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Regulatory Mechanisms in Cellulase Synthesis
During IIyphal Morphogenesis
in the Water Mold Achlya
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
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
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
--A.--DATA-FROM-CULTURES- USED IN ISOLATION OF. RIBOSOMES 84
B. EFFECT OF INHIBITORS ON BRANCHING . .. 85
C. SCINTILLATION COUNTING OF 1C-URIDINE ... 87
D. EXTRACTION OF RNA CPM OF 1C IN EXTRACTS FROM
FOUR REPRESENTATIVE SAMPLES . ... 89
BIBLIOGRAPHY .......... ............. ........ 90
BIOGRAPHICAL SKETCH ...................... 95
LIST OF TABLES
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
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)
'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
LIST OF ABBREVIATIONS
A.U. Absorbance Unit
BU Barksdale Unit
CM Cushioning Medium
DEP Diethyl Pyrocarbonate
EM Enriched Medium
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
Bernard Evan Kane, Jr.
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
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.
Achlya ambisexualis Raper is a fungus whose sexual morphogenesis
is controlled by a sequence of hormones . 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 , in protein synthesis
, and in the activity of specific enzymes .
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.
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 . The
respiratory metabolism has been studied  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-
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 .
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.
Various strains may be arranged in a linear series from pure males
through self-fertile intersexes all the way to pure females .
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
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  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
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  or of
Barksdale . 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  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  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
The chemical structure of antheridiol has been described 
and two possible isomers have been synthesized in the laboratory..
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 .
-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
. 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 . Mullins  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 , 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  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 , 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  to eucaryotic organisms.
Considering the Karlson hypothesis, Grant  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
Tata, in another review of hormonal control , 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  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  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 . 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  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 . 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.
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
of gravitational field, or 1 x 10- cm/sec/dyne/g .
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 .
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 .
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 . 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  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  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 . These reports were on ascomycetes;
none was found from phycomycetes.
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-
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 . 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 .
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 . The rate of protein synthesis
slowly decays and this decay parallels a decay in functional polysomes .
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.
This drug was chosen primarily for the present study because of
the report by Lin and Key  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
. 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.
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:
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 , 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
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].
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.
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
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
Response of E87 d to Metabolic and Sexual Induction
Thomas and Mullins  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  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.
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
0 0 i 0 04
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btb p 0 4- S 00
004, 00 4' 0r 0B
H 0- i M HP 0 a
k, o 0 k *-H H r4
J-o 0 'B D- a
H1 0 H
4) 10 = a at
0Q > *H > -H
0 0 .00
.00 CO 1- 0-H Ot I
00 I ;, 0 03
q 4) H41 0 + -HO 0 0 0
0 D 0 m P M + B0
a) 0 k 0 M 10 ";) I) o 0
>0. 000 H 0) a
,~l .S-S Sg ^ rI^ g^ a
Cs 4) 0l 9 C
20 rD4- 0. HP POP m So aS *M
0.0 1e. 4-' H 0. 00 >a0
p0 w-D 0 1-O '0
9 0 0) 40,0' 00
R0 3 H 50 0 0 b 94 D U
4- P 0 H o3
0 0'0 4'). 0i 00~ 01 W ~ '0
S0 1-fl P 0 0 00 4 -'.0 4 O P 0
; 0 S 0 or=o 0 41 0 4 W O 0 0 .:
1 -H 0 0 0 aHs C
4M 0 U Lo 0 OP
P 00 H 04 O k 400 0 4 k
0 ~ O-H 04 >H > -H00 P0 O 0
0 0 0
0 4.- 04-' 0
4 0 H OH d
oc, o g E ia
a m o 0 0 -H
Cd w 10u-
a) H z
k 1C '0 P0
N1 00 0
0 0 F, 4) 0
4-' r4 v r
P0 0 0 0; -
>. .0 0 10H 01-,> : 0 *
1-,H 0 dk ~ O k
I 0 0- *. 0
N, 0, >.H 00-D
r I da th
C CD 0 4~"
0 0 0
& 0 Hl
0 3 : OH j 4 M -
4O 0 H C CO 0 0 1 0-W 0 0 H 1 C
0, 4s v
4- mum ~
U)04 co v LO to t-0 0 0 H
which delimits the antheridium from the vegetative mycelium while the
vegetative branch lacks such a septum. Barksdale  has described
sexual branches as being more slender and spiraled than vegetative
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
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  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
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
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
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 .
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
21 28 35
.._Figure--.. Sucrose gradient profile of Achlya ribosomes from vegetative
cultures homogenized with DEP. Experiment #1, Sample A-i.
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+
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
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
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
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
o r l4
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
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
Two sets (C-1 and C-2) of mungbean seeds were germinated for
20 hr in rolls of moist paper . 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
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 , 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.
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
S 1 (a)
------ Sample C-2
I I I I I
0 10 20 30 40 50
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].
-- Sample C-3
---- Sample C-4
0 10 20 30 40 50
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).
-- Sample C-5
---- Sample C-6
0 .'0 -, -- -- ---- ,- ,
0 10 20 30 40 50
Figure 8. Sucrose gradient profiles of Achlya ribo-
somes from vegetative cultures homogenized
with DEP. Experiment #3, Samples C-5 and
and the use of a flow-cell cuvette and a recorder for measuring the
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
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  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
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 . This shows the value of
0 C0! 0 0 0
o to o o o o
0 0 U> Hr N 0
SCO N C, rI H
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,
Figure 11. Sucrose gradient profile of Achlya ribosomes from
cultures grova on enriched medium and homogenized with 20
DEP. Experiment #4, Sample D-3.
0.0 d.5 1.0 1.5 2.0 4.5
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.
C a v
T ". '
'0.0 0.5 1.0 1.5 2.0 4.5
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.
0.0 0.5 1.0 1.5 2.0
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.
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 .
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:
The grinding, cushioning, suspension and gradient media con-
tained no MgCl2.
As in E-l except the grinding medium contained 0.4% DOC.
The MgCI2 concentration of the grinding medium was 10 mM; all
other solutions were as in Experiment #4.
As in E-3 except the grinding medium also contained 0.4% DOC.
The MgC12 and KC1 concentrations in the grinding, cushioning,
suspension and gradient media were both 30 mM
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
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.
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 .
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
44 M 1
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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
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.
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 , then the failure
to inhibit branching would indicate that continued synthesis of this
species of RNA is not essential during branch induction.
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
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
Effect of Inhibitors and Antheridiol
on RNA Synthesis
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.
The rate of RNA synthesis was determined by measuring the rate
of incorporation of a radioactive precursor molecule (UL- 14C-uridine)
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
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
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
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
 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 . 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 .
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  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
12,000 X g for 20 min.
Rinse (3X) in 5 ml
10% trichloroacetic acid
at 2 C
12,000 X g for 20 min
after each rinse.
10% ethanol saturated with
sodium acetate at 2 C (2X);
95% ethanol at 2 C (1X);
12,000 X g for 20 min
after each rinse.
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
Solubilized RNA for
UV analysis and
Figure 22. Flow sheet for RNA extraction.
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.
2. Rate of 14C incorporation in cpm/pg RNA/min of exposure
3. Rate of incorporation as the percentage of the rate of a
corresponding vegetative control.
4. The percentage of the total 14C in the medium at the end
of the experiment.
5. The percentage of the total 14C incorporated into the
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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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,
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
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 .
Effect'of'Actinomycin-D on Cellulase Activity
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  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.
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
230 240 260 290 310
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 . 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
The cellulase activity was assayed viscometrically in a modifi-
cation of the method of Bell et al. . 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.
....... AD + Antheridiol
/ *....-. -
-- -- CH
0 15 30 45 60
Figure 24. Effect of AD and antheridiol on cellulase activity
released in the medium by E87 d .
...... AD + Antheridiol
0 15 30 45 60
Figure 25. Effect of AD and antheridiol on cellulase activity
in the mycelium of E87 d.
-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  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 . 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;
however, this would require that Achlya be unique in its performance
of a fundamental biological process. This is not likely, but will be
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  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-
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 .
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
, 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.
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  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  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  and Grant  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
 and Thomas and Mullins , 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.
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.
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
25 pg/ml FU+I
0 NC, NVA
2 NB, NC, NVA
6 FB, NC, NVA
12 SB, NC, NVA
22 SB, some CC,
40 NB, CC
EFFECT OF INHIBITORS ON BRANCHING
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
SB, NC FB, NC
SB, some CC, FB-GB, NC,
good veg. good veg.
SB, CC FB, NC
Recovery Growth on
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
GB, NC NB, NC,
APPENDIX B (Continued)
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
NB, some CC FB, NC
NB, CC SB, NC, slow
no NB, CC, SB, NC, NVA
growth sparse veg.
Recovery Growth on Fresh Complete Medium
NB, NC, NVA
NB, NC, NVA
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
CPM X 103 in 441 320 295 295
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
CPM X 103 in 110 164 98.5 196 187 177 123
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
CPM X 103 in 187 177 196 164
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
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
CPM X 103 in 178 160 36.3 57.8
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
1st 90% ETOH:AC
2nd 90% ETOH: AC
1st ETOH: CHLOROFORM
2nd ETOH: CHLOROFORM
2nd ETOH: ETHER
Total in Extracts
% in Extracts
% in Residue
% in RNA
Total in Mycelium
Extracts + Residue
% in Mycelium
Remaining in Media
% in Media
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