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AM. ZOOLOGIST. 6.33-41 (19G6).
Gene Activity Patterns and Cellular Differentiation
ULRICH CLEVER
Department of Biological Sciences, Purdue University
Lafayette, Indiana
SYNOPSIS. Puffing in giant chromosomes ot Diptera is considered to reflect the pattern of active gene loci in these chromosomes. In any one tissue only a relatively small
portion of the total bands (about 10 to 20%) have been observed to form a puff at
some time or another in larval development. These patterns of "potentially active"
loci are tissue specific, though greatly overlapping. The actual rate of activity at
these loci is controlled independently of each other and independently in each tissue
by factors of the extranuclear metabolism. Puffing at some loci seems to be related
to specific cellular functions, such as secretion of the salivary glands. The activity of
others may be related to more basic metabolic processes. In relation to larval development, puffing patterns may change with changing cell functions or with developmental
processes in the cells themselves. In salivary glands of Chironomus activity of DNAase
and of acid phosphatase seems to change in relation to cell breakdown at the end
of the pupal molt. Changes of acid phosphatase activity begin early in the last larval
ins tar, but the enzyme is bound to.lysosomes until metamorphosis. This suggests
that the genes specifically active during metamorphosis have to interact with a longterm control-system of development. The induction of metamorphosis is a sequential
process, gene activations being among the first steps in this sequence. The activation
of these genes by ecdysone is independent of protein synthesis. It is only the reaction
of these genes that leads to the subsequent events in the cell, including the subsequent
puff activations. This is shown by the fact that they depend on early RNA synthesis
as well as on protein synthesis. These results on puffing are discussed with regard
to the general problem of the relationships between patterns of gene activity and
differentiation.
With the breakthroughs in molecular
genetics, problems concerning the mechanisms that control the different expression
of the genome in differentiated cells have
become one of the central issues of developmental biology. DNA-dependent synthesis
of RNA and protein synthesis are now believed to form the two primary steps in
the process of information transfer from
the genome to the final character. By
studying cellular RNA and protein metabolism it seems possible, therefore, to get some
insight into the problem of control of gene
expression. Different patterns of enzyme
activities or of protein syntheses have been
discussed in terms of different gene activity
patterns. Recent evidence seems to indicate,
however, that control of enzyme amount,
synthesis, and activity in cells of higher
organisms may take place at various levels
in the cell, and consequently may not simply be a reflection of gene activity (see,
e.g., Schimke, 1964). Studies on RNA metabolism, on the other hand, have clearly
shown that there are changes in the rate
of synthesis of different RNA species in
development (cf. Brown, 1964). However,
these changes generally cannot be attributed to particular genes, or at most to loci
with some general function such as direction of ribosomal RNA synthesis. The pattern of RNA-synthesizing loci (puffs) in a
given cell type and its changes in relation
to differentiation can easily be studied in
dipteran giant chromosomes. Some general aspects of puffing in giant chromosomes
have been discussed by W. Beermann
(1963). Thus, we may concentrate on the
problem of how this pattern of "gene activity" is related to differentiation.
GENE ACTIVITY PATTERNS IN DIFFERENT
CELL TYPES
Giant chromosomes exist in a number of
dipteran tissues such as malpighian tubules, rectum, midgut, bristle-forming cells,
nurse cells and others, but most studies have
been made with larval salivary glands. As
all these tissues are highly differentiated,
a relationship between differentiation and
(33)
UI.RICH CLEVER
gene activity patterns should imply differences in puffing patterns that are related
to the different functions of these cells.
The salivary glands of many Diptera consist of several lobes that differ in their function. In Acricutojnts lucidus, e.g., there are
three lobes. Two of them apparently synthesize the amino acid, oxyproline, the
third lobe does not. This lobe, in contrast
to the two other lobes, secretes a carotinoid
during definite stages of larval life (Mechelke, 1953; Baudisch, 1963a, b). Each of
the three lobes shows a specific pattern of
Balbiani rings, a special kind of puffs, in
the giant chromosomes. Comparable results have been obtained in Trichocladhis
(Beermann, 1952b), and it seems that Balbiani rings quite generally represent gene
loci of a highly cell-type-specific activity.
As the secretion of a mucoprotein-like
product is the most important function of
the gland cells, and as the Balbiani rings
represent not only genes that are specifically active in these cells, but are furthermore the most active loci in them, it is
tempting to assume that they are involved
in the production of the secretion. This
assumption was supported by the results
of Beermann (1961). In some special cells
of the salivary glands of Chironomus pallidivittatus a granular secretion is produced;
the granules are missing in the rest of the
gland cells. In the special cells, and only
there, there is a small Balbiani ring in one
region of the IVth chromosome. In
Chironomus ten tans, a closely related species, there are no granules in the secretion
of the special cells and there is no Balbiani
ring in the homologous region of the fourth
chromosome. Cytogenetic experiments localized the mutation that is responsible for
the difference in the secretion between the
two species in exactly the region in which
there is the cell-specific Balbiani ring in
the granule-producing cells of C. pallidivitlatus.
To define more precisely in what processes puffs like this one may be involved,
a better understanding of the function of
the glands is necessary. So far, there are
very few data in this respect. In Acricotopus, oxyproline has been found only in the
secretion and in two lobes of the salivary
glands, but not in the other tissues of the
larvae nor in the food (Baudisch, 1963a).
This seems to show that oxyproline as part
of the secretion is synthesized in the gland
cells. Kato and Sirlin (1963) reported some
evidence that secretory mucoproteins are
synthesized in the glands of Brndysia. While
these results support the view that the main
function of the gland cells is to produce
the secretion, this assumption has been
challenged by Laufer (1965a). He found
that all the proteins that he was able to
detect in the secretion of Chironomus
thummi were also present in the hemolyraph of this insect. Furthermore, if he
injected foreign proteins into the hemolymph these proteins appeared later on
in the secretion of the salivary glands. He
therefore concludes that the salivary gland
cells do not produce any secretion, but
merely control the passing of the secretory
products, being synthesized somewhere else
in the larvae. He suggests that the Balbiani rings may be involved in the production
of some permease that controls the secretion process. Anyway, whether the function
of the gland cells is to produce some secretion product or to control the secretion
process, the data are consistent with the
idea that some of the puffs, and among
them probably the large Balbiani rings, are
involved in some specific cellular functions.
As even cells that only very slightly differ
in their functions show specific differences
in their puffing patterns, one might expect
much greater differences between different
organs. When Beermann (1952a) compared
the puffing patterns in various tissues (salivary glands, malpighian tubules, rectum,
and midgut) of individual Chironomus
tentans larvae, this was what he actually
observed. A comparison of a larger number of larvae, however, shows that this only
partly is due to genes with tissue specific activity. Loci that are not in a puffed condition in the salivary glands of one larva, may
be puffed in the glands of another larva.
The more larvae we examine the fewer
loci are left that are specifically active
only in one tissue (Fig. 1). Puffing patterns
in homologous tissues of different larvae
GENE ACTIVITY AND DIFFERENTIATION
TTT
I
UJ
A ...
B
-A
-B
C
D
-D
E
F
G
t-
Pattern of
potential
activity
-E
-G
B —
—
—
-A
-B
D —
—
-
-D
E
F
U
-E
©
G
FIG. 1. Diagram of the differences and similarities
in the puffing patterns of different tissues and different larvae (I-III). A-G puff forming loci. Note
that only C and F form puffs that are specific for
one of the two tissues respectively. Except for
these loci the patterns of potential activity of the
two tissues are identical.
become more similar if we experimentally
induce similar physiological conditions in
all of these larvae. If we know more specifically the physiological situation in which
a given chromosomal site is active, we can
induce the same degree of puffing at this
locus in all larvae by experimentally inducing this situation. Region 17-B of the
1st chromosome is puffed in most, but not
in all, larvae of Cliironomus tentans. In
some larvae the puff is small, in others it
is very large. In prepupae of a definite
stage the puff is always large. If we induce
metamorphosis by injecting ecdysone into
larvae with various puff sizes, puffs of uniform size will have formed a definite period of time later in all larvae (Clever,
1961, 1962). The possibility of adjusting
puffing patterns to experimental physiological situations suggests that the observed
differences of puffing patterns in homologous tissues of different larvae reflect dif-
35
ferences in the physiological activity of the
cells at the moment of fixation. The actual
activity of many loci (and thus the size
of the puffs) thus seems to vary with the
processes in which they take part. Similarly, differences in the puffing patterns
of various tissues seem partly to be due to
differences in the actual activity of particular metabolic processes in these tissues
at a given moment rather than to tissue
specificity of these processes. This situation
is understandable, as there is certainly a
large number of basic metabolic processes
such as respiration that all tissues have in
common, though their actual intensity in
a given moment may vary. The idea that
some puffs are involved in respiration finds
some support in results obtained by Ritossa
(1962), but, in general, attempts to correlate puffing with basic metabolic processes have failed so far.
CHANGES OF GENE ACTIVITY RELATED
TO DEVELOPMENT
All cells showing giant chromosomes are
highly differentiated. The organisms, however, in which these cells exist are still in
the larval stage and undergo remarkable
developmental processes: the molts, and
especially the larval-adult transition, metamorphosis. Actually, differences of puffing
patterns in larvae of different stages of
development have been found by many
investigators {cf. Beermann, 1956; Clever,
1964a, 1965a). In Chirotiomus tentans the
relation of puffing changes to larval development may be classified as diagrammed
in Fig. 2. Most puffs behave in the fashion
of scheme A-B, i.e., their activity does not
show any relation to development. Other
puffs are present only during molting periods (F, G); still other puffs are restricted
to the pupal molt, i.e., metamorphosis (H).
The next step in our analysis would be to
correlate the changes in the activity of
these genes with particular developmental
processes. A priori, one might expect two
types of processes related to development
in these cells: (1) they might have another
function during molting or metamorphosis
than before, or (2) they may undergo
developmental changes themselves.
36
ULRICH CLEVER
Puff
TL-5-A
I-W-B
i-n-B
M-B-A
M-V-Az
I-18-C
I-11-B
I-I-A
pupal
larval
molt
molt
•last larval instar
•pupa — * instar—*
-3
FIG. 2. Diagram of characteristic time patterns of F-G: puffs are formed during each molting period.
puffing during the third and the last larval instar H: a puff is formed only during metamorphosis
of Chironomus lentaiis. A-C: puffing is controlled (the pupal molt).
independently of the developmental processes.
rd
Functional changes during definite stages
of development are characteristic for a
number of larval organs in insects. The
prothoracic glands, for example, rhythmically produce the molting hormone, ecdysone (Rehm, 1952). Silk glands of many
metamorphosing lepidopteran larvae produce characteristic cocoons in which metamorphosis takes place. Similarly, the secretion produced by the salivary glands of
Chironomus differs somewhat from that
produced at earlier stages. In Acricotopits
lucidus, the front lobe of the salivary gland
secretes a carotinoid during the larval and
pupal molts, but not during the intermolt
periods (Mechelke, 1953; Baudisch, 1963b;
Panitz, 1964). In Chironomus as well as in
Acricotopus, the puffing pattern changes
more or less simultaneously with these
functional changes. However, it has not
yet been possible to correlate the activity
of particular puffs with one of them.
As a consequence of metamorphosis, the
larval salivary glands break down at the
end of the pupal molt. This breakdown
may be considered as the actual developmental process of the gland cells and may
be compared with other processes of
cellular differentiation (Clever, 1965a). At
least part of the puffing changes characteristic for this period may be supposed to
have some function in the control of cellular breakdown, possibly by controlling the
synthesis of hydrolytic enzymes responsible
for processes of degeneration. A change in
the puffing pattern which may reflect a
result of the beginning cell breakdown,
may be seen in, the decreasing activity of
most loci in the course of the pupal molt
(Clever, 1962).
Changes in enzyme activities which may
be related to cellular breakdown have been
studied by Laufer and Nakase (1965b) and
by Dr. Schin in our laboratory. Laufer
found that in salivary glands of C. thummi
the activity of DNase is very low in young
last instar larvae. The activity increases
with age, especially after beginning of the
pupal molt. Though the function of the
enzyme in salivary gland cells is not yet
understood, it seems reasonable to assume
that it plays some role in cellular breakdown. Some of the prepupal puffs, then,
may be responsible for the control of
enzymes like this one.
The results obtained so far by Schin do
not disagree with this idea, but they seem
to show that metabolic processes leading
to cellular breakdown may be controlled
in a more complicated fashion than we
had expected. Many hydrolytic enzymes
which are supposed to be responsible for
cellular breakdown, are found in cellular
organelles termed lysosomes (c/. deReuck
and Cameron, 1963, for references). One
of the lysosomal enzymes is acid phosphatase. The localization of this enzyme can
be demonstrated by means of electron microscopic cytochemistry. Acid phosphatasecontaining lysosomes are present even in
very young Chironomus tentans larvae,
long before they reach the last larval instar. Their number is, however, very small
until about the fourth day of the last instar.
GENE ACTIVITY AND DIFFERENTIATION
At this time, a remarkable increase in the
number and size of lysosomes sets in. In
old prepupae there are very many lysosomes, full of acid phosphatase. Up to
this time, however, no enzyme was ever
detectable outside the lysosomes in the
cytoplasm. This situation changes drastically after the beginning of cellular breakdown. In degenerating cells or parts of
cells, all lysosomes are ruptured and the
enzyme is freely distributed in the cytoplasm (Schin and Clever, 1965, and unpublished results). According to these results it seems that there are several mechanisms at work that control the developmental changes of enzyme function: one
that controls the increase of enzyme activity
in the early last instar; another one that
controls the additional increase in the late
prepupal stage. But not only the activity
and/or synthesis of the enzyme has to be
controlled: its localization, and thus probably the substrate availability as well. It
is this whole pattern of control processes
into which probably the changes of gene
activity, as reflected by puffing changes, are
integrated.
THE DEVELOPMENTAL CONTROL OF GENE
ACTIVITY PATTERNS, AND THE CONTROL
FUNCTION OF GENES IN DEVELOPMENTAL
PROCESSES
If differential gene activity patterns constitute an essential property of differentiated cells, and if this pattern changes parallel to differentiation, what are the developmental factors controlling the gene activity
pattern and how are its changes integrated
into the processes of differentiation? As
mentioned above, developmental changes
of the salivary gland cells are concentrated
toward the end of larval life, to metamorphosis. Insect metamorphosis is induced
by the steroid hormone, ecdysone. This
hormone has been purified and metamorphosis may be induced by its injection (cf.
Karlson, 1963). We used this technique
for studying the dependency of prepupal
puffing on the presence of this hormone.
Characteristic for normal metamorphosis, as well as for metamorphosis induced
37
by injected ecdysone, is a sequence of
puffing changes (Becker, 1959; Clever, 1961,
1962). In Chiron omits tentans, this sequence starts within less than one hour
after injection of the hormone. A priori,
the change of the puffing pattern during
the course of the molt might be explained
by one of two assumptions: either the hormonal milieu or some hormone-dependent
intracellular system might determine the
complete actual gene activity pattern at
any given moment; or, it is a chain of reactions that is induced by the hormone,
the reactions of some early induced genes
leading to the subsequently activated processes and genes.
The second idea is favored by the observations that in C. tertians a few puffs
not only react to the hormone very early,
but the same puffs in contrast to all others
respond very sensitively to changes in the
concentration of hormone (Clever, 1963).
These puffs, therefore, seem to be controlled by the hormone more directly than
the later-appearing puffs. Further evidence
supporting this idea resulted from studies
in which RNA synthesis was temporarily
inhibited after injection of ecdysone
(Clever, 1964b).
As is generally known, the antibiotic,
Actinomycin D, selectively inhibits DNAdependent synthesis of RNA. Treatment
of Chironomus tentans larvae with low concentrations of this drug for several hours
inhibits RNA synthesis in the salivary
gland chromosomes for about 15 to 20
hours. If ecdysone was injected right at
the beginning of the inhibitory period, the
first puffs appeared only after about 20
hours instead of after 30 minutes (Fig. 3a,
3b). Twenty-four hours after injection of
ecdysone RNA synthesis has resumed, and
one would expect to find all puffs characteristic for this stage, unless their activation is dependent on some earlier RNA
synthesis (that is, on some earlier induced
genes). Actually, the 24-hour puffs were
present only after 48 hours, and the 48-hour
puffs after 72 hours; the whole sequence
was delayed for exactly the same period of
time that synthesis of RNA was inhibited
(Fig. 3b). It follows that the activation of
ULRICH CLEVER
some early reacting genes is a prerequisite
for the activation of the later appearing
puffs to take place. The influence of ecdysone on the puffing pattern, therefore,
is a sequential process. The hormone affects one or a few early reacting genes, and
only the reactions of these genes lead to
the subsequent gene activations. Our results suggest the following steps in the
action of ecdysone:
Ecdysone
I
E
—
D—
—
—
—
—
C —
—
—
—
—
A —
—
-
-
—
D
1—
C —
1
—
—
—
—
—
B
B —
A —
gene activations
(I-18-C, IV-2-B?)
4RNA
i
protein
(cytoplasmic metabolism)
U
U
U
gene activation
gene
gene activation
activation
—
1—
'—
1—
—
RNA Synthesis inhib.
E
D —
C —
B —
A
I-
The life span of the RNA synthesized by
the early-induced genes does not seem to
exceed about 15 hours.' This is indicated
by the observation that RNA synthesized
before treatment with Actinomycin did
not induce the sequence after cessation of
the inhibition (Clever, 1965b).
If our scheme is correct one would expect that inhibition of protein synthesis
stops the sequence at least after the first
gene activations. Another question is
whether these first gene activations themselves depend on some protein synthesis.
We inhibited protein synthesis with cycloheximide (actidione). The result is diagrammed in Fig. 3c: the block always lies
behind the activation of the first genes.
In all of our experiments three puffs reacted to the injected ecdysone: I-18-C, and
IV-2-B which normally become activated
within less than one hour after injection,
and I-8-A which becomes activated a few
hours later. Evidence from other experiments seems to indicate that the first two
of these puffs respond independently of
each other to the hormone. The fact that
the sequence stops only after one other
—
Protein synthesis inhibited
0
I
24
48
-1
72
Hours after injection of ecdysone
FIG. 3. The effects of inhibition of RNA (b) and
protein (c) synthesis on sequential puff induction
by ecdysone. Synthesis of RNA was inhibited by
treatment with Actinomycin (0.2 jig/ml) for 6
hours. Protein synthesis was inhibited with Actidione (Cycloheximide: 10 ^g/ml). The period of
inhibition was tested autoradiographically and is
indicated in the diagrams. In all experiments
ecdysone was injected at time 0. a: represents the
normal sequence.
puff has appeared is not yet understood.
Anyway, all subsequent puffs never did
appear, their activation consequently being
dependent on some early protein synthesis.
This result is in good agreement with our
above scheme. Whether or not the effect
of ecdysone on the early-reacting puffs is
a direct one, is still an open question. Our
result seems to indicate that the synthesis
of some protein is not an intermediary step
between the primary action of the hormone
and the reaction of these loci. This supports the idea that the number of those
steps cannot possibly be very great.
GENE ACTIVITY AND DIFFERENTIATION
CONCLUSIONS
According to the present dogma, gene
activity is generally considered in terms of
synthesis of messenger RNA. It has been
shown that puffs are the sites of chromosomal synthesis of RNA, and that RNA's
of the Balbiani rings differ in their base
composition from each other as well as
from the bulk of cytoplasmic RNA. Finally, the correspondence of one of the
Balbiani ring loci with a salivary gland
mutation has been established (cf. Beermann, 1963). It may be concluded tentatively, therefore, that at least part of the
puff RNA is a messenger-type RNA.
Whether other types of RNA are also synthesized in puff regions, as Beermann (1965)
recently suggested, has still to be shown.
If we assume that it is informational or
actually messenger RNA that is synthesized
in the puffs, one of the conclusions to be
drawn from the results summarized in this
paper would be that the composition of
mRNA changes in the course of differentiation. This idea is supported by biochemical studies that indicated differences
in mRNA in different cell types or changes
with developmental processes (e.g., McCarthy and Hoyer, 1964; Kidson and Kirby,
1964a, b). The dependency of molting induction on DNA-dependent RNA synthesis
at the same time when hormone-dependent
puff-RNA is synthesized in a few loci, also
supports this view.
The studies on puffing patterns indicate
that there is a large number of cellular
processes in relation to which the activity
of single loci, and consequently the pattern
of active genes changes: part of these processes may be considered as developmental,
others may not. This shows that the establishment of actual gene activity patterns
is not only a feature of development.
Rather, it reflects the state of cellular activities in a given moment at the level of
the genome. We have mentioned that probably not all loci become active at some
time in any tissue, but that activity in any
tissue is restricted to a pattern of potentially active loci. These patterns, though
overlapping, probably differ specifically
39
from tissue to tissue. The pattern of potential activity, then, may be considered
as the result of differentiation. Probably it
is relatively stable, as is cellular differentiation in general. To put it another way:
it cannot easily be converted into the pattern of potential activity of another tissue.
The question is, where does this stability
reside in the cell? It has been discussed
that the stability of differentiation may be
a property of the genome. Genes, it is
sometimes argued, may become differentiated in a way that makes them sensitive
to a given stimulus in one tissue and not
• in another. This has been considered as
"true" or "major" differentiation, in contrast to the control of the actual activity
that has been considered as "modulation"
or "minor differentiation" (Fujita, 1965).
The relative stability of developmental differentiation in higher organisms, especially
in animals, makes it tempting to speculate
along these lines. Actually, a permanent
inactivation of parts of the genetic material
seems .to occur in cases such as the heterochromatization of one X chromosome in
mammalian females. The results obtained
so far on the control of puffing and control
of RNA synthesis in giant chromosomes,
however, do not support the view that
there is some sorting out into potentially
active and permanently inactive gene loci
during development. Fujita (1965) suggests
that puffs were formed in interband regions
and by faint bands only and that the normal bands represent the pattern of permanently repressed loci. In contrast to his
ideas, however, very thin bands may remain permanently inactive while thicker
bands may form puffs. And puffs not only
may completely disappear to form bands
(which would not even be necessary), but
some loci form a puff only very late in
development, after they have been in the
band condition for the greater part of
larval life. No one, so far, has demonstrated any difference between a band of
this type and a band that never has been
observed to form a puff.
If genes would become differentiated to
respond or not to respond to certain developmental conditions, one might expect
40
ULRICH CLEVER
that a whole set of genes (A, B, C . . . ) is
put into action by one developmental
stimulus or under one developmental condition, and another set of genes (D, E,
F . . . , or A, D, E . . . ) by another. As our
results on the induction of molting have
shown, however, it is only one or a very
few of the genes activated in a certain
stage of development that are regulated
by the factors controlling development
itself. It is not a pattern of genes that
responds (A, B, C . . . ) but it is a sequence
that is induced (A->B->C . . . ). For the
relationship of genes to the developmental
processes, this means that it is not the
genome (or the pattern of the gene activity)
that autonomously controls development
and differentiation, but that gene activations and reactions are parts of the whole
developing system—though certainly most
important ones. Properties, such as stability of differentiation, also have to be considered as properties of this system as a
whole. Thus, in our opinion we will have
to look for factors responsible for a long
term stability of the gene control systems
and for the systems controlling the final
translation of the genetic message rather
than for stable changes in the genetic material itself.
Intimately related with all these problems is, of course, the question of what
mechanisms control the synthesis of chromosomal RNA and how they may be affected by factors controlling development.
This question is directly touched in our
studies by the problem of how ecdysone
may exert its control on puffing. So far,
an answer cannot be given. In a formal
way, the effect of ecdysone might be compared with that of an effector in the JacobMonod model, provided that ecdysone primarily acts on the genome (c/. Clever,
1964a). Chromosomal activity in higher
organisms, however, is not only expressed
by a change in the rate of synthesis of RNA
at a given locus, but includes changes in
chi-omosomal structure as well. It seems
reasonable to assume that there are more
complicated control mechanisms involved
than in bacteria and that parts of chromosomal structure may have some function
in these mechanisms. There is some discussion presently whether protein synthesis
might be regulated at the level of translation at the ribosome rather than at the
level of transcription. Transcription, the
rate of RNA synthesis, it is suggested might
be controlled by a kind of feedback mechanism (Stent, 1964). According to all the
data that we have, however, the synthesis
of RNA in puffs and its control, e.g., the
control by ecdysone, seem to be independent of protein synthesis. The transportation of this RNA to the cytoplasm and its
stability, on the other hand, apparently
are influenced by the rate of protein synthesis (unpublished results). These observations again suggest that there are several
levels at which gene expression in higher
organisms is controlled. Our understanding of cellular differentiation will largely
depend on getting more information about
the mechanisms working at these various
levels.
Acknowledgments: The work reported here was
supported in part iby grants from the National
Science Foundation (GB-2639) and the Purdue
Cancer Committee.
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GENE ACTIVITY AND DIFFERENTIATION
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