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MICROBIOLOGICAL REVIEWS, Mar. 1981, p. 99-122
0146-0749/81/010099-24$2.00/0
Vol. 45, No. 1
Control of Growth and of the Nuclear Division Cycle in
Neurospora crassa
LILIA ALBERGHINA't* AND EMMAPAOLA STURANI2
Cattedra di Biochimica Comparata, Facolta di Scienze,' and Centro del Consiglio Nazionale delle
Richerche per la Biologia CeUulare e Molecolare delle Piante, Istituto di Scienze Botaniche,2 Universita di
Milano, 20133 Milan, Italy
.............................................................
and other microbial cells, both in steady states
of growth and during nutritional shifts, emerges
a predominant regulatory role for ribosomal
RNA (rRNA) synthesis; nevertheless, it is not
uncommon to find situations (for instance, in
yeast) in which the rate of protein synthesis is
regulated not only by the level of ribosomes but
also by the efficiency of ribosomes.
The intracellular levels of substrates of macromolecular syntheses (amino acids and nucleoside triphosphates) are fairly low as compared
with their rates of utilization, suggesting that
the rates of production are coordinateti with the
rates of utilization.
The coordination of growth and the nuclear
division cycle in Neurospora appears to be mediated by the achievement of a critical cell mass
required for the initiation of deoxyribonucleic
acid (DNA) replication, as reported for other
microbial cells.
We have developed a dynamic model to give
structure to the above observations. It offers a
unifying interpretation for the different cell cycle
patterns reported in eucaryotes. Relationships
between the macromolecular composition of the
cells and cell cycle timing have been developed
INTRODUCTION
Growth and cell division of heterotrophic microorganism are modulated essentially by two
environmental factors: temperature and availability of nutrients. In cells of higher organisms,
restriction devices have been superimposed on
this regulation during evolution so that cell proliferation at any given temperature is further
controlled by the presence of hornones and
other growth factors besides the availability of
nutrients. Thus, the study of the regulation of
growth by nutrients in microorganisms, which is
facilitated by the fact that microorganisms are
a very convenient experimental system for both
genetic and biochemical investigations, may
bring useful insights for the study of cell proliferation in general.
In this review an analysis of growth and nuclear division in Neurospora crassa mycelia under different growth conditions is presented.
A first point considered is how nutrients modulate the synthesis and degradation of ribonucleic acid (RNA) and protein. From a comparison of the situations observed in Neurospora
t Formerly F. A. M. Alberghina.
99
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RODUCTION
99
ANALYSIS OF NEUROSPORA GROWTH ..................................... 100
........ 100
Effects of Nutrients on Growth and Differentiation ....
Macromolecular Levels Characterizing Steady States of Growth .101
Ribosome Efficiency .102
Synthesis of the Different Ribonucleic Acid Species .102
Synthesis of ribosomal ribonucleic acid .103
Metabolism of polyadenylated ribonucleic acid .104
Nutritional Shifts ................................
105
Regulation of Protein Degradation .107
Effect of Temperature on Growth .108
Levels of Amino Acids and Nucleotides .109
NUCLEAR DIVISION CYCLE IN DIFERENT STEADY STATES OF
GROWT .110
MODELING CELL GROWTH AND DIVISION IN EUCARYOTES .111
111
Dynamic Model ...........................
Possible Different Cell Cycle Patterns .113
Critical Protein Level for the Initiation of Deoxyribonucleic Acid Synthesis
and Cell Cycle Timing .113
APPLICATION OF THE MODEL TO ESCHERICHIAI COLI ................... 114
Possible Molecular Basis for Control Elements in E. coli ...................
. 114
CONCLUDING REMA
.......................R..............E............
KS
115
LITERATURE CITED ....................
116
100
MICROBIOL. REV.
ALBERGHINA AND STURANI
which are potentially useful for cell cycle studies.
The satisfactory description of growth dynamics
offered by the model allows the hope that it may
be useful in devising experiments aimed at finding the biochemical bases of the control functions proposed by the model.
(1)
dM/dt = AM
Nevertheless, under these conditions, the cells
are not necessarily endowed with the same enzymatic activities: for instance, the specific activities of cytochrome c oxidase and of malate
dehydrogenase, which are very low in early exponential growth and much larger in mid-exponential phase, change in the course of exponential growth on glucose by two orders of magnitude (2). The rate of respiration (34) and the
adenosine 5'-triphosphate (ATP) level (152) also
increase during early exponential growth, reach
a maximum at mid-exponential phase, and then
decline. The initial concentration of glucose in
the growth medium affects the specific activity
of mitochondrial enzymes (2) and the manner of
glucose utilization (161), but it does not modify
the rate of exponential growth of the cultures.
All these findings suggest that different patterns
of cytodifferentiation and of metabolism are
compatible with the same exponential rate of
growth.
TABLE 1. Ultrastructure of N. crassa hyphae during the exponential phase ofgrowth on different mediaa
Mitochondria'
Growth medium
Minimal sucrose ...............
Supplemented sucrose ..........
Minimal acetate ................
Supplemented acetate ..........
Minimal glycerol ...............
Supplemented glycerol ..........
a From Alberghina et al. (8).
Hyphal diameter (pum)"
2.60 ± 0.13 (44)
4.30 ± 0.13 (43)
2.80 ± 0.16 (37)
4.10 ± 0.17 (20)
1.80 ± 0.11 (48)
2.50 ± 0.11 (35)
No./section
7.76 ± 0.80 (20)
10.85 ± 1.06 (20)
7.75 ± 0.90 (29)
13.72 ± 1.11 (20)
2.82 ± 0.31 (29)
5.43 ± 0.38 (30)
Area (% of total area)
23.63 ± 1.54 (20)
17.74 ± 0.84 (20)
23.92 ± 1.10 (29)
25.67 ± 0.20 (20)
16.36 ± 1.00 (29)
21.64 ± 0.90 (30)
b Average and standard error of the number of determinations indicated in each case by the
value.
parenthetical
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ANALYSIS OF NEUROSPORA GROWTH
Effects of Nutrients on Growth and
Differentiation
When N. crassa conidia are inoculated at
300C into a liquid shaken culture, a condition of
exponential growth of the culture is achieved
after a lag period (whose duration depends on
the age of conidia and on the composition of the
culture medium) during which germination of
conidia takes place (for a review, see reference
135).
The exponential growth of Neurospora mycelia reflects the fact that hyphal length increases at an exponential rate (173, 174). Hyphal
branching is strictly regulated during exponential growth: the hyphal growth unit, that this,
the total length of a hypha divided by the number of tips, is found to be relatively constant
(about 80 ,um) (25, 175). The exponential rate of
growth [expressed as the number of doublings
per hour (,) or as the rate constant of exponential growth (A, expressed per minute), A = (In 2/
60) .y] can be measured as the increase in dry
weight or, in dilute cultures, as the increase in
the absorbance of the culture at 450 nm (1). By
choosing appropriate carbon and nitrogen
sources, it is possible to elicit in batch cultures
a wide range of exponential growth rates so that
at 300C the doubling time of Neurospora cultures varies from about 1 to 8 h (7).
The ultrastructural organization of Neurospora hyphae is strictly affected by the nutrients
(8) (Table 1): the hyphal diameter is smaller in
glycerol than in glucose or acetate; the addition
of Casamino Acids to all three media makes the
hyphae become considerably larger. Analysis of
electron micrographs shows that the number of
mitochondria per section and the percentage
mitochondrial area are larger in glucose and in
acetate than in glycerol and are increased by the
addition of Casamino Acids. In fact, the formation of mitochondria in Neurospora (80) is not
regulated by catabolite repression (56), whereas,
for instance, in Saccharomyces cerevisiae aerobic growth on glucose or on other fermentable
carbon sources yields cells that are not endowed
with nornal mitochondria (94, 123, 124). Vacuoles are almost absent during exponential
growth on glucose, whereas significant vacuolation occurs during exponential growth in glycerol
and less evident vacuolization occurs in acetate
(8).
Balanced exponential growth is characterized
by a proportional increase in macromolecular
components (DNA, RNA, and protein), all of
which accumulate at a rate given by the constant
of the rate of growth times the steady-state level
of the individual component (M):
GROWTH AND NUCLEAR DIVISION CYCLE IN N. CRASSA
VOL. 45, 1981
Macromolecular Levels Characterizing
Steady States of Growth
zoopfii (126), and in the slime mold Physarium
polycephalum (124). Many data are also available on the yeast S. cerevisiae. Two groups of
workers (19, 185) have shown that RNA per cell
(or the RNA/DNA ratio) varies with the growth
rate in batch cultures, although in a nonlinear
fashion, and less sharply than it does in Neurospora. Sebastian et al. (136) have found an almost linear increase in RNA content per cell in
chemostat-growing cultures over the same range
of growth rates (u from 0.1 to 0.7).
Figure 2 reports the ribosome content on a
protein basis in Escherichia coli, Neurospora,
and yeast cells as a function of the growth rate
(A, per minute). For Neurospora as well as for
E. coli there is a direct proportionality between
the rate of growth and the ribosomal content so
that a doubling in growth rate corresponds to a
doubling in the number of ribosomes. As observed by Maal0e (100) and Maal0e and Kjeldgaard (101), it follows that in these organisms
the average ribosomal activity does not vary
with the growth rate (see the next paragraph).
In contrast, in yeast a doubling in the growth
rate results only in a relatively small increment
in the level of ribosomes, so that there is an
excess of ribosomes at the lower growth rates.
As regards tRNA in Neurospora, as in yeast
cells (185) and E. coli (131), the tRNA/rRNA
ratio decreases with the growth rate (Fig. 1), and
0-J
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2
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L.
0
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x
z
0
1.50
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0.5-
-20
0
-10
0
I
0.2
I
0.4
I
0.6
p--
1
0.8
%P
I
FIG. 1. Macromolecular composition of N. crassa mycelia cultivated at different growth rates. The molecules of tRNA are given per genome equivalent (0) and per ribosome (A). The protein level (0) is expressed
as the number of amino acids (AA) bound into protein per genome equivalent. The number of ribosomes (0)
is given per amino acid bound into protein. R-protein (U) indicates ribosomal protein, as the percentage of
total protein. The rate of exponential growth is given as ,u (number of doublings in biomass per hour). (From
Alberghina et al [161.)
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It is a constant feature of microbial growth
that during balanced exponential growth at a
given temperature (between 20 and 400C for
mesophilic organisms), the cell composition, in
terms of DNA, RNA, and protein contents, is
uniquely related to the rate of growth (7, 19, 91,
99-101, 122, 126, 185). Figure 1 gives the cell
composition of N. crassa mycelia as a function
of the rate of growth (Ja). Briefly, for ,u > 0.2
there is an appreciable linear increase in ribosome content (ribosomes per unit of protein)
with increasing growth rate. Instead, the levels
of protein (protein per genome equivalent of
DNA [33]) and of transfer RNA (tRNA molecules per genome equivalent of DNA) increase
only slightly with the growth rate. Moreover,
since the total protein level does not vary significantly with IL, whereas that of ribosomes
does, it follows that whereas at low growth rates
only about 10% of proteins are ribosomal, at the
highest rates 40% of the proteins are ribosomal
protein (7).
The total RNA content has been found to
increase with increasing growth rate in bacteria
(87, 150) and in a number of eucaryotic micros, for instance, in the protozoan Tetraorg
hymena pyriformis (91), in the alga Prototheca
101
102
ALBERGHINA AND STURANI
MICROBIOL. REV.
a
.
4.01a
Q
3.0
a
-a
w
w
Ul
2.0F
J
U)
oc
e0n
ir
5
10
15
20
30
RATE OF GROWTH (*A,min-1X103)
FIG. 2. Ribosomal level as a function of the rate of growth in different organisms. The ribosomal level, p,
is expressed as the number of ribosomes per amino acid bound into protein. A (per minute) is the growth rate.
The data of Kjeldgaard and Gausing (87) were used to estimate the value of p in E. coli Blr cells (A). The
data of Skjold et al. (150) were used to estimate the value of p in E. coli 15 T- cells (A). The data of Waldron
and Lacroute (185) (@) and of Boehlke and Friesen (19) (0) were used to estimate the value of p in yeast cells.
The data of Alberghina et al. (7) were used to estimate p values in Neurospora (U). (From Alberghina et al.
[16].)
the number of tRNA molecules per ribosome
decreases from about 35 at a low growth rate to
12 to 15 for,u > 0.5 (7).
Ribosome Efficiency
The rate of protein synthesis in Neurospora
has been calculated from the rate of accumulation plus that of degradation; this rate divided
by the ribosomal content yields the average
ribosomal activity (K2) (7, 16). This value is
relatively constant during exponential growth
over a range of growth rates (Table 2): about 480
amino acids are polymerized per min per ribosome (i.e., 8.3 amino acids per s). The rate of
protein synthesis has also been determined by
pulse-labeling experiments in a few of the considered conditions with comparable results
(103). That the average ribosomal efficiency is
constant is also confirmed by the finding that
the percentage of ribosomes associated to form
polysomes is only slightly affected by changing
growth rate: from 44% in cells grown in ethanol
to 58% in celLs growth in nutrient broth (Table
2, percent active ribosomes). Thus, the rate of
peptide elongation per active ribosome is very
much the same at the different growth rates
(about 960 amino acids per min). Moreover,
polyadenylated RNA in Neurospora is a rela-
tively constant fraction of total RNA, about 5%,
at different growth rates (162) (Table 2).
These findings are similar to those reported
for E. coli (57, 89, 115), in which the messenger
RNA (mRNA) activity per ribosome does not
vary significantly with the growth rate and in
which the polypeptide elongation rate is constant except at very low growth rates. However,
there is a marked difference between the results
for ribosome efficiency in Neurospora and S.
cerevisiae: in S. cerevisiae the percentage of
ribosomes bound into polysomes and the average ribosomal activity decrease sharply at low
growth rates, whereas the polypeptide elongation rate remains fairly constant at all growth
rates (183, 184).
Thus, whereas in Neurospora the rate of protein synthesis is limited by the number of ribosomes, in S. cerevisiae an enhancement of protein synthesis may be obtained by recruiting
inactive ribosomes into polysomes (98, 184). A
behavior similar to that of the yeast has been
described also for T. pyriformis (91).
Synthesis of the Different Ribonucleic
Acid Species
Multiple DNA-dependent RNA polymerases
(RNA nucleotidyltransferases) of nuclear origin
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ul
VOL. 45, 1981
GROWTH AND NUCLEAR DIVISION CYCLE IN N. CRASSA
103
TABLE 2. Ribosome efficiency in N. crassa
PolyadenylateGrowth medium
iAa
K2b
% Active
ribosomesc
containing
RNA
(% of
total RNA)d
R~ate of poly-
Nutrient broth .0.91
480
58
NID
Glucose plus Casamino Acids .0.65
480
53
ND
Acetate plus Casamino Acids .0.58
460
54
ND
Glucose .0.51
480
56
5.12
47
475
ND
Glycerol plus Casamino Acids .0.48
Acetate .0.41
46
500
4.49
600
51
Glycerol .0.26
5.22
420
44
Ethanol .0.13
ND
a Number of duplications per hour.
b
Average ribosomal activity, expressed as amino acids polymerized per min per ribosome (from Alberghina
et al. [16]).
,
Polyribosomes (from Alberghina et al. [7]).
dFrom Sturani et al. (162).
'Amino acids polymerized per min per active ribosome, calculated from K2 and percent active ribosomes.
f ND, Not determined.
' From Sturani (unpublished data).
have been found in yeasts and fungi as well as in
many other eucaryotes (139).
A clearly tripartite transcrptive system has
been characterized in yeasts (179), and three
different RNA polymerase activities have been
isolated. Polymerase I (or A), of nucleolar origin,
preferentially transcribes rRNA (75, 180), and
this activity (unlike other eucaryotes) is inhibited by high concentrations of a-amanitine
(179). Polymerase II (or B), of nucleoplasmic
origin, is inhibited by low concentrations of aamanitine and transcribes mRNA (179). Polymerase HI is totally unaffected by a-amanitine
and in other related eucaryotes has been shown
to transcribe 4S and 5S RNA. Structural studies
on purified polymerases I and II have shown
that three subunits, among the small-molecularweight components, appear to be common to the
two enzymes (24). It has been suggested that the
common subunits perform the same basic function during transcription. Since the production
of bacterial RNA polymerase seems to be autoregulated (62, 66, 78, 167) (see Possible Molecular Basis for Control Elements in Escherichia
coli), the possibility arises that the common
subunits of eucaryotic polymerases may be involved in control by autoregulation.
The activities of polymerases I and II have
been shown to vary independently of each other
during the cell cycle (137), as functions of the
rate of growth (136), and during nutritional shifts
(29).
The studies of RNA polymerases in Neurospora are not as extensive. In one report (169)
DNA-dependent RNA polymerases were isolated from nuclei of the Neurospora slime mutant: four peaks of activity were separated upon
chromatography on diethylaminoethyl cellulose
and were characterized on the basis of their
sensitivities to a-amanitine and ionic requirements. In a second report (171) the RNA polymerases were separated from crude preparations
of nuclei from the wild type and, when chromatographed on diethylaminoethyl-Sephadex,
only two major peaks and one very small peak
of activity were resolved. The first two peaks
were identified as polymerases I and II on the
basis of a-amanitine sensitivity and divalent ion
requirements.
Synthesis of ribosomal ribonucleic acid.
For the synthesis of cytoplasmic rRNA, which
constitutes the bulk of stable RNA in Neurospora mycelia, the following process has been
described (132). The first product synthesized in
a significant amount is a 2.4 x 106-dalton rRNA
precursor. This RNA is methylated and cleaved
to produce two species of RNA molecules: 0.7
x 106 and 1.4 x 106 daltons. The former is the
mature 17S rRNA, and the latter is cleaved to
produce the mature 1.27 x 106-dalton (25S)
rRNA and the 61,000-dalton (5.8S) rRNA (97)
of the large ribosomal subunit (27). In the maturation process, approximately 15 to 20% of the
2.4 x 106-dalton precursor is lost. As in other
eucaryotes, the 5S and 4S RNAs do not derive
from this precursor.
This pattern of maturation is quite similar to
that described for cytoplasmic rRNA of S. cerevisiae (177). The molecular weights of mature
rRNA's in Neurospora are very close to those
found in other fungi and green plants (27).
The rate of synthesis of rRNA (drR/dt) is the
same as its rate of accumulation if its rate of
degradation is negligible, as it is in exponential
growth. Thus, drR/dt can be calculated in the
different steady states of growth from the differ-
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peptide eloY
petdeon
gation'
828
906
852
858
1,008
1,086
1,176
954
104
MICROBIOL. REV.
ALBERGHINA AND STURANI
ent rRNA levels (rR) and expressed
as
nucleo-
tides polymerized into rRNA per genome equivalent of DNA per minute as follows (7):
drR/dt = XrR
(2)
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In Neurospora foru > 0.2, the rate of synthesis
of rRNA varies proportionally to the square of
,u (7), and its rate of methylation follows the
same pattern (163).
It is interesting to recall that in S. cerevisiae
it has been shown that the specific activity of
DNA-dependent RNA polymerase A increases
sharply with the growth rate, whereas that of
polymerase II is almost constant (136).
Metabolism of polyadenylated ribonucleic acid. In eucaryotes the major part of
mRNA has a polyadenylic acid region at the 3'
end of the molecule (28). Polyadenylated RNA
is ubiquitously found in all eucaryotic organisms
that have been examined as well as in bacteria
and in mitochondrial and viral transcription systems, and the length of the polyadenylic acid is
larger for the organisms evolutionarily more advanced (28). The presence of the polyadenylate
sequence has allowed the study of this fraction
of mRNA, since polyadenylated RNA can be
easily separated by means of adsorption chromatography on oligodeoxythymidylate-cellulose
(48), polyuridylate-Sepharose (92), and polyuridylate-glass fiber filters (142). In Neurospora
the presence of polyadenylated RNA associated
with ribosomes was first reported by Mirkes and
McCalley (108). It has been characterized by
means of chromatography on Sepharose 4B and
polyacrylamide gel electrophoresis (96). As in
yeast cells, it is heterogeneous in size, with no
large-molecular-weight species of the type of
heterogeneous nuclear RNA found in animal
cells, and it ranges from 2 x 105 to 1.3 x 106
daltons; its template activity in translation systems is about five times that of nonpolyadenylated RNA (96; E. Sturani, unpublished data).
The isolated polyadenylate sequences are distributed in three major classes of 30, 55, and 70
residues of adenine (96).
The structure of the 5' terminus of Neurospora polyadenylated RNA has also been analyzed (138). Two 5'-terminal cap structures
[m7G(5')-ppp(5')Ap and m7G(5')ppp(5')Gp]
have been found to be present, the former in
75% of the cases and the latter in 25%. In mammalian cells the nucleotide adjacent to the 5' cap
structure may contain the 2'-0-methyl substituent, and in addition N6-methyladenosine is
sometimes present in the mRNA. No evidence
for such additional methylation has been found
in Neurospora mycelia (65).
Polyadenylated RNA associated with protein
has been isolated from Neurospora ribosomes
dissociated by treatment with ethylenediaminetetraacetate (107). Differential elution of messenger ribonucleoprotein complexes from oligodeoxythymidylate-cellulose has been achieved
by increasing the concentration of formamide,
and the proteins associated with the particles
eluted at different formamide concentrations
have been characterized. The specificity of binding and the function of these proteins have not
yet been determined: of course, the more attractive hypothesis is that they may play a role in
the regulation of translation or of degradation of
specific mRNA molecules.
As reported in Ribosome Efficiency, the level
of polyadenylated RNA has been determined in
N. crassa mycelia growing exponentially in
three different media, i.e., glucose, acetate, and
glycerol: it represents substantially the same
fraction, 5%, of total RNA in the three media
(162).
The level of such an unstable element as polyadenylated RNA (half-lives of 20 min in S.
cerevisiae [81, 120] and 45 min in Schizosaccharomyces pombe [58]) is dependent on its rate of
degradation in addition to its rate of synthesis.
The average half-lives of polyadenylated RNA
in Neurospora have been determined in each of
the aforementioned media by means of a kinetic
analysis (162) of the data obtained from doublelabeling experiments: they are 60, 32, and 43
min, respectively, in glucose, acetate, and glycerol media (162). Thus, th average stability of
polyadenylated RNA varies in the different
growth conditions, with no direct correlation
with the rate of growth.
These values are not necessarily in conflict
with the shorter half-life of mRNA reported for
nitrate reductase (127), since, as stated before,
the average half-lives have been estimated.
The absolute rate of synthesis of a particular
RNA species cannot be directly obtained from
the rate of incorporation of a radioactive precursor, since the rate of equilibration of the pools of
nucleotide triphosphates is slow and varies with
the growth conditions (R. Zippel, L. Popolo, M.
G. Costantini, and E. Sturani, Exp. Mycol., in
press), and moreover, there is the possibility of
compartmentalization of the nucleotide pools
(88). An estimation of the relative rate of polyadenylated RNA synthesis with respect to the
rate of rRNA synthesis has been obtained by
measuring the ratio between the radioactivity
incorporated into polyadenylate-containing
RNA and that incorporated into stable RNA
after a 10-min pulse with [5-3H]uridine (162).
The relative rate of synthesis of polyadenylatecontaining RNA becomes lower at higher rates
of exponential growth (Table 3), or, in other
VOL. 45, 1981
GROWTH AND NUCLEAR DIVISION CYCLE IN N. CRASSA
TABLE 3. Relative rates of synthesis and average
half-lives ofpolyadenylate-containing RNA in N.
crassa in different steady states of growtha
Growth medium
Glucose
Acetate
.........
Ratio between rate
of synthesis of pol- Average half-life
of polyadenylyadenylate-containing RNA and ate-containing
that of stable
RNA (min)
RNA
0.15
60 4
0.26
32 ± 1
0.35
43 ± 3
a
to
I..
x
_
x
2
E
Co
up
15 °
a,
cn
.c
1
E
_
10 z
~-
words, the rate of synthesis of stable RNA becomes an increasingly larger fraction of the total
RNA synthetic rate as the growth rate increases.
From these ratios and from the known rates of
synthesis of stable RNA (7) the absolute rates
of synthesis of polyadenylated RNA (per genome equivalent) have been calculated. These
data are reported in Fig. 3 together with the
level of polyadenylated RNA (per genome) and
its rate of degradation (per genome). Whereas
the level of polyadenylated RNA per genome is
directly related to the rate of growth, its rates of
synthesis and degradation change with changes
in the rate of growth, without a clear correlation
with it.
We have suggested the hypothesis (162) that
the rate of synthesis of mRNA is a function of
the state of the promoters-as given, for instance, by their accessibility to RNA polymer
ases, which is affected by the nutritional and
physiological conditions but not related as such
to the rate of growth. Moreover, the dynamic
equilibrium existing between mRNA and ribosomes would protect against degradation a constant (relative to ribosomes) amount of mRNA,
whereas the excess would remain vulnerable to
degradation; the rate of degradation would thus
be a variable dependent on the rate of mRNA
synthesis and the amount of unprotected RNA.
According to this hypothesis, the limit to the
growth rate is given not by the rate of synthesis
of mRNA but rather by the rate of synthesis of
rRNA, which sets both the ribosomal level and
the steady-state level of mRNA.
Nutritional Shifts
Investigations on nutritional shifts further
suggest that the level of ribosomes and therefore
their rate of synthesis are central elements in
the regulation of the rate of growth in Neurospora. During a shift-down transition (from glucose to glycerol) (164), the net syntheses of
macromolecules are affected different ways (Fig.
4). Briefly, RNA accumulation almost stops for
about 2 h after glucose exhaustion; afterwards,
5__
In
an
_.
I
I
"0.2
0.3
0.4
0.5
FIG. 3. Level and rates of synthesis and degradation of polyadenylated RNA in N. crassa as a fuinction of the rate of growth. The level is expressed as
nucleotides (Nu) per genome, and the rates are expressed as nucleotides per minute per genome. Symbols: 0, rate of synthesis; 0, rate of degradation; A,
level of polyadenylated RNA. (From Sturani et al.
[1621.)
it resumes at about the same rate as that measured during growth on glycerol alone. The rates
of accumulation of DNA and protein are only
slightly affected during the transition period.
Moreover, the rate of methylation of rRNA is
largely reduced (by 85%) after glucose is exhausted, but that of tRNA is only partially inhibited (by 50%) (164).
The pattern of macromolecular net syntheses
observed during a shift up (from growth on
acetate to growth on glucose) (163) is shown in
Fig. 5. The rates of DNA accumulation and
protein accumulation remain at the preshift values for about 2 h after the addition of glucose
and then increase to the rate characteristic of
the new medium; in contrast, the rate of RNA
accumulation increases markedly 30 min after
the addition of glucose, initially at a rate substantially greater than that of the new exponential growth, which is achieved later on. The
enhanced RNA accumulation is due to an increase in the production of mature rRNA and,
less conspicuously, to an increase in the rate of
synthesis of tRNA. An increase in the relative
rate of ribosomal protein synthesis (163) is also
observed during the shift.
An observation made in both shifts in Neurospora is that the production of rRNA and that
of tRNA are not coordinated during the transitions (163, 164).
It is interesting to compare the shift up just
Downloaded from http://mmbr.asm.org/ on December 29, 2014 by guest
Glycerol ........
From Sturani et al. (162).
105
106
ALBERGHINA AND STURANI
MICROBIOL. REV.
P
*-
AAA
..--
106(
4)
A
0
O
D
..000
R
0
5.--
00
:
0
-
E
-
***000*00e@ee*
0
0
0
*
co
~05-
0&
0
.-
.
I
-1
l
-2
I
0
T
1
2
3
4
5
6
7
TIME (hours)
FIG. 4. DNA, RNA, and protein contents (D, R,
and P, respectively) of N. crassa during a shift-down
transition from glucose to glycerol. The cells were
grown in a medium containing limiting glucose (100
pg/ml) and an excess of glycerol (2%, vol/vol). D was
determined by the diphenylamine method (26) at the
times indicated. R was determined by the orcinol
method (46) as well as by measuring the incorporation
of [32P]orthophosphate into trichloroacetic acid-precipitable material (103). P was determined by the
microbiuret method (193) as well as by measuring the
incorporation of L -[carboxyl-'4C]leucine into hot tri-
chloroacetic acid-precipitable material. D, P, and R,
normalized, are plotted on a semilogarithmic graph.
The shift down (arrow) occurred at time zero. (Redrawn from Fig. 1 and 2 of Sturani et al. [164j)
AA
p
AA
A
10 -
A
AA
I
A
A
D
5described in Neurospora with similar experi&AAIA
AA
ments performed with yeast cells (98, 182).
4)
A
When Casamino Acids are added to a proto0
A
0trophic strain of S. cerevisiae, the rate of protein
accumulation is immediately enhanced (98),
whereas the rate of accumulation of RNA is
stimulated after 15 min, and the enhancement
E0 1of 25S, 18S, and 5.8S rRNA's is more precocious
of
RNAs
in
than that 5S and 4S
(98, 182). Also,
0
0
S. cerevisiae RNA is accumulated after the shift
0.5 g:for
the new
at a rate higher than that required
.
0
steady state (98). DNA synthesis begins to in.0
crease after about 100 min. Thus, S. cerevisiae
and Neurospora differ during the shift up in that
I
I
I
I.
stimulation of protein accumulation happens
5
44
0
1
2
3
-2 -1
reatively early in the yeast. It should be pointed
Time (hours)
out that in yeast cells, ribosome efficiency inFIG. 5. DNA, RNA, and protein contents (D, R,
creases with the growth rate (184), so the ob- and
P, respectively) of N. crassa during a shift-up
served immediate increase in the rate of protein transition
ofgrowth from acetate to glucose. Cultures
of
amino
be
after
addition
acids
synthesis
may
in acetate were shifted at zero
exponentially
sustained by recruiting inactive ribosomes into time (arrow) bygrowing
addition of 2% (wt/vol) glucose. Expolysomes.
perimental conditions and macromolecular levels are
In Neurospora the early and preferential in- as in the legend to Fig. 4. (Redrawn from Fig. I and
hibition (during a shift down) and stimulation 2 of Sturani et al. [1631.)
0 0
Downloaded from http://mmbr.asm.org/ on December 29, 2014 by guest
0
0
(during a shift up) of rRNA synthesis suggest
that the level of ribosomes is a key element in
the control of Neurospora growth and that a
feedback mechanism is active on ribosome synthesis. Furthermore, the partial inhibition of
ribosomal activity by low doses of cycloheximide
brings about a marked increase in the ribosome
level (16, 105a). These observations together
indicate that the intracellular concentration of
functional ribosomes influences their formation
in an autogenous fashion. However, in both Neurospora (23, 44) and yeast cells deprived of an
essential amino acid, the so-called stringent response has been observed. In particular, in yeast
cells the rates of both rRNA and ribosomal
protein syntheses are strongly inhibited (186),
whereas the synthesis of tRNA is much less
reduced (118, 147). Moreover, the coordination
of the transcription of rRNA and mRNA for
ribosomal proteins with translation has been
observed in lower eucaryotes in several experimental conditions (186).
Since it appears that the transcription of the
precursor of rRNA is not dependent on concurrent synthesis of ribosomal proteins (70, 148),
the transcription of rRNA is the primary target
of the stringent control. In conclusion, the reg-
VOL. 45, 1981
GROWTH AND NUCLEAR DIVISION CYCLE IN N. CRASSA
ulation of RNA synthesis is complex and can
separately adjust the rates of synthesis of the
different classes of transcripts and coordinate
their synthesis with the translational requirements of the cells.
diphosphate 3'-diphosphate resulting from a reduction in its rate of degradation due to energy
limitation (160). In addition, changes in the level
of another guanosine nucleotide, the so-called
phantom spot, have been proposed as part of a
servomechanism able to signal the reduction in
the capacity of the cell to synthesize ATP and
at the same time to protect against a complete
depletion of the intracellular ATP pool (64).
Neither guanosine 5'-diphosphate 3'-diphosphate nor phantom spot is detectable in Neurospora (6, 23, 104, 149). Nevertheless, in Neurospora, feedback processes that depress ATP utilization very quickly after inhibition of ATP
synthesis have been shown to be active (153).
The ATP level in Neurospora is small compared with its rate of utilization (39, 104, 152).
In fact, it has been calculated that the unreplenished ATP pool would be sufficient to sustain
the two major macromolecular syntheses (of
protein and RNA) only for a few seconds (104).
Thus, the ATP level is unlikely to have in itself
a regulatory role on macromolecular synthesis
(and degradation), whereas it seems possible
that nutritional conditions yielding a decrease in
energy production also induce inhibition of
rRNA synthesis and stimulation of protein degradation through the mediation of a metabolic
signal(s) yet unknown but different from the
one(s) that mediates a similar response in bacteria.
As far as the mechanism of enhanced proteolysis is concerned, whereas in E. coli it seems
to involve the activation of preexisting proteases
(159), in Neurospora it requires the synthesis of
new, relatively stable protein, either proteolytic
enzymes or regulatory polypeptides (104). In
effect, in Neurospora growing on limiting glucose the addition of cycloheximide before glucose is exhausted hinders protein degradation,
whereas if it is added after the onset of protein
degradation, it has no effect on it. Moreover,
when glucose is added back to glucose-starved
cultures, protein degradation is rapidly inhibited
even in the presence of cycloheximide. Protein
synthesis is thus required for the onset of protein
degradation, but not for its maintenance. Moreover, protein synthesis is not required to block
protein degradation.
In yeast cells (S. cerevisiae), protein turnover
has been found to be dependent on the carbon
source used (95). For instance, in yeast cells
growing in ethanol, a turnover rate of up to 2%/
h is observed, whereas in glucose the protein is
degraded at a lower rate (0.5 to 1%/h). Starvation for a nitrogen source increases the rate of
degradation. Removal of glucose also has a positive effect on protein degradation; however, this
occurs only if a low phosphate concentration is
Downloaded from http://mmbr.asm.org/ on December 29, 2014 by guest
Regulation of Protein Degradation
Another process that contributes to the regulation of cell growth is intracellular protein degradation (9, 68). In Neurospora the rate of protein degradation has been determined by standard pulse-chase methods, by measuring either
the radioactivity retained by proteins or the
radioactivity released into the soluble fraction
(intracellular soluble fraction plus medium) during the chase (5, 104).
In Neurospora, in which multiple intracellular
peptidases have been described (168), in different conditions of exponential growth the average
rate of protein degradation increases as the rate
of grwth decreases. It has been determined that
the rates of protein degradation in cells growing
in glucose, acetate, glycerol, and ethanol media
correspond to 3, 4, 14, and 25% of the respective
rates of protein synthesis (104). In slow-growing
cells the considerable rate of protein degradation
appears to put a heavy burden on growth metabolism, since up to one-fourth of the protein
synthetic activity is counteracted by proteolysis.
The physiological significance of this phenomenon is obscure, although it may explain the
presence of "extra" rRNA (7, 89) observed in
cells growing at very low rates.
During glucose starvation and a shift-down
transition of growth, the rate of protein degradation is greatly enhanced. In both conditions a
moderate (30%) decrease in the ATP level is
observed. If the ATP level is more severely
reduced by treatment of the glucose-starved
cells with 2-deoxyglucose (155), protein degradation is blocked. It therefore seems a general
feature of protein degradation in E. coli (68,
160), yeast (95), and Neurospora that a moderate decrease in ATP enhances therate of protein
degradation, whereas a depletion of ATP arrests
this process.
The latter effect indicates an ATP requirement for the process of protein degradation,
which has also been reported to occur in an in
vitro system of protein degradation (51). It recalls to mind similar energy requirements of
other catabolic processes, such as fatty acid oxidation and glycolysis.
A reduction in the availability of cellular highenergy phosphates may have a regulatory role
in enhancing the rate of protein degradation and
in reducing the rate of RNA synthesis. In bacteria this regulation has been proposed to be
mediated by an accumulation of guanosine 5'-
107
108
ALBERGHINA AND STURANI
MICROBIOL. REV.
present. Removal of glucose from a medium
containing 50 uM phosphate does not cause
changes to protein degradation.
In yeast cells (95), protein degradation is not
affected when the intracellular ATP concentration falls from 4 to 1 mM. However, when the
ATP concentration falls below 0.3 mM, protein
degradation stops completely.
I
I
I
I
0.4 w
!-
0.2
0.1 0
0.8
0
I
8
s
%
00
0.41
0.05 o
rl:4
a
0
0.21
IL.
0.1
[
0
0
I
z
z
* oW\t~~~
I
I
I
I
0.05
0
3.2 3.3 3.4 3.5 3.6
1/ABSOLUTE TEMPERATURE (X10$)
FIG. 6. Arrhenius plots of the rates of growth of N.
in different media. Growth was measured in
glucose medium (0) and in glucose medium plus 2%
Casamino Acids (0) (left ordinate), in acetate medium (5), and in acetate medium plus 2% Casamino
Acids (U) (right ordinate).
crassa
L cells the polypeptide initiation reaction berate limiting (41), whereas in differentiated reticulocytes, low temperature appears to
slow down both polypeptide initiation and elongation (42).
In Neurospora the average ribosome efficiency is much lower (down to 20%) at 80C
compared with that at 250C, whereas the percentage of polysomes is the same at both temperatures, suggesting that there is not a preferential inhibition of the initiation reaction of protein synthesis. The reduction in the rate of
growth at low temperature is due not solely to
the decreased efficiency of the ribosomes, but
also to a severe inhibition of the rate of rRNA
accumulation (103). If the inhibition of the rate
of rRNA accumulation reflects a parallel change
in the rate of synthesis of rRNA, and not an
enhancement of its degradation, at least two
possible mechanisms can be hypothesized: a
temperature effect on the rate of processing of
precursor to mature rRNA, as observed for mitochondrial rRNA (90), or an effect of temperature on the promoter efficiency of the rRNA
genes (172).
comes
Downloaded from http://mmbr.asm.org/ on December 29, 2014 by guest
Effect of Temperature on Growth
Temperature is the other environmental factor besides nutrient availability that affects microbial growth. Hyphal elongation and branching in wild-type N. crassa follow similar pattems
over a wide temperature range. The hyphal
growth unit is not appreciably modified by temperature changes between 15 and 300C. In contrast, in a temperature-sensitive colonial mutant
its value is sharply reduced at 300C (157). The
specific growth rate of the hyphae varies proportionally with the mean extension rate; thus,
temperature modifies the rate at which the hyphal growth unit is duplicated, but not its length.
The Arrhenius plots of the growth rate constants of N. crassa cultures growing in glucose
and in acetate media (1, 3, 103) are shown in Fig.
6: maximal rates are reached in both media
around 36 to 380C. A sharp deviation in the plot
occurs at about 200C. The activation energy
calculated from the slope in the high temperature range (20 to 3700) is 7.8 kcal/mol (ca. 32.8
kJ/mol) in both media. In the low temperature
range (below 2000) the activation energy is
about 25 kcal/mol (ca. 105 kJ/mol) for both
cultures. At 300C the addition of Casamino
Acids to both glucose and acetate media causes
a sharp increase in the rate of growth; by lowering the temperature the percent stimulation
becomes smaller, and for temperatures of 200C
and below it is no longer observable. At temperatures below 200C the growth rate is the same
in glucose and in acetate, with or without the
addition of Casamino Acids. The activation energy in the high temperature range is 15.8 kcal/
mol (ca. 66.4 kJ/mol) in glucose and in acetate
media supplemented with Casamino Acids (see
Levels of Amino Acids and Nucleotides for the
effects of amino acids and temperature on RNA
synthesis).
The low-temperature deviation in the Arrhenius plot of the growth rate is generally interpreted as determined by the cold denaturation
ofmacromolecular components, limiting the rate
of growth. In E. coli low temperature has been
shown to inhibit the initiation reaction of protein
synthesis, so by lowering the temperature to 8°C
a rapid runoff of polysomes is observed (43, 61).
In mammalian cells the effect of low temperature is more complex. In rapidly growing mouse
TEMPERATURE (centigrade )
40 30
20
10
VOL. 45, 1981
GROWTH AND NUCLEAR DIVISION CYCLE IN N. CRASSA
TABLE 4. Amino acid levels in N. crassa at
different steady states ofgrowtha
Amino acid levelb in cells grown
in:
Amino acid
Leucine ......
Isoleucine .............
Phenylalanine .........
Valine ................
Proline ................
Alanine ...............
Glycine
..
Glucose
Glucose
300CC
0.38
0.24
0.28
0.65
0.65
8.20
2.28
Glucose
plus
Casamino
Acids
200Cc 80Cd 300Cc 200Cc
0.27 0.27 1.10 0.81
0.25 0.18 0.58 0.23
0.24 0.17 0.33 0.28
0.72 0.59 4.37 1.60
0.43 0.36 0.82 0.32
8.50 8.70 8.77 10.15
1.71 0.94 2.18 2.45
Lysine, arginine, and
histidine ............ 5.76 7.70 11.40 7.73 7.53
The rates of growth (u, number of duplications per hour)
were 0.51 in glucose at 30°C, 0.29 in glucose at 200C, 0.05 in
glucose at 80C, 0.65 in glucose plus Casamino Acids at 30°C,
and 0.30 in glucose plus Casamino Acids at 20°C.
'Determined according to Hartley (76). The data are expressed aa nanomoles per unit of absorbance at 450 nm of
culture.
c From Martegani et al. (106).
d
From Martegani and Alberghina (103).
reaction catalyzed by purified synthetases (113,
141, 156). On the other hand, since the cytoplasmic pool is small, it is still possible that the
amino acid pool available for protein synthesis
may fluctuate more widely and rapidly than the
average intracellular concentration. The average
intracellular concentration of total tRNA has
been estimated in Neurospora in different
steady states of growth to range between 0.2 and
0.4 mM and to be slightly higher in faster-growing cells (7). Each specific tRNA should therefore be present at concentrations that do not
exceed the order of 10 ,uM. Since the Km values
of purified synthetases for tRNA are in the order
of 1 to 0.01 uM (133, 136, 141), the level of at
least some tRNA (and of its cognate amino acid)
may not be saturating.
The degree of charging of tRNA has long been
thought to have a regulatory role for enzyme
activity (156) and for RNA and protein synthesis
in both procaryotes and eucaryotes (37, 63, 73).
The formation of guanosine 5'-diphosphate 3'diphosphate in bacteria (63) and that of diadenosine tetraphosphate in mammalian cells (129)
may both be considered as parts of mechanisms
able to measure the relative amounts of charged
tRNA and uncharged tRNA (that are the outcome of variations that have occurred in the
energy or in the anabolic metabolism of the cell)
and to adjust macromolecular synthesis accordingly.
The addition of Casamino Acids (at a concentration between 2 and 0.005% [wt/vol] to a minimal medium at 300C) increases the rate of
Downloaded from http://mmbr.asm.org/ on December 29, 2014 by guest
Levels of Amino Acids and Nucleotides
It has been observed that cells growing in
different media (for instance, Neurospora cultures growing in glucose or in glycerol plus Casamino Acids) may grow at the same rate of
exponential growth, although of course they differ in their intermediary metabolisms. In addition, the enzymatic setup and the manner of
utilization of the carbon source may be modified
without affecting the rate of growth (see Effects
of Nutrients on Growth and Differentiation).
Nevertheless, no matter how different the intermediary metabolism patterns may be, since the
rate of macromolecular synthesis is the same,
the rate of production of their substrates (nucleotides, amino acids) also has to be the same.
It therefore seems possible that the rates of
production of the substrates for macromolecular
syntheses are closely coupled to the rates of
their utilization and that in each given growth
condition the rate of production of a given particular substrate may become the rate-limiting
step for the rate of growth. Infornation on this
point has been sought by analyzing the relationships between the intracellular levels of amino
acids (106) and ribonucleoside triphosphates
(39) and the rates of protein and RNA syntheses.
The intracellular contents of several amino
acids measured during exponential growth on
minimal media are shown in Table 4 (106). They
are very similar to other reported amino acid
levels in Neurospora mycelia (134, 166). For
cells growing on glucose, the intracellular level
of amino acids remains constant even if the rate
of growth and hence the rate of protein synthesis
is reduced to 1/10 by reducing the temperature.
The rate of utilization of amino acids may be
very high compared with their intracellular
levels. For instance, in cells growing on minil
glucose at 300C, the unreplenished pools would
last from about 1 min (for leucine, isoleucine,
and phenylalanine) up to about 10 min (for
alanine). This may very well be an overestimation, since the amino acid pool utilized for protein synthesis is only part of the intracellular
content. In fact, in Neurospora, amino acids are
compartmentalized into two pools (166, 189, 190,
192). A relatively small pool is utilized for protein synthesis and quickly equilibrates with the
extemal amino acids. A second one, localized in
vesicles, which for several amino acids accounts
for the bulk of their intracellular content, is
expandable and exchanges slowly with the cytoplasmic pool.
The average intracellular concentration of the
amino acids present at the lowest levels (isoleucine, leucine, and phenylalanine) is around 0.2
mM in glucose-growing cells, well above the 10
to 0.1 uM values of Km for the tRNA charging
109
110
ALBERGHINA AND STURANI
division cycle is an important aspect of the regulation of cell proliferation (30, 77, 83, 109),
although the relationships between the two processes are not yet completely understood.
Whereas the growth metabolism of Neurospora
has been extensively characterized as levels and
rates of syntheses of protein, rRNA, tRNA, and
polyadenylate-containing mRNA and protein
degradation as discussed above, the information
available in the literature on the nuclear division
cycle in Neurospora is much less abundant (105,
140).
When young hyphae of N. crassa stained with
Giemsa or Feulgen and crystal violet are observed by optical microscopy, different nuclear
shapes are distinguishable: uniformly colored
globular nuclei, ring-shaped nuclei double-ring
nuclei, and horseshoe nuclei (105, 187, 188). Evidence obtained by arresting the progression of
the cell cycle by treatment with picolinic acid
has indicated that compact globular nuclei are
most likely in the G1 phase and ring nuclei are
most likely in the S and G2 phases, whereas
double-ring and horseshoe nuclei are premitotic
or mitotic shapes (105). The frequency of compact globular nuclei is much higher in hyphae
growing at lower rates, whereas that of ring
nuclei increases when the hyphae are growing at
higher rates (105). Taking into account the age
distribution function of the nuclei (102, 125), the
durations of the G, and the S plus G2 phases at
various duplication times have been calculated
(Table 7) (105). First estimations of the durations of the various nuclear division cycle phases
in Neurospora have thus been obtained: they
indicate that an increase (up to 3 times) in the
NUCLEAR DIVISION CYCLE IN
length of the duplication time (T) is achieved by
DIFFERENT STEADY STATES OF
increasing (up to 15 times) the length of the G1
GROWTH
phase, whereas the lengths of the S plus G2
The coordination between growth (mainly phases appear to remain more or less constant,
protein and RNA synthesis) and the nuclear following a pattern that is quite common also to
other eucaryotic cells (17, 85, 151). These results
TABLE 5. Effect of amino acids on growth rate and have been recently confirmed in our laboratory
by using several other experimental approaches
RNA content of N. crassaa
(see Table 7 and E. Martegani, F. Tome, and F.
RNA/
J. Cell. Sci., in press). The length of the
Trezzi,
DNAc
Growth
rate
Temp
(wt/wt)
Medium
(,)b
(0C)
S phase in Neurospora has been determined to
ratio
be about 30 min, by measuring DNA synthesis
in mycelia whose nuclei have been synchronized
30
0.51 ± 0.07 (35) 35.5 ± 2.5
Glucose ..........
Glucose plus Casain G1 by treatment with picolinic acid (105). This
30
0.65 ± 0.03 (12) 44.9 ± 1.9
mino Acids
value
compares with S phase durations of 20
0.29 ± 0.08 (8)
36.6 ± 2.3
20
Glucose ..........
min in Aspergillus nidulans (85), 10 to 15 min
Glucose plus Casain S.pombe (20), and 20 to 25 min in S. cerevisiae
36.8 ± 3.4
20
0.30 ± 0.10 (8)
mino Acids ...
(77, 151).
a From Martegani et al. (106).
bThe average value and standard error are given. The
MODELING CELL GROWTH AND
number of determinations for each growth condition is given
DIVISION IN EUCARYOTES
in parentheses.
' The average value and standard deviation are given. Each
As discussed above, considerable progress has
value is the average of six determinations. RNA and DNA
were determined by standard colorimetric techniques (26,46). been made in the description of the macromo-
Downloaded from http://mmbr.asm.org/ on December 29, 2014 by guest
growth of Neurospora cultures and their ribosomal level (106). The stimulatory effect is temperature dependent, since it is abolished at 200C
(Table 5 and Fig. 6), and seems to be due to
neutral amino acids, since the additions of basic
and acidic amino acids have no effect (106).
It seems improbable for the moment that the
lack of stimulation of stable RNA synthesis observed at 200C is due to a reduced uptake of
amino acids, since there are no great differences
in the intracellular amino acid pools between 30
and 200C (Table 4). The experimental conditions just described may be useful to study the
relationships between the availability of nutrients and the rate of stable RNA synthesis.
The intracellular levels of the four ribonucleoside triphosphates (ATP, guanosine 5'-triphosphate, uridine 5'-triphosphate, and cytidine 5'triphosphate) in several conditions of exponential growth of Neurospora cultures are shown in
Table 6 (39). The rate of utilization of the nucleotides is very high compared with their levels:
for instance, the amount of cytidine 5'-triphosphate in 2% glucose would support total RNA
synthesis for about 1 min (39). The level of
nucleotides does not appear to be strictly related
to the rate of RNA synthesis: for instance, the
nucleotide levels are significantly different in 2%
and in 0.01% (initial concentrations) glucose,
although the rates of RNA synthesis are very
similar. Moreover, during a shift-down transition
their level shows large oscillations, whereas
RNA accumulation is almost completely
blocked (39).
MICROBIOL. REV.
GROWTH AND NUCLEAR DIVISION CYCLE IN N. CRASSA
VOL. 45, 1981
111
TABLE 6. Nucleoside triphosphate levels during exbonential growth of N. crassaa
Nucleoside tnphosphate level (nmol/unit of absorbance at 450 nm)
Growth medium
(initial compnsition)
ATP
Glucose (2%) ..
Glucose (0.01%) .
Acetate ..
..
..
Glycerol ....
a From Costantini et aL (39).
Guanosine 5'-tri-
2.462 ± 0.065
1.080 ± 0.102
1.213 ± 0.204
1.234 ± 0.101
crassa
G,
Medium
(minT b phase
(mn,b(min)b
S+G2
phaes
Pae
(min)b
450
370
80
Ethanol ............
3.62
220
132
88
Glycerol ............
3.57
100
20
Glucose .............
80
3.52
8
72
80
3.62
Supplemented glucose
'
Duplication dme, duration of a cell cycle (In 2/A).
b From L Alberghina and L Mariani (in DeBernardi, ed.,
Biooical and Matmatia Aspects in Population Dynamics, Mem. Ist Ital. Idrobiol., suppl. 37, in prem).
Critical protein content rquired for the onset of DNA
replication, expresed as protein (tens of billions of amino
acids) per gepnome equivalent. P. values were calculated from
equation 9 with data obtained from Alberghina et al. (7) and
Martepni et aL (105) and assuming a constant length of TR
(time required for DNA replication) equal to 30 min.
lecular metabolism which characterizes different
growth conditions in Neurospora as well as in
other microorganisms, such as E. coli and S.
cerevisiae. Notwithstanding, our knowledge on
the regulation of cell growth in bacteria and
eucaryotic cells is still poor. More and more it
appears that the reductionistic approach, which
has been so useful for the description of the
molecular components of a cell and of more
simple regulatory systems (regulation of enzymatic pathways, control of gene -expression in
bacteria), is not adequate for the study of highly
integrated systems, such as those controlling
growth and cell division (10, 71), for which it
seems necessary to develop an integrated approach. Mathematical models may very well be
useful for this undertaking. In fact, they allow
quantitative descriptions of both the interrelations among the relevant variables of the phenomenon and the dynamics of the events under
consideration. Comparison of the predictions of
the model with experimental results allows verification of the validity of the assumed functional
or causal links. In any event, it is obvious that
the isolation of the phenomenon from its context
and the choice of the variables and their interactions are to a certain extent arbitrary. Different models can describe the same phenomenon,
and each of them may be more useful than
0.523 ± 0.024
0.255 ± 0.020
0.291 ± 0.053
0.255 ± 0.016
0.681 ± 0.019
0.395 ± 0.060
0.425 ± 0.056
0.277 ± 0.031
Cytidine 5'-triphos-
phate
0.300 ± 0.035
0.150 ± 0.020
0.155 ± 0.031
0.133 ± 0.015
others for a given specific purpose. Thus, each
model can only describe partial aspects of a
phenomenon. With these limitations in mind, we
have developed a mathematical model of cell
growth and division (9, 11; L. Alberghina and L.
Mariani, Mem. Ist. Ital. Idrobiol. Pallanza Italy,
in press). Since the events of cell growth and
division have their origin in a remote evolutionary past that is common to all eucaryotic organisms, a fair degree of evolutionary stability of
the elements and relations that are geared in the
regulatory process may be expected, and therefore a model common to eucaryotic cells is pro-
posed.
Dynamic Model
For the control of cell proliferation, the interactions between the growth cycle (mainly RNA
and protein synthesis) and the nuclear division
cycle (made up of a sequence of events: DNA
replication, G2 phase, nuclear division, and cell
division) have to be considered. A large number
of findings obtained in various lower eucaryotes
suggest that a cell size control is effective over
DNA replication and nuclear division (52-54,83,
109, 170).
A model has thus been developed that attempts to give structure to these relations (9, 11)
(Fig. 7). The model is divided into two subsystems: the first, whose state variables are ribosomes (R) and protein (P), determines the dynamics of the entire system; the second is activated by two P thresholds, one controlling DNA
synthesis (P.) and the other controlling nuclear
division (Pm). The rate of protein accumulation
sets the dynamics of the DNA division cycle,
since DNA replication can only start when the
P level reaches a P. threshold, and mitosis can
take place only after the P level reaches a Pm
threshold (Pm > P.).
rRNA synthesis and ribosome accumulation
correlate with enhanced cell proliferation, and it
has been suggested that rRNA metabolism may
have a regulatory role for the progression of the
cell cycle (84). This point is, of course, very
relevant for the proposed structure of our model.
Experiments have been performed in our laboratory to dissociate the rates of accumulation of
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TABLE 7. Nuclear division cycle parameter8 in N.
Uridine 5'-triphos-
pahate
112
ALBERGHINA AND STURANI
8TG*wivsio
2k pm
FIG. 7. Model of the cell cycle. The increase in the level ofprotein (P) is determined by the bakmce between
the rate of synthe8is (given by the product of the number of ribosomes [RI and their average efficiency in
amino acid polymerization [KO]) and the rate of degradation. The increase in R is due, in a similar manner,
to the balance between the rate of synthesis and that of degradation. The rate of synthesis is determined by
a more complex relationship (which is expressed as K1 [pP - R], if the actual value of R is less than the
desired value pP [p is the required ribosome level per unit amount of PI; otherwise the rate of synthesis is
zero). When the level ofprotein exceeds 2%p, (2k is the number ofhaploid genome present; P. is the critical P
value required for the onset of DNA replication), DNA replication starts at a linear rate and stops at the end
of TR (time required for DNA replication). After a further TG,mmi (the incompressible time between the end of
TR and the beginng ofmitosis) period and after the protein level has surpassed Pm (critical P value required
for nuclear division), mitosis takes place, and, followingTo; (time between the end of mitosis and cell division),
cell division occurs (Tm + TG; = Tc). j2, comparator; r, threshold. (From Alberghina et al. [13].)
protein and ribosomes and to investigate to
which of the two rates the rate of DNA synthesis
appeared to be coupled. These experiments indicated that in S. cerevisiae the rate of bud
formation (which is coincident with the initiation of DNA replication [77]) is reduced when
protein accumulation is inhibited by low doses
of cycloheximide, which, in contrast, have no
effect on the rate of rRNA accumulation (L.
Popolo, M. Vanoni, and L. Alberghina, manuscript in preparation). The assumption that a
critical protein level controls cell cycle progression therefore appears to be substantiated by
these results, whereas the ability to produce
ribosomes does not seem to be strictly necessary
for the initiation of DNA synthesis (see also
references 74 and 110).
The first subsystem models the controls and
the rates of ribosome and protein synthesis (11)
that yield an exponential increase (with the
same rate constant) of both macromolecular
levels during a cell cycle (49, 50, 59). At time ti,
the protein level (P), which increases exponentially from an initial P0 value (protein content of
a newbom cell), reaches the threshold value
2kP., i.e.:
P(tO) = 2eP8, P. = constant, k = 0, 1, 2, ... (3)
where 2k is the number of haploid genome sets
present in the cell, and DNA replication starts
at a constant rate. It stops, after TR (time required for DNA replication), at time t2:
t2
=
ti + TR
(4)
After a further interval, TG2 (time between the
end of TR and the beginning of nuclear division),
which cannot be shorter than TG2 mi (incompressible period between the end of TR and the
beginning of nuclear division), if (and only if)
the protein level P has exceeded the threshold
level:
2kPm = constant, Pm > P.
(5)
nuclear division occurs, requiring the time period TM (time required for mitosis). After a further constant delay, rTG; (which is usually very
short in most eucaryotic cells but may be considerable in yeast celLs [112] and during which
the cells are binucleate, each nucleus having a
presynthetic content of DNA), cell division takes
place, giving two identical daughter celLs, and a
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2
MICROBIOL. REV.
VOL. 45, 1981
GROWTH AND NUCLEAR DIVISION CYCLE IN N. CRASSA
T2-TR + TG2n + TM + TG;=Tri
(6)
where Tm., is the minimal time between the
beginning of TR and cell division.
The first two patterns are characterized by
the fact that during the cell cycle both threshold
levels (P. and Pm) are crossed, and cycles with
all four classical phases (G1, S, G2, and M) thus
originate. The third pattern, which is less common, is characterized by the fact that the P.
level is larger than the P. leveL and DNA replication therefore starts at the beginning of the
cycle; a cell cycle is thus generated that lacks
the GI phase.
The parameters of the nuclear division cycle
are likely to be structural determinants of the
system and therefore genetically determined; for
instance, in S. pombe, mutants have been described in which Pm appears to be no longer
operative (55). However, nutritional conditions
seem able to modulate the values of at least Pm
and P8. In fact, there is experimental evidence
that the critical cell size for nuclear division in
S. pombe is set by the growth conditions and is
quickly reset when they change (54). The critical
cell size for budding (coincident with the initiation of DNA replication) in S. cerevisiae appears
also to be modulated by the growth conditions
(82, 176).
Critical Protein Level for the Initiation of
Deoxyribonucleic Acid Synthesis and Cell
Cycle Timing
The model discused so far is deterministic,
and its parameters are taken to be time invariant
and constant, unless they are changed by modifications of the environment. Furthermore, if
one assumes that during a steady-state condition
of growth a cell divides at the end of the cycle
exactly into two parts (so that each new cell is
identical in composition to the original one), it
is possible, for instance, to determine P., the
critical protein level per genome required for the
initiation of DNA replication, from a few parameters readily measurable on exponential cultures
for organisms that follow one of the first two
patterns of the cell cycle. In fact, it has been
shown (12) that the average protein level per
cell (P) in balanced exponential cultures is related to P. as follows:
P = In 2 x 2 x P. x 2T
(7)
where T is the time between the beginning of TR
and cell division.
As shown in Table 7, in Neurospora the critical protein level (P.) required for the initiation
of DNA replication is not modified by the
growth rate, contrary to the pattern observed in
S. cerevisiae (82, 176). The possibility of determining P. is important both to identify mutants
of cell cycle regulation and to investigate the
biochemical basis of such a control.
The protein level per cell at the beginning of
the cycle (PO) is univocally related to P as follows
(12):
P.=P/(2xIn2)
(8)
Furthernore, if one knows the P. value from
equation 7, it may be possible to estimate the
relative duration of the S phase (TRIT) by measuring the average DNA content (D)) of an exponential culture and by using the following
relation (12):
D
P
2.08
P-
1 - 2-TRIT
TR/T
(9)
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new cycle begins, following the same rules.
For Neurospora this general model has to be
slightly modified to account for the lack of cell
division in hyphal growth (14).
Possible Different Cell Cycle Patterns
Analysis of the cell cycle patterms observed
experimentally indicates that they can be divided into three groups: (i) one in which only a
size control over S is active and in which the
subsequent G2 phase has a constant duration;
(ii) one having a size control over S, but in which
the length of the G2 phase varies, depending on
the rate of growth; and (iii) one for which the
cycle lacks the G1 phase and the size control is
operative only over mitosis. Several lower eucaryotes, such as Physarum (117), the wild-type
strain of S. pombe (20), and Amoeba (119, 130)
follow the third cell cycle pattern. The attribution of a cell cycle to the first or the second
pattern requires knowing whether or not the
observed TR + TG2 period is expandable by
changing the growth conditions and hence the
duration of the cell cycle. An example of the first
pattern is Neurospora (Table 7). Examples of
the second pattern are given by S. cerevisiae (in
which, by changing the growth rate, a moderate
expansion of the Tr, period is observed [17,
151]) and A. nidulans (85).
It has been shown (12) that all three different
patterns can be interpreted by the previously
presented model, since they are particular cases
that derive from the values assumed by the cell
cycle length, T (duplication time [In 2/1]), which
depends on the first subsystem, and by the parameters P., Pm, TR, Tr,m TM, and TG*l of the
second subsystem, under the firther assumption
that:
113
114
ALBERGHINA AND STURANI
MICROBIOL. REV.
APPLICATION OF TH MODEL TO
T < Tmlin,
(10)
that is, there are cell cycles shorter than 62 mi
(40 min for C and 22 min for D); rapidly growing
cells have, in fact, duplication times between 60
and 20 min (36).
From the analysis of the model, relations are
derived that determine, for any possible cell
cycle, the time of initiation and that of termination of DNA replication and the number of
chromosomes involved in both processes (lla).
These relations offer a new and general way to
describe the kinetics of DNA replication in E.
coli from any given value of the parameters of
the DNA synthesis and division cycle.
Possible Molecular Basis for Control
Elements in E. coli
Although the model proposed for E. coli aims
only to suggest a possible logic for the regulation
of macromolecular biosyntheses, without making any commitment on the molecular basis of
such a regulation, nevertheless, it may be interesting to discuss some experimental findings that
are in qualitative agreement with the proposed
control circuits.
In the model two elements are assumed to
control ribosome production: a positive feedback, driven by the protein level in the cell, and
a negative feedback that blocks their production
when a ribosome surplus occurs. Molecular elements present in E. coli cells could perform
these functions. The rate of ribosome formation
is blocked in E. coli by the increase in the
intracellular concentration of guanosine 5'-diphosphate 3'-diphosphate (31). This nucleotide
is produced when ribosomes are unable to continue protein elongation (31, 63), and the increase in its concentration could indicate that
more ribosomes are present in the cell than are
able to work properly. Thus, guanosine 5'-diphosphate 3'-diphosphate signals the occurrence
of a surplus of ribosomes to their synthetic machinery, acting as a key element of a negative
feedback control loop.
Furthermore, the rate of ribosome synthesis
is primarily dependent on the relative amount
and specific activity of RNA polymerase (45,
100, 111, 114). A clue to how the RNA polymerase activity may increase with the increase in
total protein (positive feedback) comes from the
following observations. The number of RNA
polymerase molecules engaged in RNA synthesis (per unit of cell mass) is found to increase
with increasing growth rate, whereas the amount
of unengaged RNA polymerase is relatively constant and independent of the growth rate (100).
If the unengaged RNA polymerase acts as an
autorepressor (69), a simple feedback control
that keeps its concentration relatively constant
will increase the total number of RNA polymerase molecules with the increase in the protein
level. The fact that a number of experimental
reports indicate that the synthesis of RNA polymerase is under autogenous regulation (62, 66,
78, 167) may give some further support to this
suggestion.
CONCLUDING REMARKS
Three different metabolic domains are apparent when cell growth and division are consid-
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ESCHERICHIA COLI
The cell division cycles of procaryotes and
eucaryotes are clearly distinguishable by a number of features. In eucaryotic cells, DNA synthesis is a periodic activity during the cell cycle,
whereas it may be a continuous one in bacteria
(36). Eucaryotic cells are unable to synthesize
DNA during mitosis and cell division, whereas
bacteria are able to do so. Eucaryotic cells cannot initiate multiple rounds of DNA replication,
whereas bacterial cells very often have multiple
DNA initiations and may begin a new round of
replication before the previous one has finished
(36, 79). The DNA content per cell and the
chromosome configurations are therefore much
more variable in bacteria than in eucaryotic
cells. All these differences may well be related
to nuclear organization, which represents the
major difference between these two groups of
cells. In fact, the complex process of mitosis
probably does not allow more than two sister
chromatids, thus barring the possibility of complex chromosome configurations. In addition,
the DNA condensation into chromosomes during mitosis is not likely to be compatible with
continued DNA synthesis. Nevertheless, there
are two important similarities between procaryotic and eucaryotic cell cycles: a critical cell
mass appears to be required for DNA initiation
for both types of cells (47, 109), and there is an
analogy between the C and D periods of the
bacterial cell cycle and the S and G2 phases of
the eucaryotic cell cycle.
There is a growing awareness that it is possible
to explain the cell division cycles in both procaryotes and eucaryotes by a unifying model (11,
lla, 35). If a few provisions are taken to account
for the indicated differences between the two
kinds of cells, the model discussed previously is
also able to describe growth and cell division of
bacterial cells (lla, 15).
The real major difference between the two
models is that for eucaryotic cells T 2- Tin, (see
equation 6), whereas in bacterial cells it is also
possible that:
VOL. 45, 1981
GROWTH AND NUCLEAR DIVISION CYCLE IN N. CRASSA
115
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ered: (i) the intermediate metabolism, which for starting DNA synthesis appears at random
converts the nutrients to the substrates of mac- in the cytoplasm and may be transient (22). The
romolecular synthesis; (ii) the processes of RNA threshold behaves therefore as a point of instaand protein synthesis; and (iii) the events of the bility that generates a fluctuation. The deternuclear division cycle, whose landmarks are ministic representation of cell growth and diviDNA replication, the G2 phase, mitosis, and cell sion discussed in this paper, which is able on the
division. These domains are connected by fluxes average to describe the behavior of a population,
of metabolic products or by regulatory signals. is no longer reliable at the level of the threshold
An example of metabolic flux is given by the for individual cells, and thus the dynamics acproduction of amino acids and ribonucleoside quires a stochastic behavior (143, 146, 154). The
triphosphates, which appears to be closely re- mathematical model discused in this paper may
lated to the rates of protein and RNA syntheses. be useful in further studies on the functional
The production of polyamine by intermediate properties of the threshold mechanism, since it
metabolism may also be considered as a meta- allows prediction of the kinetics that result from
bolic flux (or facilitating reaction) required to the dispersion of the threshold and permits their
sustain proper ribosome and DNA synthesis. comparison with the experimental results (L.
The high content of spermidine found in Neu- Alberghina, L. Mariani, and E. Martegani, in M.
rospora mycelia during exponential growth on
Rotenberg, ed., Biomathematics and Cell Kiglucose (181) indicates that a large production netics, in press).
of spermidine and polyamines is generally
In any case, for a cell population the rate of
geared to the normal pathway of ribosome for- proliferation is given by the activity of the first
mation. It is not surprising, therefore, that the subsystem, i.e., by the rate of protein accumurate of polyamine production correlates with the lation. When the critical cell mass is achieved,
rate of growth, which depends on the rate of the nuclear division cycle, of relatively constant
RNA synthesis. The request for specific proteins duration (T), is activated. It follows that a cycle
in the program of the nuclear division cycle is without the G1 phase is generated when T (set
also sustained by a metabolic flux between the
by the activity of the first subsystem) is equal to
domain of RNA and protein synthesis and that T (11). Thus, the G1 phase is not specifically
of the nuclear cycle.
needed for the initiation of DNA replication,
An example of a regulatory signal between since the conditions required for that are continintermediary metabolism and a process of the uously built up from the previous initiation of
nuclear division cycle in eucaryotic cells is given DNA replication (35). The G, phase represents
by the specific activation of DNA polymerase a period of silence in the activity of the nuclear
(DNA nucleotidyltransferase a), the putative en- division cycle due to the fact that the conditions
zyme of DNA replication (32, 72), by diadenosine
required for its activation take longer to be gen5',5"'-P4_,P-tetraphosphate. Many more of these erated in the cell than the duration of one of its
regulatory signals are likely to be discovered cycles. The interplay between the value of T and
when the details of the regulation of cell growth the parameters of the nuclear division cycle sets
and division are unraveled. For the moment, the the average cell composition, as indicated in
major regulatory relation in growth metabolism Possible Different Cell Cycle Patterns.
appears to be the activation of the nuclear diviAn intriguing observation has recently been
sion cycle program by the attainment of a critical made in both yeast and mammalian cells (82,
cell mass or, more likely, a critical protein level. 191): older cells are bigger than younger cells, as
in the cell. The threshold mechanism able to if the value of the threshold increases or its
detect the attainment of the critical protein level mechanism becomes ineffective in senescent
seems also endowed with the following propercells. This hypothesis finds support in results
ties: (i) it is able to integrate other informations obtained by cell fusion that indicated that the
on the metabolic conditions of the cell in addisenescent phenotype involves a block which pretion to that on the critical protein level (77, 178), vents cells in the G1 phase from entering the S
(ii) it assures a relatively constant mass distri- phase (158). The resulting alterations in the
bution of a cell population in each given growth nuclear/cytoplsinic mass ratio may cause a
condition (83), and (iii) it allows some variability metabolic collapse and hence death of senescent
in the cell mass for the initiation of DNA syn- cells.
thesis among individual cells of a population (86,
The rates of RNA and protein syntheses (and
144).
degradation) are closely connected to the activThe synthesis of a phase-specific protein of ity of the intermediate metabolism, which proshort half-life seems required for entry into the vides the substrates for macromolecilar syntheS phase in both yeast (145) and mammalian (21, sis. The rates of production of the substrates are
93) cells. For the latter, the signal responsible generally adjusted to their rates of utilization
116
ALBERGHINA AND STURANI
ACKNOWLEDGMENTS
We thank our friends and colleagues M. G.
Costantini, L. Maniani, E. Martegari, L. Popolo, F.
Tome, F. Trezzi, and R. Zippel for helpful discussions.
The editing and preparation of the manuscript by
Betty Johnston is gratefully acknowledged.
This review was partially supported by a grant from
the Consiglio Nazionale delle Ricerche to L.A.
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VOL. 45, 1981
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