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1986 Characterization of Messenger Ribonucleoprotein Particles in Dormant Sporangiospores of the Fungus Mucor Racemosus (Messenger-Rna). Charles Patrick Chapman Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Chapman, Charles Patrick, "Characterization of Messenger Ribonucleoprotein Particles in Dormant Sporangiospores of the Fungus Mucor Racemosus (Messenger-Rna)." (1986). LSU Historical Dissertations and Theses. 4290. https://digitalcommons.lsu.edu/gradschool_disstheses/4290
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Chapman, Charles Patrick
CHARACTERIZATION OF MESSENGER-RIBONUCLEOPROTEIN PARTICLES IN DORMANT SPORANGIOSPORES OF THE FUNGUS MUCOR RACEMOSUS
The Louisiana State University and Agricultural and Mechanical Ph.D. Col. 1986
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University Microfilms International CHARACTERIZATION OF MESSENGER RIBONUCLEOPROTEIN PARTICLES IN DORMANT SPORANGIOSPORES OF THE FUNGUS MUCOR RACEMOSUS
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Microbiology
by
Charles Patrick Chapman B.S., Louisiana State University, 1981 M.S., Louisiana State University, 1982 December 1986 DEDICATION
To my most loyal and d u tifu l wife, G loria.
11 ACKNOWLEDGEMENTS
First, I would like to thank my father, William F. Chapman, for
bringing me to this country, adopting me as his son, and giving me an
opportunity to succeed. I especially would like to thank him for
instilling in me the "can do” attitude. I also would like to thank
him for giving me much moral support and encouragement in completing my work.
My sincere thanks also go to Phyllis and Jeff for their warm and kind-hearted support for my work and for being truly loyal family members.
I would like to express my heartfelt thanks to Dr. Orlowski for
giving me much help in completing my work. Without his assistance
this project would not have been completed.
At the professional lev el, Dr. Orlowski is the apotheosis of what a major professor ought to be. He was always available and extremely generous with his time in solving my research problems. Dr. Orlowski showed me how to be organized, methodical, patient, frugal, neat and
tenacious in conducting research.
At the personal level, he has shown me over the years much k in d n ess, g e n e ro sity , and thoughtfulness. I sought out and received much valuable personal advice from him. I spent many hours with him sharing his experiences. Dr. Orlowski has been a professor, a counselor, and a friend to me. I will deeply miss his friendship and
company which he provided.
Finally, X am very grateful to Dr. Orlowski for the help he gave to me in the writing of this dissertation. Without his advice,
i i i direction, and editing the task could not have been completed. I am
especially grateful that he allowed me to use his not-yet-published
review on Mucor dimorphism as well as unpublished research papers as major source materials in the literature review.
I would like to thank Dr. Braymer for showing his personal
interest in my research progress: oftentimes he appeared unexpectedly
and quizzed me, all for my benefit. His sincere, compassionate, and
thoughtful guidance helped me enormously. I also thank him for his day-to-day light-hearted kidding, which helped make my stay at LSU a very pleasant time.
I want to thank Dr. Srinivasan for sharing his unique philosophies of life with me. I shall ever appreciate his unswerving realism. I also wish to express ray appreciation to him for his service on my research committee. His advice on scientific matters was always impeccable.
I wish to thank Dr. Socolofsky for coming through with summer support money when I needed it greatly. His services as a chairman will be sorely missed by the Microbiology Department. I also wish to thank him for serving on my dissertation committee.
I would like to thank Dr. Grodner and Dr. Hembry for serving on my dissertation committee. I appreciate their performing this vital function in the completion of my degree.
X wish to thank the rest of the Microbiology faculty and my fellow graduate students for providing me an amicable millieu in which to pursue my goals. I appreciate Mary Schramke, Geun-Eog Ji, and
Young-Hwan Ko for their thoughfulness and friendship. Especial thanks are given to John Linz for his help and concern and for blazing the
iv research trail In front of me.
Finally, I would like to thank my wife, Gloria, for being so
patient and loyal for all these years. She sacrificed much without wishing anything in return. In addition to holding down a full time
job, she did the laundry, cooking, and numerous other household
chores. I f i t had not been for her sac rifice s, my work would not have
been completed. I also want to thank her for giving me endless
encouragement and moral support when I needed them. Whatever this degree may bring, Gloria deserves a lot of credit. I hope to repay her for years of sacrifice, loyalty and devotion shown me.
v TABLE OF CONTENTS
Page Dedication ...... i i
Acknowledgements ...... i l l
Table of Contents ...... * ...... vi
List of Tables...... v iii
L ist of F igures...... ix
A bstract...... x ii
Literature Review...... 1
Mucor; Background and P ractical Importance ...... 1 Fungal Dimorphism...... 3 The General Phenomenon...... 3 Mucor Dimorphism...... 4 Regulation of Mucor Dimorphism...... 14 Macromolecular Synthesis and Mucor Dimorphism ...... 17 Mucor Dimorphic Development from Sporangiospores ...... 21 Messenger Ribonucleoprotein Particles ...... 25 Perspective...... 25 General Properties of Ribonucleoprotein Particles ...... 26 Physicochemical Properties of RNP's from Animal C ells ...... 29 Physicochemical Properties of RNP's from Fungus C e lls...... 43
M aterials and Methods ...... 47
Organism and C ultiv atio n ...... 47 Sporangiospore Production ...... 47 Breakage of Sporangiospores...... 48 Isolation of mRNP’s from Sporangiospores ...... 49 Radioisotopic Labelling of Sporangiospores ...... 52 [%]Polyuridylic Acid Hybridization to Polyadenylated RNA...... 54 Assay of Incorporated Radioisotopes...... 55 Sedimentation Velocity (Rate Zonal) Centrifugation in Sucorse Density Gradients ...... 57 Sedimentation Equilibrium (Isopycnic) Centrifugation in CsCl G radients ...... 60 Sedimentation Equilibrium (Isopycnic) Centrifugation in Metrizamide Gradients ...... 62 Protein Assays ...... 62 Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis ...... 63 Photography...... 64 Purification and Analysis of mRNA from mRNP's ...... 64
v i TABLE OF CONTENTS (Continued)
R esults...... 67
Location of Pre-formed mRNA in Dormant and Germinating Sporangiospores* • , ...... 67 Isolation of Poly(A) RNA-containing RNP's with 01igo(dT)-cellulose Columns...... 70 Size Distribution of mRNP's by Sedimentation Velocity Centrifugation ...... 86 Iso latio n of 40S Ribosomal Subunits ...... 94 Bouyant Densities of mRNP's by Sedimentation Equilibrium Centrifugation in CsCl Solution ...... 94 Bouyant Densities of mRNP's by Sedimentation Equilibrium Centrifugation in Metrizamide Gradients ...... 99 SDS-PAGE Analysis of mRNP Protein Components ...... 114 Size Distribution of RNA Population Isolated from mRNP's ...... 118
Discussion ...... 121
Literature Cited ...... 137
V ita ...... 156
v i i LIST OF TABLES
Table Page
1 Sedimentation C oefficients and Bouyant D ensities of cmRNP's from Various Animal Systems ...... 41
2 Protein Components of mRNP’s from Mucor and Neurospora...... 132
v i i i LIST OF FIGURES
Figure Page
1 Types of spores formed by Mucor spp ...... 6
2 Biphasic morphogenesis and alternative developmental fates of sporangiospores from dimorphic Mucor spp ...... 8
3 Photomicrographs of the germination sequence of Mucor racemosus sporangiospores under air (aerobic) or 100% nitrogen (anaerobic) atmospheres ...... 10
4 t^H]Poly(U) hybridization to poly(A)*RNA from Mucor racemosus sporangiospore extracts fractionated by sucrose density gradient centrifugation ...... 68
5 Analytical oligo(dT)-cellulose affinity chromatography of ^P-labelled particulate fraction from Mucor racemosus sporangiospore extract ...... 73
6 Analytical oligo(dT)-cellulose affinity chromatography of L-[35s ]methionine-labelled particulate fraction from Mucor racemosus...... 75
7 Analytical oligo(dT)-cellulose affinity chromatography of non-radioactive particulate fraction from Mucor racemosus sporangiospore extract ...... 77
8 [ H]Poly(U) hybridization to poly(A) RNA- containing particles from Mucor racemosus sporagngiospore extracts eluted from an analytical oligo(dT )-cellulose column by a standard sequence of b u ffe rs...... 79
9 Preparative oligo(dT)-cellulose affinity chromato graphy of 32P-labelled particulate fraction from Mucor racemosus sporangiospre extract: H2-eluted mRNP’s ...... 82
10 Preparative oligo(dT)-cellulose affinity chromato graphy of 32p-labelled particulate fraction from Mucor racemosus sporangiospore extract: H3-eluted mRNP's ...... 84
11 Analysis of Mucor dormant sporangiospore mRNP’s eluted from oligo(dT )-cellulose column by buffer H2 (low ionic strength) by sedimentation velocity centrifugation ...... 88
ix LIST OF FIGURES (Continued)
Figure Page
12 Analysis of Mucor dormant sporangiospore mRNP's eluted from ollgo(dT)-cellulose column by buffer H3 (50% formamide) by sedimentation velocity cent rifugation ...... 90
13 Analysis of material from Mucor dormant sporangio spore extracts not binding to oligo(dT)-cellulose at high ionic strength by means of sedim entation velocity centrifugation ...... 92
14 Purification of 40S ribosomal subunits by sucrose density gradient centrifugation: initial gradient separation of components in 15,000-xg supernatant fraction of Mucor hyphae ...... 95
15 Purification of 40S ribosomal subunits by sucrose density gradient centrifugation: re-sedimentation of 40S material from the separation depicted in Fig. 14...... 97
16 Isopycnic centrifugation in CsCl solution of 32p-iabelled Mucor sporangiospore mRNP's eluted from oligo(dT)-cellulose in buffer H2 (low ionic strength) ...... 100
17 Isopycnic centrifugation in CsCl solution of 32p-iabelled Mucor sporangiospore mRNP's eluted from oligo(dT)-cellulose ip buffer H3 (50% formamide)...... 102
18 Isopycnic centrifugation In CsCl solution of 32p-iabelled 40S ribosomal subunits purified from hyphae of Mucor racemosus...... 104
19 Isopycnic centrifugation in metrizamide solution of 32p-iabelled Mucor sporangiospore mRNP's eluted from oligo(dT)-cellulose in buffer H2 (low ionic strength)...... 106
20 Isopycnic centrifugation in metrizamide solution of 32p-labelled Mucor sporangiospore mRNP's eluted from oligo(dT)-cellulose in buffer H3 (50% formamide)...... 108
x LIST OF FIGURES (Continued)
Figure Page
21 Isopycnic centrifugation in metrizamide solution of 32P-labelled AOS ribosomal subunits purified from hyphae of Mucor.racemosus...... 110
22 Isopycnic centrifugation in metrizamide solution of non-radioactive AOS ribosomal subunits purified from hyphae of Mucor racemosus...... 112
23 Protein composition analysis of mRNP's from dormant sporangiospores of Mucor racemosus by SDS-PAGE: a comparison with protein populations in other cell fra c tio n s ...... 115
2A Analysis of RNA from dormant Mucor sporangiospore mRNP’s by sedimentation velocity centrifugation: distribution on sucrose density gradients of 32p-iabelled material recovered in phenol extraction of pooled H2-eluted fractions derived from oligo(dT)-cellulose chromatography of spore extract particles ...... 119
x i ABSTRACT
Extracts of sporangiospores of Mucor racemosus contained RNA that 3 readily hybridized with [ H]polyuridylic acid. Prior to germination,
this RNA was in a form sedimentlng at <80S. Within 10 minutes after
initiating germination, most of this RNA sedimented with polyribosomes and 80S monoribosomes. Particulate material from spore extracts bound to oligo(dT)-cellulose at high ionic strength and was assumed to contain messenger ribonucleoprotein particles (mRNP's). A portion of the mRNP's was released from the column by lowering the ionic strength. Other portions were eluted stepwise in buffer containing
50% and 90% formamide and in 0.1-N NaOH. Identical elution patterns 32 were observed whether monitoring incorporated P-orthophosphate or 32 L-[ S]methionine, absorbance at 280 nm, or hybridization of 3 [ H ]p o ly u rid y lic a c id . mRNP's from the f i r s t two f r a c tio n s were analyzed. A bimodal population of particles was detected in sedimentation velocity and sedimentation equilibrium centrifugation.
Particles eluted at low ionic strength demonstrated a sedimentation coefficient distribution of 20S-to-80S, with a mean of 55S. Particles eluted in formamide demonstrated a sedimentation coefficient distribution of 20S-to-60S, with a mean of 40S. Particles eluted at low ionic strength displayed two peaks in CsCl centrifugation, with bouyant densities of 1.37 gm/cc and 1.59 gm/cc. Particles eluted in formamide displayed a single peak with a bouyant density of 1.61 gm/cc. Particles eluted at low ionic strength and centrifuged in metrizamide solution formed two bands having bouyant densities of 1.15 gm/cc and 1.30 gm/cc; formamide-eluted particles banded only at the
x i i higher density. Mucor AOS ribosomal subunits banded at 1.56 gm/cc and
1.28 gm/cc in CsCl and metrizamide solution respectively. Analysis of mRNP fractions by sodium dodecylsulfate-polyacrylamide gel
electrophoresis characterized protein components of these particles which helped explain their bimodal sedimentation behaviour. Each mRNP
fraction contained a unique spectrum of proteins not shared by ribosomes or soluble cell proteins. The formamide-eluted particles
contained two proteins, whereas the particles eluted at low ionic strength possessed 12 proteins. A 2A,000-dalton protein was the predominant form in both. The denser mRNP’s were smaller and lacked proteins found in the larger less dense particles. The former had a protein/RNA ratio of A5/55; whereas the later possessed a value of
90/10. Messenger RNA extracted from the mRNP’s sedimented between
8S-to-20S.
x i l i LITERATURE REVIEW
Mucort Background and Practical Importance
Mucor racemosus Is a fungus belonging to the class Zygomycetes
(Alexopolous and Mims, 1979). These fungi are characterized as producing primitive coenocytic (aseptate) hyphae and structures of sexual recombination called zygospores. They include many well-known genera such as Rhizopus» Phycomyces, Pilobolus, Cunninghamella ,
Absidia, Thamnidium and Mucor (Ross, 1979). Most of these genera grow in only a single vegetative form, the coenocytic hyphal morphology.
Three genera within the order Mucorales, however, include species capable of growing as either coenocytic hyphae or as spherical multipolar budding yeasts. This property is called dimorphism and will be considered in more detail later. The genera within
Zygomycetes displaying dimorphic species are Mycotypha, Cokeromyces, and, most importantly, Mucor. Species within the genus Mucor are of considerable importance to human beings for many practical reasons which I shall briefly summarize.
Mucor species are usually saprophytes, living off decaying animal and vegetable matter in the soil (Alexopolous and Mims, 1979).
However, p ractically any species can cause an opportunistic in fe c tio n called mucormycosis. The degree of severity of the infection can range from a mild dermatophytic colonization of the skin, nailbed or hair follicle to a deep infection of the lungs or brain which are
Invaded by penetration of hyphae directly through the nasal passages
(L eh rer, 1980; Emmons et a l . , 1977). The more su p erficial forms of infection can usually be cured with antibiotics or antiseptics; however, the deep mycoses usually result In death even when treated with potent fungicides such as amphotericin B (Lehrer, 1980; Emmons et al •, 1977). Fortunately it is usually only patients that are extremely debilitated to begin with that acquire Mucor infections, such as diabetics, burn victims, cancer patients on chemotherapy, organ transplant recipients on immunosuppressants, AIDS patients and others with weakened immune systems (Rippon, 1982).
Mucor species are notorious spoilage agents in the food industry.
They can grow on practically any substrate providing a simple or complex sugar or a protein as the main carbon source. They can grow at a low temperature, a low water a c tiv ity , and a low pH. Hence they are ubiquitous despoilers of meat, poultry, dairy products, citrus f r u its , and baked goods (F razier, 1958; Jay, 1978).
On the other hand, Mucor species have been used to produce many food products consumed by man. For example, many alcoholic beverages are fermented from rice in the Orient by the action of Mucor rouxii
(H e n ric i, 1930). Sufu and tempeh are fermented soybean curd products also made in the Orient by the action of Mucor (Jay, 1978). In the
West, the proteolytic enzyme rennin is recovered from culture supernatants of Mucor meihei and used in cheesemaking (Ayres et a l.,
1980).
Several industrial products and processes have employed some species of Mucor. Ethyl alcohol has been produced commercially in
France by the fermentative action of Mucor rouxii on grain (Henrici,
1930). The enzyme alpha-amylase and the antibiotic fusidic acid are commercially recovered from Mucor cultures (Ayres et al., 1980).
Mucor species are reportedly involved in the retting of flax and hemp and in the ripening of snuff (Henrici, 1930). The organism is a nuisance in causing the rot of leather products (Henrici, 1930).
Mucor has proven to be an important laboratory organism in
studies on mating (Gooday, 1974), macromolecular synthesis (O rlow ski,
1981), cell wall formation (Bartnicki-Garcia, 1981) and morphogenesis
(Sypherd et al., 1978). The role of trisporic acid in sexual
attraction and zygospore formation was elucidated in several
zygomycete species including Mucor mucedo and Mucor hiemalis (Gooday,
1974). The response of ribosome function to growth rate and nutrition
has been studied in Mucor racemosus by Orlowski and his co-workers
(Orlowski, 1981; Ross, 1983). The structure and function of the
chitosome, a newly-described organelle for making chitin, was
initially reported in Mucor rouxii by Bartnicki-Garcia (1981). Many
d ifferen t aspects of Mucor dimorphism have been studied as a model of morphogenesis mainly in Mucor rouxii, Mucor racemosus and Mucor
genevensis in about two dozen laboratories around the world.
Fungal Dimorphism
The General Phenomenon
Dimorphism is a common occurance among fungi. A large number of fungi, belonging to diverse taxonomic groups, have the ability to grow in alternative morphological forms. Most commonly these organisms can grow in the form of yeasts (round, budding cells) or hyphae (long, branching structures). Many of these dimorphic species are severe pathogens which cause systemic or deep mycoses. Examples of pathogenic species include Coccidiodes jimnitis, the etiological agent of coccidiodimycosis; Histoplasma capsulatum, the cause of histoplasmosis; Blastomyces dermatitidis, the agent of blastomycosis; and Candida albicans, which causes candidiasis. Histoplasmosis and blastomycosis are diseases endemic to the lower M ississippi River
Valley and so are of great concern in Louisiana and other south- central states. It is characteristic of these dimorphic pathogens that only one morphological form of the organism (usually the yeast or spherule form) is found in infected tissue. Human infection is generally an opportunistic occurance in a debilitated host. All the mentioned organisms are found primarily in the soil living saprophytically in the mycelial form (Alexopolous and Mims, 1979;
Ross, 1979; Deacon, 1980; Jawetz et a l . , 1970).
Mucor Dimorphism
Since cell morphology seems intimately related to pathogenicity in many fungi, an understanding of the molecular mechanisms regulating dimorphism may provide a basis for treatment or prevention of the diseases caused by these fungi. Mucor provides us with a relatively safe system in which to study the regulation of fungal dimorphism.
What we learn from the Mucor system may someday be applied to control of the more dangerous dimorphic fungal pathogens and also may add to our knowledge of developmental biology and biological control mechanisms in general.
In addition to the low pathogenicity of Mucor, several other properties of this fungus account for its growing popularity as a laboratory model of dimorphism. The organism is very easy to grow in the pure form of either morphology. A very rapid and synchronous conversion of yeasts to hyphae or hyphae to yeasts can be caused by simply changing the gaseous environment of the cells. This conversion
is quite reversible by changing the system back to the original gas.
A gross alteration in nutritional conditions is not necessary for this
dimorphic conversion, as is sometimes the case in other dimorphic
genera (Sypherd et a l., 1978). The conversion can occur in complex
(Larsen and Sypherd, 1974) or defined (Peters and Sypherd, 1978)
liquid media. Mutants can be made and selected for in either
morphological form on agar plates (Peters and Sypherd, 1978; Roncero,
1984). Genetic exchange is possible by means of zygospore formation
(Gauger, 1965) or heterokaryon formation following the fusion of
protoplasts made by treating cells with chitosanase to digest the
outer wall (Genthner and Borgia, 1978; Lasker and Borgia, 1980). Any
one of several environmental stimuli can induce the morphological
transition. Growth is an obligate part of morphogenesis, as it is in
the case of higher organisms.
In all species of Mucor growth on a solid medium in air gives
rise to the production of sporangiospores contained within sporangia
(sacs) at the ends of sporangiophores (aerial hyphae) (Fig. 1). Upon
introduction to liquid medium the ellipsoidal spores swell, become
spherical, and, if under air, form hyphal germ tubes (Figs. 2 & 3).
The hyphae rapidly elongate and branch many times (Fig. 3). Hyphal
growth is by apical extension of the cell wall. Upon depletion of
carbon or nitrogen sources in the medium the hyphae fragment into many approximately spherical arthrospores which resemble yeasts except that
they do not possess buds. If the sporangiospores are from a dimorphic species of Mucor (several species exhibit only hyphal growth) and are under an anaerobic atmosphere, multiple buds at random locations Figure 1. Types of spores formed by Mucor spp. A) Zygospores of
Mucor hiemalis. These cells are the products of genetic recombination between heterothallic mating types. Some species of Mucor are homothallic, but all can produce these spores. B) Arthrospores of
Mucor racemosus. These structures serve as a survival mechanism under conditions of environmental duress. All species can produce them.
C) Sporangiospores of Mucor racemosus. These spores serve to disseminate the organism through the environment. All species can produce them. All specimens are displayed at the same magnification.
The marker bar represents 20 yam in each case. 7 Figure 2. Biphasic morphogenesis and alternative developmental fates of sporangiospores from dimorphic Mucor spp. Development of the yeast morphology requires the presence of a hexose sugar in the growth medium as well as an anaerobic atmosphere. 9
ANAEROBIC ATMOSPHERE
0cl — D> Q. — 0 .Cb Yaaat* S p o rv 0 - ~ o — n r Sphorloat Growth G3— cr—
AEROBIC ATMOSPHERE Figure 3. Photomicrographs of the germination sequence of Mucor racemosus sporangiospores under air (aerobic) or 100% nitrogen
(anaerobic) atmospheres. A—>B) Spherical growth under either atm osphere. C—>E) Development of hyphae under a ir. F—>H) Develop ment of yeasts under nitrogen. All specimens are displayed at the
same magnification. The marker bar represents 20 urn in each case. around the surface of the swollen spore are formed (Figs. 2 & 3). A
single mother cell may possess in excess of 10 buds, although 3 or 4 is more typical (Fig. 3). Growth in either the yeast or hyphal form w ill continue indefinitely unless the environmental conditions are changed. The specific environmental stimuli for morphogenesis will be discussed below. In any yeast-to-hypha transition the mother cells will first shed all buds. Afterwards, hyphal outgrowths are formed on
80-to-90% of the spherical cells and growth by apical extension and branching ensues. In any hypha-to-yeast transition, spherical cells bud from the sides and tips of the hyphae. Some hyphal structures always remain in the culture even after long periods, making the shift in this direction less quantitative and therefore much less frequently studied (Sypherd et a l . , 1978; Inderlied et a l . , 1985; C ihlar, 1985).
Mucor dimorphism may be influenced by the Interaction of several environmental factors, including atmosphere, nitrogen sources and carbon sources. Yeast morphology is directly influenced by the concentration of fermentable hexoses. High hexose concentrations are required for yeast development, whereas low concentrations of hexose favor hyphal development. Other carbon sources such as mannitol, various disaccharides, pentoses and two- and three-carbon compounds, such as glycerol, acetate and pyruvate, elicit only hyphal growth.
The importance of hexoses can be underscored by the necessity of a hexose carbon source for the morphopoietic action of various chemical agents. Phenethyl alcohol maintains yeast morphology In Mucor rouxii regardless of the atmosphere, but fails to prevent hyphal morphogenesis in the absence of a hexose (Terenzi and Storck, 1968).
Sim ilarly, the antibiotic chloramphenicol favors yeast development in Mucor rouxii and Mucor genevensis regardless of atmosphere, but is
also dependent on the presence of hexoses (Clark-Walker, 1973;
Zorzopolous et al., 1973). Cyclic AMP also induces yeast development
in Mucor rouxii and Mucor racemosus, but will not do so in the absence
of a hexose (Larsen and Sypherd, 1974). It has been claimed that EDTA
induces growth exclusively in the hyphal form (Zorzopolous et al.,
1973), however, this observation is not repeatable with all species of
Mucor (Orlowski, 1986).
Atmosphere also Influences Mucor dimorphism. Bartnicki-Garcia
and Nickerson (1962a) reported that a CC^ atmosphere favors yeast
development on both liquid and solid media, whereas air stimulates
hyphal growth. Temperature and pH did not affect morphology according
to their study, proving that acidification of the media by COg is not a factor. However, the effect of CC^ is modified by hexose concentration and completely cancelled by the presence of oxygen. A nitrogen atmosphere was originally thought to favor hyphal growth in
Mucor rouxii (Bartnicki-Garcia and Nickerson, 1962b). Later, Mooney and Sypherd (1976) claimed that c e ll morphology was dependent upon the flow rate of nitrogen gas through the culture: the greater the volume of gas flow, the greater the proportion of yeast forms in the culture.
However, Phillips and Borgia (1985) have recently shown that both previous results were spurious, being due to minute quantities of contaminating oxygen that is able to penetrate the gas delivery tubing
(normally made of tygon or teflon) at low nitrogen flow rates. Any truly anaerobic culture produces only yeast cells, provided a hexose is present. Regulation of Mucor Dimorphism
A large number of biochemical and physiological properties of the
cell have been examined in an attempt to discover the ultimate
determinant(s) of Mucor morphology. The cell wall and wall
synthesizing enzymes have been studied for years by Bartnicki-Garcia and his co-workers. The gist of what they have found is that the
chemical composition of the wall is not very different in Mucor yeasts
and hyphae, being composed mainly of chitin and chitosan microfibrills
(Bartnicki-Garcia, 1968). They think that Mucor vegetative morphology is determined by the pattern of wall polymer deposition and that this pattern is governed largely by the distribution of chitin synthesizing vesicles called chitosomes (Bartnicki-Garcia, 1981). The enzyme which polymerizes chitin from N-acetylglucosamine, called chitin synthetase, is synthesized in an inactive (’’zymogen'') form (Ruiz-Herrera and
Bartnicki-Garcia, 1976). Its activity within the chitosomes appears to be modulated by a balance between the levels of a proteolytic activator and a heat stable, dialyzable inhibitor present in the cell cytoplasm (Ruiz-Herrera and Bartnicki-Garcia, 1976). The activities of chitin deacetylase, to create chitosan from chitin, (Davis and
Bartnicki-Garcia, 1984); various wall lytic enzymes, such as chitinase to allow insertion of new wall fragments, (Humphreys and Gooday,
1984a,b); and several enzymes responsible for synthesizing the various
"glue-like” polyuronide matrix components of the cell wall (Dow and
Rubery, 1977; Dow and V illa, 1980; Dow et al., 1983) must also be regulated in an exquisitely coordinated fashion in order to effect wall assembly in the precise vectorial pattern typical of hyphae or in the diffuse generalized pattern typical of yeasts. Microfilaments and microtubules (Sterwart and Rogers, 1978) and intracellular ion
currents (Bartnicki-Garcia, 1973; Kropf et al., 1983) have been
proposed as mechanisms to direct chitosomes to the appropriate sites
of chitin deposition, however, no definitive evidence yet exists to
support these notions (Orlowski, 1986).
Redirection of the pattern of wall deposition may be the most
immediate determinant of a morphological change in Mucor and may lead
one to believe that the process of Mucor morphogenesis is conceptually very simple. However, the number of enzymes, activators, inhibitors, sub-cellular organelles, and substrates involved in wall construction are considerable and may be directly or Indirectly affected by a diversity of physiological parameters. There may be an extensive chain or network of molecular events and signals that ultimately
relate atmospheric composition and the pattern of wall deposition. In addition to the obvious gross morphological differences, Mucor yeasts and hyphae may be considerably differentiated from one another in terms of many of their cytological, physiological and metabolic properties. Most properties of the organism are not yet well- characterized in either yeasts or hyphae. Therefore, comparative studies of Mucor yeasts and hyphae have not stopped at the cell wall but have encompassed an examination of carbon and energy metabolism, nitrogen metabolism, lipid synthesis, enzyme composition and properties, effects of several endogenously synthesized small molecules and macromolecular synthesis (Orlowski, 1986). In addition, several gene products have been investigated with respect to their potential roles in Mucor dimorphism by means of modern molecular genetics (Orlowski, 1986). To put the matter most concisely, after a long period of study by many laboratories (Bartnicki-Garcia and Nickerson, 1962a,b; Rogers et a l., 1974; ilerenzi and Storck, 1968; Zorzopolous et al., 1973;
Faznokas and Sypherd, 1975; Inderlied and Sypherd, 1978; Phillips and
Borgia, 1985; Borgia et al., 1985) it must be concluded that the pathways of carbon and energy metabolism are Irrelevant to Mucor dimorphism (O rlow ski, 1986). The same conclusion was recently drawn from the extant literature by Orlowski with respect to nitrogen metabolism in his soon-to-be-published review on Mucor dimorphism
(Orlowski, 1986). Changes in phospholipid synthesis have been suggested to affect membrane composition and the activity of wall- synthesizing enzymes (Ito et al., 1982; Lopez-Roraero et al., 1985), but a dearth of actual data must make these conclusions rather preliminary. Changes in enzyme activities and enzyme properties abound during Mucor morphogenesis (Orlowski, 1986). Distinguishing the changes causally related to morphogenesis from those casually accompanying morphogenesis remains one of the major obstacles to understanding Mucor dimorphism.
An assortment of endogenously synthesized small molecules fluctuate in amount in characteristic patterns during Mucor morphogenesis. Some of these, such as cyclic AMP, have a definite effect on cell morphology (Larson and Sypherd, 1974; Orlowski and
Ross, 1981). Others, such as the polyamines (Garcia et al., 1980) and
S-adenosylmethionine (SAM) (Garcia and Sypherd, 1984), offer evidence of a strong but not yet definitive correlation with morphogenesis.
Cyclic AMP is a small molecule that has been well characterized as a regulatory element in many other systems, acting primarily as an effector of protein kinases in eukaryotes (Robison et al., 1971). As will be discussed below, cyclic AMP may act in such a role to regulate the protein synthesizing system in Mucor (Larson and Sypherd, 1979).
The polyamines also normally act as effectors of the protein synthesizing apparatus in prokaryotic and eukaryotic cells (Abraham and P ih l, 1981; A lgranati and Goldemberg, 1977). SAM is universally a methyl donor within the cell and, in Mucor, may modulate the protein synthesizing machinery through key methylations of a critical component of th is system (H iatt et a l . , 1982; Fonzi et a l ., 1985). In the next Introductory section I shall discuss the known facts of macromolecular synthesis in Mucor and in general as a prologue to my own research.
Macromolecular Synthesis and Mucor Dimorphism
Macromolecular synthesis is essential to any morphogenetic change in Mucor. If RNA or protein synthesis is blocked with an inhibitor or brought to a stop by starvation of an auxotroph all developmental change ceases (Orlowski, 1986). Aerobic hyphae grow and synthesize macromolecules at much faster rates than CX^-grown yeasts; however, they do so at only slightly faster rates than Ng-grown yeasts
(Orlowski, 1981). Orlowski and Ross (1981) have discredited the notion that Mucor morphology Is an obligate correlate of the growth rate or of the steady-state rate of protein synthesis. Evidence does suggest that a number of critical events take place at the level of macromolecular synthesis during Mucor morphogenesis.
Under conditions conducive to a yeast-to-hypha conversion, there are immediate but transient increases in the rates of RNA (Orlowski 18
and Sypherd, 1978b) and protein (Orlowski and Sypherd, 1977)
synthesis. Orlowski and his colleages determined that the rates of
protein synthesis are controlled by a dynamic balance between i) the
cellular ribosome concentration, ii) the percentage of ribosomes
recruited into the translation process, and iii) the rate of
polypeptide chain elongation (Orlowski and Sypherd, 1978a,c; Orlowski,
1981; Ross and O rlow ski, 1982a,b). The l a t t e r two param eters are
adjusted immediately upon the stimulation of morphogenesis by a change
of atmosphere. The specific activities of several different enzymes have been documented to increase at th is time (Orlowski, 1986) and an analysis of radioisotopically-labelled proteins by means of two- dimensional polyacrylamide gel electrophoresis (2-D PAGE) and autoradiography (O'Farrell, 1975) suggested that several proteins are newly synthesized during the dimorphic conversion whereas the
synthesis of several other proteins ceases at this time (Hiatt et al.,
1980; P h illip s and Borgia, 1985).
Several parameters that are known to affect the quantity and quality of messenger RNA translation in other biological systems show major changes in Mucor at the same time that alterations in the rates and id e n titie s of proteins synthesized are being displayed. The same correlations are noticed whether one considers a yeast-to-hypha conversion or the development of hyphae from sporangiospores. The development of yeasts has not yet been studied with regard to these parameters. Specifically, the parameters of interest are i) the p hosp h o ry latio n of ribosom al p ro te in S6 (Larsen and Sypherd, 1979;
1980), ii) intracellular levels of the polyamine putresclne (Garcia et a l ., 1980) and of the enzyme o rn ith in e decarboxylase (In d e rlie d et 19
al., 1980) which is responsible for making putrescine, and iii) the
degree of methylation of protein synthesis elongation factor EF-lot and
intracellular levels of the methyl donor SAM (Garcia et al., 1980;
Hiatt et al., 1982).
Ribosomal protein S6 shows a very low level of phosphorylation in
Mucor yeasts and in sporangiospores (less than one mole of phosphate
per mole of protein). When hyphal germ tubes emerge from yeast mother
cells or from swollen sporangiospores protein synthetic activity is at
its highest and S6 carries from 3 to 4 covalently bound phosphate molecules per protein molecule (Larsen and Sypherd, 1979; 1980).
Nothing more has been studied in Mucor of this phenomenon but a
relationship between the degree of S6 phosphorylation and the hormonal induction of specific gene products has been noted and studied in s e v e ra l anim al systems (Kruppa et a l . , 1983; Martin-Perez and Thomas,
1983; Mailer et al., 1986). Cyclic AMP-specific protein kinases have been found to play a role in several of these systems. It is possible that the role of cyclic AMP in Mucor morphogenesis is as an effector of a protein kinase that specifically phosphorylates ribosomal protein
S6. Passeron Is presently studying the properties of protein kinases and phosphoprotein phosphatases in Mucor (Moreno et al., 1977; Moreno and Passeron, 1980; Pastor! et al., 1981; Moreno et a l., 1983;
Seigelchifer and Passeron, 1984; 1985), but no one is studying the potential of S6 as a developmentally-specific substrate.
The polyamines are known to enhance both the amount and the f i d e l i t y of p ro te in s sy n th esiz ed in v itro (Abraham and P ih l, 1981).
Also, bacterial mutants with reduced levels of polyamines make many mistakes in protein synthesis iji vivo (Algranati and Goldemberg, 20
1977). Right at the time the rate of protein synthesis peaks during
Mucor hyphal development intracellular levels of putrescine also rise
to a maximum (Garcia et a l. , 1980). The level of ornithine
decarboxylase, which makes putrescine, shows the same time course of
activity (Inderlied et al., 1980). Spermine and spermidine, in
contrast, do not change in amounts at this time. It has been
hypothesized that putrescine may act as a subtle regulator of protein
synthesis during Mucor morphogenesis (Orlowski, 1986).
Elongation factor-la (EF-lot) is found in all eukaryotes and, as
the name suggests, functions In nascent polypeptide chain elongation
(Moldave, ^986). EF-lCt had been known as a phosphorylated protein but
it was first shown to be variably methylated in Mucor racemosus (Hiatt
et al., 1982). It has more recently been documented as methylated in
other systems as well (Fonzi et a l., 1985). Analysis by 2-D PAGE
demonstrated that EF-la represents the most abundant of all proteins
in Mucor hyphae (Hiatt et al., 1982). It is present in much greater amounts in Mucor hyphae than in yeasts or sporangiospores. It is also much more highly methylated In hyphae than in yeasts or
sporangiospores, correlating with protein synthetic activity. EF-la is almost totally non-methylated in dormant spores. The degree of methylation increases considerably during germination and hyphal outgrowth, attaining a maximum of approximately nine mono-, di-, or tri-methylated lysine residues per EF-la molecule in developing hyphae. The activity of EF-la, described in terms of phenylalanine polymerized in vitro on a polyuridyllic acid template, increases
seven-fold during hyphal development from sporangiospores. Using cDNA probes it was determined that the increase in factor activity happens without any increases in the amount of mRNA specifying EF-la or in
concentration of EF-la protein. Factor activity appears to be
regulated exclusively by the level of methylatlon (Fonzi et al.,
1985). Again, using cDNA probes, Linz and Sypherd have shown that at
least three different genes at quite distinct sites within the Mucor
genome specify EF-la activity (Linz et al., 1986). These show
substantial homology with the known gene from Saccharomyces. It is not known if the presence of th is gene in m ultiple copies carries any
developmental significance. In fact, it is not yet known if all
copies of the gene are transcribed into mRNA (Linz et al., 1986).
Intracellular levels of SAM and SAM synthetase were shown to rise and
fall in direct proportion to the degree of EF-la methylatlon, the rate of protein synthesis, and the extent of hyphal development from yeasts
(Garcia et a l., 1980; Garcia and Sypherd, 1984; Inderlied et al.,
1980) leading to formulation of the hypothesis that all these parameters may play roles interrelated in a global mechanism regulating Mucor morphogenesis (Orlowski, 1986).
Mucor Dimorphic Development from Sporangiospores
Assuming i t is from a dimorphic species, the Mucor sporangiospore may develop into e ith er the yeast or hyphal morphology (Figs. 2 and 3) depending upon only the nutritional and gaseous environments. The re la tio n s h ip s between environment and morphology are exactly the same as in the case of vegetative interconversions. Incubation of the spores under an anaerobic atmosphere in the presence of a hexose yields yeast cells, whereas the presence of oxygen in the atmosphere
Induces the development of hyphae. This dual potentiality of the Mucor sporangiospore makes it very rare, if not unique, among the many and varied microorganisms studied as developmental systems. This property may make it a more valid model of metazoan development into specialized cell types than, for example, the unidirectional morphogenesis observed in the commonly studied Saccharomyces or
Bacillus systems. It is also much easier to obtain large homogenous populations of the same cell type at the same stage of development during Mucor spore germination than it is in those few popular systems in which differentiation into multiple cell types does take place, such as in the slime mold Dictyostelium or the nematode
Caenorhabditis.
Much work has been done on the development of the cell wall during Mucor sporangiospore development into yeasts or hyphae by
Bartnicki-Garcia and his collaborators. The sporangiospore wall is a specialized structure, largely proteinaceous in composition, that is fractured, penetrated and shed by the developing vegetative cell. The vegetative cell wall develops de novo during germination beneath the spore wall and has essentially the same chemical properties in yeasts and in hyphae as I have described earlier (Bartnicki-Garcia, 1968;
1973).
Studies on the machinery and processes of macromolecular synthesis during Mucor sporangiospore development have mainly been done in M. Orlowski's laboratory at LSU. The sporangiospores of Mucor racemosus were demonstrated to possess a pool of stable polyadenylated messenger RNA which is made during spore formation but is not utilized as a template for p ro tein synthesis u n til spore dormancy is broken by exposure of the spores to nutrient medium (Linz and Orlowski, 1982). Prior to the induction of germination most ribosomes are in the form
of free inactive 40S and 60S subunits (Orlowski and Sypherd, 1978c).
Within 10 min of spore introduction into nutrient medium approximately
85% of the ribosomes have been recruited into polyribosomes actively
engaged in translation (Linz and Orlowski, 1982). RNA synthesis does not take p lace during the f i r s t 20 min of germination and newly-made mRNA is not available for translation until at least 30 min have elapsed. A substantial number of proteins are synthesized from the stored messenger RNA molecules during this period (Linz and Orlowski,
1982).
An analysis of the proteins made ^n vivo during formation and germination of Mucor sporangiospores was carried out by means of radioisotopic labelling, 2-D PAGE, and autoradiography. Linz and
Orlowski (1984) determined that the population of proteins made during formation of the spores differ considerably from those made during germination of the spore. Several proteins are made during spore formation but not during germination. On the other hand, several proteins are made during the first 30 min of germination but are not manufactured during spore formation. The latter observation is noteworthy in that the messenger RNA encoding these proteins must be synthesized and stored, yet not translated, as the dormant spore is being assem bled. A p o s t- tr a n s c r ip tio n a l regulatory mechanism that directs selective translation apparently exists in the maturing spore.
Many proteins, although made throughout germination, show major changes in the individual rates of their synthesis during this period.
At least one protein was reported to be subject to post-translational modification during the initial hour of germination (Linz and Orlowski, 1984).
Messenger RNA populations were analyzed in dormant and germinating sporangiospores of Mucor racemosus by means of ^n vitro translation, 2-D PAGE, and autoradiography (Linz and Orlowski, 1986).
Genes expressed in the form of mRNA molecules were compared with gene products actually appearing in the form of protein in the living cell at various stages of development. It was found that i) most of the differential gene expression displayed at the level of protein synthesis during germination results from concomitant changes in functional mRNA levels, ii) some of the stored mRNA species may be activated and others inactivated by post-transcriptional processing mechanisms, and iii) a small population of gene products may be regulated at the level of selective translation of pre-existing messages (Linz and Orlowski, 1986).
Radioisotopically-labelled In vivo translation products were recovered from Mucor racemosus sporangiospores germinating under an a i r or a n itro g en atm osphere and were compared by means of 2-D PAGE and autoradiography (Linz and Orlowski, 1985). The population of stable pre-formed messenger RNA species available for translation within the first 30 min of germination is, of necessity, identical in each case. Most of the proteins synthesized within the first hour of germination are identical in the two systems. However, there is a small number of proteins manufactured that are unique to each system.
Synthesis of these unique proteins continues throughout germination and is characteristic of the ultimate vegetative morphology that develops. It is not yet known whether the observed differences are specifically linked to morphology or to aerobic versus anaerobic metabolism. In either case, it is clear that at least some
differential gene expression is regulated during the first stages of
germination by selective translation of a specific subset of the total
set of pre-formed mRNA molecules stored in the dormant sporangiospore.
The Mucor sporangiospore may maximize its developmental options by
storing mRNA's essential to the formation of both yeasts and hyphae
but expressing only those appropriate to the morphogenetic sequence
dictated by the environment.
Messenger Ribonucleoprotein Particles
Perspective
The findings by Linz and Orlowski (1982) that Mucor racemosus
sporangiospores contain a large pool of stable pre-formed messenger
RNA and that this mRNA does not reside in polyribosomes but yet must
be instantly available for inclusion into polyribosomes and
utilization in the translation process prompted an investigation into
the site of mRNA storage in the dormant spore. Any suggestion that
mRNA's may be s to r e d fre e in the cytoplasm or n o n -s p e c ific a lly
associated with the inner face of the rough endoplasmic reticulum must
be rejected since it has become clear over the years that no nucleic
acid exists as a naked molecule. Rather, these lengthy polyanionic molecules are normally associated with a number of specific proteins.
These are commonly basic proteins or proteins with major basic domains
(Saenger, 1984). The histone proteins associated with DNA in very
specific spatial relationships within nucleosome subunits of the
eukaryotic chromosome (Kornberg, 1974) and the five dozen or so unique
proteins bound to specific regions of four particular RNA species within the large and small subunits of the ribosome (Wittmann, 1983;
Lake, 1985) are the best known examples. Without their protective
coating of proteins, nucleic acids would be hydrolyzed, certainly
irreparably damaged, within minutes by the ubiquitous nucleases found within living cells. Being single stranded, RNA is even more
susceptible to damage by nucleolytic cleavage than DNA.
The mRNA in Mucor sporangiospores is not stored in association with 80S monoribosomes, for this fraction of ribosomal particles is very small (Linz and Orlowski, 1982) or completely lacking (Orlowski
and Sypherd, 1978c) in these spores. Most of the ribosomes are in the
form of 40S and 60S subunits. One possibility that existed before
this project was performed was that the stable mRNA is stored in 40S
ribosomal initiation complexes. This model might have explained the
extremely rapid mobilization of pre-formed mRNA's into polyribosomes.
All that might be necessary to initiate translation could be the
joining of 60S subunits into the complex (or lifting an inhibition to
Buch an event). As will be made clear in the results of this study,
th is model was shown to be fa lla cio u s. The major a lte rn a tiv e s ite of mRNA storage in Mucor sporangiospores would have to be the messenger ribonucleoprotein particles (mRNP's) first described in the 1960's by the Russians Georgiev (Samarlna et a l., 1965) and Spirin (Spirin et al., 1965).
General Properties of Ribonucleoprotein Particles
Despite in itial resistance to and skepticism of the concept of mRNP's, these structures have been described in many different b io lo g ic a l system s ( S p ir in , 1969; Spohr et a l . , 1970; Blobel, 1972; Lindberg and Sundquist, 1974; Kumar and Pederson, 1975; M irkes, 1977;
Jaworski and Stumhofer, 1981). In fact, several classes of
ribonucleoprotein particles (RNP’s) have come to be differentiated
depending upon the roles they play in the complex process of mRNA
maturation in eukaryotic cells.
Following its initial synthesis by transcription, eukaryotic mRNA
must be extensively altered before it can be translated. Eukaryotic
genes contain many introns, or intervening sequences of nucleotides
not coding for any genetic information, interspersed throughout their
length. These introns are transcribed into the form of RNA and must
be cut out. The primary RNA transcripts still carrying the introns
are c a lle d heterogeneous n u clear RNA's (HnRNA's). HiiRNA's already
carry a complement of many specific proteins and such complexes have
been described as HnRNP's (Martin et a l ., 1980; Nevins, 1983; Padgett
et al., 1986).
Several things must be done to the HnRNP's before they become
mRNP’s. First, the introns must be cut out. This is accomplished by
base pairing of specific pieces of small nuclear RNA molecules
(SnRNA’s) to short complementary nucleotide sequences on both sides of
the intron forming a "lariat" structure. The lariat, containing the
intron, is cut out by specific endonucleases and the open ends of the
pre-mRNA are spliced back together by another specific ligase enzyme.
The SnRNA’s are not naked nucleic acids, but are complexed to specific proteins, some of which may have specific RNA cutting and splicing a c tiv itie s (Busch et a l . , 1982a,b; Wooley et a l ., 1982; Chabot et a l.,
1985). These complexes are called small nuclear ribonucleoprotein particles (SnRNP’s). The introns are carried off by the SnRNP1 s and presumably degraded to their nucleotide components. SnRNP's are
smaller (approximately 10-12S) than mRNP's and lack the polyadenyllic
acid tails of the latter which help in their purification. The term
"spliceosome" has recently come into use to designate a 60S RNP
comprising a complex of an SnRNP and an HnRNP in the process of intron
excision and exon ligation (Brody and Abelson, 1985; Pikielny and
Rosbash, 1986). SnRNP’s were not collected or characterized in this
study.
After excision of introns from HnRNA within the HnRNP's both the
5' and 3' ends of the RNA molecule must be modified. A 7-methyl
guanoslne 5'-triphosphate moeity is added in a 5'-5' linkage to the 5'
end of the RNA strand. A chain of polyadeny llic acid approximately
200 residues in length is added to the 3' end of the RNA molecule.
£ There may be one or two methylations of internal adenines at the N
position of the purine ring. With these alterations maturation of the
HnRNA into mRNA is completed. Most (>70%) HnRNA's that do not mature
into mRNA's are not polyadenylated. Conversely most (>70%) mRNA's are
polyadenylated. This provides for a conveniant way of isolating mature mRNA's or mRNP's with the use of oligo(dT)-cellulose or
poly(U)-sepharose.
Further distinction between HnRNP's and mRNP's can be made in
animal cells wherein it is relatively easy to isolate intact nuclei and cytoplasmic fractions. HnRNP's are found only in the nucleus whereas mRNP's are in both the nucleus and the cytoplasm, in the latter case being mainly associated with polyribosomes. Since in
Mucor sporangiospores we cannot gently break open the cells to recover intact nuclei and since polyribosomes are totally absent from these cells, we lack the criteria to make a distinction between HnRNP's and mRNP's. However, since most HnRNP's that are polyadenylated are m ainly an immature form of siRNP's, and since there are thought to be
no synthetic processes going on in the dormant sporangiospore, perhaps
HnRNP's are absent from Mucor sporangiospores. If there is a
significant amount of pre-mRNA stored in the form of HnRNP's that are not yet polyadenylated it will unfortunately not be recoverable using oligo(dT)-cellulose. Therefore, one must caution that it may not be possible to recover all genetic information stored in the form of RNA from Mucor sporangiospores nor will all recoverable material necessarily be destined for expression by the developing cell. The same caveats hold true for all systems heretofore studied, no matter how "clean" they are thought to be.
The pro tein components of mRNP's a re thought to perform th re e potential functions: i) to protect the mRNA from nuclease attack, ii) to transport the mRNA from the nucleus via the nuclear pores onto the cytoplasmic face of the rough endoplasmic reticulum membrane, and i l l ) to exert regulation of gene expression by facilitating or denying entry of the mRNA into an initiation complex with a 40S ribosomal subunit. Regulation might also be effected by establishing whether th e i n i t i a t i o n complex w ill be made w ith a f r e e or membrane-bound ribosome—a level of control that may precede expression of a signal peptide (Blobel, 1977). Roles i) and ii) are all but certainties; role iii) is more speculative at this time.
Physicochemical Properties of RNP's from Animal CellB
Nearly all of the existing literature on the subject of RNP's deals with these structures as they are isolated from animal cells.
Very little information exists with regard to RNP's from protozoa,
plants, or fungi. The main reason for this has to do with the
relative ease of rupturing animal cells and releasing the internal
structures relatively undamaged and unchanged. As suggested in the
previous section, RNP's are quite differentiated with respect to the
subcellular fraction from which they are isolated. It has not proven
easy to recover undamaged nuclei, polyribosomes or membrane-bounded
internal organelles from cells with a thick, rigid outer wall. If
these cell fractions can be recovered following osmotic rupture of
protoplasts generated with wall lytic enzymes, they are often
substantially altered during the long period of incubation under
conditions certainly s tre ssfu l and probably damaging to the c e ll. The
RNP's housed in these various cellular locations are highly dynamic
structures with the RNA content in particular being subjected to
excisions, splicings, chemical modifications, translation and
degradation, the complete sequence of which can occur on a smaller
time scale than can digestion of the cell wall. Furthermore, as we
find even in work with animal systems, the mRNA sequestered within
RNP’s loses much of its protection and becomes highly susceptible to nucleolytic cleavage once the cell is broken open or severely
perturbed physiologically.
That the eukaryotic cell has a high degree of internal
organization and is not just a membrane bounded solution of freely
diffusible molecules (i.e., a "bag of enzymes") is a lesson constantly underscored by the latest observations. Apparently mRNA experiences a profoundly changing but strictly defined millleu of structural 31
proteins, enzymes, and other molecules as its odyssey through the cell
unfolds. Exposure of BNP’s to Inappropriate nucleases displaced from
their proper cytological niche in the course of cell disruption seems
to inevitably nick their RNA. Even in animal systems, it is generally
the exception to be able to isolate intact, in vitro-transl.atable mRNA
from purified native RNP’s. Nor can one recover translatable mRNA
from purified mRNP's fixed with formaldehyde or glutaraldehyde as is
done to recover the stru ctu res in CsCl gradients (S pirin, 1969; Martin
et al., 1980). Most of our knowledge of mRNA processing in eukaryotes
was gained using RNA samples phenol extracted directly from whole
cells or isolated nuclei, not from RNP's (Martin et al., 1980).
For the reasons elucidated above, most analytical studies of
animal RNP's have focused not upon th e ir RNA component but mainly upon
their protein composition, shape, size, bouyant density and other
physicochemical properties. The literature in these regards is truly
a jungle of numbers. The variability of most measured parameters is
high both within a single system and among different systems. This
great variability of reported results undoubtedly accounts for the
disparity one often notes in textbook descriptions of RNP's (Thorpe,
1984; Becker, 1986; Lewin, 1984; Sheeler and Bianchi, 1983). However,
if one gives credence to the hypothesis that the protein components of
RNP's may serve to regulate expression of the mRNA component of these
organelles, one might expect to see a significant heterogeneity in the
protein make-up and other corresponding properties of the particles.
After all, even a structure as universal in its function and organization as the ribosome shows noteworthy diversity between eukaryotic species and apparent heterogeneity within a species 32
(Bielka, 1982).
In spite of the wide variance in data reported, a generalized picture of RNP's from animal systems has been quite convincingly
•presented (Martin et al., 1980). There are several distinct classes of RNP's, most of which were less formally alluded to e a rlie r in th is dissertation. The easily recognized classes are: i) HnRNP's, ii)
SnRNP's, iii) Spliceosomes, iv) poly(A)NP's, v) cmRNP' s (free cytoplasm ic mRNP's), and v i) pmRNP' s (polyribosom al mRNP's). The first three forms of RNP's exist solely in the nucleus whereas the latter two inhabit only the cytoplasm. Based on the previous regulatory arguments, one should expect to see heterogeneity within the latter two of these particles and, in fact, diversity is observed within both the RNA and protein components of all these structures found in a given organism. An additional reason for heterogeneity may be accounted for by the fact that HnRNP's are eventually converted to cmRNP's and pmRNP's and intermediate forms may persist long enough to turn up in purified RNP preparations.
As related in the previous section, HnRNP's represent the first incarnation of mRNA, being formed within the nucleus as tra n s c r ip tio n yet proceeds. These structures vary from about 30S to 45S in size
(Martin et al., 1980). Variations in the literature values of the sedimentation coefficients of these structures are reportedly not merely the result of imprecise measurements. Simply pelleting and resuspending HnRNP's from mouse tumor cells alters their sedimentation c o e f f ic ie n t from 34S to 40-45S (Martin and McCarthy, 1972; B illin g s,
1979). Mild ribonuclease treatment also, paradoxically, increases the value of this parameter (Stevenin et al., 1976; 1977). HnRNP's are classically isolated in CsCl gradients following fixation with an
aldehyde. They display bouyant densities in CsCl of 1.39-to-1.43
gm/cc and co n tain 75-80% p ro te in and 20-25% RNA by one estim ate
(S pirin, 1969) and 85-90% protein by other estim ates (Hamilton, 1971;
Billings and Martin, 1978). Attempts to recover these structures in density gradients of the non-ionic, non-denaturing solute metrizamide have given mixed results, including particle dissociation (Karn et al., 1977; Gattonl et al., 1977). As isolated, these complexes are thought to contain 700-1000 nucleotides of RNA and approximately 10^ daltons of protein, however, there is sometimes great departure from these v a lu es. The length of RNA isolated from HnRNP's may sometimes approach 1000 nucleotides (Samarina et a l., 1968) although it is u s u a lly much sm aller, commonly on the order of 50-100 nucleotides
(Kinniburgh and Martin, 1976).
Evidence suggests that the 30-45S particles may be identical subunits of a much larger aggregate structure with a sedimentation coefficient on the order of 200S (Martin et al., 1980). Although it cannot be recovered intact, it is thought that the RNA in these massive structures represents the HnRNA (pre-raRNA) before excision of introns and splicing of exons. The polyadenylate tails are not found in this structure but apparently reside within the nucleus in separate
15S RNP particles [poly(A)NP' s ] until they are ligated to the mRNA just prior to its export from the nucleus. A very small percentage of
HnRNA's ever receive poly(A) tails so HnRNP's cannot be recovered by binding to oligo(dT)-cellulose.
Although HnRNA cannot be recovered intact from isolated HnRNP's there is plenty of indirect evidence to conclude that the HnRNA extracted directly from nuclei with phenol does reside in the HnRNP’s.
It is similar to cellular DNA in base composition and hybridizes to homologous DNA in a manner sim ilar to pulse-labelled HnRNA (Parsons and McCarthy, 1968). The kinetics of sy n th e sis and tu rn o v er of the pulse-labelled RNA of 30-45S HnRNP's are consistent with values obtained for HnRNA (Martin and McCarthy, 1972). Hybridization-
competltlon experiments show that pulse-labelled RNA of 30-45S HnRNP's co n tain nucleus re s tric te d sequences (Martin and McCarthy, 1972). In fact, experiments in which 30-45S HnRNP-RNA has been hybridized with cDNA transcribed from cytoplasmic polyadenylated mRNA suggest that only 5-10% of HnRNP-RNA is homologous to cytoplasmic mRNA but that a ll mRNA sequences are represented in nuclear HnRNP's (Kinniburgh and
Martin, 1976). These results are consistent with the hypothesis that most of the HnRNA is sequestered in HnRNP's but only a small percentage is processed to mRNA. Labelling of HnRNP's with radioactive RNA precursors is prevented by inhibitors of HnRNA synthesis, such as a-amanitin (Stunnenberg et al., 1978), but not by
Inhibitors of rRNA synthesis, such as actinomycin D at low concentration (Pederson, 1974).
The protein composition of HnRNP's is highly variable depending upon the biological source and displays heterogeneity within a single source evidenced by the ratios of specific proteins. From 4 to 12 polypeptides in the size range of 35,000- to 40,000-daltons are generally associated with 30-45S HnRNP's (Martin et al., 1979;
B illings and Martin, 1978; Karn et a l ., 1977). The m ajority of these proteins are mildly basic and have pi's in the range of 8-9 (Billings,
1979). The relative stoichiometry of these proteins varies from 35
tissue to tissue and with the physiological state of the tissue
(Billings, 1979). Amino acid analyses of these proteins show that
they are all relatively high in glycine and low in cysteine. Unusual
amino acids are present, particularly dimethylarginine (Billings and
Martin, 1978). It has been suggested that these proteins may be from
a closely related gene family and may play a structural role similar
to hlstones in the nucleosome (Martin et al., 1980). Proteins
isolated from larger HnRNP aggregates resolve into 50 or more bands of
10.000- to 200,000-daltons upon sodium dodecylsulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) (Pederson, 1974). Tissue-specific
patterns are observed; however, contamination with extraneous proteins
is feared because of the way the HnRNP*s are prepared (Martin et al.,
1980). The poly(A)NP's are reported to contain five major proteins of
57.000-, 63,000-, 86,000-, 120,000-, and 140,000-daltons (Billings and
M artin, 1978) or two polypeptides of 74,000- and 86,000-daltons (Kish and Pederson, 1977) depending upon the study.
Based upon immunochemical studies, it can be said that the proteins of HnRNP’s are never found in the cytoplasm. The cmRNP's and pmRNP's do not cross react with antisera raised against proteins from
HnRNP * s (Martin et a l., 1979). Thus there appears to be a complete exhange of RNP proteins at some point during the maturation of mRNA or its transport through the nuclear membrane.
Electron microscopic observations have been made of RNP's on both sides of the nuclear membrane and in the process of traversing the membrane through the nuclear pores. There is a clear change in the ultrastructure of these particles upon making this journey. In the insect Chironomus tentans transcripts of the Balbiani ring (BR) genes 36 are packed into a series of well-defined ribonucleoprotein structures of increasing complexity: a 10-nm fiber, a 19-nm fiber, a 26-nm fiber and a 50-nm granule. The basic 10-nm element is revealed in "Miller spreads." The in situ structure of the transcription products and RNA compaction estimates suggested that the 10-nm fiber is packed into the
19-nm fiber as a tight coil. The transition of the 19-nm fiber into the 26-nm fiber is accompanied by a major change of the basic 10-nm fold into a non-coiled structure. Finally, the 26-nm fiber makes a one and one-third left-handed turn forming the final product, the BR granule. Upon translocation through the nuclear pore the BR granule becomes rod-shaped, which most lik e ly corresponds to a re la x a tio n of the highest-order structure into a straight 26-nm fiber (Skoglund et al., 1983). More recently this three dimensional structure has been corroborated by means of electron microscope tomography (Skoglund et al., 1986). The twist in the highest level structure ends up bringing the 5' end and 3’ ends of the HnRNA into close proximity.
SnRNP's, as mentioned earlier, are nuclear RNP's containing small pieces of RNA (4-8S in size) that base pair with nucleotide sequences flanking introns on the HnRNA creating so-called "lariat" structures
(Padgett et al., 1984). Endonucleolytic cleavages excise the intron- containing lariats and the remaining exons are ligated together.
SnRNP's are approximately 10-12S in size (Busch et a l., 1982a,b).
They contain proteins that have not yet been characterized and several classes of well-characterized SnRNA's. Six of these classes are designated Ul through U6 because they are high in uridylic acid residues. They also possess a 5' cap containing 3-methylguanosine triphosphate in a 5'-5' linkage. Each class displays chain length 37 variability, the longest example being 214 and the shortest 107 nucleotides in length. About 10 other classes are not capped and not enriched in uridylic acid residues. (7S)SnRNA is the largest of these at 295 nucleotides (Busch et a l ., 1982a,b).
When the SnRNA in an SnRNP base pairs with the HnRNA in a 30-453
HnRNP to form a lariat for the purpose of intron excision, the hybrid
60S particle is called a spliceosome (Brody and Abelson, 1985;
Pikielny and Rosbash, 1986). Internal secondary structures of the
RNA's in both SnRNP’s and HnRNP's are apparently important for proper base pairing and at least one has been proposed in the case of human
U5-SnRNA (Chabot et al., 1985). The proteins in the spliceosome appear to be equally important to bring about the correct cuts and s p lic e s of pre-mRNA. Ln v itr o s p lic in g of an raRNA p recu rso r was inhibited by a monoclonal antibody to the "C proteins" of the HnRNP's.
This antibody, 4F4, inhibited cleavage at the 3' end of the upstream exon and formation of the intron la r ia t. However, i t did not prevent the ATP-dependent formation of the 60S spliceosome. The "C proteins" remained present in such structures, but if removed from HnRNP's by immunoabsorption with 4F4 splicing activity was lost (Choi et a l.,
1986).
Mature eukaryotic mRNA is found only in mRNP's situated in the cell cytoplasm. As indicated earlier, these may be found in polyribosomes in which case the mRNA is being actively translated into protein and the structure^ are referred to as pmRNP's. Alternatively, the particles may be found free in the cytoplasm, always on the internal side (never on the lumenal side) of the rough endoplasmic reticulum membrane, ln which case they are called cmRNP’s. Genetic or 38
regulatory distinctions that may exist between the two populations of mRNP's are still largely a matter of speculation. It has been opined
that craRNP's are not merely mRNP's caught in transit between the
nucleus and some rlbosomal subunit depot where they will be converted
to pmRNP's lickety-split, but may sequester messages whose translation
is initiated at a lower efficiency by design or by fortuity (Martin et
a l., 1980; Adams et a l ., 1981).
cmRNP' s were first discovered by Spirin and his co-workers in early stages of developing fish embryos, before rRNA synthesis is
initiated, and were referred to as "informosomes" (Spirin, 1969).
These structures sedimented at 20-75S in sucrose gradients (Spirin,
1969), although some cmRNP's have recently been reported as large as
120S In size (Bag and Sarkar, 1976). After fixation with formaldehyde or glutaraldehyde they were determined to have bouyant densities between those of free protein and free ENA and distinct from those of ribosomes or ribosomal subunits. These structures appear to be ubiquitous in embryos and eggs of numerous vertebrate and Invertebrate
species and appear to represent stored genetic Information of maternal o rig in . The devices by which th is stored mRNA Is "masked" u n til the appropriate stages of development are of considerable interest to developmental biologists and are thought to somehow involve the proteins of the cmRNP's.
cmRNP's have since been discovered in a large number of fully differentiated tissues from a variety of organisms including human
(Perry and Kelley, 1968; Henshaw and Loebenstein, 1970; Spohr et a l . ,
1970). Since It became clear that most mRNA molecules are polyadenylated, poly(A)+RNA has been reported in a ll cmRNP fractions 39
examined (Ouellette et al., 1976; Perry and Kelley, 1976). The
fraction of total cytoplasmic poly(A)+RNA found in cmRNP's, as opposed
to pmRNP's, varies from 15% to 30% (Martin et a l ., 1979b; McMullen et
al., 1979). Specific mRNA's have been detected in cmRNP's by means of
in vitro translation in some earlier experiments (Olsen et al., 1975)
or, more recently, by hybridization with cDNA probes (Sinclair and
Dixon, 1982).
By 1980, four cmRNP's containing specific mRNA's had been
partially purified (Martin et al., 1980). Three of these (carrying
mRNA encoding duck globin, chicken actin and trout protamine) were
isolated on the basis of their very small size and one (carrying mRNA
coding for chicken myosin) on the basis of its very large size (120S).
The proteins of these cmRNP's were analyzed by SDS-PAGE and found to
be quite different in each case. The number of proteins per cmRNP
varied from two to eight and the molecular weights of the proteins
varied from 15,000- to 98,000-daltons. Many other studies have
analyzed the proteins from total unfractionated cmRNP populations
collected by adherence to poly(U)-sepharose or oligo(dT)-cellulose
affinity columns. Using this method, Barrieux et al. (1975) reported
three major polypeptides of 34,000-, 52,000-, and 78,000-daltons along with four other minor proteins in Ehrlich ascites cmRNP's. In
contrast, Jeffery (1978) found proteins of 56,000-, 67,000-, 71,000-,
and 81,000-daltons in both cmRNP's and pmRNP's of E h rlic h a s c ite s
cells. For comparative purposes, results of other recent studies have
revealed 1) seven major proteins of 23,500- to 87,000-daltons in cmRNP's of b rin e shrimp (S ieg ers et a l . , 1981); i i ) e ig h t major proteins of 43,000- to 150,000-daltons in cmRNP’s of trout testes 40
(Sinclair and Dixon, 1982); iii) 15-20 major proteins of 40,000- to
100,000-daltons and 15-23 minor proteins of 22,000- to 190,000-daltons
in cmRNP's of sea urchin eggs (Moon et a l . , 1980); and iv) ten major
proteins of 40,000- to 100,000-daltons in cmRNP's of chicken muscle
(Jain and Sarkar, 1979). Among these results it is significant that
proteins of approximately 52,000- and 78,000-daltons are always found.
These are also always found in pmRNP's (Jain and Sarkar, 1979;
Jeffery , 1978) and th e ir putative role w ill be discussed below.
Also displayed in this introduction for later comparative purposes is a table including recently measured sedimentation
c o e f f ic ie n ts and bouyant d e n s itie s of cmRNP's from various animal
systems (Table 1). mRNA recovered from cmRNP's has been reported to
display a heterodisperse size range from 8-14S (Siegers and Kondo,
1977) to 8-30S (Jain and Sarkar, 1979).
pmRNP's although having some features in common with cmRNP's are quite distinguishable from them. They are released from polyribosomes by treatment with GDTA. They contain both poly(A)"*RNA and protein.
These structures vary in size from approximately 14S to greater than
80S. Their bouyant densities in CsCl range from 1.35-to-1.55 gm/cc, suggesting a protein content of greater than 50%. Isolated pmRNP's have been successfully translated in vitro. Isolated cmRNP's, in contrast, have some block or defect which has heretofore prevented t h e ir in v itr o tr a n s la tio n ( S in c la ir and Dixon, 1982; Liautard and
Egly, 1980). The poly(A)+RNA isolated from pmRNP's of Ehrlich ascites tumor cells shows exactly the same size distribution (4-18S) as poly(A)+RNA from cmRNP's of the same source, and in vitro translation can be carried out on either template (Barrieux et a l. , 1975). 41
Table 1: Sedimentation C oefficients (S) and Bouyant D ensities (p) of cmRNP's from Various Animal Systems
Organism S p (CsCl) p(Metrizamide) P(Sucrose) Reference
Brine Shrimp 5-30 1.38-1 .40 1.26-1.27 (1)
Brine Shrimp 20-30 1.39 1.205 1.27-1.28 (2)
Trout Testes 14 1.35-1,.37 - - (3)
Sea Urchin Eggs 60-65 1.38-1,.47 - - (4)
Sea Urchin Eggs 60-70 1.38-1,.47 1.22 - (5)
Chicken Muscle 20-80 1.41-1,.43 1.205-1.22 (6)
Bouyant densities are expressed in terms of gm/cc.
References (See "Literature Cited" for complete citations):
(1) Siegers et al., 1981
(2) Siegers and Kondo, 1977
(3) Sinclair and Dixon, 1982
(4) Kaumeyer et a l . , 1978
(5) Moon et a l . , 1980
(6) Jain and Sarkar, 1979 However, the former mRNP particles can direct in vitro translation whereas the latter cannot (Barrieux et al., 1975). Protein analysis reveals that the cmRNP*s contain many more proteins than pmRNP’s
(Barrieux et al., 1975). The pmRNP's display three Intense protein bands on SDS-PAGE gels of 34,000-, 52,000-, and 78,000-daltons.
Several minor bands are also visible on the gels. The cmRNP's display these same three major bands plus several other major bands.
Studies of pmRNP’s from a large number of organisms have consistently noted proteins of approximately 52,000- and
78.000-daltons (Morel et al., 1971; Blobel, 1972; Bryan and Hayashi,
1973; Burns and Williamson, 1975). Lindberg and Sundquist (1974) reported that EDTA-released pmRNP's from KB cells isolated by oligo (dT)-cellulose affinity chromatography contain four major proteins of 56,000-, 68,000-, 78,000- and 130,000-daltons. pmRNP's from adenovirus-infected KB cells contain an additional protein of
110.000-daltons. The oligo(dT)-cellulose method has been used to isolate pmRNP's from many cell types for protein analysis including:
HeLa (Kumar and Pederson, 1975), KB cells (Van der Marel et a l.,
1975), Ehrlich ascites cells (Jeffery, 1977) and mouse kidney (Irwin et al., 1975). Although these investigators have reported different numbers of major proteins (three to six) and a variety of molecular weight distributions, the 52,000- and 78,000-dalton proteins are in v a ria n t.
It is thought that the 78,000-dalton protein binds the polyadenylic acid tail on the 3* end of the mRNA (Jeffery and
Brawerman, 1974; Adams et a l . , 1981; Jain and Sarkar, 1979; Siegers et al., 1981). This function holds true for both cmRNP's and pmRNP's, 43
explaining why the 78,000-dalton protein is found in both. An
interesting observation of unknown significance at this time is that
the length of the polyadenyllc acid tail on mRNA's decreases from
about 65 residues in cmRNP's to approximately 15 residues in pmRNP's
(Adams et al., 1981). Certainly many interesting questions remain unanswered about all of the various particles in which mRNA finds
itself domiciled at one time or another.
Physicochemical Properties of RHP's from Fungus Cells
The literature on fungal RNP's is very scant, encompassing a
couple of papers on the acellular slime mold Physarum (Adams et al.,
1980; 1981), one on the cellular slime mold Dictyostelium (F irtel and
Pederson, 1975), one on the water mold Blastocladiella (Jaworski and
Stumhofer, 1981), and one on the ascomycete Neurospora (Mirkes, 1977).
The role of SnRNP's in HnRNA splicing has been investigated in a few studies on Saccharomyces (Brody and Abelson, 1985; Pikielny and
Rosbash, 1986).
Physarum polycephalum grows vegetatively as a plasmodium which is essentially a multinucleate mass of protoplasm lacking a rigid cell w a ll and bounded by only a ty p ic a l plasma membrane. The plasmodium can be easily lysed and fractionated into nuclear and cytoplasmic components. cmRNP's and pmRNP's have been isolated from the cytoplasmic fraction in Jeffery's laboratory (Adams et al., 1980;
1981). The polyribosomal fractio n released RNP's upon EDTA treatment that sedimented at 18-45S with a mean of about 25S whereas the non— polyribosomal fraction contained structures sedimenting between 18-60S with a mean of about 40S. RNA derived from these structures was heterodisperse in size, ranging from about 8S to 30S with a mean value
of perhaps 20S. After RNase treatment the resistant poly(A)NP
sedimented at 10-15S. A protein analysis of these particles was not performed. However, i t was determ ined th a t the cmRNP's contained poly(A) tails 65 residues in length whereas the pmRNP's contained poly(A) tails only 15 residues long.
Dictyostelium discoideum which grows vegetatively as an amoeba
lacking a rigid cell wall was lysed and nuclei were isolated. HnRNP's were purified from the nuclei (Firtel and Pederson, 1975). These structures were somewhat larger than the corresponding particles from animal cells, displaying a sedimentation coefficient of 55S. No large aggregates, such as occur in animal cells, were recovered. RNA within the HnRNP's was polyadenylated, homologous with mRNA (based upon hybridization kinetics), and sedimented at a mean value of approximately 15S. From 16-20 major proteins ranging in size from
15,000- to 150,000-daltons appear to be associated with the HnRNP's.
One protein with a molecular weight of 72-74,000-daltons was found to be associated with the poly(A) tails of the RNA component.
The oomycete Biastocladiella emersonii constructs a dormant structure called a zoospore in which protein synthesis does not take place until germination because the ribosomes are segregated from the mRNA in a specialized structure called the nuclear cap. When germination commences a flagellum is formed on the zoospore and the ribosomes initiate translation from a pool of pre-formed stored polyadeny lated RNA (Lovett, 1975). Jaworski has characterized the poly(A)+RNA population before and after germination (Jaworski, 1976;
Jaworski and Thomson, 1980) and also the RNP's in which they are sequestered (Jaworski and Stumhofer, 1981). Most of the poly(A)+RNA
in Blastocladiella zoospores had been reported in particles which
sediment at just greater than 80S in sucrose gradients (Johnson et
al., 1977). mRNP's were isolated on metrizamide gradients and
identified by binding to poly(U)-fliters (Jaworski and Stumhofer,
1981). They displayed a bouyant density of 1.27 gm/cc in metrizamide gradients and had a size range of 20-80S on sucrose gradients.
Lighter particles were found to contain different proportions of the
protein components identified on SDS-PAGE gels than heavier particles.
The five major protein components had molecular weights of 42,000-,
56.000-, 64,000-, 105,000-, and 120,000-daltons. Heavier cmRNP's have more of the 120,000-dalton protein than do lighter particles. The
105.000-dalton protein is mostly absent from pmRNP's.
pmRNP's were isolated from vegetative hyphae of Neurospora crassa freeze-fractured by grinding in a mortar and pestle under liquid nitrogen (Mirkes, 1977). This procedure liberates intact polyribosomes which were collected on sucrose step gradients. The polyribosomes were treated with EDTA to liberate pmRNP's which were collected by binding to and elution from an ollgo(dT)-cellulose column. Increasing amounts of the hydrogen bond-disrupter formamide were included in the elution buffer to dissociate the poly(A) tails of the pmRNP-RNA's from the oligo(dT)-cellulose. The isolated pmRNP complexes exhibited sedimentation coefficients ranging from 15S to greater than 60S. RNA isolated from these structures sedimented in sucrose gradients between 4S and 40S, w ith broad peaks a t 15S and 24S.
The bouyant density of pmRNP's eluted with 25% formamide was 1.42-1.44 gm/cc, whereas for pmRNP's eluted with 50% formamide i t was 1 .4 8 -1 .5 0 gm/cc. Six polypeptides, with molecular weights of 14,000-, 19,000-,
24,000-, 31,000-, 44,000-, and 66,000-daltons, were associated with pmRNP's eluted with 25% formamide. The pmRNP’s eluted with 50% formamide had but one associated protein of 27,000-daltons. All of the protists mentioned in this section are phylogenetically quite distant from Mucor, however, Neurospora is probably most closely re la ted . MATERIALS AND METHODS
Organism and Cultivation
Mucor racemosus (M. lusitanlcus, 11. circinelloldes) ATCC 1216B
was used ln all experiments. Stock cultures were maintained on a
solid growth medium (YPG agar) composed of 2% (wt/vol) glucose, 1%
(wt/vol) peptone, 0.3% (wt/vol) yeast extract, and 3% (wt/vol) agar.
Dehydrated media components were obtained from Difco Laboratories,
Detroit, MI. The medium was adjusted to pH 4.5 with concentrated
sulfuric acid. A small amount of aerial hyphae containing
sporangiospores was inoculated with a standard bacteriological loop
onto the center of YFG agar plates (100-mm diameter) which were
incubated in a ir at room tem perature (approxim ately 22°C) for two weeks, at which time stock cultures were transferred to fresh medium.
SporangioBpore Production
Sporangiospores were produced in amounts suitable for biochemical
fractionation in sterile pyrex or Corning Ware baking dishes 2 (approximately 500 cm surface area) containing 250 ml of YPG agar or 2 in standard Petri plates (78.5 cm surface area) containing 20 ml of
YPG agar. A standard inoculum was prepared by adding 5 ml of sterile
distilled water to a week-old stock culture of M. racemosus. Scraping
the submerged mycelial surface with a s te r ile bent glass rod re le ase d
the sporangiospores from their sporangia into suspension, leaving the hyphae and sporanglophores still firmly attached to the agar surface.
A small quantity of this standard spore suspension (50 yul per pyrex
dish or 20 yil per Petri plate) was evenly distributed over the agar 48
surface using a sterile glass rod or was streaked accross the agar
surface in closely spaced (2 cm apart) parallel rows. The cultures
were incubated in air at room temperature for a period of 7 to 10 days
at which time the agar surface was completely covered with aerial
hyphae bearing sporangia containing the black sporangiospores to be
used as experimental material.
The spore-containing dishes or Petri plates were quickly flooded
with an aqueous solution of sodium azide (10 mM), sodium fluoride (10
mM), and cycloheximide (1 mg/ml). This solution of inhibitors had
previously been shown to immediately stop all energy metabolism and macromolecular synthesis in Mucor sporangiospores (Linz and Orlowski,
1982). Sporangiospores were released into suspension with a glass rod
as described above, sedimented at 10,000 xg for 10 min at 4°C in a
S o rv a ll RC2B r e f r ig e r a te d cen trifu g e with a GSA ro to r, washed twice with the above-mentioned inhibitor solution, and collected by vacuum filtration onto cellulose acetate/cellulose nitrate membrane discs
(Millipore Corp., Boston, MA; Type AA; lQO-mm diameter, 0.8-jim pore size). Suction was continued until a semi-dry cake with the consistency of thick tar was obtained. This dry spore cake was immediately frozen with liquid nitrogen and stored at -70°C until further use.
Breakage of Sporangiospores
Frozen sporangiospores were retrieved from storage at -70°C and placed into a pre-chilled porcelain mortar that had been sterilized by baking at 110°C for 24-48 hours. The mortar was filled with liquid nitrogen (temperature, -196°C) and the spores ground without abrasive using a sterile porcelain pestle. Vigorous grinding was carried out
for 10-15 min, replenishing the liquid nitrogen when necessary. After
grinding, residual liquid nitrogen was allowed to evaporate off and
the broken spores were scooped from the mortar with a sterile piece of
stiff but flexible paper (Millipore filter spacers work optimally).
The broken spores were immediately placed into cold (0°C) sterile
buffer of the appropriate composition (see below).
Isolation of mHHP's from Sporangiospores
Sporangiospores broken under liquid nitrogen, as described above,
were resuspended in 10-15 ml of buffer H, containing 20 mM
N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 500 mM
KC1, 10 mM magnesium acetate (MgAc), 1 mM phenylmethanesulfonyl
fluoride (PMSF), 1 mM disodium ethylenedlaminetetraacetate (Na 2 EDTA),
0.01% (wt/vol) heparin and 2.0% (vol/vol) Triton X-100 at pH 7.2. The
broken spore suspension was placed into a sterile 30-ml Corex
centrifuge tube (Corning Glass Works, Corning, NY) pre-coated with a
silicon film (Sigma-Cote; Sigma Chemical Co., St. Louis, M0) to retard
adherence of nucleic acids to the glass surface. The suspension was
thoroughly homogenized with a Dounce homogenizer (Wheaton S cien ctific,
Millville, NJ). The extreme viscosity of the homogenate, indicating
the presence of much DNA, suggested that most of the spore nuclei were
probably ruptured. The homogenate was centrifuged at 12,000 xg for 10
min at 4°C in a Sorvall RC2B refrigerated centrifuge equipped with an
SS-34 rotor. The pellet containing mostly unbroken spores, spore walls, and mitochondria, was discarded. The supernatant solution,
containing the spore cytoplasm and nucleoplasm, was retained. 50
The supernatant solution was layered above a one-ml shelf of
s t e r i l e b u ffe r HS, containing 20 mM Hepes, 500 mM KC1, 10 mM MgAc, 1
mM PMSF, 1 mM Na2 EDTA, 0.01% heparin and 1 M sucrose at pH 7.2, in
5-ml ultraclear centrifuge tubes (Beckman Instruments Inc., Palo Alto,
CA). The biphasic suspensions were centrifuged at 150,000 xg for 90 min at 4°C in a Beckman Model L-50 preparative ultracentrifuge equipped with an SW50.1 rotor. Particles substantially smaller than
30S rlbosomal subunits but not as small as 4S tRNA molecules should be pelleted under these conditions. The supernatant fractions were discarded. Material transiting the shelf and forming a pellet on the bottom of the tubes was collected. The pellets were resuspended in ste rile buffer HI, containing 20 mM Hepes, 500 mM KC1, 10 mM MgAc, 1
mM PMSF, 1 mM Na2 EDTA and 0.01% heparin at pH 7.2, and resedimented under the previously described conditions. The second pellet was suspended in sterile buffer HI and incubated with 0.5 gm (for analytical purposes) or 2.5 gm (for preparative purposes) of oligo(dT)-cellulose (Sigma) overnight at 4°C with constant stirring.
Poly(U)-sepharose 4B (Sigma) was used in place of oligo(dT)-cellulose for comparative purposes in a few runs. No differences in results were noted.
The oligo(dT)-cellulose slurry was poured into a 0.7 cm x 10 cm
(in analytical runs) or 2.5 cm x 20 cm (in preparative runs) sterile
Econo-column chrom atography column (Bio-Rad L aboratories, Richmond,
CA) and allowed to settle by gravity. The initial supernatant buffer
HI was allowed to percolate through the oligo(dT)-cellulose packing and was discarded upon elution from the column. In analytical runs,
20 ml of s t e r i l e b u ffe r HI were p e rc o la te d through the column and 20-drop (1-ml) fractions were collected into sterile 13-mm x 100-mm disposable test tubes using a Gilson Model FC-80 Microfractionator
(Gilson Medical Electronics, Middleton, WI). This was followed by 10 ml each of s te r ile buffer H2, composed of 20 mM Hepes, 1 mM PMSF, 1 mM
Na2 EDTA and 0.01% heparin at pH 7.2; s te r ile buffer H3, composed of 20
mM Hepes, 1 mM PMSF, 1 mM Na2 EDTA, 0.01% heparin and 50% (vol/vol) deionized formamide at pH 7.2; sterile buffer H4, containing 20 mM
Hepes, 1 mM PMSF, 1 mM Na2 EDTA, 0.01% heparin and 90% (vol/vol) deionized formamide at pH 7.2; and sterile 0.01 N NaOH. In each case
20-drop (1-ml) fractions were collected. Much greater volumes (50-60 ml) of sterile buffers H2 and H3 were used in the preparative isolation of mRNP's for further analyses. Just prior to use, formamide was autoclaved and deionized with Amberlite MB-1 ion exchange resin (Rohm and Haas Co., Philadelphia, PA).
Portions (100-1000 fi 1) of each fraction were assayed for 3 incorporated radioactivity, protein content or [ H]poly(U)-binding activity as described elsewhere in this section. In preparative runs, fractions containing a significant amount of mRNP material were pooled. For density gradient analyses (see below), the pooled fractions in buffer H2 were used as collected. The pooled fractions in buffer H3 (volume approximately 10-12 ml) were dialyzed versus 2 one-liter volumes of sterile buffer H2 at 4°C for 12 hours. Most density gradient centrifugation procedures (see below) were performed in buffer H2. For protein analyses (see below), the pooled fractions in either buffer H2 or H3 were centrifuged at 150,000 xg for 12-24 hours at 4°C. Pellets of mRNP material were resuspended in appropriate volumes of electrophoresis sample buffer (see below), 52
before addition of 2-mercaptoethanol, and assayed for protein by the modified Folin-phenol procedure (Lowry et a l., 1951) of Linz and
Orlowski (1984).
Radioisotopic Labelling of Sporangiospores
Aerobic hyphae of M. racemosus were grown and sporangiospores allowed to develop in the presence of [ 32 Pjphosphoric acid, which radioactively labels primarily nucleic acids including the RNA component of RNP's, or L-[ 35 S]methionine, which radioactively labels cell proteins including the protein fraction of RNP's. Twenty-five 32 mCi of carrier-free [ P]phosphoric acid (ICN, Irvine, CA; specific radioactivity: 27 ,930 Ci/mmol), suspended in 2.5 ml of sterile distilled water, were distributed in 50-^il aliquots onto the agar surface of each of 50 standard Petri plates (100-mm diameter) of YPG agar. The radioisotope (0.5 mCi/plate) was uniformly distributed accross the agar surface with a disposable sterile plastic spreader
(West Coast Scientific, Inc., Emeryville, CA) . Each plate was inoculated at the center with 5 yul of a standard spore suspension (see above) in sterile distilled water. Alternatively, 1 mCi of 35 L-[ S]methionine (New England Nuclear; specific radioactivity: 400
Ci/mmol), suspended in 0.5 ml of sterile distilled water, was uniformly spread from 50-jil aliquots upon the agar surface of 10 YPG agar plates (100-mm diameter) by means of a sterile disposable plastic spreader. These plates were inoculated with a drop of standard spore suspension at their center.
Hyphal growth and spore formation were allowed to proceed in air at room temperature for 7-to-10 days. Growth and morphogenesis 53
occurred at a normal rate and with the formation of normal cell types
in spite of the high radioactivity. The plates were each sealed with
tape and placed into p la stic boxes which in turn were stored within a
plexiglass hood and behind additional plexiglass shields throughout
the duration of the incubation period. Radioactive spores were
collected within the sealed hood by flooding each plate with
approximately 5 ml of the previously described azide-fluoride-
cycloheximlde inhibitor solution and dislodging the spores from their
sporangia with a disposable plastic spreader. After the
sporangiospores of 50 plates were pooled, they were pelleted by
centrifugation, washed and packed into a semi-dry cake by vacuum
filtration as described above. The semi-dry spore mass was
immediately frozen under liquid nitrogen and stored until further use
or the spores were broken immediately by grinding them under liquid
nitrogen with a mortar and pestle.
It is essential to keep these highly radioactive materials under
containment. Aerosols are significant sources of contamination during
spore collection from plates and spattering disperses much radioactivity during grinding under liquid nitrogen. Therefore these procedures were carried out within the sealed plexiglass hood referred to above. Potential contamination was constantly monitored with a hand-held Geiger-Muller counter (Mini-Instruments, Ltd, Essex,
England). Whenever working with radioactivity laboratory personnel always wore lab coats, two thicknesses of vinyl plastic gloves, partlcle-fliter masks, safety glasses and Landauer personal radiation dosimetry badges and rings provided by the LSU Radiation Safety O ffice. 54
3 [ H]Polyuridylic A d d Hybridization to Polyadeny lated RNA
mRNP’s, which contain mRNA in the form of poly(A)+RNA, were
identified on the basis of the binding of their RNA component to 3 [ H] polyuridylic acid [poly(U)]. The experimental protocol consisted
of three major steps: i) recovery of RNA from appropriate column or 3 density gradient fractions, ii) hybridization of [ HJpoly(U) with polytA^RNA, and iii) detection and quantitation of poly(U)-poly(A) hybrids.
i) Extraction of RNA fron column or gradient fractions. This procedure was based upon that of Siegers and Kondo (1977). Eight-drop
(400-yil) fractions were collected directly into sterile 1.5-ml microcentrifuge tubes containing 200 ul of 6% (wt/vol) SDS. The tubes were agitated with a vortex mixer, then incubated at room temperature for 15 min. Three hundred yil of water-saturated phenol were added to each tube, and the layers were mixed using a vortex mixer. The tubes were incubated on ice (0°C) for 5 min, after which 300 yul of chloroform were added to each and mixing was again performed with a vortex mixer. These homogenates were centrifuged for 5 min at room temperature in an Eppendorf Model 5414 microcentrifuge (Eppendorf,
Hamburg, West Germany). Five hundred yul of the upper aqueous phase 3 were collected from each tube for hybridization with [ H]poly(U).
ii) [^H]Poly(U) hybridization with poly(A)+RNA. Five hundred yul of sterile distilled water were added to the 500 of the aqueous 3 upper phase from the previous step. Fifty nCi of [5,6- HJsodium polyuridylate (New England Nuclear; specific radioactivity: 3.2
Ci/mmol UMP) in 10 yil of sterile distilled water were added to this solution which was heated to 65°C for 10 min. Annealing of poly(U) 55
with poly(A) sequences was allowed to proceed for 14 hours at 30°C.
I l l ) D etection and quantitation of poly(U)-poly(A) hybrids. The
incubation mixtures were chilled on ice (0°C) and ribonuclease A,
which hydrolyzes single-stranded but no-t double-stranded RNA, was
added to each at a final concentration of 25 yig/ml (10 of a
2.5-mg/ml solution). Incubation was continued at 0°C for 30 min after
which 1 ml of ice-cold 10% (wt/vol) trichloroacetic acid (5%, final
concentration) was added to each mixture to precipitate non-hydrolyzed
RNA. The mixtures were kept on ice for 20 min, after which
precipitated RNA was collected on cellulose acetate/cellulose nitrate
membrane discs (Millipore; Type HAWP; 25-mm diameter; 0.45-yim pores)
by vacuum f il tr a tio n . Each f i l t e r disc was washed three times with 1
ml of ice-cold 10% (wt/vol) trichloroacetic acid followed by three
washes with 1 ml of ice-cold 95% ethanol. The discs were dried under
a heat lamp, placed into the liquid scintillation counting solution
described elsewhere in this section, and assayed for radioactivity
with a Beckman Model LS-200 liquid scintillation spectrometer.
Assay of Incorporated Radioisotopes
When appropriate, radioactive protein was precipitated in hot
(90°C) 5% (wt/vol) trichloroacetic acid for 20 min or radioactive nucleic acid was precipitated in cold (0°C) 5% (wt/vol)
trichloroacetic acid for 20 min. Either kind of sample was collected on cellulose acetate/cellulose nitrate membrane filters (M illipore, type HAWP) by vacuum filtration after chilling on ice for 10-15 min.
The filters were washed with 3 one-ml volumes of ice-cold 5% (wt/vol) trichloroacetic acid and 3 one-ml volumes of ice-cold 95% ethanol. 56
They were dried under a heat lamp and placed into 5 ml of a scin tillation counting solution composed of 0.4% (wt/vol)
2,5-diphenyloxazole (Sigma), 0.01% (wt/vol) 2,2'-p-phenylenebis-
(5-phenyloxazole) (Sigma), and scintillation grade toluene (J.T.
Baker, Phillipsburg, NJ) and contained within 20-ml disposable polypropylene vials (Wheaton Scientific, Millville, NJ). The vials were assayed for radioactivity in a Beckman Model LS-200 or a Beckman
Model LS-6800 scintillation spectrometer. This procedure was followed 32 35 to determine the fate of P- and S-labelled material in analytical 3 oligo(dT)-cellulose affinity chromatography and in [ H]poly(U)- + poly(A) RNA hybridizations.
In most density gradient runs the gradient fractions were collected directly into 20 ml of the aqueous scintillation counting solution of Miller and Sypherd (1973) or into 20 ml of Aquasol-2 (New
England Nuclear). In preparative oligo(dT)-cellulose column runs 32 (with P as label), the bottom of each 13-mm x 100-mm test tube containing an eluted fraction was held in contact with the protective screen covering the thin mica window of the Geiger-Muller counter probe. The large surface-area, thin-window probe (Mini-Instruments
Model 5-10EL) used in this work gives a highly sensitive quantitative response for emission energies down to the level of ^C-beta emissions
(Product Catalogue, Research Products International Corp., Mount
Prospect, IL). Radioactivity levels, expressed as counts per second, were read d irectly from the display meter and recorded. 57
Sedimentation Telocity (Rate Zonal) Centrifugation in Sucrose Density
Gradients
This method was used to i) follow the cellular distribution of poly(A)+RNA during sporangiospore germination, ii) determine the sedimentation coefficient distribution of isolated mRNP's, iii) characterize the material not retained by oligo(dT)-cellulose at high ionic strength, iv) determine the sedimentation coefficient distribution of RNA molecules extracted from mRNP's, and v) isolate
40S ribosomal subunits and SOS complete ribosomes for comparison with mRNP's. The essential features of each experimental protocol are described in turn below. In general, the procedures were much the same, with the exceptions noted.
i) Dormant sporangiospores and sporangiospores germinated for 10 min in liquid YPG medium were exposed to the azide-fluoride- cycloheximide inhibitor solution described above. The spores were collected on membrane filters (M illipore; type AA) by vacuum filtration, washed with the inhibitor solution, and broken by grinding under liquid nitrogen as previously described. The broken spores were suspended in sterile buffer HI containing cycloheximide (250 yjg/ml).
The suspension was centrifuged at 15,000 xg for 10 min at 4°C. The supernatant liquid was collected and measured for absorbance at 260 nm in a Beckman Model 35 UV-Vis spectrophotometer. A volume of the supernatant fraction containing 8 ^ 6 0 u n its was la y ered on top of
11.0-ml linear 10-to-40% (wt/vol) sucrose density gradients buffered with HI and resting on 0.8-ml 2-M sucrose cushions. The gradients were centrifuged at 150,000 xg for 100 min at 4°C in a Beckman Model
L2-65B ultracentrifuge equipped with an SW41 rotor. Gradients were 58
scanned at 254 nm with an ISCO Model 640 density-gradient fractionator
(Instrumentation Specialties Co., Lincoln, NE). Concomitant with
scanning, 8-drop fractions were collected with a Gilson Model FC-80
M icrofractionator. RNA was recovered from each fraction by
phenol/chloroform extraction and incubated with [ H]poly(U) as
described above. Ribonuclease A-refractory poly(U)-poly(A) duplexes + indicate the presence of poly(A) RNA (mRNA).
i i ) Fractions eluted from oligo(dT )-cellulose columns co n tain in g
mRNP's in buffer H2 were pooled and assayed for radioactivity. A
volume of eluate containing a minimum of 250,000 counts per min (CPM)
was applied to the top of 10-to-40% (wt/vol) linear sucrose density
gradients buffered in H2. A volume of non-radioactive Mucor
sporangiospore extract (15,000 xg supernatant fraction) containing 8
^260 un*ts was a3-80 applied to the gradients as a source of size markers. The gradients were centrifuged at 77,000 xg for 14 hours at
4°C in a Beckman Model L8-70M u ltra c e n trifu g e equipped w ith an SW41
rotor. Gradients were scanned at 254 nm as above. Fractions (6
drops) were assayed for radioactivity directly in aqueous
scintillation counting solution as described above. 32 iii) P-Labelled cellular material not binding to oligo(dT)-
cellulose at high ionic strength was analyzed on sucrose density
gradients exactly as above. A Mucor sporangiospore extract (15,000-xg
supernatant fraction) in buffer HI was passed through an oligo(dT)-
cellulose column several times to remove any polyadenylated m a te ria l.
A volume of eluate containing a minimum of 250,000 CPM was applied to
10-to-40% (wt/vol) linear sucrose density gradients buffered in HI.
Non-radioactive markers were included as above. Centrifugation, 59 absorbance measurement, fractionation and radioactivity assay were performed as before.
iv) RNA was extracted from mRNP's eluted in buffer H2 as indicated below. The RNA was redissolved in buffer ANE (10 mM sodium acetate, 100 mM NaCl, and 1 mM Na2EDTA at pH 5.3) containing 50%
(vol/vol) formamide as a denaturant. A volume of this solution containing 3 A ^ q units was layered on top of 5-to-20% (wt/vol) linear sucrose density gradients also in ANE with 50% formamide.
Centrifugation was carried out at 77,000 xg for 18 hours at 4°C in a
Beckman Model L8-70M ultracen trifu g e equipped with an SW41 ro to r. The gradients were scanned for absorbance at 254 nm and fractionated as described above. Radioactivity was measured in each fraction, as described elsewhere in this section, after precipitation of RNA in cold trichloroacetic acid and collection of the precipitates on membrane f i l t e r s .
v) For preparation of 40S ribosomal subunits, a 27,000-xg supernatant fraction of broken Mucor hyphae was prepared in buffer TMK
(50 mM Tris-[hydroxymethyl]aminomethane (Tris)-HCl, 10 mM MgAc, and
500 mM KC1 at pH 7.2). In order to stimulate dissociation of 80S monoribosomes and polyribosomes to 60S and 40S ribosomal subunits, 0.1 mM puromycin-HCl was added to the e x tr a c t, which was then incubated for 20 min at room temperature. Approximately 4 ml of extract containing 200 A ^ q units was layered onto each of six 33-ral 10-to-40%
(wt/vol) linear sucrose density gradients buffered in TMK and resting on 2-ml 2-M sucrose cushions. The gradients were centrifuged at
100,000 xg fo r 9 hours at 4°C in a Beckman Model L8-70M u l t r a centrifuge equipped with an SW28 rotor. Fractions (12 drops) were 60
collected from the bottoms of the tubes using a Beckman Fraction
Recovery System and a Gilson Model FC-80 M icrofractionator. The A 2 ^q
of each fraction was measured with a Beckman Model 35 UV-Vis
spectrophotometer. Fractions determined to contain 40S ribosomal subunits were pooled, diluted with TMK buffer, and centrifuged - on
sucrose gradients again under the identical conditions. The gradients were again fractionated and the 40S ribosomal material was identified and pooled. The 40S ribosomal subunits were sedimented at 150,000 xg for 12-24 hours at 4°C in a Beckman Model L-50 ultracentrifuge equipped with an SW50.1 ro to r, washed once with buffer H2, sedim ented again under identical conditions, and resuspended finally in buffer
H2. Thorough resuspension required the use of a Dounce homogenizer.
The 40S ribosomal subunits were assayed for protein by the method of
Lowry et al (1951) and stored frozen (-20°C) in H2 until used in
SDS-PAGE analyses. Preparation of SOS ribosomes was carried out in a similar fashion, except that puromycin was excluded from the system and a minute quantity ribonuclease A was included in order to convert all polyribosomes to 80S monoribosomes by cutting strands of mRNA lying unprotected between the ribosomes.
Sedimentation Equilibrium (Isopycnic) Centrifugation in CsCl Gradients
mRNP's e lu te d from oligo(dT )-cellulose columns in buffers H2 and
H3, and 40S ribosomal subunits purified from sucrose gradients, were analyzed on CsCl gradients to determine their bouyant densities.
Buffer H2 served as the suspending medium in all such centrifugation runs. Buffer H3 eluates were first extensively dialyzed against buffer H2 (10 ml versus 2 x 1 liter) before use in CsCl 61
centrifugation. Ribosomal subunits were resuspended in H2 after
sedimentation from TMK (see above). Prior to introduction into CsCl
solutions all samples were fixed for 30 min with 6% (vol/vol) aqueous
formaldehyde (formalin) which had been neutralized to pH 7.0 with 1 N
NaHCOg. A minimum of 200,000 CPM of 32P-labelled material was loaded
per gradient. For most runs, the CsCl solution was made up to an
in itial uniform density of 1.4226 gm/cc, which developed into the mid-point value of the gradient upon attainment of equilibrium. The
appropriate weight of CsCl (5.69 gm) per volume of water (10 cc) to
achieve this density was obtained from the Handbook of Chemistry and
Physics. 64th edition (Weast, 1984). This volume was divided between
two 5-ml polyallomer or ultraclear centrifuge tubes (Beckman). The
gradients were defined by centrifugation at 200,000 xg for 40-48 hours at 20°C in a Beckman L-50 or L8-70M ultracentrifuge equipped with an
SW50.1 ro to r.
Fractions (6 drops) were collected from the bottoms of the tubes using a Beckman Fraction Recovery System and a Gilson Model FC-80
Microfractionator. Densities were determined gravimetrically by measuring the weight of 20 ^il (+/- 0.5%) of solution in tared capillary pipettes (Kimble, Toledo, OH) on a Mettler Model H20T analytical balance (Mettler Instrument Corp., Hightstown, NJ).
Radioactivity was measured as described above after mixing each fraction with 20 ml of aqueous scintillation fluid in a plastic scintiallation vial. In some runs, non-radioactive 40S ribosomal subunits were recovered by side puncture of the tube at the site of a visible band and the bouyant density was determined by measuring the weight of the first 20 ^il of liquid drawn from this site of puncture 62
by a tared capillary pipette.
Sedimentation Equilibrium (laopycnic) Centrifugation in Metrizamide
Gradients
mRNP’s and 40S ribosomal subunits from the same sources as
indicated in the previous protocol were analyzed on metrizamide 32 gradients. At least 200,000 CPM. of P-labelled material were loaded
into each pre-gradient mixture. In all runs, the sample (contained in
buffer H2) was homogeneously suspended in 40% (wt/vol) metrizamide
(final concentration) dissolved in buffer H2. The 10-ml total volume was divided into two 5-ml u ltra c le a r centrifuge tubes (Beckman). The
gradients were formed by centrifugation at 200,000 xg for 48 hours at
20°C in a Beckman L-50 or L8-70M u ltr a c e n tr if u g e equipped with an
SW50.1 rotor.
Fractions were collected by bottom puncture of tubes and
densities determined gravimetrically as in the case of CsCl gradients.
In most runs, particles were located by measuring their radioactivity as in the case of CsCl gradients. In some runs, non-radioactive 40S
ribosomal subunits were detected by means of the Bradford assay for protein (Bradford, 1976). In other runs, non-radioactive 40S ribosomal subunits were recovered by side puncture and their bouyant density determined by measuring the weight of a known volume (20 yjl) of the accompanying liquid.
Protein Assays
Protein in mRNP's and other cell fractions of M. racemosus sporangiospores was detected and quantitated by i) measuring the 63
absorbance at 280 nm, ii) performance of the Bradford assay, which is
based upon the alteration in the absorption spectrum of the basic dye
Coomassie Blue R-250 when it binds protein (Bradford, 1976), iii)
performance of the Folin-phenol assay (Lowry et al., 1951) or iv)
performance of the Folin-phenol assay after precipitation of protein
with trichloroacetic acid (Linz and Orlowski, 1984).
Sodium Dodecylsulfate-Polyacrylamlde Gel Electrophoresis (SDS—PAGE)
The conditions used for SDS-PAGE were essentially those of
Laemmli (1970) applied in a S tu d ier-ty p e a p p aratu s (CBS S c i e n t i f i c ,
Del Mar, CA). The sample was heated for 3 min at 100°C in a sample
buffer composed of 2.5% (vol/vol) 2-mercaptoethanol, 1% (w t/v o l) SDS,
25 mM Tris-HCl (pH 7.0), and 10% (wt/vol) sucrose. This solution,
containing 125 ^jg of protein, was applied to the top of a stacking gel
(2 mm in width, 2 cm in length) containing 4% (wt/vol) acrylamide,
0.1% (wt/vol) methylenebisacrylamide, 160 mM Tris-HCl (pH 7.0), and
0.007% SDS. The separating gel (2 mm in width, 8 cm in length) contained a linear 8-17% (wt/vol) acrylamide (0.21-0.45% bisacrylamide) gradient, 0.1% SDS and 0.375 M Tris-HCl (pH 8.8). The electrode buffer contained 0.1% SDS and 25 mM Tris-glycine (pH 8.2).
The electric current was held constant at 15 mA while the sample m igrated through the stacking gel, and a t 25 mA u n til the Bromphenol
Blue tracking dye left the separating gel. The temperature was ambient and the run time approximately four hours. The gels were fixed and stained overnight in a solution of 0.05% (wt/vol) Coomassie
B rilliant Blue R-250 (Sigma) in methanol/acetic acid/water (5/4/1 by volume). The gels were destained in a solution of methanol/acetic 64 acid/water (10/7/83 by volume).
Photography
Coomassie Blue-stained polyacrylamide gels from electrophoretic protein separations were photographed in transmitted light through a
Cokin #29A orange filter (Paris, France) on Polaroid type 55
Positive/Negative film using a Polaroid MP-3 industrial camera. The negatives were treated with 12% (wt/vol) sodium sulfite, washed with water, rinsed with appropriately diluted Photo-flo 200 solution
(Kodak, Rochester, NY), and dried in a ir . Kodak P o ly c o n tra st Rapid
II photographic paper was exposed to the negative images through a
Durst Laborator S-45 enlarger (Bolzano-Bozen, Italy), developed by immersion for 1 min in a 50% aqueous Dektol solution (Kodak), passed through a Kodak Stop Bath for 5 sec, fixed for 5 min in half-strength
Kodak Rapid Fixer, washed in flowing water for 30 min, dried in air overnight, and pressed with a Fotoflat dry mount press (Derby, CT).
Photographs of Mucor spores and vegetative cells were taken on
Kodak Plus-X pan black and white film (ASA 125) under a Leitz Ortholux
I p h a se -c o n tra st m icroscope (W etzlar, West Germany). The film was developed in Kodak D-19 developer, fixed in Kodak Rapid Fixer, washed and dried. Positive prints were made on Kodak Polycontrast Rapid II
RC photographic paper as described above. Composite plates were composed, rephotographed on Polaroid type 55 P/N film as previously described, and printed on Kodak paper as related above.
Purification and Analysis of iJW* from rnRHP's 32 Ten ml of pooled P-labelled mRNP-containing fractions in buffer H2 collected from a preparative oligo(dT)-cellulose column were
subjected to phenol extraction and ethanol precipitation as follows.
Purified unlabelled Mucor RNA (8 A^q units) was added to the pooled
fractions as a carrier. An equal volume of redistilled phenol
containing 0.1% (wt/vol) 8-hydroxyquinoline and saturated with buffer
H5 (20 mM Hepes and 1 mM Na 2 EDTA at pH 7.2) was added to the pooled
fractions along with SDS at a final concentration of 1% (wt/vol). The
biphasic suspension was mixed thoroughly but gently with a sterile
spatula to avoid shearing mRNA. The mixture was allowed to stand on
ice for 10-15 min after which it was centrifuged at 10,000 xg for
10 min at 4°C to separate the aqueous and phenol layers. The upper
aqueous phase was carefully collected with a sterile pipette and
subjected to re-extraction with phenol twice more as described above and once with a chloroform/isoamyl alcohol (25/1 by volume) mixture.
Ammonium formate was added to the final aqueous phase at a final
concentration of 0.3 M followed by two volumes of cold absolute
ethanol. After mixing by stirring with a sterile spatula, RNA was precipitated from the mixture at -20°C overnight. The precipitated
RNA was collected by centrifugation at 30,000 xg for 20 min at 4°C.
The residual alcohol was removed using a V irtis Freezemobile-6 lyophilizer (Virtis Corp., Gardiner, NY). The visually-imperceptible pellet was resuspended in ANE buffer for sucrose density gradient analysis.
A volume of the solution of purified RNA containing 3 A ^ q u n its was brought to 50% (vol/vol) formamide, heated to 55°C for 10 min and layered on top of a 5-20% (wt/vol) linear sucrose density gradient
(11.0-ml volume) which was made up in a solution of 50% (vol/vol) formamide in ANE buffer and which rested on a 0.8-ml cushion of 2 M
sucrose dissolved in the same solution. The gradients were
centrifuged at 77,000 xg for 18 hours at 4°C in a Beckman Model L8-70M ultracentrifuge equipped with an SW41 rotor. The gradients were scanned at 254 nm using an 1SC0 Model .640 density gradient fractionator equipped with a Model UA-5 absorbance monitor (ISCO,
Lincoln, NE). Fractions (6 drops) were collected into one ml of ice-cold 10% (wt/vol) trichloroacetic acid, incubated on ice for 30 min, filtered, washed, and assayed for radioactivity as described above. RESULTS
Location of Fre-forned ntKHA in Dormant and Germinating Sporangiospores
Dormant sporangiospores of M. racemosus contained no poly
ribosomes and only a small amount of potentially active 80S ribosomes .
Most ribosomal material identified on scans of sucrose density
gradients was in the form of inactive 40S and 60S ribosom al sub u n its
(Fig. 4A, dotted line). When fractions of such gradients were assayed
for poly(A)+RNA (putative mRNA) by hybridization with [^H]poly(U),
large amounts of message were observed, n early a l l of i t in a form
smaller than 80S in size. Most of this appeared to be in the region
of 30S-to-70S on the gradient, with peaks at about 40S and 55S (Fig.
4A, solid line). When dormancy of M. racemosus sporangiospores was broken by exposure to liquid YFG medium for no more than 5-10 min, m ost of th e rib o s o m a l p a r tic le s w ith in the c e ll moved in to 80S monoribosomes and polyribosomes (Fig. 4B, dotted lin e ). Accompanying 3 th is change was a m ig ratio n of most of the [ H]poly(U)-hybridizable material (mRNA) into the same regions of the sucrose density gradients
(Fig. 4B, solid line). The simplest interpretation of these data would be that the dormant spore contains a large cache of stable poly(A)+RNA, not associated with active ribosomes, which is immediately mobilized for translation by polyribosomes upon hydration of the spore and resumption of active metabolism. This is consistent
With the findings of Linz and Orlowski (1982, 1984, 1986) that stored mRNA is immediately translated upon Initiation of germination.
It is significant that hardly any of the poly(A)+RNA was found associated with the small 80S ribosome peak. The pre-formed mRNA is
67 3 + Figure 4. [ H]Poly(U) hybridization to poly(A) RNA from Mucor
racemosus sporangiospore extracts fractionated by sucrose density
gradient centrifugation. Centrifugation of an initial spore-free
15,000-xg supernatant fraction was for 100 min at 150,000 xg through a
10-to-40% linear sucrose gradient. Fractions were collected from the gradient and concomitantly scanned at 254 nm. Each fraction was 3 phenol extracted and the RNA recovered was incubated with [ H]poly(U).
After destroying single stranded RNA with RNase A, each incubation mixture was passed over a cellulose nitrate filter. Radioactivity in double-stranded RNA adhering to the filter was assayed by liquid scintillation spectroscopy. Specific details of each procedure are described in the text. A) Extract from dormant sporangiospores.
B) Extract from spores exposed to nutrient medium for 10 min.
Symbols: ( ...... ), ^Radioactivity trapped on filter, as CPM (counts per minute). 0.8 0.7* -7 0.6 62 c0.5 6 0 S 60S s|
0.3 ,80S 0.2 0.1
FRACTION NUMBER
VO 70 not stored in a state of translational arrest on complete ribosomes.
Neither is it stored free of associated macromolecules, most likely proteins. The size range of poly(A)+RNA-containing material suggested by the rate of sedimentation in sucrose gradients significantly exceeds that determined for naked poly(A)+RNA (6S-20S) by Linz and
Orlowski (1982). The data in the present experiment could not establish whether the pre-formed mRNA existed in translation in itia tio n complexes with 40S ribosom al su b u n its or in fre e RNP's.
The following experiments resolve this issue and characterize the form in which poly(A)+RNA is stored in dormant sporangiospores.
Isolation of Poly (A)+RNA—containing RHP's with 01igo(dT)-cellulose
Columns
Subcellular particles in the same general size range as previously characterized RNP's were c o lle c te d by c e n tr ifu g a tio n and fractionated on oligo(dT)-cellulose columns as described in the methods section. The spores from which these structures were recovered had been radloactively labelled with [ P]orthophosphate32 to 35 identify nucleic acid or with L-[ S]methionine to identify protein.
Preparation from spores not isotopically labelled were used in 3 [ H]poly(U) hybridization studies or in identification of proteinaceous material by absorbance at 280 nm. 32 Figure 5 shows that most of the P-labelled m aterial, which was eluted in buffer HI, did not bind to oligo(dT)-cellulose at high ionic strength. This implies the absence of poly(A)-containing material in these fractions. The absence of poly(A) sequences was corroborated in the experiment depicted in Fig. 8. Very little, if any, of the material eluting at high ionic strength in buffer HI contained an RNA 3 component that hybridized with [ H]poly(U). These results are
entirely reasonable and, in fact, expected. The great bulk of 32 P-labelled (i.e., essentially nucleic acid-containing) material in
any cell iB the ribosome fraction. All ribosomal particles are in the size range of structures collected here and no ribosomal particles c o n ta in poly (A) sequences. Most of the m aterial in a ll preparations initially loaded on oligo(dT)-cellulose columns represents ribosomes.
Since no deoxyribonuclease treatment was carried out at any time, some of this material may also represent DNA which pelleted in the form of nucleosomes. DNA does not carry significant poly (A) sequences and would not bind oligo(dT)-cellulose. The amount of DNA in most cells is generally no more than 10% of the total RNA content. Since most of the RNA in HnRNP’s from characterized systems is non-polyadenylated, one would predict that HnRNP's may also be found in this non-binding fraction, at a small percentage of the total.
Four additional peaks of 32 P-labelled material are observed elu tin g from the oligo(dT )-cellulose column in Fig. 5. The f i r s t and largest peak was eluted in buffer H2, representing a dramatic reduction in ionic strength which causes most hydrogen bonds between poly(A) and oligo(dT) sequences to be weakened and the duplexes to 32 fall apart. A further amount of P-labelled material was released in buffer H3 which contains 50% (vol/vol) formamide. This substance further disrupts hydrogen-bonding in both nucleic acids and proteins causing denaturation of both types of molecules. The extent of denaturation is a function of the concentration of formamide, with
100% formamide often employed in gel electrophoresis or density 72
gradient separation demanding complete denaturation (Rickwood and
Hames, 1982). Increase of the formamide concentration up to 90%
(vol/vol) in buffer H4 released yet more 32 P-labelled material but
never enough to analyze fu rth er. Most secondary, te rtia ry , and
quartenary structure should be abolished at this concentration. One 32 might have expected all P-labelled RNA to be released from the
column and, in fact, dissociated from its protein component under
these conditions. However, a rather sizeable amount of 32 P-labelled material was yet released from the column when all RNA was finally hydrolyzed and eluted with NaOH. This is commonly observed (Lindberg
and Sundquist, 1974; Mlrkes, 1977) and may be a consequence of nucleic acid molecules getting trapped or tangled among packed cellulose fibers. The NaOH-eluted material could not, of course, be further analyzed.
The material eluted by buffers H2, H3, and H4 all contained + 3 poly(A) RNA based upon hybridization of [ H]poly(U) to the RNA
extracted from each fraction (Fig. 8 ). The extent of hybridization 32 was approximately proportional to the quantity of P (=[mRNA])
recoverable in each fraction (compare Fig. 5 and Fig. 8 ). 35 Figure 6 displays the content of L-[ SJmethionine (“[protein]) in material fractionated on oligo(dT)-cellulose columns. The distribution is somewhat similar to that previously presented in the case of RNA (Fig. 5). Again, most material did not bind the column at high ionic strength in buffer HI. This undoubtedly represents the protein fraction of ribosomes, nucleosomes and HnRNP's. As before, four peaks were observed eluting in buffers H2, H3, and H4 and in
NaOH. The ratios of the protein peaks eluted in buffers H2 and H3 73
Figure 5. Analytical oligo(dT)-cellulose affinity chromatography 32 of P-labelled particulate fraction from Mucor racemosus sporangiospore extract. The preparation was applied to the column in buffer HI and followed with the several elution buffers indicated on the figure. Fractions were collected and assayed for radioactivity by liquid scintillation spectroscopy. Buffer compositions and other experimental particulars are described in the text. Abbreviation:
(CPM), radioactivity as counts per minute. 74
NaOH
0 10 20 30 40£ SO ] 60 FRACTION NUMBER Figure 6 . Analytical oligo(dT)-cellulose affinity chromatography
of L-[ 35 S]methionine-labelled particulate fraction from Mu cor
racemosus sporangiospore extract. The preparation was applied to the
column in buffer HI and followed with the several elution buffers
Indicated on the figure. Fractions were collected and assayed for radioactivity by liquid scintillation spectroscopy. Buffer compositions and other experimental particulars are described in the
text. Abbreviation: (CPM), radioactivity as counts per minute. CM
CPM x 10 n 160 40 80 20 30 10 H1 0 0 50 0 4 0 3 20 10 RCIN NUMBER FRACTION H2 H3 i J H4 ------NaOH NaOH I i -I— ► I J 60 __ 77
Figure 7. Analytical oligo(dT)-cellulose affinity chromatography
of non-radioactive particulate fraction from Mucor racemosus
sporangiospore extract. The preparation was applied to the column in
buffer HI and followed with the several elution buffers indicated on
the figure. Fractions were collected and their absorbance at 280 nm was assayed with a spectrophotometer. Buffer compositions and other
experimental particulars are described in the text. 280 0.04- 0 0 . . 18 2 - - 0 0 5 0 4 0 3 0 2 10 RCIN NUMBER FRACTION 1 NaOH 78 79
3 + Figure 8 . [ H]Poly(U) hybridization to poly(A) RNA-containing p a rtic le s from Mucor racemosus sporanglospore extracts eluted from an analytical oligo(dX)-cellulose column by a standard sequence of buffers. Affinity chromatography was performed as indicated in
Fig. 5. RNA hybridization was performed as indicated in Fig. 4. See text for experimental details. Abbreviation: (CPM), radioactivity as counts per minute. 10 CPM x 103 11 5 6 3-H1 9 4 7 8
10 RCIN NUMBER FRACTION H2 20 30 40 50 l l l l 60
80 were similar to those for the previously depicted RNA. peaks. However, much less protelnaceous material was eluted in buffer H4 and in NaOH.
Similar observations were made when protein was monitored by measuring absorbance at 280 nit, except that the amount of material released from
the column by NaOH was again higher. The low recovery of protein in buffer H4 may signify that most proteins were removed with eluted
RNP's and were totally stripped from non-eluted RNA's in buffer H3. A comparison of proteins from mRNP's eluted in buffer H2 versus buffer
H3 presented later in this study will bear out that several proteins were dissociated from RNA in the presence of formamide (see below).
Sodium hydroxide may solubilize or hydrolyze proteins trapped in the cellulose matrix.
Figures 5 through8 display the results of analytical runs on oligo(dT)-cellulose columns. The analytical fractionation of labelled cellular material on poly(U)-sepharose columns yielded results indistinguishable from these (not shown). These analytical runs employed re la tiv e ly small amounts of column packing (0.5 gm) and relatively limited amounts of cellular material (<3 &26Q un*ts containing approximately 500,000 CPM), well below the binding capacity of the column. Eluate in buffer HI contained little, if any, labelled material that bound to the column upon re-passage, establishing that binding capacity was not exceeded. Figures 9 and 10 display the results of preparative runs on oligo(dT)-cellulose columns. These employed much greater amounts of oligo(dT)-cellulose (2.5 gm) and cellular material (about 50 A£gQ units containing approximately
7 10 CPM) for fractionation. The loaded column was extensively washed with buffer HI (>100 ml) to maximize removal of non—polyadenylated 82
Figure 9. Preparative oligo(dT)-ce1lulose affinity 32 chromatography of P-labelled particulate fraction from Mucor
racemosus sporangiospore extract: H2-eluted mRNP 1 s. The standard
particulate fraction was applied to a large capacity column in
buffer HI. After extensive washing of the column with buffer HI,
poly(A) RNA-containing material was eluted from the column with
buffer H2. Fractions were collected, assayed for radioactivity with a high-sensitivity Gelger-Muller counter, and pooled if containing
substantial radioactivity. This material represents what is referred
to in the text as particles (mRNP's) eluted at low ionic strength.
Buffer compositions and other experimental details are described in
the text. Abbreviation: (CPS), radioactivity expressed as counts per second. 83
POOLED
H2-ELUTED mRNP's 70*
60
50
O l 40
30
10
10 20 30 40 50 60 FRACTION NUMBER 84
Figure 10. Preparative oil go(dT)-ce1lulose affinity
chromatography of 3 2 P-labelled particulate fraction from Mucor
racemosus sporanglospore extract: H3-eluted mRNP's. The standard
particulate fraction was applied to a large capacity column in
buffer Hi. After extensive washing of the column first with buffer HI, then with buffer H2, poly(A) RNA-containing material was eluted from the column with buffer H3 containing 50% formamide.
Fractions were collected, assayed for radioactivity with a high- sensitivity Geiger-Muller counter, and pooled if containing substantial radioactivity. This material represents what is referred to in the text as formamide-eluted particles (mRNP’s). Buffer
compositions and other experimental details are described in the text.
Abbreviation: (CPS), radioactivity expressed as counts per second. 85
H3-ELUTED mRNP's 70
60
50
Q. 40 POOLED
30
20
10
0 10 20 3040 50 60 FRACTION NUMBER material. Eluate in buffer HI was not re-passed over the column to
ascertain loading capacity, but was discarded. Approximately 50 ml of buffer H2 was passed over the column, eluting substantial amounts of
labelled material in about 1 0 one-ml fractions which were pooled for
further analyses (Fig. 9). Another 50 ml of buffer H3 was passed over
the column, eluting labelled material in about 1 0 one-ml fractions, in somewhat lesser amounts than the previous material (Fig. 10). These fractions were pooled for further analyses. Such a small amount of labelled material was eluted in buffer H4 that this preparative procedure could not produce enough for further analyses.
Size Distribution of mRHP's by Sedimentation Telocity Centrifugation
Figure 11 shows the sedimentation coefficient distribution in a 32 sucrose gradient of purified P-labelled mRNP's eluted from an oligo(dT)-cellulose column in low ionic strength buffer H2. The presence of p articles varying in size from about 20S to 80S, with a mean value of approximately 55S, was indicated. Figure 12 displays the sedimentation distribution in a sucrose gradient of purified 32 P-labelled mRNP’s eluted from an oligo(dT)-cellulose column in buffer H3 at low ionic strength with 50% (vol/vol) formamide. A different size distribution of particles was displayed, varying from about 20S to 60S, with a mean value of approximately 40S. Taken together, the two purified mRNP fractions appeared to encompass all of 3 the [ H]poly(U)-binding material depicted in Fig. 4, with the exception of the low molecular weight material at the top of these gradients. This material would be too small to sediment in the in i t i a l preparative step of mRNP iso latio n . Although one can only 87
speculate at this time, this material may represent the poly(A)NP's
characterized as 10-15S in other systems (Martin et al, 1980; Adams et
al, 1980, 1981). In light of data to be presented later in this work
dealing with the protein compositions and bouyant densities of the
separately isolated mRNP populations, it becomes clear that the
H3-eluted material lacks a population of mRNP's present in the
H2-eluted preparation. A comparison of Figs. 11 and 12 will show that
the H3-eluted preparation (Fig. 12) contained mostly lighter
(approximately 20-50S) particles and was missing a population of
heavier (approximately 50-80S) particles, whereas the H2-eluted
preparation (Fig. 11) contained both light and heavy populations.
Figure 13 displays the sedimentation profile in a sucrose 32 gradient of the P-labelled material that did not bind to oligo(dT)-
cellulose at high ionic strength in buffer HI. This material lacked
poly (A) sequences and did not bind to oligo(dT)-cellulose even after
several passes over the column. In this experiment, soluble m aterial
was not removed from the extract by centrifugation prior to oligo(dT)-
cellulose chromatography, so tRNA's and poly(A)NP's remained in the
preparation. The distribution of material here, whether monitored as
^254 or as ^ncorPorate^ radioactivity (cold trichloroacectic acid- precipitable CPM), indicated only the presence of 4-5S tRNA's, 40S and
60S ribosomal subunits, and 80S ribosomes. All mRNP's and poly(A)NP's were apparently absorbed on the column and there was no trace of them
evident here (assuming they could be detected against the high
background of the non-polyadenylated nucleic acid). If HnRNP' s or nucleosomes were present in any sizeable quantity in the non-binding 32 fraction of P-labelled material, they are not apparent as discrete 88
Figure 11. Analysis of Mucor dormant sporangiospore mRNP’s
eluted from oligo (dT)-cellulose column by buffer H2 (low ionic
strength) by sedimentation velocity centrifugation. Distribution of
32P -labelled m aterial pooled from preparative column on a sucrose density gradient. Centrifugation was at 77,000 xg for 14 hours through a 10-to-40% linear sucrose gradient in a Beckman SW41 rotor.
Fractions were collected and concomitantly scanned at 254nm.
Radioactivity in each fraction was quantified by liquid scintillation spectroscopy. Specific experimental protocols are provided in the
text. Symbols: ( ...... ), A2 5 4 of RNA size standards; (——— — ), radioactivity expressed as CPM (counts per minute). 89
0.8 5S A 0.6 6 0 T“ 60S 5 X 80S,] 40S 3o 0.2
10 20 30 40 50 60 FRACTION NUMBER 90
Figure 12. Analysis of Mucor dormant sporangiospore mRNP's eluted from oligo(dT)-cellulose column by buffer H3 (50% formamide) by 32 sedimentation velocity centrifugation. Distribution of P-labelled material pooled from preparative column on a sucrose density gradient.
Centrifugation was at 77,000 xg for 14 hours through a lO-to-40% linear sucrose gradient in a Beckman SW41 rotor. Fractions were collected and concomitantly scanned at 254nm. Radioactivity in each fraction was quantified by liquid scintillation spectroscopy.
Specific experimental protocols are provided in the text. Symbols:
( ...... } A * 254 of RNA size standards; ( ------), radioactivity expressed as CPM (counts per minute). 91
Mrl
CM 0 .4 * CPM CPM x 1Q3
FRACTION NUMBER 92
Figure 13. Analysis of material from Mu cor dormant
sporangiospore extracts not binding to oligo(dT)-*cellulose at high
ionic strength by means of sedimentation velocity centrifugation. The 32 distribution of non-binding P-labelled material is shown on a
sucrose density gradient. Centrifugation was at 77,000 xg for 14
hours through a 10-to-40% linear sucrose gradient in a Beckman SW41
rotor. Fractions were collected and concomitantly scanned at 254nm.
Radioactivity in each fraction was quantified by liquid scintillation
spectroscopy. Specific experimental protocols are provided in the
te x t. Symbols: ( ...... ), ^Radioactivity
expressed as CPM (counts per minute). 93
0.8
0.7 5S 0.8 E0.5 60S c So.4 80S CM < 0 . 3 40S 0.2
0.1
FRACTION NUMBER entities on this figure. If they were present they could be concealed within the AOS peak.
Isolation of AOS Ribosomal Subunits
Figures 14 and 15 i l l u s tr a te the purification of AOS ribosomal subunits for analysis and comparison with isolated mRNP's. Figure 1A represents a sucrose density gradient fractionation of a Mu cor cell-free extract (27,000-xg supernatant fraction). The most
outstanding features are A 2 0 Q peaks of A-5S tRNA's, AOS and 60S ribosomal subunits, and 80S ribosomes. It should be noted that, unlike mRNP profiles on sucrose gradients, the AOS ribosomal subunit peak was highly compact making i t rath er u n lik e ly th a t the AOS ribosomal particle represents the true identity of mRNP's, although it cannot be excluded on this basis that mRNP's might not be modified forms of AOS ribosomes which have gained or lo st sp ecific pro tein s.
When the fractions containing AOS material were pooled and re-run on another sucrose gradient a single sharp peak was obtained (Fig. 15).
This was collected and the AOS ribosomal subunits recovered by centrifugation.
Bouyant Densities of mRNP's by Sedimentation Equilibrium
Centrifugation in CsCl Solution
Figures 16 through 18 represent the behaviour of mRNP fractions and AOS ribosomal subunits in CsCl isopycnic centrifugation. Figure
16 demonstrates that the H2 (low ionic strength) oligo(dT)-cellulose column eluate contained two populations of mRNP particles, one with a bouyant density of about 1.37 gm/cc and another with a bouyant density 95
Figure 14. Purification of AOS ribosomal subunits by sucrose density gradient centrifugation: initial gradient separation of components in 15,000-xg supernatant fraction of Mucor hyphae. Hyphal supernatant fractions were prepared and subjected to centrifugation through 10-to-40% linear sucrose gradients at 150,000 xg for 9 hours in a Beckman SW28 rotor. Fractions were collected, assayed for absorbance at 260 nm in a spectrophotometer, and those occurring w ithin the known region of AOS ribosomal particles were pooled for further purification. 96
0.3 4 -5 S 40S 60S 80S
0.2
CJ POOLED
0.1
0 10 20 30 40 . 50 60 FRACTION NUMBER 97
Figure 15. Purification of 40S ribosomal subunits by sucrose density gradient centrifugation: re-sedimentation of 40S material from the separation depicted in Fig. 14. Pooled fractions from the
40S peak were subjected to centrifugation through 10-to-40% linear sucrose gradients at 150,000 xg for 9 hours in a Beckman SW28 rotor.
Fractions were collected, assayed for absorbance at 260 nm in a spectrophotometer, and those occurring within the known region of 40S ribosomal p artic le s were pooled and la te r subjected to high-speed centrifugation to collect the particles in a pellet. 98
.20 4 -5 S 40S 60S 80S
.15
.10
.05
0 10 20 30 40 50 60 FRACTION NUMBER 99 of approximately 1.59 gm/cc. In contrast, Fig. 17 demonstrates that the H3 (low ionic strength plus 50% formamide) oligo(dT)-cellulose column eluate contained a single population of mRNP particles having a bouyant density of 1.61 gm/cc. Based on this parameter, it may be speculated that the denser particle may be identical or very similar in the two preparations. On the basis of this parameter it can be seen that the AOS ribosomal subunit, which displayed a bouyant density of approximately 1.56 gm/cc in CsCl solution (Fig. 18), represents an entity distinct from the mRNP's.
Bouyant Densities of mRNP's by Sedimentation Equilibrium
Centrifugation in Metrizamide Solution
Figures 19 through 22 represent the behaviour of mRNP fractio n s and AOS ribosomal subunits in metrizamide isopycnic density gradient centrifugation. Metrizamide is a tri-iodinated derivative of
2-deoxyglucose which, in contrast to CsCl, is non-ionic and non-denaturing and does not require sample fixation with an aldehyde before a run. Thus biological samples can be separated presumably in their native forms (Rickwood, 1978; Price, 1982).
Figure 19 shows that the H2 (low ionic strength) oligo(dT)- cellulose column eluate, as in the previously depicted results, contained two populations of mRNP particles, one with a bouyant density of about 1.15 gm/cc and another with a bouyant density of approximately 1.30 gm/cc. Figure 20 again presents the contrasting results for the H3 (formamide-containing) oligo(dT)-cellulose eluate which possessed a single population of mRNP's having a bouyant density of 1.35 gm/cc. Once again i t appears that the same or sim ilar dense 100
Figure 16. Isopycnic centrifugation in CsCl solution of 32 P-labelled Mucor sporangiospore mRNP's eluted from oligo(dT)- cellulose in buffer H2 (low ionic strength). Particles were fixed with formaldehyde prior to centrifugation at 200,000 xg for 48 hours at 20°C in a Beckman SW50.1 rotor. Fractions were collected dropwise after bottom puncture of tubes. Densities were measured gravimetrically and radioactivity was assayed by liquid scintillation spectroscopy. Experimental details are provided in the text.
Symbols: (open squares), density in gm/cc; (open circles), radioactivity as CPM (counts per minute). 101
8 E o to z Ul 0 1 > 3 O to 20 25 30 35 (top) FRACTION NUMBER (bottom) 102
Figure 17. Isopycnic centrifugation In CsCl solution of 32 P-labelled Mucor sporangiospore mRNP's eluted from oligo(dT)-
cellulose in buffer H3 (50% formamide). Particles were dialyzed against buffer H2 and fixed with formaldehyde prior to centrifugation at 200,000 xg for 48 hours at 20°C in a Beckman SW50.1 rotor.
Fractions were collected dropwise after bottom puncture of tubes.
Densities were measured gravimetrically and radioactivity was assayed by liquid scintillation spectroscopy. Experimental details are provided in the text. Symbols: (open squares), density in gm/cc;
(open circles), radioactivity as CPM (counts per minute). BOUYANT DENSITY (gm/cc) ( 0 5 0 5 0 5 0 45 40 35 30 25 20 15 10 FRACTION NUMBER NUMBER FRACTION (bottom)
CPM x 102 ( 103 104
Figure 18. Isopycnic centrifugation in CsCl solution of 32 P-labelled AOS ribosomal subunits purified from hyphae of Mucor racemosus. Ribosomes were suspended in buffer H2 and fixed with
formaldehyde prior to centrifugation at 2 0 0 , 0 0 0 xg for 48 hours at
20°C in a Beckman SW50.1 rotor. Fractions were collected dropwise after bottom puncture of tubes. Densities were measured gravimetrically and radioactivity was assayed by liquid scintillation spectroscopy. Experimental details are provided in the text.
Symbols: (open squares), density in gm/cc; (open circles), radioactivity as CPM (counts per minute). BOUYANT DENSITY (om/cc) (o „)
cZ"
CD CPM x IQ’ ( 106
Figure 19. Isopycnic centrifugation in metrizamide solution of 32 P-labelled Mucor sporangiospore mRNP's eluted from oligo(dT)-
cellulose in buffer H2 (low ionic strength). Particles were subjected
to centrifugation at 200,000 xg for 48 hours at 20°C in a Beckman
SW50.1 rotor. Fractions were collected dropwise after bottom puncture
of tubes. Densities were measured gravimetrically and radioactivity was assayed by liquid scintillation spectroscopy. Experimental
details are provided in the text. Symbols: (open squares), density
in gm/cc; (open circles), radioactivity as CPM (counts per minute). O (a s) (gm/cc) CPM X CPM 103 X (» BOUYANT DENSITY DENSITY BOUYANT
(top) FRACTION NUMBER (bottom) 108
Figure 20. Isopycnic centrifugation in metrizamide solution of
3 2 P-labelled Mucor sporangiospore mRNP's eluted from oligo(dT)- cellulose in buffer H3 (50% formamide). Particles were dialyzed against buffer H2 prior to centrifugation at 200,000 xg for 48 hours at 20°C in a Beckman SW50.1 rotor. Fractions were collected dropwise after bottom puncture of tubes. Densities were measured gravimetrically and radioactivity was assayed by liquid scintillation spectroscopy. Experimental details are provided in the text.
Symbols: (open squares), density in gm/ccj (open circles), radioactivity as CPM (counts per minute). BOUYANT DENSITY (gm/cc) ( 1.4 1.5 1.7 (top) RCIN NUMBER FRACTION (bottom) 20 18
°L 109 110
Figure 21. Isopycnic centrifugation in metrizamide solution of 32 P-labelled 40S ribosomal subunits purified from hyphae of Mucor racemosus. Ribosomes were suspended in buffer H2 prior to centrifugation at 200,000 xg for 48 hours at 20°C in a Beckman SW50.1 rotor. Fractions were collected dropwise after bottom puncture of tubes. Densities were measured gravimetrically and radioactivity was assayed by liquid scintillation spectroscopy. Experimental details are provided in the text. Symbols: (open squares), density in gm/cc;
(open circles), radioactivity as CPM (counts per minute). BOUYANT DENSITY (gm/cc) (a— o)
- fo iii ii 0 1 ip t — i— i— i— i— i— I— I— i— i— i— J— r a
ro Z o
L I I . I I L. -I—I___ I I I L ro Oi CD ro CPM x 103 (—•) 111 112
Figure 22. Isopycnic centrifugation in metrizamide solution of non-radioactive 40S ribosomal subunits purified from hyphae of Mucor racemosus. Ribosomes were suspended in buffer H2 prior to
centrifugation at 200,000 xg for 48 hours at 20°C in a Beckman SW50.1
rotor. Fractions were collected dropwise after bottom puncture of
tubes. Densities were measured gravimetrically and the presence of protein was detected spectrophotometrically by the dye-binding assay of Bradford (1976). Experimental details are provided in the text.
Symbols: (open squares), density in gm/cc; (open c i r c l e s ) , of protein complexed with Coomassie Blue R-250. BOUYANT DENSITY (gm/cc) 0
A 5 9 5 ( « ) 113 114
particles were found in both preparations of mRNP*s. Again, purified
40S ribosomal subunits displayed a different bouyant density from that
of either mRNP population. The value was essentially the same (about
1.28 gm/cc) whether the ribosomes were monitored on the basis of
incorporated radioactivity (Fig. 21) or protein/Coomassle Blue color
complex formed in the Bradford assay (Fig. 22).
All particles displayed lower bouyant densities in metrizamide
gradients than in CsCl gradients. This was not unexpected, since
proteins and nucleic acids exist in a state of greater hydration at
lower ionic strength (Scopes, 1982; Saenger, 1984). The apparent
bouyant densities of all the mRNP populations suggest that these
structures contain both protein and nucleic acid. The lower density
particle would contain a greater and the higher density particle a
lesser percentage of protein than 40S ribosomal subunits (Spirin,
1969).
SDS-PAGE Analysis of mRHP Protein Components
Figure 23 displays a one-dimensional SDS-polyacrylamide gel electrophoretic separation of proteins from various cell fractions including isolated mRNP fractions. Lane 1 depicts the protein
molecular weight standard bovine serum albumin ( 6 6 , 0 0 0 -daltons), ovalbumin (45 ,000-daltons ) , bovine pancreas trypsinogen
(24,000-daltons) and egg white lysozyme (14,300-daltons). Lane 2 represents proteins from the soluble portion of the cell (i.e., proteins that remain in suspension in a cell-free extract after ribosomes, RNP's and other particulate material has been sedimented at approximately 200,000 xg). Lane 3 represents the protein composition 115
Figure 23. Protein composition analysis of mRNP 1 s from dormant sporangiospores of Mucor racemosus by sodium dodecyl sulfate- polyacrylamide gel electrophoresis: a comparison with protein populations in other cell fractions. mRNP's, ribosomes and soluble cell proteins were prepared and subjected to SDS-PAGE as described in the text. Gels were stained with Coomassie Blue R-250 and photographed in transmitted light as indicated in the methods section.
Identities of separated materials are as follows: Lane 1) protein standards; lane 2) soluble cell proteins; lane 3) 80S ribosome proteins; lane A) AOS ribosomal subunit proteins; lane 5) proteins
from mRNP's eluted in buffer H2 (low ionic strength); lane 6 ) proteins from mRNP's eluted in buffer H3 (50% formamide). Molecular weights of the standard proteins (bovine serum albumin, ovalbumin, trypsinogen, _ 3 and lysozyme) are represented x 1 0 on the far left of the electrophoretograra. 116 117
of SOS ribosomes. Lane 4 depicts the protein components of the 40S
ribosomal subunit. Lane 5 shows the proteins present in mRNP's eluted
from oligo(dT)-cellulose with buffer H2. Lane 6 shows the proteins
contained within mRNP's eluted from oligo(dT)-cellulose with buffer
H3. The first appropriate remark to make about the data would be to
caution that, because this procedure separates proteins on the basis
of molecular weight, a given stained band on the gel may represent
more than a single gene product. This fact aside, it is clear that
each major cell fraction represented possessed a unique set of
proteins.
Both mRNP populations possessed proteins not detectable in the
other cell fractions. They also lacked most of the proteins found in
the other fractions, especially the most prominent proteins. Most
noteworthy among the population of mRNP proteins is a band
corresponding to a molecular weight of approximately 24,000 daltons.
This band was the most predominant one, it was found in both
populations of mRNP's and was not detectable in all other cell
fractions. Several other major protein bands, totalling about twelve
in number, were also found in the H2-eluted mRNP's (Lane 5). These
displayed a molecular weight range of about 13,000- to 71,000-daltons, with most clustered between 20,000- to 42,000-daltons. Aside from the
24,000-dalton protein, only one other major protein of 32,000-daltons was found in the H3-eluted mRNP's.
This finding that the population of formamide-eluted mRNP's lack
specific proteins found in the mRNP's eluted at low ionic strength is
consistent with the lower sedimentation coefficients and higher bouyant densities of the former structures relative to the latter. 1X8
These parameters had already suggested that the formamide-eluted
particles are smaller and possess a higher proportion of nucleic acid
than the other population. It is possible that the formamide itself
stripped the missing proteins from the particles. However, a large
portion of the mRNP fraction eluted at low ionic strength had
sedimentation coefficient and bouyant density values roughly the same
as observed for these protein-deficient structures suggesting that the
latter may occur naturally in the cell. The significance of a bimodal
population of mRNP’s can only be speculated upon at this time. It
could possibly represent the existence of immature, damaged or
specialized structures. It is clear from the present data that neither population of mRNP's represents 40S ribosomal subunits in
either a native or modified form.
Size Distribution of RNA Population Isolated from mRNP's
Figure 24 displays the sedimentation coefficient distribution in 32 a sucrose density gradient of P-labelled RNA purified from mRNP's that had been eluted from an oligo(dT)-cellulose column in buffer H2.
It can be seen that the recovered RNA molecules ranged in size from about 10S to 23S, with a mean value of approximately 18S. This is X approximately the same size range reported for poly (A) RNA isolated from M. racemosus sporanglospores by Linz and Orlowski (1982) and similar to literature values for mRNA from other organisms as well
(F irte l and Pederson, 1975; Mirkes, 1977; Martin et a l, 1980). 119
Figure 24. Analysis of RNA from dormant Mucor sporangiospore mRNP's by sedimentation velocity centrifugation: distribution on sucrose density gradients of 32 P-labelled material recovered in phenol extraction of pooled H2-eluate fractions derived from oligo(dT)- cellulose chromatography of spore extract particles. Centrifugation of the purified RNA was for 18 hours at 77,000 xg through a 5-to-20% lin e a r sucrose g ra d ie n t in a Beckman SW41 r o to r . F ra c tio n s were collected and concomitantly monitored for absorbance at 254nm.
Radioactivity was assayed by liquid scintillation spectroscopy.
Experimental details may be found in the text. Symbols: ( ...... ),
^254 c a r r ie r RNA w ith known siz e d i s t r i b u t i o n ; ( ------), radioactivity expressed as CPM (counts per minute). 120
0 10 20 30 40 50 60 FRACTION NUMBER DISCUSSION
In its proper context, the present work should be considered as
part of the foundation of a much more expansive project investigating
the mechanisms of differential gene expression used to control Mucor
sporangiospore germination and yeast/hypha dimorphism. It was made
clear by John Linz and Michael Orlowski that the first protein products appearing during sporangiospore germination are synthesized
from genetic information stored in the form of mature, potentially
translatable mRNA (Linz, 1983). This mRNA is made during spore
formation but not used in protein synthesis at that time. Many of the proteins made during germination are novel, not appearing in the
developing or mature spore. The pre-formed message exists in a stable but unused form in the dormant spore, unassociated with ribosomes or ribosomal subunits. As soon as germination is initiated by introduction of the spores into nutrient medium (or rehydration with distilled water, for that matter) specific messenger RNA populations within the total pool of stored mRNA are somehow mobilized for translation. Ribosomal subunits are recruited into polyribosomes and proteins appropriate to yeast or hyphal development are synthesized depending upon the gaseous environment of the spores.
Many questions remained to be answered about the sequence of events just related. Foremost among the major unresolved issues would be the physical form in which the pre-formed mRNA Is sequestered.
Understanding this issue is a necessary prerequisite to understanding why the stored mRNA is stable and not degraded, why it is not translated during dormancy, what causes the initiation of translation
121 122 upon breaking dormancy, and how only certain mRNA populations are selected for translation. It is this issue that the present study has focused upon.
Progress has been made in this endeavor and some elementary information gleaned about the form in which mRNA is stored in the dormant sporangiospore. Facts have been gathered about the structures in which the mRNA is sequestered and other hypothetical associations have been disproven. The reassortment of subcellular particles in association with mRNA has been observed to occur on a large scale during development, however, certainty about the original residence of some of these is in doubt. Indeed, a systematic investigation of changes in the interactions of mRNA and its associate macromolecules as a function of development has not yet been attempted. A major accomplishment is that we now have a point of reference—a physicochemical characterization of the mRNP's in dormant sporangiospores—and are able to address the question "where do we go from here?” I shall next consider in more specific terms our current experimental beachhead and speculation on potential future laboratory f orrays.
The first contribution of this study was to corroborate the finding of Linz and Orlowski (1982) that mRNA, in the form of 4* poly(A) RNA, does exist in the dormant sporangiospore. This was done 3 by hybridizing [ H]poly(U) to RNA extracted from cellular material that had been fractionated by sucrose density gradient centrifugation
(Fig. 4). This was a different experimental approach than the oligo(dT)-cellulose column chromatography of phenol-extracted total cellular RNA post-transcriptionally labelled with [ H] dimethyl 123
sulfate, which had been employed by the previous researchers. In + addition to demonstrating the mere presence of poly (A) RNA, the new method allowed a size determination for whatever form in which it
occurred. Most of this RNA was found in the 30-70S region of the
g ra d ie n ts w ith peaks at about 40S and 55S. Since i t had been shown
that purified poly(A)+RNA from dormant sporangiospores is on the order
of 6S-to-20S in size (Linz and Orlowski, 1982), native mRNA must exist
in aggregates with other macromolecules, most likely proteins
according to the extant literature.
This seminal experiment also reiterated a previous finding
(Orlowski and Sypherd, 1978c; Linz and Orlowski, 1982) that polyribosomes are totally absent from dormant sporangiospores.
Moreover, the few 80S monoribosomes found in the spores are apparently neither active in translation nor arrested in the act of translation because they have no poly (A) RNA associated with them. Inactive 40S and 60S ribosomal subunits often condense together to form 80S particles in the absence of mRNA or fail to dissociate after completion of translation. Dissociation of such structures can usually be forced by high salt concentration (Martin, 1973).
Accordingly, the buffer in this experiment was 500 mM with respect to
KC1, a concentration that is usually effective but may not have been completely so here.
A third contribution that this experiment made was to demonstrate that the stored mRNA is immediately used in protein synthesis upon the initiation of sporangiospore germination. Previous work had shown that polyribosomes immediately form upon hydration of the spores (Linz and Orlowski, 1982) and that the proteins synthesized at this time by 124
and large reflect the composition of the mRNA pool in the dormant
spore, as determined by 2-D PAGE analysis of radioactively-labelled
proteins made in vivo and comparison with those translated jLn vitro
from purified mRNA (Linz and Orlowski, 1984). Furthermore, neither
poly(A)"*RNA nor RNA of any sort is made at this time, adding to the
evidence that the stored mRNA must serve as the template for the
proteins that appear de novo in early germination. The present
experim ent showed that the polyribosomes and 80S monoribosomes formed
within minutes after immersing the spores in liquid YPG medium carry 3 with them most of the [ H]poly(U)-hybridizable material that had been
concentrated in the 30-70S region of the gradient during dormancy.
In the future it will be of great Interest to isolate the
structures (pmRNP's) in which the polyribosome-associated mRNA resides
free from a l l ribosomal m aterial by EDTA treatment and to compare them with the free structures (most likely cmRNP’s but perhaps also
HnRNP's) in which the mRNA resides in the dormant spores. I t would be
of considerable importance to extract and compare by in vitro
translation the population of raRNA's found in pmRNP's with those found
in cmRNP ’ s. ■ We already know that translation at this stage of
development is selective (Linz and Orlowski, 1985), but we do not know how this is accomplished at the molecular level. For example, we do not know if all mRNA’s are indiscrim inately mobilized into polyribosomes and some translations are not initiated, some translations are arrested or aborted, or some protein products are degraded while still nascent; or if, alternatively, specific mRNA's are not recruited into polyribosomes unless the environment so dictates. Determining the simple question of whether the populations 125 of mRNA's in pmRNP' s are the same or different from those in cmRNP's is precedurally straightforward, involving phenol extraction of mRNA from polyribosome and soluble cytoplasmic cell fractions followed by in vitro translation of each and 2-D .PAGE and autoradiography. The only lim itation may be the minute quantities of praRNA or cmRNA availab le.
The present study has demonstrated that a particulate sporangiospore fraction can be prepared and structures sequestering poly(A)+RNA isolated from it by means of olido(dT)-cellulose or poly(U )-sepharose a ffin ity column chromatography (Figs. 5-10). These structures contain both protein (detectable as incorporated 35 L—[ S]methionine or A 2 gQ) and nucleic acid (detectable as
[ 32 P]orthophosphate or A2^ ) and they hybridize with [ H]poly(U). 3 As expected, most of the particulate material in the cell represents ribosomes. This material does not bind to oligo(dT)-cellulose and can be identified on sucrose density gradients (Fig. 13). Some contribution from nucleosomes and HnRNP's may also exist in the particulate fraction but we had no way of readily assaying for these structures. The presence of the former particles would be expected but, because they do not bind to oligo(dT)-cellulose, would not interfere with the present study of RHP*s. The presence of the latter particles could complicate the interpretation of our results, especially if the hypothetical HnRNP's were polyadenylated and present in large quantities. We could not break open the tough-walled Mucor sporangiospores without also rupturing the nuclei, which could result in releasing any HnRNP's and mixing them in with the cytoplasmic mRNP's. 126
Me have no hard evidence to disprove that our preparations
contained HnRNP's, but we feel that such p o te n tia l co n tam in atio n was
minimal for several reasons. First, most HnRNP's are reportedly not
polyadenylated (Martin et al., 1980). Addition of the poly(A) tail is
the last event in the processing of the mRNA in HnRNP's. Soon
thereafter, an RNP containing poly(A)+RNA is transported out of the
nucleus and can be considered a cmRNP. Most HnRNA in HnRNP's is, in
fact, never polyadenylated at all. Most of it is degraded before it
reaches that point (Padgett et al., 1986). Second, HnRNP's contain mostly immature mRNA which s till possesses introns and is much longer
than the fin a l p ro d u c t. In v itr o t r a n s la t io n of th ese HnRNA's or
their sheared fragments would yield many anomolous spots in 2-D PAGE analysis of the products. Linz and Orlowski (1984, 1985, 1986) never observed such anomolous spots. Practically every spot representing a protein synthesized Jri vitro could be matched with a spot representing a protein made fn vivo. There were no anomolously large products
(translation of HnRNA's) or small products (fragments of HnRNA's) formed in the jLn vitro system. Just about all of the ^n vitro translatable poly(A)+RNA extracted from the dormant sporangiospore generated protein products indistinguishable from those synthesized from bone fide mRNA in the intact living cell. We make the tentative interpretation that most of this material was, in fact, genuine mRNA rather than immature HnRNA. We further make the tentative assumption that this mRNA is resident in mRNP's rather than HnRNP's. Since we have already shown that there are no pmRNP's in the dormant spore, we suggest that the structures we have characterized are most likely cmRNP' s . 127
We realize that this assertion cannot be substantiated until
someone devises a technique for recovering a high percentage of intact
nuclei from Mucor sporangiospores. Intact nuclei have been recovered
at low yields from Mucor hyphae after digestion of the cell wall with
chitlnase and chitosanase and subsequent gentle rupture of the
osmotically fragile protoplasts (P.S. Sypherd, personal
communication). However, in addition to chitin and" chitosan, the
spore wall contains large amounts of uncharacterized proteins, glucans
and melanin among other compounds. The large collection of hydrolytic
enzymes required to digest this assemblage of macromolecule may very well damage released nuclei. Certainly the long period of time that would be required to sufficiently hydrolyze the thick multilayer spore wall could allow many changes to occur in the nucleus, including both
the continuation of normal mRNA processing and aberrant reactions provoked by the acutely stressful conditions. Although protoplasts are commonly obtained from the v e g e ta tiv e c e l l s of fu n g i t h e i r generation from fungal spores is very rare (Peberdy and Ferenczy,
1985). Although the conidlospores of a few species of ascomycetes and deuteromycetes have yielded protoplasts there are no reports of protoplast production from the ungerminated sporangiospores of any zygomycete (Peberdy and Ferenczy, 1985).
Assuming that what we have isolated from dormant sporangiospores of M. racemosus are in fa c t cmRNP*s, we can proceed to compare and contrast the properties of these structures with those reported for cmRNP*s from other systems. Measurements of sedimentation coefficient distributions in sucrose density gradients first suggested the existence of two populations of cmRNP * s, one with a distribution of 128
approximately 20-50S and a mean size of 40S and another with a
distribution of about 50-70S and a mean size of perhaps 55S. These
values are larger than some reported for various animal and yet
smaller than others (Table 1). They most closely approximate the
values ascribed to chicken muscle cmRNP’s (Jain and Sarkar, 1979)
among the animal systems. Values for the Mucor structures more
closely approximate those reported for other protists. The oomycete
Blastocladiella (Jaworski and Stumhofer, 1981) showed the same overall
size distribution (20-80S) for these particles as the zygomycete
Mucor. The ascomycete Neurospora (Mirkes, 1977) and the myxomycete
Physarum (Adams et al., 1980; 1981) had only slightly smaller
particles, displaying a range of 15-60S and 18-60S respectively.
Bimodal populations of cmRNP’s were describedfor both Blastocladiella
and Neurospora and, as in the Mucor system, they did not display a
distinct separation on sucrose gradients. InNeurospora, as in Mucor,
the different populations were made evident by the conditions of elution from an oligo(dT)-cellulose column. Particles eluted in 50% formamide were smaller and lacked proteins found in the particles eluted at low ionic strength, facts which will be expounded upon at
greater length below.
The bimodality of cmRNP' s from Mucor sporangiospores was most apparent in isopycnic centrifugation runs. Whether the separation was effected on formaldehyde-fixed particles in CsCl gradients or non denatured (native) particles in metrizamide gradients, two peaks were always observed in H2-eluates. These had bouyant densities of 1.37 gm/cc and 1.59 gm/cc in the former case and 1.15 gm/cc and 1.30 gm/cc in the latter instance. Only the denser particles were observed in H3 129
(formamide)-eluates and corresponded to the smaller particles on the sucrose gradients. Lower limit bouyant densities in CsCl solution recorded for animal systems are about the same as observed in Mucor, varying from 1.35 gm/cc in trout testes to 1.41 gm/cc in chicken muscle (Table 1). Upper limit values of about 1.47 gm/cc in sea urchin eggs are somewhat lower that the Mucor values (Table 1). The only other values available for a protist system were collected for the fungus Neurospora. These vary from a low density of 1.42 gm/cc to a high value of 1.50 gm/cc. In metrizamide solution, the animal particles are midway between the two Mucor peaks (Table 1). This parameter has been measured in only one other fungus, the oomycete
Blastocladiella (Jaworski and Stumhofer, 1981). The value of 1.27 gm/cc measured in this system is closer to the higher value recorded fo r Mucor.
It is a simple consequence of the laws of physical chemistry that a higher bouyant density in CsCl solution implies a lower proportion of protein to RNA in any ribonucleoprotein structure. Conversely, a lower bouyant density mandates that there is a greater percentage of total mass that is proteinaceous in composition. This is because pure protein is much less compact and dense than pure RNA under these conditions of reduced hydration (Tanford, 1961). Various authors have attempted to compute the ratio of protein/RNA based upon the bouyant den sities of RNP's in CsCl so lu tio n , however, there is quite a b it of discrepency in the calculations made and in the base-line values employed. For example, a ribonucleoprotein particle with a density of about 1.41 gm/cc has been variously calculated to contain 75% protein
(Spirin, 1969), 80% protein (Perry and Kelley, 1966) and 85% protein 130
(Hamilton, 1971), The bouyant density of purified RNA which is taken
into the calculations has been variously reported as 1.66 gm/cc
(Kaumeyer et al., 1978; Moon et al., 1980), 1.87 gm/cc (Irwin et al.,
1975) and >1.9 gm/cc (Rickwood, 1978) in CsCl solution. Resolution of
this issue was only confounded when direct measurements of RNA and
protein content by the orcinol and Lowry methods yielded significantly
higher estimates of the protein/RNA ratio, on the order of 90% or
higher (Karn et a l., 1977; Billings and Martin, 1978; Martin et al.,
1980). Even the thoroughly studied ribosome, which has a well
established bouyant density in CsCl solution of about 1.56 gm/cc in most eukaryotic cells, has been variously described as being composed
o f tw o - th ir d s (L ak e, 1985), 50-60% (N o lle r, 1984) and 50% (McConkey,
1974) RNA. Based upon the bouyant densities measured in CsCl solution
for Mucor ribosomes (approximately 1.56 gm/cc), the present data seem
reliable. This figure compares with values of 1.56 gm/cc and 1.58 gm/cc recently measured for ribosomes of Artemi a salina (Siegers and
Kondo, 1977) and Neurospora crassa (Mirkes, 1977) in CsCl solution.
Using the data collected in this study and employing the equation of
Hamilton (1971), which states that
% Protein = (1.87) - (peq ) / (0.0040) x (peq ), it was calculated that the larger mRNP's contain about 90% protein
(10% RNA) and the smaller denser mRNP's comprise approximately 45% protein (55% RNA). The 40S ribosomal subunits of Mucor theoretically c o n t a i n r o u g h ly 50% p r o t e i n (50% RNA) b a s e d upon t h e a b o v e relationship. These calculated values will be seen to compare quite favorably with the protein analyses displayed below. 131
Isopycnic banding of native mRNP's on metrizamide gradients has
generally shown the same qualitative relationships as observed on CsCl
gradients (Martin et al., 1980). The same was true in the Mucor
system. The ribonucleoprotein particles are non-denatured and more
completely hydrated in metrizamide solution than they are in CsCl
solution. Therefore they display lower bouyant densities. However, a
bimodal distribution of particles, with one population more and the
other less dense than ribosomes, is s till observed. There seems to
have been little or no attempt in the literature to quantify the ratio
of protein/RNA in RNP's based on measured bouyant densities in
metrizamide gradients and we have made no attempt to do so here.
Since the source of mRNP samples were the same here as in the CsCl
runs, one would predict the same protein/RNA ratios in each of the two
peaks as previously observed.
The protein composition of Mucor sporangiospore cmRNP' s is
displayed on the SDS-polyacrylamide electrophoretogram shown in Fig.
23. It is notable that the 52,000- and 78,000-dalton proteins usually
(but not always) found in cmRNP's from animal cells are absent in
these structures from Mucor. There is a 71,000-dalton protein in
H2-eluted sporangiospore cmRNP*s and perhaps it represents the fungal
form of this gene product. It was claimed that a 72,000-dalton
protein was associated with poly(A) tails of mRNA (the putative role
of the 78,000-dalton protein in animal RNP's) in the slime mold
Dlctyostelium (Firtel and Pederson, 1975). The five major proteins
described in cmRNP1 s of B lastocladiella, however, do not include a molecule in this size range, the closest being 64,000-daltons
(Jaworski and Sturahofer, 1981). 132
Upon examination of the current meager literature, the organism
possessing a distribution of mRNP proteins most similar to that found
in Mucor would be Neurospora (Mirkes, 1977). The following table
attempts to compare the protein components of the two major populations of mRNP's from Mucor and Neurospora (Table 2).
Table 2: Protein Components of mRNP's from Mucor and Neurospora
Mucor Neurospora
Large Particles Small Particles Large Particles Small Particles
71,000 66,000 42,000 44,000 32,000 32,000 31,000 26,000 27,000 24,000 24,000 24,000 21,000 20,000 19,000 14,000 14,000 13,000
Proteins are characterized in terms of their molecular weights. Values in bold-face type represent major proteins. "Large particles” were eluted from oligo(dT)-cellulose at low ionic strength. "Small particles" were eluted from oligo(dT)-cellulose with 50% formamide as recounted in the text.
It is evident that there is roughly the same number and size distribution of mRNP-proteins in the two organisms. Furthermore, the major proteins are of roughly the same size, as are the minor proteins. Moreover, the particles eluted at 50% formamide in both systems display a drastic reduction in the number of proteins from that observed in the particles eluted at low ionic strength. Only two proteins remain in the Mucor particles and but one in the Neurospora mRNP's. In the Mucor system these remaining proteins are clearly identical to two molecules present in the larger mRNP's. The 24,000- 133 dalton protein Is clearly the predominant form in both cases. In
Neurospora, only a 27,000-dalton protein remains in 50% forraamide- eluted mRNP's. This protein may be identical to the 24,000- or
31,000-dalton molecules, which are co-predominant proteins in the larger Neurospora particles.
In both the Mucor and Neurospora systems these protein analyses make it quite clear why the 50% formamide-eluted mRNP's are smaller and denser than the particles eluted at low ionic strength. They have many fewer proteins and, assuming that the length of mRNA is not drastically different in the two types of particles, they have a lower protein/RNA ratio and a consequent greater bouyant density in CsCl s o lu tio n .
In neither the Mucor nor Neurospora system can It be certain that the smaller particles are not artifacts caused by formamide-induced dissociation of proteins from RNA. Just as formamide releases poly(A) sequences from poly(U) or oligo(dT) sequences by disrupting hydrogen bonding, so it can free proteins from association with nucleic acids.
In fact, at a high enough concentration of formamide (ca 90%), all proteins and nucleic acids become completely denatured into linear rod-like forms. Arguing against this interpretation is the presence of the smaller, denser protein-deficient particles even in the low ionic strength eluate lacking any formamide. Lest one be made overly complacent by the last comforting argument, a final caution must be raised about the well-known ability of high salt concentrations to dissociate weakly-bound proteins from nucleic acids. It is an inescapable fact that the conditions required to cause poly(A)+RNA to bind to oligo(dT)—cellulose (approximately 0.5 M) could conceivably 134
cause some protein components to dissociate from mRNP’s. This cannot
be corrected if binding to oligo(dT)-cellulose is to remain the major
criterion for defining and recovering mRNP's. However, one must
strive to become cognizant of all lim itations of methodology and
realize that most biological systems are recognized and defined on an
operational basis.
An important contribution of the foregoing protein analysis, not
to be overlooked, is the establishment of non-identity between protein
populations from ribosomes and mRNP’s. These findings refute the possibility that the structures we have recovered on oligo(dT)-
cellulose columns are merely 80S ribosomes arrested in the act of
translation or 40S ribosomal subunits in translation initiation
complexes with mRNA. We therefore discount such hypothetical mechanisms as possible strategies Mucor might employ to store mRNA in the dormant sporangiospore and to preclude its premature utilization
in protein synthesis.
According to current thought (Martin et al., 1980) it is the protein component of mRNP’s which controls expression of their mRNA content. The present study has elucidated the protein composition of only non-polysomal mRNP's. Another logical extension of the present work would be to do a comparative analysis of the protein compositions of pmRNP' s and cmRNP’s, especially during the initial appearance of the former early in germination. This could be accomplished using the simple 1-D SDS-PAGE method we have employed here, or, if the protein populations seem somewhat complex, 2-D PAGE could be attempted. The simpler system has proven adequate in most reported studies because there are rather few proteins in any of these structures, as has been 135
noted. No one has yet been able to definitively ascribe a function to
any specific protein in an mRNP or an HnRNP, though there are certin
"core proteins" that regularly occur. It has been hypothesized that
these may play a structural role, like some of the histones present in nucleosomes (Martin et a l., 1980). The frequently observed
78,000-dalton protein is apparently associated with the poly(A) tail of the mRNA, but its functional role is still a mystery. We have no
illusions about quickly and dramatically uncovering specific roles for
these proteins in the Mu cor system while the more heavily exploited
systems remain Impervious to analysis. Our modest hopes are to detect any changes that correlate with morphogenesis, and then to discern with what else they may correlate. For example, we know that mRNA's are selectively expressed during sporangiospore germinaton (Linz and
Orlowski, 1985). We may also find that mRNP's with different protein components may sequester different mRNA populations. Making such a discovery, one would then have to address the issues of how such a molecular relationship comes about and what are the consequences of it in terms of gene expression.
We cannot say based upon the present data whether the sequestered mRNA's differed between the two populations of cmRNP's. We did not recover enough material to analyze the size distribution of mRNA's in the formamide-eluted cmRNP fractions and we did not recover enough of any material to attempt 1^ vitro translation. The sucrose density gradient data gathered using mRNA extracted from cmRNP's eluted at low ionic strength (Fig. 24) showed no difference in size distribution from total mRNA directly phenol extracted from unfractionated broken sporangiospores (Linz and Orlowski, 1982). This size distribution 136
(8-20S) Is typical of that described for mRNA in several other biological systems (Mirkes, 1977; Firtel and Pederson, 1975; Siegers and Kondo, 1977) and could conceivably be in-vitro translated to do th e com parative study of pmRNP's v i s - 'a - v i s cmRNP's suggested e a r l i e r .
The only potential drawback in this matter would be obtaining a large enough yield of purified mRNA to translate. The quantities analyzed here were exceedingly minute and required much unlabelled carrier RNA to recover. The problem is not theoretically intractable.
Essentially, a massive scale-up of material is required. Perhaps this
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1198-1204. VITA
C harles P a tric k Chapman was born on August 23rd, 1956, In S e o u l,
South Korea. He attended St. Clement of Rome Elementary School in
M etairie, Louisiana, and graduated from Brother Martin High School in
New Orleans, Louisiana, in 1975. He attended Louisiana State
University in Baton Rouge, Louisiana, and received the Bachelor of
Science degree in Microbiology in May, 1981, and the Master of Science degree in Microbiology in August, 1982. He is married to the former
Gloria Marie Kimball of Plaucheville, Louisiana. He is presently a candidate for the Doctor of Philosophy degree with a major in
Microbiology and a minor in Food Science.
156 DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate: C harles P a tr ic k Chapman
Major Field: Microbiology
Title of Dissertation: Characterization of Messenger Ribonucleoprotein Particles in Dormant Sporangiospores of the Fungus Mucor racemosus
Approved:
Major Professor and Chairman
te School
EXAMINING COMMITTEE:
V v\tJT -S'W'1
Date of Examination: 25 August 1986