processes. Two such

inhibitors were used in this study, actinomycin-D (AD) and 5-fluoro-

uracil (FU).

The primary effect of AD is the inhibition of DNA-dependent

RNA synthesis. The proposed mechanism is a steric interference caused

by binding of AD to guanine in the DNA, thereby preventing translation

via RNA polymerase [53]. The inhibition of RNA synthesis may be as

high as 95% at a concentration of 1 to 10 pg/ml in some plant tissues,

but smaller inhibitions may occur at higher concentrations in others [23].

AD was chosen for use in this research because in plant tissues

it allows protein synthesis to continue for several hours after RNA

synthesis has been inhibited [24]. The rate of protein synthesis

slowly decays and this decay parallels a decay in functional polysomes [31].

The action and use of FU has been recently reviewed by Mandel

[33). This drug is a base analog of uracil and replaces uracil during

transcription. This replacement has a variety of effects, but two of

the more direct ones are: (1) to prevent the formation of new ribo-

somes, and (2) to block DNA replication.







12



This drug was chosen primarily for the present study because of

the report by Lin and Key [31] comparing the effects of FU with that

of AD. They found that, although FU inhibited the synthesis of new

ribosomes, it did not prevent protein synthesis or cell elongation, or

reduce the level of functional polysomes in the roots of intact soybean

seedlings. AD, however, in levels that only partially inhibited RNA syn-

thesis, completely prevented cell elongation. These data were inter-

preted to indicate that continued synthesis of ribosomal RNA was not

necessary for cell elongation and that the inhibition was of a select

class of RNA (probably mRNA).

















PLANT MATERIALS AND CULTURE METHODS


The two strains of A. ambisexualis used in this study were

E87 "c and 734 $ Both were obtained from stock cultures provided by

Dr. J. T. Mullins. Details of the origin of these cultures are given

by Raper [461.

When treatment of cultures with exogenous antheridiol was

desired, an aqueous solution containing 103 BU/ml was used. This

solution was provided by Dr. J. T. Mullins and had been prepared by

dilution from an acetone base stock solution of crude natural anther-

idiol containing 106 units per ml.

The cultures were maintained in 100 X 15 mm plastic Petri

dishes on a nutrient agar medium developed by Mullins and Barksdale

[37]. The composition of this medium is given in Table 1. During

preparation the pH of the media was adjusted to 6.9 by the addition

of H2SO This medium will be referred to as complete medium.

Three types of liquid media were used, depending upon the

experiment performed. Most studies were made, using the complete

medium in Table 1 without agar. In some experiments, a variation of

this medium was used in which the supply of nitrogen (monosodium

glutamate) was reduced by one-half. For convenience, the latter medium

will be referred to as N/2 medium. A third medium, used in some of the

studies of ribosomes, was chosen to provide for a very rapid growth.

13














TABLE 1

COMPLETE MEDIUM



Monosodium Glutamate 0.4 g

Glucose 2.8 g

Tris hydroxymethyll) aminomethane 1.2 g

Combined Liquid Stocks 17.5 ml

Distilled Water to 1.0 1

Agar 20.0 g


aCombined Liquid Stock:

1-methionine (15 mg/ml in 10% HC1) 10.0 ml

KC1 (2M) 10.0 ml

MgSO4 7H20 (0.5M) 10.0 ml

CaCI2 (0.5M) 10.0 ml

HEDTA (10 mg/ml) 20.0 ml

KH2 PO (1M) 15.0 ml

Metal Mix #4 (2mg/ml)b 100.0 ml


bMetal Mix #4:

Grind together:

Fe(NI4)2 -(SO4)26H20 28.9 g

Zn(SO ).72 0 8.8 g

Mn(SO4).-H20 3.1 g

As used this metal mix provides:

Zn, 1.0 mg/l; Fe, 2.0 mg/l; Mn, 0.5 mg/l.










This medium was designed by extrapolation from data compiled by

Barksdale [4], which indicated optimal carbon and nitrogen quantities

and ratios. The composition of this medium is listed in Table 2 and

is referred to as enriched medium.

Liquid cultures were inoculated by use of a sporulation medium

(Table 3). Plugs were taken with a number 5 cork borer from the grow-

ing margin of cultures maintained on plates of complete medium. Four

such plugs were placed in 50 ml of sporulation media in a Morton 250 ml

culture flask and incubated at 28 C for 16 to 18 hr with gentle agita-

tion on a reciprocating shaker. At this time the liquid contained

numerous vegetative spores produced by the mycelium on the agar plugs.

The liquid was decanted into a 500 ml Erlenmeyer flask containing

200 ml of the desired culture medium. This newly inoculated culture

was then incubated on a reciprocating shaker at 28 C for 46 to 60 hr.

At this time each flask generally contained from 2 to 8 g fresh weight

of mycelium.

All of the culturing procedures described above were performed

aseptically. Periodic tests were performed to be certain that the

cultures were free from bacterial or fungal contaminants.

Cultures were harvested by pouring the entire contents of the

culture flask through a 9.0 cm #1 Whatman filter paper on a Buchner

funnel with gentle suction. In some experiments the mycelium was

weighed and used directly. In others, as will be indicated, the

harvested mycelium was divided into portions of the desired weight and

resuspended in a known volume of the filtrate. The filtrate has been

shown to be a superior suspension medium for studying the various

responses to the hormone [65,70].





















TABLE 2

ENRICHED MEDIUM


Casein Hydrolysate 1.5 g

Monosodium Glutamate 0.5 g

Glucose 14.0 g

Tris hydroxymethyll) aminomethane 1.2 g

Combined Liquid Stocka 17.5 ml

Distilled Water to 1.0 1

(pH adjusted to 6.9 with 12M H2SO4)



aSee Table 1.





















TABLE 3

E87 d SPORULATION MEDIUM



Edamin 40.0 mg

Dextrin 240.0 mg

Calcium Glycerophosphate 3.0 mg

Potassium Bicarbonate 20.0 mg

CaCl2 (0.5M) 0.1 ml

MgS0o47H20 (0.1M) 0.1 ml

Distilled Water to 1.0 1






18



The resuspended cultures were maintained on a reciprocating

shaker at room temperature for 1 hr prior to being subjected to any

further experimental procedures. This 1-hr equilibration period was

used to allow the mycelium to recover from any trauma of the initial

harvesting procedures. Following experimental treatment, the cultures

were harvested as before, reweighed to obtain the final fresh weight

and quick frozen prior to further analysis.

















EXPERIMENTS AND RESULTS


The problem of hormonal mechanisms was approached in several

ways. Each required special techniques. The variations in procedure

were often an important part of the experiment. The purpose of each

approach and the special techniques used will be discussed with each

experiment.


Response of E87 d to Metabolic and Sexual Induction

Purpose

Thomas and Mullins [65] has shown that the cellulase produced

with metabolic inducers, such as casein hydrolysate, had different

extraction properties, metabolic stability, and thermal stability than

that produced following hormone treatment. This suggests that there

might be two sites for induction of branching, one which responds to

the hormone and one which responds to metabolic induction.

Thomas has also shown [63] that a decline in cellulase activity,

which begins 3 or 4 hr after hormonal induction, was not affected by

a second treatment with hormone given 1 hr after the first. Thomas

interpreted this as indicating that the decline was the result of some

control mechanism other than mere depletion of the hormone.

If two sites for induction of branching did exist it seemed

plausible that one branching response could be superimposed on another

by sequential treatment with two types of inducers. A test of this











was made by treating cultures of E87 d first with antheridiol, allow-

ing the sexual branching response to begin, and then treating the

cultures with casein hydrolysate to see if a second burst of branch-

ing could be stimulated. The reciprocal experiment was also performed,

i.e., first inducing branching with casein hydrolysate and then treat-

ing with antheridiol.


Method

Cultures of E87 d were cultivated on N/2 liquid medium and

harvested. Thirteen 0. 1 g portions of mycelium were resuspended in

10 ml aliquots of filtrate in 60 X 15 mm Petri dishes and allowed to

equilibrate as previously described. Treatment with antheridiol

(5 BU/ml) was done by addition of 0.05 ml of the stock solution

(5 BU/ml of medium). Treatment with casein hydrolysate (0.2%) was

by addition of 1.1 ml of a 2% solution of casein hydrolysate.


Results and Discussion

The results were recorded in terms of morphological responses

and are shown in Table 4.

Interpretation of this table requires that the investigator

recognize certain limitations. The first is that the initial branch-

ing response, the formation of numerous branch initials (referred to

as "pegs"), in the region behind the growing tip, has a similar appear-

ance regardless of whether the induction is by antheridiol or casein

hydrolysate. It is not until the branches mature that an absolute

distinction can be made between sexual and vegetative branching.

This distinction is that antheridial (sexual) branches form a septum









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which delimits the antheridium from the vegetative mycelium while the

vegetative branch lacks such a septum. Barksdale [7] has described

sexual branches as being more slender and spiraled than vegetative

branches.

In the absence of a female, the antheridial initials can be

further distinguished because they generally form secondary branches

and fail to elongate, taking on a dendritic appearance. Vegetative

branches, by contrast, continue to elongate by apical growth and seldom

form secondary branching.

The drawback to interpretation of the data in Table 4 then is

that such differences are qualitative, not quantitative, and the inter-

pretation is at least partly subjective.

It appeared that if casein hydrolysate treatment followed

induction by antheridiol, the final response was predominantly vege-

tative, the sexually induced branches being modified by the change in

substrate availability (cultures 1 and 2, 8 hr). In the reciprocal

case, the treatment by antheridiol following induction with casein

hydrolysate was that the response continued to be vegetative with per-

haps a very reduced sexual response (cultures 7 and 8).

The purpose of cultures 5, 6, 11, and 12 was to provide con-

trols, receiving only one of the two inducers, whose morphology could

be used for comparison with the experimental cultures. Similarly,

cultures 3, 4, 9, and 10, which received two treatments, both with the

same inducers, were also used for morphological comparison.

Culture 13 provided an untreated control which was treated

with 734 filtrate to be certain that the E87 c would respond to

antheridiol.











It is not possible to draw a firm conclusion as to whether both

a vegetative and a sexual site for branch induction exist. The exper-

ment does indicate that a good sexual response depends upon a sub-

strate that is low in the nutrients provided by the casein hydrolysate.

That the organism would continue to be predominantly vegetative in the

presence of a rich substrate is not surprising; a general principle

proposed by Klebs [28] is that sexual and asexual reproduction in fungi

is not likely to be induced in a mycelium which is vigorously growing

on a rich substrate. Because there was no apparent second burst of

branching in any of the treatments, I suggest that there is probably

one mechanism for branch induction which responds to inducer sub-

stances, but that the development of the branches is determined by

other factors. Specifically, these would include the presence of a

female and the condition of the environment with respect to substrate

availability. Furthermore, it seems that substrate availability can

modify the sexual response.



Examination of Ribosomes on Sucrose Gradients


Purpose

There were several reasons for studying isolated ribosomes

from Achlya. First, a number of recent studies [13,32,38,62,68] on

plants, where protein synthesis is enhanced by hormones, have shown

marked changes in nucleic acid synthesis and in the distribution

of ribosomes between monosomal and polysomal fractions. Further-

more, there was the possibility that a stable polysomal fraction

could be obtained which would be useful for studying the effect of










antheridiol on cellulase synthesis in vitro. Finally, although ribo-

somes have been examined extensively in bacteria, animals, and higher

plants, few reports were found from fungi. The few that were found

were studies of ascomycetes [21,29,61] and were primarily consider-

ations of sedimentation velocities of the monosome, not attempts to

isolate stable polysomes.

The potential rewards seemed worth the effort to develop the

techniques for examination of ribosomes on sucrose gradients.

The first step was to spend a week in residence at the

University of Georgia in the laboratory of Dr. J. L. Key, a pioneer

in the study of hormonal effects on nucleic acid synthesis. Here is

assembled a team of skilled researchers with the necessary facilities

for routine isolation of ribosomes.

An initial experiment (Experiment #1, below), using their

routines and Achlya cultures brought from Gainesville, Florida, was

performed, both for training and to provide preliminary information for

future attempts.


Experiment #1

Methods

Three culture flasks of E87 d were grown on complete liquid

medium and harvested as previously described, except that enroute to

the University of Georgia they were subjected to above normal growth

temperatures. Growth in the flasks was slower than usual and when

harvested at 48 hr the fresh weight of mycelium was 3.0 g, which is

less than half the expected amount.










This was divided into two 1.5 g samples (A-1 and A-2) and

was frozen.

A standard medium (SM) was used with various modifications for

the several steps involved in isolating the ribosomes. This medium

contained: Tris-HCl, pH 7.5, 50 mM; MgCl2, 5mM; KC1, 15 mM. Various

amounts of sucrose (ribonuclease-free) and other reagents were added

to this SM as desired. Three common variations were:

Homogenizing Medium HM 250 mM sucrose in SM

Cushioning Medium CM 1.5 M sucrose in SM

Suspension Medium SM+T 1% Triton-X in SM

The frozen mycelium was homogenized in 8 ml of HM made 0.67 mM

in dithiothreitol, using a Willem Polytron PT 20st tissue homogenizer

at high speed for 8 seconds. Diethyl pyrocarbonate (10 X) was added

to sample A-i immediately before homogenization.

The homogenate was centrifuged at 20,000 x g for 15 min and

the supernatant filtered through one layer of Miracloth (Calbiochem,

Los Angeles, California). The ribosomal pellet was prepared from the

filtered supernatant by layering the supernatant over 3.0 ml of CM

and centrifuging at 229,414 X g for 85 min in a Spinco type 65 rotor

at 0 C.

The ribosomal pellet was rinsed once with SM and suspended in

0.4 ml SM+T. The absorbance at 260 nm of this suspension was esti-

mated by measuring the absorbance of a 0.04 ml aliquot in 2.0 ml dis-

tilled H20 with a Gilford 240 spectrophotometer. From this estimate

a fraction of the suspension containing 16 Absorbance Units (A.U.) was

layered on a 35-ml 10-34% w/v linear sucrose gradient.



An Absorbance Unit is the amount of material which in a
volume of 1 ml will have an absorbance of 1.00.










The gradients were prepared in advance by successively layer-

ing 7 ml aliquots of 34%, 28%, 22%, 16%, and 10% w/v of sucrose in SM

into the centrifuge tubes and allowing them to stand for 1 hr at room

temperature. They were then stored in a refrigerator overnight.

After centrifuging, the gradients were scanned for the

absorbance profile at 254 nm using an ISCO model D gradient fraction-

ator and absorbance monitor with a light path 10 mm long. The flow

rate of the gradient through the absorbance monitor was 3.5 ml/min.

The absorbance profile of the gradient. was automatically recorded on

an ISCO chart recorder.


Results and discussion (Experiment #1)

The profiles are reproduced in Figures 1 and 2. A large peak,

centered about 10 ml from the top of the gradient, was the dominant

feature in both samples. This compared reasonably well to the loca-

tion of the 80S monosome in samples of mungbean hypocotyl prepared in

the same way [1].

Ribonuclease is usually present in ribosomal preparations and

degrades the single-stranded mRNA component, thereby converting poly-

somes to monosomes. Some protection against this is provided by per-

forming all operations at 0 C. Diethyl pyrocarbonate (DEP) has also

been reported [15,71] to protect polysomes from this enzyme and was

added to sample A-1 for that purpose. A disadvantage of DEP is that

it causes disassociation of monosomes into their subunits.

Some differences can be seen in samples A-1 and A-2. For

example, in A-2 there is a small peak at 7.0 ml, which is interpreted

as the 60S ribosomal subunit. Absorbance in this same region in












Monosome Peak


21 28 35


.._Figure--.. Sucrose gradient profile of Achlya ribosomes from vegetative
cultures homogenized with DEP. Experiment #1, Sample A-i.













Monosome Peak


Figure 2. Sucrose gradient profile of Achlya ribosomes from vegetative
cultures homogenized without DEP. Experiment #1, Sample A-2.


) 7 14 21 28. 35










sample A-1 is off the scale of the chart recorder, although there is

a very small cleft between this region and the adjacent 80S monomer.

The greater amount of 60S absorbance in sample A-1 is attributed to

the negative action of DEP on the monosome.

There were slight shoulders in A-i at about 12 ml, 15 ml, and

17 ml which may represent small amounts of di-, tri-, and tetrasomes,

or aggregation of monosomes with Mg+


Experiment #2

Methods

Nine flasks of E87 d were grown on N/2 medium in the usual

manner for 50 hr. Three flasks were treated with antheridiol, 5 BU/ml

(1.25 ml of stock solution flask) for 15 min, combined and harvested

(sample B-l); three were treated with casein hydrolysate at 0.1% (10 ml

of 2.5% solution flask) for 15 min, combined and harvested (sample

B-2), and three were combined and harvested untreated as controls

(sample B-3).

The general design of the procedures for isolation of ribo-

somes was an attempt to duplicate those used in Dr. Key's laboratory.

.Significant modifications were necessary because of the different

facilities available.

The frozen mycelium was ground to a powder with a mortar and

pestle that had been precooled on dry ice. The powder was homogenized,

using 8 ml IIM with 6 mMl P-mercaptoethanol and 20 X DEP added just

prior to homogenization. The homogenate was transferred to a Kontes

ground-glass tissue homogenizer and ground five strokes with four

half turns of the rod at the end of each stroke. The homogenate










was centrifuged at 20,000 X g for 15 min in a Sorvall S-34 rotor at

0 C. The supernatant was then filtered through one layer of Miracloth.

The ribosomal pellet was prepared by layering the sample over 3,0 ml

CM and centrifuging at 105,000 X g for 3 hr in a Spinco type Ti50 rotor

at 0 C.

The pellet was rinsed once with SM and suspended in 0.5 ml SM.

The absorbance at 260 nm of this suspension was estimated by measuring

the absorbance of a 0.010 ml aliquot in 2 ml distilled H20 with a

spectrophotometer. The entire remaining suspension (20-25 A.U.) was

then layered onto a 4.5 ml 10%-34% sucrose w/v linear sucrose gradient

and centrifuged at 58,000 X g for 2 hr in a Spinco type SW39L rotor

at 0 C, using 1/2" X 2" tubes.

The sucrose gradients were prepared in advance by successively

layering 0.9 ml aliquots of 34%, 28%, 22%, 16%, and 10% w/v sucrose

in SM and allowing them to stand for 1 hr at room temperature. They

were then refrigerated for at least 1 hr prior to use.

The gradients were fractionated manually by means of a siphon.

A blunt 3-1/2-inch, 20-gauge hypodermic needle was connected by a

20-cm piece of plastic tubing (1 mm bore) to a 21-gauge thinwall needle.

The centrifuge tube containing the gradient was centered and clamped

directly under a 1-inch, 18-gauge needle. The 20-gauge needle was

gently lowered through the 18-gauge guide needle, into the sucrose

gradient all the way to the bottom of the centrifuge tube. The siphon

was started by applying suction to the attached 21-gauge needle and the

gradient was slowly removed from the tube. Five or six drop fractions

(as noted in individual results) were collected in 13 X 100 mm test

tubes each containing 2.0 ml distilled H20.










The contents of the tube ,ere mixed, using a Vortex-Genie

device (Scientific Industries, I: ) and the absorbance of each frac-

tion at 260 nm was measured with pocctrophotometer using standard

quartz cuvettes and a light patl nun long.


Results and discussion (Experimen.t #2)

The absorbance of each fraction was plotted against the frac-

tion number to form a gradient absorbance profile for each of the three

samples (Figures 3, 4, and 5).

As in Experiment #1 (Figures 1 and 2), these profiles are also

dominated by one large peak, presumably repri.renting the 80S monomer.

There was little evidence of a 60S subunit, except that in all three

samples the sides of the peaks are sloping. These profiles are asym-

metrical, having more area under the part of the curve to the right of

the 0OS peak than to the left. This suggests that polysomes may have

been present.

There were no significant differences between the antheridiol-

treated samples (Figure 3), the casein hydrolysate-treated sample

(Figure 4), and vegetative control (Figure 5).

The sloping sides of the peaks and the lack of resolution of

any minor peaks showed that the procedures must be improved if com-

parisons of profiles between cultures receiving different treatments

-were to be made.

Several variations were tried in an attempt to improve the

resolution of the gradients. They were:

1. Layering fewer absorbance units onto the gradient.

The problem was to use enough to give significant


















































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absorbance readings when the gradient is divided into

50 fractions but not so much that resolution of peaks

is masked by an overload of absorbing material.

2. Increasing the length of the gradients. Centrifuge

tubes had a nominal volume of 5 ml. Gradients of 4.5 ml

were used at first to allow room for the ribosomal

suspension. The actual capacity was larger than the

nominal capacity so that gradients of 5 ml could be

used and still have room for the sample.

3. Selecting centrifuge tubes with smooth inside bottoms.

The tubes are molded from cellulose nitrate and there

is considerable variation in the smoothness of the

bottom. This could cause turbulence and thus mixing in

the gradient during fractionation.

4. Increasing the ratio of pellet size to suspending medium

to provide a suspension containing 10 A.U. in 0.2 ml or

less. A narrow band of ribosomal suspension gives better

resolution than a large one because all of the particles

start sedimenting from nearer the same place.

5. Changing the culture medium. In previous trials N/2

or complete medium was used. Since polysomes are asso-

ciated with rapid protein synthesis, an enriched medium

was developed to provide maximum growth rate. This medium

provided approximately 5.6 g fresh weight of mycelium per

culture flask in 36 hr, while N/2 medium produced approx-

imately 2.0 g per flask in 48 hr.










6. Changing the length of the siphon and the gauge of the

needles used in order to decrease the time required to

fractionate the gradient from about 20 to 10 min per

gradient.

7. Homogenizing the tissues less vigorously by not using

the ground-glass homogenizer.

8. Increasing the amount of homogenizing medium to 15 ml

for 5 to 6 g of tissue and trying various amounts of DEP.

9. Isolating ribosomes from mungbean hypocotyls (Phaseolus

aureus Roxb.) to provide a standard control.

Not all of these variations were tried at once. Various

combinations of procedures were tried on partial or complete experi-

ments to see which modifications were most feasible. Finally, the

procedures which seemed best were combined in a complete experiment

with duplicates of each treatment so that the reproducibility could

be estimated.


Experiment #3

Methods

Two sets (C-1 and C-2) of mungbean seeds were germinated for

20 hr in rolls of moist paper [30]. Each sample consisted of 300

hypocotyls harvested after they reached a length of 0.75 to 1.0 cm.

Four culture flasks of E87
the usual manner for 36 hr. Two cultures were treated with 20 BU/ml

antheridiol (4.8 ml stock solution per flask) for 100 min and harvested

(samples C-3 and C-4). Two were left untreated as vegetative controls

(samples C-5 and C-6).










The frozen tissues were powdered in mortars and pestles pre-

cooled on dry ice. This was accomplished by first crumbling the

tissue by tapping it 25 times with the pestle and then grinding it to

a powder, using 50 circular grinding motions.

This powder was then homogenized, using 15 ml HM made 0.67

IM in dithiothreitol, by 50 more circular grinding motions to produce

the homogenate. Just prior to homogenization 30 X DEP was added.

A ribosomal pellet was prepared from this homogenate as described in

Experiment #2, except the use of the ground-glass tissue homogenizer

was omitted.

The pellets were rinsed once with SM and suspended in 0.6 ml

SM+T. The absorbance at 260 nm of this suspension was estimated by

measuring a 0.02 ml aliquot in 2 ml distilled H20 with a spectrophotom-

eter. From this estimate a fraction of the suspension containing 10

A.U. was layered on a 5.0 ml 10-34% w/v nonlinear sucrose gradient and

centrifuging as in Experiment #2.

The gradients were prepared in advance by successively layer-

ing 0.9 ml of 34%, 0.9 ml of 28%, 1.15 ml of 22%, 1.15 ml of 16%, and

1.00 ml of 10% w/v sucrose in SM into centrifuge tubes selected for

smooth bottoms. These were allowed to stand for 2 hr and then chilled

for at least 1 hr prior to use.

The gradients were fractionated by siphon as before, except:

(1) the collecting needle was a 3-1/2-inch long, 18-gauge needle;

(2) the tubing was 25 cm; and (3) 7-drop fractions were collected.

The fractions were then processed as in Experiment #2.










Results and discussion (Experiment #3)

The gradient absorbance profiles, plotted as in Experiment #2,

are shown in Figures 6, 7, and 8.

The mungbean controls (Figure 6) exhibited an 80S peak in

fraction 13 from the top of the gradient. Both samples show a shallow

trough preceding a broad flattened polysomal region beginning between

fractions 16-20 and extending toward the bottom of the gradient.

This profile corresponds well with the profiles obtained by

Anderson and Key [1], using the system described in Experiment #2 (see

inset, Figure 6), except that the 60S subunit peak and the peaks of

some of the first polysomal elements are more clearly resolved in the

Anderson and Key system than in this system.

The Achlya profiles (Figures 7 and 8) are dominated by a single

large peak occurring between fractions 13-14 for all four samples.

This corresponds very well with the position of the 80S peak in the

mungbean samples. Nevertheless, there still was no indication

of polysomes in Achlya.


Experiment #4

Methods

At this stage it was apparent that an isolation procedure that

was well suited for obtaining polysomes in several higher plants [1,71]

produced predominantly monosomes in Achlya. Very high levels of ribo-

nuclease could be responsible for this. Therefore, another trial was

made, using very high levels of DEP in some samples.

Several mechanical changes were made in the methods used in

Experiment #3. This included a new technique for preparing gradients










0.2-









0
0.4-










0.0


Monosome Peak
+


S 1 (a)


Sample C-I

------ Sample C-2


I I I I I
0 10 20 30 40 50
Fraction Number


Figure 6. Sucrose gradient profiles of mungbean ribosomes from hypo-
cotyls homogenized with DEP. Experiment #3, Samples C-1
and C-2 (replicates), (a) Data from [I].


Monosome Peak


'I
















Monosome Peak

I


-- Sample C-3

---- Sample C-4


0.1*
.0





0. 1





0.0 .....---
0 10 20 30 40 50
Fraction Number




Figure 7. Sucrose gradient profiles of Achlya ribo-
somes from antheridiol-induced cultures
homogenized with DEP. Experiment #3,
Samples C-3 and C-4 (replicates).





















Monosome Peak

.$


-- Sample C-5

---- Sample C-6


0I 1






I I
0.1





0 .'0 -, -- -- ---- ,- ,
0 10 20 30 40 50
Fraction Number





Figure 8. Sucrose gradient profiles of Achlya ribo-
somes from vegetative cultures homogenized
with DEP. Experiment #3, Samples C-5 and
C-6 (replicates).










and the use of a flow-cell cuvette and a recorder for measuring the

absorbancy profiles.

Two samples of mungbean hypocotyls (D-1 and D-2) were pre-

pared exactly as in Experiment #3 for controls.

Four cultures of Achlya were grown on EM for 28 hr, harvested,

frozen and processed to obtain a ribosomal pellet as in Experiment #3,

with the single exception that two cultures, C-3 and C-4, were homog-

enized with 20 X DEP, while the other two were homogenized with 100 X

DEP.

The pellets were resuspended in 1.2 ml of SM. The absorbance

at 260 nm was estimated by using a 0.1 ml aliquot in 3.0 ml SM. From

this estimate a fraction containji. 2.0 A.U. was layered onto a 5 ml

10-34% nonlinear sucrose gradient I d centrifuged as in Experiment #3

for 2 or 3 hr, as indicated in the data.

The gradients were prepared in advance, using a simple two-

chambered mixing device designed and described by Noll [39] that can

be assembled from common laboratory glassware. The gradients are

formed by the controlled mixing of a concentrated sucrose solution with

a more dilute one. One of the advantages of making gradients this way

is-that actual test gradients could be prepared by placing a dye solu-

tion in one of the chambers and then measuring the absorbance of the

gradients formed. The increase in sucrose will be exactly propor-

tional to the increase of the absorbance as the solution with dye is

added to the solution without dye. Such gradients can be used to

determine the quality of the gradient preparation and fractionating

procedures.










After centrifugation the gradient profiles were formed, using

a Beckman fraction recovery system (Spinco Division, Beckman Instru-

ments) and a Gilford model 240 spectrophotometer equipped with a

model 203 flow cell, model 244 Autocuvette positioner and model 2453

recorder, at a setting of 260 nm and a light path of 5 mm. The gra-

dients were pumped through the flow cell by the bottom puncture, top

recovery method using a 50% w/v sucrose solution to displace the gra-

dient. The 50% sucrose was pumped into the bottom of the centrifuge

tube containing the gradient by gravity from a reservoir consisting of

a 50-ml hypodermic syringe barrel connected by a 50-cm plastic tube

to the bottom puncture needle of the Beckman recovery system.

The delivery from the fraction recovery system to the flow

cell was by means of a 20-gauge, 22-cm length of Teflon tubing. The

outlet from the flow cell was attached to a vertical glass tube cali-

brated in 0.1 ml divisions from 0.0 to 5.0 ml.

The rate of sample flow through the cell was adjusted by the

reservoir height. As the viscosity of the sample increased, the

height of the reservoir was changed by calibrated amounts to provide

a nearly constant flow of approximately 1.0 ml/3.4 min. Because the

flow rate was not exactly linear, the recorder chart was marked man-

ually as each 0.5 ml was delivered through the flow cell. These marks

provided the scale used on the gradient profiles.


Results and discussion (Experiment #4)

The profile of a test gradient prepared with the Noll gradient

maker is shown in Figure 9. Plotted with this is the viscosity in

centipoises of such a gradient at 0 C [59]. This shows the value of

















'(do) 4TS-00STA

0 C0! 0 0 0
o to o o o o
0 0 U> Hr N 0


44
0


4.1

0

of



m








4'
CO




0

0
o N









0
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SCO N C, rI H

asoaons %












such a nonlinear gradient. Although the gradient in sucrose concen-

tration is steep at first and shallow near the last, the viscosity is

nearly linear. Since centrifugal force increases directly with the

radius of the particle from the center of the rotor, the particle

velocity in this type of gradient is more nearly linear than in a

gradient that is linear with respect to concentration. This figure

also shows that the gradients being made were smooth and continuous,

and thus were ideal.

The profiles of the mungbeans, the Achlya with 20 X DEP and

the Achlya with 100 X DEP and centrifuged for 2 hr, are shown in

Figures 10, 11, and 12, respectively.

The mungbean control profile was excellent. The 80S monosomes

dominated the gradient, but excellent resolution of the components of

the polysome were obtained up through the pentasome with a hint of

elements even larger (Figure 10).

Achlya samples treated in the same way (Figure 11) yielded

nothing but a single well-resolved monosome peak. There was no evi-

dence of polysomes. When treated with 5 times the amount of DEP

_(Figure 12), the only difference was a slight degradation of the mono-

some, as evidenced by the small shoulder, presumably the 60S subunit,

preceding the 80S peak.

-When replicates of these cultures were centrifuged for 3 hr on

the gradients, one result was a movement of all of the elements further

down the gradient and a wider spread between them (Figures 13, 14,

and 15).




















4.

InC











5 E
S08




03a
A












S$4







4H


0o 1



\ Co


M
C
4)
C)
4)
2 -
0


eounqaosqV















Monosome Peak


1.0 1.5


Figure 11. Sucrose gradient profile of Achlya ribosomes from
cultures grova on enriched medium and homogenized with 20
DEP. Experiment #4, Sample D-3.





















Monosome Peak


,0



1.








0.,









0.0 d.5 1.0 1.5 2.0 4.5
ml



Figure 12. Sucrose gradient profile of Achlya ribosomes from
cultures grown on enriched medium and homogenized
with 100 X DEP. Experiment #4, Sample D-5.





















B0




N

4I







o 0
0*






C 0

4.'
0
0
0-






o m




C!







I
0
-H
q-4
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0








, g
* 0

















0
SI
T ". '
o vg








o1*


aouLtq.losqv


*a
0


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0























Monosome Peak

I


0.5







0.
'0.0 0.5 1.0 1.5 2.0 4.5
ml




Figure 14. Sucrose gradient profile of Achlya ribosomes from cultures
grown on enriched medium and homogenized with 20 X DEP.
Experiment #4, Sample D-4.




















Monosome Peak


0






1.0








0.5








0.0
0.0 0.5 1.0 1.5 2.0
ml






Figure 15. Sucrose gradient profile of Achlya ribosomes from cultures
grown on enriched medium and homogenized with 100 X DEP.
Experiment #4, Sample D-6.










A pronounced and peculiar additional change in the mungbean

sample was a change in the height of the monosome peak. There is

no certain explanation for this.

The ribosomal pellets in these replicates were stored in

tubes, on ice, while the first three were centrifuged for 2 hr on

gradients (the Spinco type SW39L Rotor used has a capacity of 3

samples). The change in the mungbenn sample is as though elements of

the polysome were being assembled from the monosome during this stor-

age. The relative amount of polysome is greater in the second sample.

A similar, though not as pronounced, effect occurred in Experiment #3

(Figure 6). Again the profile containing the greatest proportion of

polysome was from a sample stored for 2 hr on ice.


Experiment #5

Methods

two final procedures were tried in an attempt to obtain poly-

somes from Achlya. One of these was to vary the concentrations of Mg

and K+ in the SM used to prepare the homogenate, the gradients, and the

ribosomal suspension. The second was to use deoxycholate (DOC), which

has a mild detergent effect, in the homogenizing medium. DOC is used

to release ribosomes that are bound to endoplasmic reticulum in prepar-

ations of polysomes from certain animal tissues. In combination with

divalent cations it can cause aggregation of certain macromolecules by

formation of coordination compounds [60].

Six cultures of E87 e were grown on enriched medium for 24 hr,

harvested and frozen as before. All techniques for obtaining gradient

profiles were the same as in Experiment #4, except as follows:











Sample E-l

The grinding, cushioning, suspension and gradient media con-

tained no MgCl2.

Sample E-2

As in E-l except the grinding medium contained 0.4% DOC.

Sample E-3

The MgCI2 concentration of the grinding medium was 10 mM; all

other solutions were as in Experiment #4.

Sample E-4

As in E-3 except the grinding medium also contained 0.4% DOC.

Sample E-5

The MgC12 and KC1 concentrations in the grinding, cushioning,

suspension and gradient media were both 30 mM

Sample E-6

As in E-5 except the grinding solution also contained 0.4% DOC.


Results and Discussion (Experiment #5)

The profiles of samples E-l through E-6 are given in

Figures 16-24.

Sample E-l, which has no Mg++ and no DOC added, apparently was

unable to maintain 80S ribosomes. The only peak exhibited is in the

region of the gradient which should contain 40S and 60S subunits.

These two components are not resolved into separate peaks but that is

the best interpretation of this profile.

The profile of E-2 shows a definite peak in the region of an

80S monosome, plus a broadly flattened or double peak which may be

assumed to represent the 40S and 60S subunits.




















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++
Samples E-3 and E-4 (Figures 18-19) both have 10 mM Mg treat-

ment, show no subunit absorbance and have strong 80S peaks, followed

by a shoulder that may represent a disome in E-3 and an even more pro-

nounced shoulder in E-4, which was homogenized with DOC.

When samples were treated with 30 mM Mg plus 30 mM K as in

samples E-5 and E-6 (Figures 20 and 21), the number of peaks beyond

the monosome increases still further.

For several reasons, the additional peaks beyond the monosome

may be interpreted as the result of aggregation, rather than the isola-

tion of intact polysomes. First, the literature contains no accounts

of functional polysomes isolated in the presence of Mg+concentrations

higher than 20 mM. Second, when functional polysomes are isolated

(polysomes that can participate in in vitro protein synthesis) the rela-

tive amount of material in the polysome region is greater than in the

monosome. Finally, the stairstep shape of these Achlya profiles is

characteristic of aggregation [41].

Polysomes supposedly form wherever mRNA is being translated

into protein. For reasons that are not clear, the techniques that

are usually adequate for demonstrating the presence of polysomes have

been unsuccessful in the present study with Achlya.

Several possible explanations for the absence of polysomes may

be considered: (1) there may be exceptionally high levels of ribo-

nuclease in Achlya; (2) the ribonuclease present may be insensitive to

DEP; (3) polysomes in Achlya may be bound so tightly to membranes that

they are removed in the 20,000 g pellet; (4) the attachment of ribo-

somes to the mRNA may be especially fragile, so that it is not main-

tained during conditions of isolation; (5) perhaps only one ribosome







57









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61

associates with each messenger molecule during translation, in contrast

to the situation believed to be common to all other organisms.

All of these possibilities could be investigated, using

ribonuclease assays, pulse labeling techniques with RNA precursors,

and polyacrylamide gel electrophoresis. Such experiments were not

designed or attempted as a part of this research, but are potential

areas for future investigations.

(Supplementary data from experiments 1-5, indicating culture

media, weights, ribosomal yields, etc., are recorded in Appendix A.)


Screening Selected Inhibitors for Effect on Branching


Purpose

In order to make use of selected inhibitors in later experi-

ments, it was necessary to perform screening experiments to determine

the optimal inhibitor concentrations. The objective was to find levels

of inhibitors that would prevent branching but not be fungicidal.

It would be especially desirable to find levels of inhibitors that

-_could prevent sexual induction but allow normal vegetative growth.

A restriction placed on the screening experiments was that the

effect of a wide range of inhibitor concentrations needed to be tested

--while using as little inhibitor as possible because of the prohibitive

cost, especially of AD.


Experiment #1

Methods

Plugs of agar containing mycelium from E87 d and 734 $ cultures

were used to inoculate Petri dishes of complete agar medium. Two plugs,

one d and one 9, were placed in each dish separated by approximately










6.0 cm. After 72 hr incubation the d and ? hyphae were separated

by approximately 2 cm and no sexual differentiation was evident.

At this time 50 X aliquots of inhibitor solutions containing 10, 4,

2, 0.5, or 0.1 pg, respectively, of inhibitor were applied to the

agar surface between the two hyphal mats. Forty-eight hr after the

addition of the inhibitor the cultures were examined for the effect

on vegetative growth, appearance of the cytoplasm, and the degree of

mating reaction that had occurred.


Results and discussion (Experiment #1)

The results are given in Table 5.

This method of examining for inhibitor effects has the advan-

tage of requiring only very small amounts of inhibitor, but because

the exposure is quite long, it is not a test of the consequences of

a temporary alteration in control mechanisms in the organism. A further

disadvantage is that the inhibitors diffuse at unknown rates, so that

neither the final concentration nor the area affected by them is known.

Also, one can only guess to what extent the organism is accumulating

inhibitor during long-term exposure.

In spite of these limitations, this experiment did show that

the organism was quite sensitive to long exposures to either inhibitor

in the highest levels used. Vegetative growth and branching were

inhibited in a distinguishable area with both inhibitors.

The highest concentration of AD used iThibited both growth

and branching, while the two lower concentrations had no apparent

effect. The normal appearance of the cytoplasm in areas where branch-

ing was inhibited indicated that AD could possibly inhibit branching

without being fungicidal.










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Extensive cytoplasmic clumping occurred when FU was used,

especially in the male mycelium. Where branching and cytoplasmic

clumping occurred together, it is presumed that the former preceded

the latter. If the action of FU in Achlya inhibits synthesis of func-

tional rRNA, as has been shown in soybean roots [31], then the failure

to inhibit branching would indicate that continued synthesis of this

species of RNA is not essential during branch induction.


Experiment #2

Methods

Cultures were grown on N/2 medium and harvested as described

previously. Two-tenth -g aliquots of mycelium were placed in small

Petri dishes, each containing 4 ml of filtrate, and allowed to equili-

brate for 1 hr on a reciprocal shaker. Inhibitors were added 20 min

prior to addition of antheridiol. The antheridiol was added at a time

designated "zero"; 15 min later the reciprocal shaking was discontinued,

and the cultures were examined at 6, 12, 22, and 40 hr. After 40 hr

a very small portion of the mycelium was removed from the medium con-

taining the inhibitor, rinsed by dipping it briefly into fresh medium,

placed on complete agar medium, and observed after 24 hr for recovery

growth.


Results and discussion (Experiment #2)

The complete observations are given in Appendix B, but for the

sake of clarity a summary of the inhibitor effects is described here.

AD at concentrations of 25 pg/ml and 5 pg/ml completely inhib-

ited branching. The higher concentration also stopped vegetative

growth, caused extensive cytoplasmic clumping in 22 hr, and after










40 hr exposure the mycelium was dead. The 5 pg/ml treatment did not

cause extensive cytoplasmic clumping until 22 hr, and after 40 hr the

mycelium showed excellent recovery when removed from the inhibitor.

At 1 pg/ml only slight branching was observed after the hormone treat-

ment and the cytoplasm remained normal at 40 hr.

When FU was used at concentrations of 25 pg/ml and 5 pg/ml,

fair branch induction was observed by 6 hr. At 22 hr both concentra-

tions caused some cytoplasmic clumping and by 40 hr the clumping was

extensive; after 40 hr at 25 pg/ml the mycelium was dead, while recov-

ery growth from the 5 pg/ml treatment was "good" compared with a judg-

ment of "excellent" for untreated controls. FU at 1 pg/ml had little

effect when compared with controls.

AD had the desired effect of inhibiting branching in levels

which did not kill the organism. On the basis of this experiment,

I decided to use 15 pg/ml of AD in experiments on the inhibition of

RNA synthesis and induction of cellulase activity. Five pg/ml was

sufficient to inhibit branching in this experiment, but because a

shorter exposure (15 to 60 min) was to be used in the experiments

planned, a higher concentration seemed desirable.

..- .-._._-Ucaused cytoplasmic clumping and death in. levels which did

not prevent branching. It was therefore difficult to select an

optimal concentration of this inhibitor based on these experiments.

I decided arbitrarily to use the same concentration selected for AD

(15 pg/ml).










Effect of Inhibitors and Antheridiol
on RNA Synthesis


Purpose

The purpose of this experiment was to determine the rate of

RNA synthesis in Achlya and to examine the influence of antheridiol,

AD, and FU on this rate.


Methods

The rate of RNA synthesis was determined by measuring the rate
14
of incorporation of a radioactive precursor molecule (UL- 14C-uridine)

into RNA.

The cultures of E87 d were incubated on complete liquid medium

for 48 hr and then harvested. Portions of the mycelium weighing 0.25 g

or 0.50 g were resuspended in filtrate and allowed to equilibrate as

previously described.

When desired, 20-min pretreatments of 15 pg/ml of AD or

15 pg/ml of FU were given to these resuspended cultures. The start of
14
the incorporation was begun by the addition of UL- C-uridine (specific

activity 160 mCi/mM, International Chemical and Nuclear Corporation)

to the cultures. The amount of label added was varied from 0.9 pCi to

3.3 pCi in the four experiments performed. Antheridiol (15 BU/ml) was
14
added simultaneously with the 1C-uridine where indicated. The cultures

were incubated for the desired intervals, harvested in the usual manner,

reweighed and frozen.

Prior to harvesting, small portions of the mycelium were set

aside in filtrate without inhibitors for morphological observation










to determine whether the cultures were viable and whether branch induc-

tion occurred in those cultures receiving antheridiol.

For extraction of RNA, the frozen mycelium was processed

exactly according to the method (Figure 22) of Holdgate and Goodwin

[18] to obtain a powder free of lipid and insoluble in cold trichloro-

acetic acid. The dried powder was then hydrolyzed in 0.5 N KOH at

30 C for 12-15 hr to solubilize the RNA. Protein, DNA, cell wall

material, and KC104 were precipitated by acidifying the KOH digest to

pH 2 with 1 M HC10O and making the solution 2 mM in Mg+ by the

addition of MgCl2 [58]. Two volumes of cold 95% ETOII were added and

the precipitate removed by.centrifuging at 15,000 X g for 10 min.

The amount of RNA in the supernatant was determined by ultra-

violet spectrophotometry, using the method of Key and Shannon [27].
14
After determining the RNA content, the incorporation of 1C-uridine

was measured by scintillation counting.

The sample was placed in a scintillation vial and evaporated

to 0.4 ml. Then 6.0 ml of 100% ETOH and 10.0 ml of scintillation

cocktail [10] were added. The cocktail was composed of:

4.0 g PPO (2,5-diphenyloxazole)

0.1 g POPOP (p-Bis[2-(5-phenyloxazolyl)]-benzene

1.0 1 toluene (reagent grade).

Counting was done in a Packard Tri-Carb Liquid Scintillation Counter.

Aliquots of all supernatants in the procedure for extracting

the ribonucleic acid, of the filtrate to which the label was added, and

of the HC104 precipitate were saved in selected samples for liquid

scintillation counting as a means of measuring the distribution of the










Frozen Mycelium

Homogenized in
85% methanol
12,000 X g for 20 min.


Resi


due

Rinse (3X) in 5 ml
10% trichloroacetic acid
at 2 C
12,000 X g for 20 min
after each rinse.


Residue

10% ethanol saturated with
sodium acetate at 2 C (2X);
95% ethanol at 2 C (1X);
ethanol:chloroform (3:1)(2X);
ethanol:ether (1:1)(2X);
ether (lx);
12,000 X g for 20 min
after each rinse.


I
Supernatants

Discard


Supernatants
Disc
Discard


Hydrolyze in 0.5 N KOH
at 30 C 12-15 hr;
chill to 0 C; acidify
with HC104 to pH 2
+ 2 mM Mg+;
2 volumes 95% ethanol.


DNA, protein, cell wall,
and KC10O in
Residue


Supernatant

Solubilized RNA for
UV analysis and
scintillation counting


Figure 22. Flow sheet for RNA extraction.


Residue

Air Dry


I
Supernatant

Discard


3I


!


I


I










14C-uridine between the media, the mycelia and the ribonucleic acid.

All scintillation counting was corrected for background.

From the raw data (presented in Appendices C and D) the

following values were calculated for each sample:

1. gg RNA/g fresh weight.
14
2. Rate of 14C incorporation in cpm/pg RNA/min of exposure

to 14C-uridine.

3. Rate of incorporation as the percentage of the rate of a

corresponding vegetative control.
14
4. The percentage of the total 14C in the medium at the end

of the experiment.
14
5. The percentage of the total 14C incorporated into the

RNA extract.

The purity of the RNA extracts was tested by measuring the

absorbance of the samples over the range 230-310 nm.


Results and Discussion

The cultures, pretreatments with inhibitors, treatment with

antheridiol, and the calculated values are listed in Table 6.

Hormone-treated cultures showed an average rate of RNA syn-

thesis 14% greater than the rate observed in vegetative controls.

This increased rate was quite variable, ranging from no increase in

-one case (cf., samples 10 and 11), to a 37% increase in another

(cf., samples 3 and 4). Undoubtedly experimental error was the cause

of some of this variation.

The most striking feature of these experiments was the inhib-

ition of the rate of incorporation of label when AD was used. In five













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- 4




~a
N S



a a)
0g '.
(00



B -P



'.4

a)l-4
(00
(0H 0
a) a) u
I. ;









t ii I
0 Irl

h
-0 1+4
04 6-
00(0^
o (>>

0.04)f










of the six treatments with this inhibitor, the incorporation rate was

only 31 to 38% of vegetative controls, and in the sixth, 48% of vege-

tative controls. Treatment of AD-inhibited cultures with anther-

idiol did not increase the rate of incorporation above the rate

observed in an AD-inhibited vegetative control (cf., samples 5, 6,

and 7).

The effect of FU on incorporation was less clear than the

effect of AD. It was only used in three samples (2, 8, and 9).

In one case of a hormone-treated culture there was inhibition to 78%

of controls; in another there was no effect. The one trial with a

vegetative culture resulted in a slight inhibition to 92% of a con-

trol (cf., 9 and 11).

The observations of the mycelia set aside in inhibitor-free

filtrate at the end of this incorporation experiment may be summarized

as follows:
Vegetative Branching
Pretreatment Treatment Appearance Response

None --None Normal None

None HA Normal Good in 6 hr

FU None Normal None

FU HA Normal Fair to good
in 6 hr

AD None Normal None

AD HA Normal None in 6 hr
Fair in 22 hr


The results of the screening experiment had shown that con-

tinued exposure to AD suppresses the branching response. This experi-

ment shows that it also inhibits the rate of RNA synthesis by more










than 50%, yet the mycelium can recover and show a delayed branching

response when removed from the AD.

The absorbance profiles of the RNA extracts were examined as

a means of determining that the extract did contain material with

an absorbance profile expected for hydrolyzed RNA. A typical profile

is given in Figure 23. All profiles were virtually replicates of the

one illustrated. These curves were comparable to those obtained by

Holdgate and Goodwin, using the same techniques [18].



Effect'of'Actinomycin-D on Cellulase Activity


Purpose

The previous experiments had shown that AD could be used to

inhibit both the branching response of Achlya to antheridiol and the

synthesis of RNA. Mullins [35] had described an increase in cellulase

activity following treatment with antheridiol and proposed that this

increase is a prerequisite to branching. The purpose of this experi-

ment was to determine whether inhibition of transcription of AD affects

hormone-induced cellulase activity.


Methods

E87 c was grown 48 hr on complete liquid medium and harvested

in the usual manner. Fourteen 1.0 g portions of the mycelium were

resuspended in 20 ml aliquots of filtrate and allowed to equilibrate

for 1 hr.

Twenty-minute pretreatments with 15 pg/ml of AD were adminis-

tered as needed. Selected cultures were treated with 10 BU/ml of

antheridiol (0.2 ml of the stock solution) or 0.2% casein hydrolysate

























0.8.





0.6"





S0.4"
0
o.


0.2




0.0


230 240 260 290 310
Wavelength (nm)



Figure 23. Typical absorbance profile of RNA extract.










(0.8 ml of a 5% stock solution) at the beginning of the experiment

(T=0). Treatments were terminated by harvesting and freezing the

mycelium at the desired intervals.

Cellulase was extracted and assayed from both the mycelium

and the filtrate. The extraction of the cellulase was according to

the laboratory protocol developed by Mullins [36]. The medium was

extracted for 5 min by 5 volumes 95% ethanol with 0.25 g NaCl added

with stirring. The protein pellet was formed by centrifuging at

17,000 X g for 20 min in a Sorvall GSA Rotor at 0 C and decanting the

ethanol supernatant. This pellet was resuspended in 2 ml H20 and

centrifuged to remove denatured protein. The supernatant volume was

adjusted to 2 ml so that an assay of cellulase activity in 1 ml of this

supernatant corresponded to 0.5 g of original fresh weight of mycelium.

The frozen mycelium was extracted by first grinding it to a

powder with a mortar and pestle precooled on dry ice and then homog-

enizing it thoroughly in 2 ml of 5% NaC1. After decanting into centri-

fuge tubes the mortar and pestle were washed with an additional 2 ml

of 5% NaC1 which was then combined with the previous homogenate and

centrifuged at 17,000 X g for 10 min. This supernatant containing the

cellulase was then extracted in the same manner as was used for the

medium.

The cellulase activity was assayed viscometrically in a modifi-

cation of the method of Bell et al. [9]. One ml of extract was mixed

with 5.0 ml of substrate in series 300 Ostwald-Cannon-Fenske viscometer

tubes at 30 C in a water bath. The substrate used was 1.2% carboxy-

methyl cellulose (a soluble cellulose derivative; cellulose Gum Type

7MP, Hercules Powder Company, Wilmington, Delaware) in 0.018 M










citrate-NaOHI buffer, pH 5 with 0.05% merthiolate added as a preserva-

tive. Immediately after mixing a flow time was measured. This time

was recorded and compared with a flow time after 1 hr incubation.

Cellulase activity was expressed as cellulase units. One unit was

defined as the amount of cellulase in 1 ml of extract which causes a

10% decrease in flow time of the enzyme-substrate mixture after incu-

bation for 1 hr at 30 C.


Results and Discussion

The cellulase activities in the medium from controls, casein

hydrolysate treated, antheridiol-treated, and antheridiol-treated

following pretreatment with AD are shown in Figure 24. The cellulase

activities from the mycelia of the same cultures are shown in Figure 25.

All cellulase activities are expressed in terms of activity per g fresh

weight of mycelium, and are averages of results from two experiments.

One hr following treatment with antheridiol, a dramatic

increase in cellulase activity was apparent both in the mycelium and

the medium unless the culture was pretreated with AD. Cultures treated

with this inhibitor maintained essentially the same level of activ-

ity as was found in vegetative controls.

The data in this experiment strongly suggest that transcription

is necessary for the induction of cellulase activity and therefore also

necessary for expression of sexual morphogenesis in Achly.

Whether the transcription is of mRNA coding for cellulase or

some other type of RNA is not shown by these experiments.













Antheridiol

....... AD + Antheridiol
5-
Vegetative Control








3 /




/ *....-. -
-- -- CH






1-

0 15 30 45 60
Time (min)


Figure 24. Effect of AD and antheridiol on cellulase activity
released in the medium by E87 d .














---- Antheridiol

...... AD + Antheridiol

Vegetative Control





m 3,








+ 7.






0.








0 15 30 45 60
Time (min)






Figure 25. Effect of AD and antheridiol on cellulase activity
in the mycelium of E87 d.
0
0-




























-in the mycelium of E87 dS.

















SUMMARY AND CONCLUSIONS


The data suggest that only one site for induction of branching

exists in Achlya. Using two kinds of inducers, vegetative and sexual,

the attempt to determine whether more than one site exists was incon-

clusive; a second response did not occur to treatment with a second

kind of inducer. Failure to prove the existence of two sites, however,

does not prove that there is only one.

Thomas [65] had shown that different kinds of cellulase are

produced in response to different types of induction. This was the

basis for supposing that there may be two sites for control of cellu-

lase induction. The different cellulases produced may be related to

substrate availability. Achlya may have an array of endocellulases,

produced in different proportions; variations of the kinds and number

of cellulases produced under different conditions may be a mechanism

for adapting the rate of hyphal tip growth and the extent of the branch-

ing response to variations in growth conditions.

Repeated attempts to demonstrate the existence of polysomes in

Achlya were unsuccessful. If these had been successful, in vitro cellu-

lase synthesis would have been tried, using the methods of Davies and

Maclachlan [13]. Such a system would presumably have provided an

ideal means for determining whether antheridiol works at the transla-

tional or transcriptional level. There is a possibility that an in

vitro system for protein synthesis could be formed, using monosomcs;

78










however, this would require that Achlya be unique in its performance

of a fundamental biological process. This is not likely, but will be

examined later.

The distribution of ribosomes on sucrose gradients shows that

Achlya's ribosomes sediment at a rate reasonably close to that shown

by higher plants and that polysomes, if they do exist, are unstable

under the conditions of isolation used.

Studies on the effects of inhibitors provided the clearest and

most definite results of this work. FU did not provide any clear

evidence as to the level or mechanism of hormonal control. Male and

female strains showed differences in sensitivity to this inhibitor.

Cytoplasmic clumping was revealed to be more extensive in the male

E87 d strain than in the female 734 ? strain. Barksdale [5] reported

that male strains take up antheridiol much more rapidly than females;

the cytoplasmic clumping might therefore be related to differences in

permeability or transport between males and females and not be an effect

of different sensitivity of the mechanisms of transcription and trans-

lation.

The females were prevented from forming oogonia at very low

levels of FU concentration. When oogonia did form., they failed to

develop oospheres. This could be a specific effect of inhibition of

DNA replication preceding gametangial meiosis [56].

Treatment with FU did not inhibit branching in Achlya even in

concentrations fatal to the organism, and in the few incorporation

experiments where it was used it had a quite varied effect upon rate of

transcription. If the assumption is made that FU selectively prevents












synthesis of normal rRNA in Achlya, as it does in some higher plants

[31], then it would appear that branching is not dependent upon new

synthesis of this type of RNA.

The effects of AD suggest that transcription is necessary for

induction of cellulase activity and branching in this way:

1. AD simultaneously inhibited the induction by antheridiol

of cellulase activity and branching and reduced the rate

of transcription by more than one-half.

2. AD-pretreated, hormone-induced cultures maintained the

same level of cellulase observed in untreated vegetative

controls. This is interpreted as evidence that the inhib-

ition of induced cellulase activity was not by virtue of

a general toxic action on enzyme activities, but by pre-

vention of increases in the endogenous levels.

3. AD-pretreated, hormone-induced cultures showed a delayed

branching response if removed from the inhibitor. This

showed that the system for branch induction was still

functional and that the effect of AD was not prevention of

uptake of antheridiol.
14
4. In C-uridine incorporation experiments the percentage of

label remaining in the medium was similar in AD-treated

cultures to that of non-treated cultures. This is further

evidence that endogenous metabolic functions, such as

uptake of metabolites, were not impaired.











The small increase in the rate of transcription in cultures

treated with antheridiol might be considered as weakening the evi-

dence for transcription as a site of control, but this is not the

case. Most of the RNA in cells is ribosomal. A small increase in

mRNA coding for a specific protein (i.e., a cellulase or a polymerase)

might be undetectable as a percentage increase in total RNA, yet have

a profound effect upon the organism.

Thomas [63] has shown that protein synthesis is required for

response to antheridiol. Because increases in rate of amino acid

incorporation were detected after an induction period of only 1 min,

Reiskind [54] suggested that antheridiol acts directly at the level of

translation, perhaps by some action on preformed mRNA.

Evidence presented here indicates that transcription is neces-

sary for increasing cellulase activities. The two concepts do not

conflict with each other. As Tata [62] and Grant [17] pointed out,

there may be several sites of primary hormone action. Thus, while some

hormone responses may prove to be mediated by a rather direct effect

of the hormone at the level of transcription, other responses might

result from action of the hormone at the level of translation.

This study, when considered together with the work of Mullins

[35] and Thomas and Mullins [65], provides evidence supporting the

following hypotheses of the mechanism of hyphal morphogenesis in Achlya:

(1) increased cellulase activity is a prerequisite for branching in

response to vegetative or sexual induction, (2) both vegetative and

sexual induction are controlled by one site, and (3) hormonal control

of cellulase activity is exercised at the level of transcription of RNA.






82



Further proof that the level of control is transcriptional

would be provided if an mRNA fraction could be isolated from hormone-

treated cultures that would enhance cellulase synthesis in an in vitro

system of protein synthesis isolated from vegetative cultures.





































APPENDICES











APPENDIX A

DATA FROM CULTURES USED IN ISOLATION OF RIBOSOMES


Growth No. of Incubation Fresh
Sample Organism Medium Flasks Time (hr) wt (g)


A-1 Achlya C
A-2 "


complete 1.5
S 1.5

N/2 3
3

3
300
seeds


EM 1
'" 1
1

1

300
seeds


EM 1

1
1

1

1
1

1

1
1
1


A.U. of
Ribosomal
Pellet

5.32

5.92

24.3

23.7

20.6

31.7

32.8

39.3

29.4
42.2

28.2

17.9

23.0

24.6

17.5

20.9



28.8

18.0
43.9

23.2
39.6
18.2


A.U./g
Fresh wt


3.54

3.95

3.80

4.08

3.27

13.8
14.3

7.42
5.44
8.80

5.76

7.78

8.20

5.93

4.16
5.36



9.93

6.00
13.3

7.48
17.20
7.28


Mungbean


Achlya






Mungbean


Achlya






hlya


B-l

B-2

B-3

C-1

C-2

C-3
C-4

C-5
C-6

D-1

D-2

D-3
D-4
D-5
D-6

E-l
E-2
E-3

E-4
E-5
E-6





















Time
(hr)

25 pg/ml FU+I


0 NC, NVA

2 NB, NC, NVA


6 FB, NC, NVA
some growth

12 SB, NC, NVA


22 SB, some CC,
some slight
veg. growth


40 NB, CC


APPENDIX B

EFFECT OF INHIBITORS ON BRANCHING


Treatment


IA 5 pg/ml FU+HA 1 pg/ml FU+HA


NB, NC, NVA NB, NC, NVA

NB, NC, NVA NB, NC, NVA


FB, NC, NVA FB, NC
good growth

SB, NC FB, NC


SB, some CC, FB-GB, NC,
good veg. good veg.
growth growth


SB, CC FB, NC


HA
only


NVA

SB


FB-GB
NC

GN, NC


GB, NC


Veg.
Control


NVA

NB, NC,
NVA

NB,NC,
NVA

NB, NC,
NVA

NB, NC,
NVA


Recovery Growth on


None Good


Guide to Abbreviations:
1. Branching: GB = Good
FB = Fair
SB = Slight
NB = No branching

2. Cytoplasm: NC = Normal
CC = Clumped


Fresh Complete Medium


Poor to Excel- Excel-
Good lent lent



3. Apparent Vegetative Growth:
NVA = Normal vegetative
appearance, or
descriptive adjective
used.


GB, NC NB, NC,
NVA NVA


~1~1_

















APPENDIX B (Continued)


Treatment


l ADtHA 5 pg/ml AD+HA 1 pg/ml ADiHA


NB, NC, NVA NB, NC, NVA

NB, NC, NVA NB, NC, NVA


veg. NB, NC, very SB, NC, NVA,
wth slight veg. normal growth
growth

NB, some CC FB, NC

NB, CC SB, NC, slow
veg. growth

no NB, CC, SB, NC, NVA
growth sparse veg.
growth


Recovery Growth on Fresh Complete Medium


Excellent Excellent


Time
(hr)


-25 pg/m:


NC, NVA

NC, NVA


NC, no
gro,


NB, CC

NB, CC


NB, CC,


Veg. Control


NVA

NB, NC, NVA


NB, NC, NVA


NC, NVA

NC, NVA


NC, NVA


Excellent


None












APPENDIX C
14
SCINTILLATION COUNTING OF 1C-URIDINE


Experiment #1: 3.3 pCi/sample. Counting Efficiency 60%
Data corrected for background of 30 CPM

Sample 1 2 3 4


Pretreatment AD FU -

Treatment HA HA HA Veg

Time (min) 30 30 30 30

Fresh wt (g) 0.25 0.24 0.20

RNA (pg) 102 75 113 107

CPM X 103 22.8 56.7 95.4 58.2
in RNA

CPM X 103 in 441 320 295 295
Medium


Experiment #2: 1.07 pCi/sample. Counting Efficiency 60%
Data corrected for background of 100 CPM

Sample 5 6 7 8 9 10 11


Pretreatment AD AD AD FU FU -

Treatment HA HA Veg HA Veg HA Veg

Time (min) 30 30 30 30 30 30 30

Fresh wt (g) 0.34 0.29 0.34 0.26 0.32 0.30 0.33

RNA (pg) 194 154 168 142 149 183 200

CPM X 103 7.56 5.17 7.05 15.8 15.4 20.4 22.4
in RNA

CPM X 103 in 110 164 98.5 196 187 177 123
Medium












APPENDIX C (Continued)



Experiment #3: 1.27 pCi/sample. Counting Efficiency 62%
Data corrected for background of 40 CPM

Sample 12 13 14 15


Pretreatment AD AD -

Treatment HA HA HA Veg

Time (min) 30 60 60 60

Fresh wt (g) 0.33 0.40 0.37 0.33

RNA (pg) 202 226 143 215

CPM X 103 7.40 16.6 36.2 51.2
in RNA

CPM X 103 in 187 177 196 164
Medium


Experiment #4: 0.9 pCi/sample. Counting Efficiency 58%
Data corrected for background of 50 CPM

Sample 16 17 18 19 20 21 22 23

Pretreatment -

Treatment HA Veg HA Veg HA Veg HA Veg

Time (min) 15 15 15 15 30 30 60 60

Fresh wt (g) 0.31 0.29 0.29 0.29 0.31 0.32 0.32 0.30

RNA (pg) 178 182 183 175 184 192 194 198

CPM X 103 16.7 16.8 17.4 13.5 34.1 33.0 58.4 50.4
in RNA

CPM X 103 in 178 160 36.3 57.8
Medium












APPENDIX D

EXTRACTION OF RNA CPM OF 14C IN EXTRACTS
FROM FOUR REPRESENTATIVE SAMPLES
1 2 -7 8

HA + 1C U Veg + 14 U HA + 1C U Veg + 1C U
15 min 15 min 60 min 60 min
CPM Added 1,135,000 1,135,000 1,135,000 1,135,000


M ETOH
1st TCA
2nd TCA
3rd TCA
1st 90% ETOH:AC
2nd 90% ETOH: AC
95% ETOH
1st ETOH: CHLOROFORM
2nd ETOH: CHLOROFORM
1st ETOH:ETHER
2nd ETOH: ETHER
ETHER

Total in Extracts
% in Extracts

Residue
% in Residue


RNA
% in RNA


190,108
169,253
24,765
5,087
1,153
1,696
295
256
112
232
77
86

393,120
34.64

9,015
0.79


169,172
136,580
17,435
2,701
911
646
610
91
390
339
.121
406

329,402
29.02

8,910
0.785


16,659 16,757
1.467 1.476


410,880
132,750
19,157
3,467
3,969
3,466
849
428
142
237
356
189

575.890
50.74


191,568
202,950
26,393
3,765
3,493
3,371
785
284
226
235
134
151

433,355
38.18


31,959 25,488
2.815 2.245

58,362 50,430
5.143 4.443


Total in Mycelium
Extracts + Residue
+ RNA
% in Mycelium

Remaining in Media
% in Media

% Unaccounted


418,794
3... 6.90


355,069
31.28


177,633 160,457
15.65 14.137

47.45 54.58


666,231
58.70

36,345
3.202

38.10


509,273
.44.87

51,786
5.091

50.04
















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