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THE EUKARYOTIC
719
CHROMOSOME
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JOHNE. H~-ARST1 ANDMICHAELBOTCHAN
Department of Chem~try and Group in Biophysics
University of California, Berkeley, California
CONTENTS
INTRODUCTION
..........................................................
] ........
CHEMICAL
COMPOSITION
OF METAPHASE
CHROMOSOMES
...........
TITRATIONANDMAGNESIUM
ION ]~INDINGTO METAPHASE
CHROMOSOMES
...
CHROMATID
SUBUNITS
ANDSTRANDEDNESS
................................
STRUCTURE
OF THEMETAPHASE
CHROMOSOME
............................
THEREPLICATION
UNIT OF THE~¢~AMMALIAN
CHROMOSOME
................
ATTACHMENT
OF THECHROMOSOME
TO THENUCLEAR
MEMBRANE
............
ANALOGIES
IN THECONTROL
SYSTEMS
(~F PROKARYOTES
AND~.UKARYOTES
....
COARSE
CONTROL
........................................................
FINECONTROL
..........................................................
BASE SEQUENCE REDUNDANCY IN THE EUKRRYOTIC
CHROMOSOME ............
151
152
158
159
160
16~.
162
163
166
173
177
INTRODUCTION
The chromosomes of the eukaryotic cell are different
in many ways
from the chromosomes of bacteria. The DNAof the eukaryotic cell is always found chemically associated with a complex class of proteins. Just
prior to cell division, at mctaphase, the chromosome is condensed into a
well-defined structure and then is mechanically separated into two parts, each
daughter cell receiving one-half. Most prokaryotic cells have a linkage number of one, i.e., contain only one chromosome.The eukaryotic cell generally
has many chromosomes and is frequently diploid. It follows that there are
a number of properties unique to the genetic material of the higher organism.
Most of these are not understood at present.
This review will concentrate on what is known about the chromosome
of the eukaryotic cell. Manyother review articles have been written on chromosomal proteins (1-3) and metaphase chromosome structure
(4-6).
hope this review will serve as a bridge between these two areas of research.
The detailed information presently available on isolated metaphase chromosomesis very modest, probably in part because they have only been available in the isolated state for a short period. There is also a paucity of original ideas being generated by those groups that have been isolating the chromosomes. Most of the experiments which have been done have been repeated in a large fraction of the laboratories having isolated chromosomes.
Examples of this phenomenon are: (1) Separation of chromosomes into
size groups by sucrose gradient sedimentation has been performed by five
"John Simon Guggenheim Memorial Fellow--1969.
151
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HEARST
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152
& BOTCHAN
laboratories
(7-11). (2) The RNAof metaphase chromosomes has
demonstrated to be rRNAin three laboratories (12-14).
There is a growing feeling that the advantages of working with isolated
metaphase chromosomeswill not be realized until they can be isolated from
an organism whose genetics have been thoroughly studied and for which
many mutants are available. With this in mind, many laboratories have been
reported to be attempting to isolate metaphase chromosomes from Drosophila melangaster. With a haploid number of four, separation of different
chromosomes of Drosophila should be possible. The wealth of genetics will
make these chromosomes valuable for hybridization studies and for the observation of chemical changes associated with cell differentiation.
Although
this advance is imminent, it has not been reported as of this writing.
Work on the structure
of the metaphase chromosome has been rather
disappointing thus far, although of high quality. A commonbias of most
people is the expectation of a highly ordered chromosomestructure at metaphase. Such a structure has not been observed. In fact the best evidence says
quite the contrary. The metaphase chromosome appears to be made of a tortuously wound 230 A fiber (4). There is considerable controversy regarding
the existence of substructure in the 230 A fiber which is not yet resolved
(6). In fact there is even controversy regarding the existence of the 230 A
fiber (5). It is our feeling that muchof this controversy will only be resolved when the chromosomes are studied under highly controlled chemical
conditions which are not yet understood. This perhaps is one of the best arguments for very detailed studies on the chemistry of isolated chromosomes.
Muchof the latter portion of this review is concerned with the mechanisms of control of transcription
in the eukaryote. Although there remain
many anomalies in the experimental evidence, the position is taken that
there are many levels of control of transcription and that manyif not all of
the mechanisms in the literature may be correct in part. The diversity of the
bacterial control systems makes such an argument plauslble (15). Furthermore, in view of the permanent repression of most of the eukaryote genome,
its complexity, and size, it is likely that different types of regulation have
evolved. Wehave divided the discussion of control of transcription into two
parts, coarse control which deals with condensation of large regions of the
chromosomeinto a repressed state, and fine control which deals with individual gene control in the noncondensed chromosome.
CHEMICAL
COMPOSITION
OF
METAPHASE
CHROMOSOMES
Isolation procedures for metaphase chromosomes have been reported by
eight laboratories (10-14, 16-18). All but one of these procedures use acidic
solutions for the disruption of the cells. Since the chromosomes are large
relative to the other stable organelles of the cell, they are readily separated
from cytoplasmic material by centrifugation.
The separation of chromosomes from intact nuclei has been accomplished by differential
centrifugation (17), zone centrifugation through 2.2 Msucrose (10, 13, 14), and filtra-
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THE
TABLE 1.
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Preparation
EUKARYOTIC
153
CHROMOSOME
METHODS O¥ METAPHASE CHROMOSOME ISOLATION
Cantor
& Hearst
Huberman
& Attardi
Maio
& Schildkraut
Salzman, Moore
& Mendelsohn
Cell type
Mouseascites
tumor
HeLa
Cell wash
Hank’s
balanced
salt solution
0.137 MNaCl
Earle’s balanced Eagle’s minimum Eagle’s minimum
0.005 M KCI
salt solution
medium
medium
0.001 M NaH2PO~
0,025 MTris,
pH 7.4
Hypotonic
buffer for
swelling cells
1/4 strength
Hank’s solution
0,1 Msucrose
7 X 10-~ M CaCh
3X10-4M MgCh
15 volumes
0.02 MTris,
pH 7.0
10-s M CaCh
10-" M MgCls
10"~ M ZnCI
0.25 to 1.0%
sodiumcitrate
1.0% radium
citrate
5X10-4MMgCh
5 X 10-4 MCaCh
Homogenizing
solution and
method
0.01 Mformate
buffer, pH 3.7
10~ M MgCh
Virtis
homogenizer
0,1 Msucrose
7 X10-4 M CaCh
3X10-4 M MgCh
3.3X101 M HCI
3 volume~
Final pH ~3.0
Motor-driven
Potter-Elvehjem
homogenizer
0.05% Saponin
in above
Dounce
homogenizer
2.5% Citric acid
0.1 Msucrose
pH 2.1
Hand shaking
then 0.1%
between 80
2.5% Citric acid
5X10-4M MgCh
5X10-4 M CaCh
Shaking, 30 rain
Sucrose gradient
sedimentation,
pellet through
2.2 Msucrose
Differential
centrlfngatlon,
pellet through
2.2 Msucrose
Filtration on
stainless
steel filters
Layered on
60%sucrose,
differential
sedimentation
Method of
Differential
separation o~ centrlfugation
chromosomes
from nuclei
and cytoplasm
HeLa
HeLa
Sy~a~ hamster
Chinese hamster
MoaseL cells
Franceschini
& Giacomoni
HeLa
tion on stainless steel filters (12). Table 1 summarizes the conditions used
by the various laboratories in isolating chromosomes. There is already good
evidence that the conditions required ~or isolating metaphase chromosomes
depend on the cell system being studied (19). The homogenizing buffer used
in this laboratory (17) for mouse ascites tumor cells is 0.01 Mformate,
3.7, and 0.001 M Mg++. For I-Ida cells, Huberman & Attardi use pH below
3.3 (13), a condition which causes gel formation about the chromosomes
from the mouse ascites cells. Nonionic detergents such as Tween 80 and
Triton X 100 (11, 12) have also been used to aid in dispersing the chromosomes and to avoid aggregation. Maio & Schildkraut (14) have described
homogenization condition which is neutral. The ionic strength of this solution is apparently high enough to keep the chromosomes condensed in spite
of the high pH (19). The neutral pH should be viewed as a major advantage over the acidic extraction conditions used by others, especially those
below pH 3 where proteins (20) are extracted ~rom nucleo~aistone and hydrolysis becomes more likely. This neutral procedure fins been successful
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154
HEARST & BOTCHAN
with several tissue culture cell lines and with mouse ascites tumor cells
without modification, so the procedure is apparently more general than the
others described in Table 1. The procedure has not been adopted in this laboratory because the isolation requires the use of the detergent saponin,
which introduces a different kind of uncertainty into the chemistry of the
product. It is rather likely that all the pH changes and detergents have an
influence on the internal structure of the chromosomes. They seem to have
no effect on the gross morphology and only small effects on the chemical
composition of the isolated chromosomes.
There is reasonable agreement in the gross chemical composition of all
isolated metaphase chromosome samples independent of the method of extraction. However, the fraction of the protein in HeLa chromosomeswhich is
soluble in 0.2 MHC1decreases as the pH of the homogenizing solution decreases. This indicates that someacid-soluble proteins are extracted from the
chromosomeswhen isolated under acidic conditions. Table 2 presents the results of the analyses presently available. A comparison of these results with
the composition of chromatin shows that the metaphase chromosomes contain a large amount of acid-insoluble protein which is not a constituent of
chromatin or nucleohistone isolated from interphase cells. They also have a
much higher RNAcontent than chromatin.
The major portion of the RNAfound in the metaphase chromosomes
has the properties of rRNA. The 28S and 18S RNAs are found in approximately equimolar amounts and these RNAshave a base composition identical to the corresponding RNA(12-14). Experiments have been performed
by Salzman, Moore & Mendelsohn (12) which they choose to interpret
evidence for that the rRNAin the chromosomes arises from contamination.
They added radioactive ribosomes to the cell suspension prior to homogeniization and measured the specific activity 0f the RNAon the isolated chromosomes from this homogenate. The specific activity
of the RNAin the
metaphase chromosomes was 85% of the specific activity of the total RNA
in the homogenate, which suggests that the ribosomes are contamination.
Interpretation
of the Salzman experiment is impossible. Ribosomes precipitate at pH below 5 (21) and as a result, added ribosomes can precipitate
on the surface of chromosomes in proportion to the number of ribosomes
already present on the chromosomes. This is possible since precipitation
is
an aggregation phenomenon. Furthermore, ribosomes can precipitate
on
other nonchromosomal sites in an analogous fashion. Consequently it is not
unlikely that the final specific activity is identical to the starting specific activity because of such an aggregation.
Even at neutral pH this type of experiment is not unambiguous. Exchange between bound and unbound ribosomes must be ruled out if this type
of experiment is to be interpreted in the manner of Salzman. It is clear that
the ribosomal material is bound strongly since it cannot be washed off (22).
However, this is a kinetic statement and does not preclude a moderately fast
exchange. Finally if the added ribosomal material is labeled to a sufficient
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THE EUKARYOTIC
CHROMOSOME
155
extent, an increase in total ribosomes hound could adventitiously account for
identities
of the specific activities
of homogenates and isolated chromosomes. This suffleieney is dependent upon the state of the nonehromosomal
ribosomes. If they are not bound to membranes and can compete with the
added material., then the amount of label needed to maintain activities
is
much greater than if the only ribosomes that can hind are the radioactive
ones. This criticism, however, can be tested by doing the same experiment at
a series of concentrations of added ribosomes.
Although it is clear that the majority of metaphase chromosomal RNAis
18S and 28S (12-14) rRNA, it has not been demonstrated that these RNAs
are on the chromosomeas ribosomes. In fact it is possible that if the RNA
is a natural constituent of chromosomes, the RNAcomes from the disappearance of the nueleolus before metaphase. The nucleolar material may incorporate itself into the structure of the chromosomes.If this is the ease the
RNAwould not be present as ribosome per se. The fact that the 18S and
28S RNAs are present in equimolar amounts makes this suggestion less
likely.
The presence of small amounts of 45S RNAin the chromosomes,
however, should not be ignored (13). Three lines of evidence point to nonartifactual
presence of RNAon chromosomes. (1) The RNApresent in all
chromosomepreps (see Table 2) normalized to DNAconcentration is surprisingly close considering the range of various conditions used (e.g., detergents, ionic strength, and pH) and the variability of the source material.
(2) The existence of a "chromosomal RNAcycle" has been known to cytologists
since Kaufmann (23) noticed it in 1948. At prophase chromosomes become deeply staining to specific RNAstains. By metaphase if one
stains for RNAthe metaphase plate is clearly distinguished by a heavily
stained line (24). The RNAappears to be shed by the chromosomes by telophase. (3) Treatment of chromosomes with RNase has no effect on morphology in isolated material as observed by a number of authors (16, 25,
26). Chorazy et al. (16) find that RNase alone does not reduce the
content of chromosomes. They have noticed that treatment of chromosomes
with pepsin plus RNase does destroy morphology while treatment with pepsin alone has no effect on the phase contrast morphology. Since the aromatic
amino acid composition of the chromosomes is low (18), pepsin alone probably has the minimal effect of exposing the RNAof precipitated
RNP
particles, which are normally impervious to RNaseactivity (27).
The problem of how much RNA(and for that matter nonhistone protein) naturally occurs on the condensed chromosomeis still unanswered. It
is hoped that a more carefully controlled experiment similar to the one done
by Salzman’s group can help answer this question.
The purity of the chromosomes after isolation cannot be determined to
any degree of accuracy at present. The criteria that are used to measure the
success of a preparation are good metaphase morphology under the light microscope, absence of visible contamination using phase contrast microscopy
and various stains, and constancy of gross chemical composition. In addi-
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156
HEARST & BOTCHAN
TABLE2. CHEMICAL
ANALYSISOF METAPHASE
CHROMOSOMES
ANDINTERPHASE
CHROMATIN
Protein soluble
in
Preparation
DNA RNA Protein
0.2 M 0.2 M
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HC1
Cantor & Hearst--Mouse ascites
metaphase chromosomes
13.5
13.5
68
Huberman& Attardi--HeLa cell
metaphase chromosomes
15.7
10.4
73.9
42.6
Maio & Schildkraut--Metaphase
chromosomes
HeLa
Chinese hamster
Syrian hamster
L cell
16.4
15.7
16.7
17.0
11.6
15.3
15.0
13.4
72.0
69.0
68.3
69.6
57.8
76.0
72.2
50.0
Sadgopal--HeLa metaphase
chromosomesisolated using
methodof:
Huberman&Attardi pH 3.0
Maio & Schildkraut pH 7.0
15.6
16.2
8.7
10.9
75.7
72.9
56.8
Salzman, Moore & Mendelsohn-HeLa metaphase chromosomes
20
14
66
25.8
Sadgopal--HeLa interphase
chromatin
Sucrose sedimentation method
Unsheared
Sheared
++ precipitation method
Mg
25.2
35.1
19.4
3.2
3.5
14.0
71.6
61.4
66.6
J. Bonner--Pea seedling interphase 36.5
chromatin
9.6
47.9
H2SO,
18.0
29.6
45.3
58.8
30.6
79.0
tion, because of extensive differential
centrifugation
at low centrifugal
fields, the preparations are free of both molecules and debris from the cell
which sediments more slowly than the chromosomes (in International
PR-2
Centrifuge, 2000 RPMfor 45 rain). Separation from interphase nuclei is obtained by differential centrifugation, sucrose gradient, zone eentrifugation,
or in one case, filtration through a porous stainless steel filter with 5/z mean
pore size. Thus any cytoplasmic contamination which irreversibly
binds to
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THE EUKARYOTIC
CHROMOSOME
157
the chromosomescan be present in all these preparations. The similarity between the chemical analyses in Table 2, in spite of different isolation conditions, suggests that the problems with contamination are not severe. A more
definite statement will require much more knowledge of the chemistry of the
metaphase chromosomes including a detailed characterization
of the proteins and RNAcontained in these structures.
Fractionation of metaphase chromosomes into size classes has been reported by Huberman & Attardi (HeLa) (8), Maio & Schildkraut (L
(7), Mendelsohn et al. (HeLa) (9), and Regimbal & Mel (Chinese
ster) (11). In all cases zone centrifugation
in sucrose gradients was
method, and the fractionation was impressive although isolation of a single
chromosome type has not yet been realized. Maio & Schildkraut have shown
that mouse satellite
DNAis evenly distributed in all chromosomes in proportion to the amount of DNAin each chromosome fraction.
It has also
been shown that rDNAis enriched with that fraction of chromosomes enriched in the nucleolar organizer.
Sadgopal (18) has made some detailed comparisons between the composition of interphase chromatin and metaphase chromosomes from Hela cells.
He concludes that the ratio of histone to DNAin metaphase chromosomes
is the same as in chromatin (0.82). When chromatin is treated with 0.2
H2SO4about 80% of the extracted protein is histone, the remainder being
acid-soluble nonhistone protein. When metaphase chromosomes are treated
with 0.2 N H2SO4,62% of the extracted protein is histone, indicating the
much larger amounts of acld-soluble nonhlstone protein in metaphase chromosomes. If the extraction is performed with 0.2 N HC1, a very large increase in the amount of extracted acid-soluble nonhistone protein occurs for
metaphase chromosomes but not for chromatin. For chromatin treated with
0.2 N HC1, 80% of the extracted protein is still histone, but for metaphase
chromosomes the percentage of histone in this extract drops to 32%. This
phenomenonis independent of the isolation procedure used for obtaining the
chromosomes(either an acidic preparation or a neutral preparatiofi).
Thus
there is a large amount of nonhistone in metaphase chromosomes which is
soluble in 0.2 N HC1 which is not present in chromatin. The amount of
acid-soluble nonhistone protein indicated is twice the amount of the histone.
He concludes that less than 6% of this acid-soluble nonhistone protein can
be the result of ribosomes bound to the chromosomes, so the majority of this
protein has some other source.
Sadgopal compared the chromatographic and clectrophoretlc
properties
of all the histone bands obtained from chromatin and metaphase chromosomes. This experiment was designed to detect chemical differences in the
proteins of the two states. Amongthe differences which could not be detected were phosphorylation, acetylation, and methylation. However, isolation of histones in 0.2 N acid might hydrolyze any of these substitutions.
Changes in the proportion of lysine-rich histones and arginine-rich histones
between the two states were observed.
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HEARST & BOTCHAN
Sadgopal also reports that the sulfhydryls of histone III are reduced in
interphase chromatin and partially oxidized to disulfides in metaphase chromosomes. The same statement is made about the large number of sulfhydryls in nonhistones of metaphase chromosomes. It is suggested that the
state of oxidation of these SH groups is important to the condensation of
chromosomesinto their metaphase structure.
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TITRATIONAND MACNESIUM
ION BIND~C TO METAPHASE
CHROMOSOMES
Cantor & Hearst (19) have studied the titration
behavior of waterwashed chromosomes. Several important features of these titration
curves
make them unique when compared to the titration
curves of chromatin or
nucleohistone recently published by Walker (28). When washed in the absence of divalent ions, the chromosomesolution has an isoionic pH of 5.6 to
5.7. If the chromosomes are then titrated
with OH- they are observed to
have a sharp irreversible
transition centered at pH 6.7 and occurring between pH 6.2 and 7.2. The difference between the forward and reverse titration curves at pH 6.25 shows that 3.3 × 10-4 mmoles/mg of OH- are taken
up by the chromosomesduring the transition.
These titration curves are not
equilibrium titration curves for the authors have shown that if the chromosomes are held at pH 6.2 at room temperature in water, a slow first-order
reaction
occurs which absorbs 3 ~< 10 ~ mmoles of OH-/mg of chromosomes. The time constant for this reaction is 33 rain under these conditions.
After the completion of this slow first-order reaction, the titration curve
of the chromosomes is identical to the curve obtained with chromosomes
which have experienced the sharp irreversible
transition.
In both of these
cases the subsequent titration behavior of the chromosomesis reversible and
similar to the titration
curves of nucleohistone determined by Walker. The
titration behavior is not changed by the presence of low concentrations of
NaC1 (10 -~ M).
When a solution of water-washed chromosomes is made 10-* ~
M in Mg
the chromosomes rapidly release hydrogen ion. If one starts with chromosomes in water at pH 6.2 and adds the magnesium ion, 3.4 × 10-* mmole of
OH-/mg chromosomes has to be added to the solution to maintain its pH at
++ the titration curves of the chromosomes
6.2. In the presence of 10-3M Mg
do not exhibit the irreversible transition observed in water or 10-8 +,
M Na
but looks very similar to the post-transltion titration curve of the chromosomes in water. This effect is clearly demonstrated to be associated with divalent cations and not ionic strength. The transition in water is accompanied
by a swelling of the metaphase chromosomesto at least ten times their condensed volume, a fact which is verified by both light and electronmicroscopy.
This swelling does not occur in the presence of divalent cation, so although
the titration curves for post-transition
chromosomes in water and chromo+* are the same, there are differences as well.
somes in the presence of Mg
Cantor & Hearst (19) have also done magnesium ion binding studies
with the chromosomes. The binding constant for the Mg~ to the chromo-
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THE EUKARYOTIC CHROMOSOME
159
-1
somes is 1.06 X 105 (moles/liter)
and the number of binding sites on the
chromosomes is 1.6 X 10-6 mmoles/mg of chromosomes. Combining this
number with the number of protons released upon binding of Mg**reveals
that about 20 hydrogen ions are released for each magnesium ion bound.
This indicates a highly cooperative change is occurring in the structureof
the chromosomes when the Mg++ is bound.
Finally, circular dichroism studies of these transitions indicate that
major structural changes are occurring at the molecular level in the protein
components of the chromosomes. They also provide further evidence for
the fact that post-transition
chromosomesin water, and chromosomesat the
++, are structurally different from one another.
same pH in the presence of Mg
CHROMATID SUBUNITS
AND STRANDEDNESS
The question of the number of DNAmolecules in one chromatid has not
yet been fully answered. The matter can be discussed at two independent
levels. At low spaeial resolution (0.1/x) one can determine if replication
the DNAin the metaphase chromatid is conservative, semiconservative, or
more complex. Taylor (29) has demonstrated by using tritium-labeled
thymidine that the replication
of DNAin each chromatid is semieonservative
and that the two chromatids of a given chromosome are separated during
mitosis. This experiment has been done with plant cells such as Vicia, animal cells such as HeLa and Chinese hamster, and insects (grasshopper).
These experiments proved there are no independently segregating subunits in the chromatlds. By far the simplest interpretation of these results is
that each chromatid contains one and only one molecule of DNAwhich replicates semiconservatively
according to the Watson-Crick replicating
scheme. This interpretation
is supported by autoradiographic experiments
where lengths of DNAwere found in mammalian tissue culture cells of
0.5 mm[Cairns (30) ], 1.8 mm[ttuberman & Riggs (31) ], and 2 cm [Sasaki
& Norman (32)]. Such long pieces of DNArepresent a substantial fraction
of all the DNAin one chromatid. They do not, however, rule out linkers
(29) which are not attacked by pronase. Such linkers would have to maintain
a linear relationship
between the linked pieces of DNAto account for the
very long strands of DNAfound by autoradiography.
Numerous exceptions
to semlconservative DNAreplication such as sister chromatld exchange and
isolabelling have been observed. Sister chromatid exchange is clearly associated with rare crossover events. Isolabelling is either caused by crossing
over or polyteny.
At a resolution of 20 A the nature of the packing of the DNAin the
metaphase chromosomehas not been revealed. In reading the next section on
chromosome structure the discussion of structures with local polystrandedness do not necessarily imply more than one DNAmolecule per chromarid. At present the connection between structural studies and genetic evidence is very fragile.
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HEARST & BOTCHAN
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STRUCTURE
OF
THE
METAPHASE
CHROMOSOME
Very beautiful electronmicrographs of whole metaphase chromosomes
from honey bee (33) and HeLa (34) have been obtained, which show the
chromosome to be a very compact and tortuous bundle of a fiber about 230
A in diameter. This fiber has been observed in interphase nuclei and swollen
metaphase chromosomes by many workers (4,25, 35, 36).
Substructure in the 230 A fiber is still very uncertain. Ry digestion with
trypsin (37), DuPraw observes a single DNase-sensitive fiber in each 230 A
fiber and argues that each fiber contains only one D N A molecule. Ris &
Chandler (38) have observed the 230
fiber to split into as many as four
subfibers. Elcctronmicroscopy on metaphase chromosomes and chromatin
has been reviewed extensively (4-6) elsewhere and since the question of
substructure in the 230 A fiber is unresolved we will say no more on the
subject.
The following are a set of rules from Cantor & Hearst (19) regarding
conditions required for the stabilization of the metaphase configuration.
1. Metaphase configuration is stable in acidic solutions ( p H < 5.5) in
the absence o r presence of divalent ions.
2. Metaphase configuration is not stable in basic solutions (pH > 7.5) in
absence of divalent ion.
3. Metaphase configuration is stable in basic solutions (7.5 < pH < 8.5)
if at least 10-3 M divalent cation is present.
Figure 1 shows a metaphase chromosome from a mouse ascites tumor
cell which has been swollen in 0.01 M Tris, p H 8.0 to about ten times its
a
FIG. 1. A typical post-transition chromosome. Buffer was 0.01 M Tris, p H 8.0
(10,400~).
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THE EUKARYOTIC CHROMOSOME
FIG
161
2. Fiber structure 01 the same post-transition cl~roiiiosomeat high tnngtiification. Buffei- same as Fig. 1 (92,000s).
condensed volurnc. Figurc 2 is a high magnification picture of this chromosome which clearly shows the typical 230 A fiber structure observed in both
interphase nuclei and metaphase chromosomes.
Keeping in mind that without careful chemical characterization, structural analysis of chromatin may have little to do with the structural State in
the nucleus o r chromosomes, there are a number of statements made about
chromatin which we enumerate below.
1. A 100 A fiber (39, 40) is the most commonly observed structural component of chromatin.
2. From spacings observed in an X-ray analysis (41, 42) of chromatin, it
has been postulated that the primary nucleoprotein fiber has a diameter of
35 A and is wrapped in a helical structure with a repeat distance of 110 A.
The 110 A spacing disappears and reappears upon stretching and relaxing
the chromatin fiber, a result consistent with this spacing being associated
with a helical repeat distance.
3. Flow dichroism of chromatin in the ultraviolet (43) is about 0.4 that
of oriented DNA, indicating some possible sirperroiling o f t h e DNA in
chromatin. This result is frequently misinterpreted as evidence of a nonordered structure for DNA in chromatin ( 4 ) . A reduction of dichroism merely
indicates that the planes of the DNA bases in oriented chromatin fibers are
not perpendicular to the fluid flow as they are in oriented DNA.
4. It is generally believed that the lysine-rich histones are primarily associated with the condensation of chromatin fibers into more condensed structures such as metaphase chromosomes and do not have a n effect on thc secondary structure of DNA in nucleohistone. T h e rather weak evidence in-
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HEARST & BOTCHAN
162
dudes changes in sedimentation coefficients
upon removal of the very lysine-rich histones (44) and the fact that removal of the very lysine-rich histones does not affect the optical rotatory dispersion of nucleohistone (45).
Furthermore, Mirsky (46) et al. have shown that extraction of histones
from metaphase chromosomes causes a dispersion
of material.
Clumped
material can be obtained again by addition of the lysine-rieh histones, while
addition of the arginine-rich histones has no effect. Littau (47) et al. have
noticed this same phenomenonin interphase chromatin.
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THE REPLICATION
UNIT
OF THE MAMMALIAlq
CHROMOSOME
Replication of DNAhas been studied in Chinese hamster cells by Taylor
(48) using the density label [~H] bromodeoxyuridine and the inhibitor
thymidylate synthesis,
fluorodeoxyuridine.
He concludes that the DNA
chains are replicated at a rate of 1 to 2/z per rain and that there may be
replicating units as small as 1 to 2/z which are joined end-to-end. He sets the
maximumsize of a unit of DNAreplication
at 180 to 360/~. Huberman &
Riggs (31) have used autoradiography and pulse-labeling to investigate the
rate and mechanism of DNAreplication
in Chinese hamster cells and Hela
cells. These authors used ["H] thymidine for their radioactive label and fluorodeoxyuridine to reduce the thymidine pool in their cells. They came to a
number of very interesting
¢oncluslons.
The rate of DNAsynthesis per
growing point appears to be constant during DNAsynthesis. The regions of
DNAsynthesis at any instant in time are not distributed equidistantly along
the DNAmolecule. The DNAfibers consist of tandcmly joined replicating
(30) units. The fibers are not broken by proteolytic enzymes so they probably contain no protein linkers. At each initiation point for DNAsynthesis,
the replication
occurs in both directions.
The maximumrate of DNAsynthesis is 2.5/, per rain and the len~h of the replication unit is 7 to 30
There are thus a number of properties associated with the chromosome
replication
of the higher organism which are not a property of bacterial
chromosome replication.
The linear rate of DNAreplication
is one-tenth
that observed in the bacteria. Replication units are probably less commonin
bacteria but examples such as episomes and the sex factor exist. Replication
units are clearly an important feature of the replication
of DNAin the
higher organism. Replication in two directions from an initiation site is apparently a unique feature of the higher organism. It has been demonstrated
that DNAreplication
occurs in only one direction and probably from one
initiation point in E. coli (49-51) and B. subtilis (52, 53). The mechanism
of DNAreplication for virus particles is more complex.
ATTACHMENT
OF THE
CHROMOSOME TO THE NUCLEAR
MEMBRANE
Substantial evidence has been presented in recent years for the attachment of the E. cull chromosome to the cell membrane and for the growing
point being firmly attached to the membrane.
In B. subtilis,
Ryter & Jacob (54, 55) were able to take electronmi-
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THE EUKARYOTIC
CHROMOSOME
163
crographs of the DNAattached to an invaginated
membrane structure
called the mesosome. This attachment has also been visualized in E. coli
(56). When Cuzin & Jacob (57) demonstrated that the episome and
chromosome do not segregate independently, it was postulated that attachment to the same membranestructure explained this non-independent segregation; the membranesite serves as a "linker" in effect. Though no metabolic interconnections
between membrane synthesis and nucleic acid synthesis have been made, it seems as if the membraneis intimately connected
with the replicating point in bacteria. Ganesan & Lederberg (58) have been
able to isolate the growing point along with a lipid (membraneous) fraction, by pulse labeling; furthermore, the label can be chased away from the
membranes indicating
attachment of the growing point to the membrane.
There is some evidence, as well, that indicates membraneattachment for the
termination site of DNAsynthesis in B. subtilis (59). The chemical nature
of the attachment site is unknown though ionic detergents seem to free the
DNA from membranes.
In a similar fashion autoradiographic studies of pulse-labeled thin-sectioned I-IeLa cell nuclei have shown (60, 61) that the chromosomeis attached
to the nuclear membrane, but the growing point is definitely
not on the
membrane but migrates from an initiation
site at the membranetoward the
center of the nucleus. Furthermore it has been shown with mealybug cells
that chromosomes are attached to the nuclear membrane even when DNA
synthesis is not occurring (62).
Combining these observations with what is known of the replication unit
discussed in the previous section, a very probable physical model for the interphase chromosome in the eukaryote is a single DNAmolecule which is
attached to the nuclear membraneat contour spacings between 7 and 60 /~.
(See Figure 3.) DNAsynthesis is initiated at these attachment points for
replication in both directions. The termination of DNAreplication may either occur at the membrane, in which ease only every other attachment
point is an initiation site, or in the center of the DNAloop, in which case
every attachment point is an initiation site.
The DNAis, of course, covered with a variety of histone and nouhistone
proteins which somehowdetermine the transcriptional
activity of the chromosome.A reasonable extension of this model, if it is correct, is that the
attachment sites on the nuclear membraneare also mecIaanistically important for the collapse of the chromosome into its metaphase configuration.
The disappearance of the nuclear membrane at metaphase suggests this may
be the ease. Despite these structural insights into the replication of the eukaryotie chromosome, the biochemical control of the replication of DNAis
poorly understood.
IN THE CONTROL
SYSTEMS
OF PROKARYOTES
AND EUKAI1YOTES
Experiments and models generated to explain trauscriptional
control in
eukaryotes have been influenced by two basic lines of research. It is temptANALOCIES
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HEARST
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FIG. 3. Twomodels for the attachment of the eukaryotic chromosometo the
nuclear membraneduring interphase. The I’s designate the initiation sites for DNA
synthesis and the T’s designate the termination sites for DNAsynthesis.
ing to categorize these lines as schools, but since it appears to us that neither group has proposed ideas that are mutually exclusive, and since it is
our belief that hierarchies of control probably exist even at the level of
RNAtranscription, we will assume that differences in emphasis in the literature are a reflection of the authors’ level of experimental observation.
Cytologists and cell biologists have noticed gross structural changes in
genetic material. The cell’s mitotic cycle from chromosomesto chromatin defines in one sense the limits of such structural changes. From the work of
several authors (63-66) it is believed that chromosomes are metabolically
inert; unfolded chromosomes or chromatin are of course metabolically active, depending on the state of the cell. Within this range, variations have
been found in the states of condensation of chromatin. Euchromatin or dispersed chromatin is found to be metabolically active and heterochromatin,
or tightly condensed material, is found to be relatively inert (67-70).
Some biochemists on the other hand, motivated in part by the relative
simplicity of RNAregulation in prokaryotes, have looked for classes of
molecules or combinations of molecules that can specifically recognize base
sequences and by doing so repress or activate genes.
It is hoped that an isolated metaphase chromosome system will help to
combine these approaches. It should be possible to reproduce the conversion
of chromosome into chromatin by treating the chromosomes with the proper
enzymes and environmental conditions and by so doing investigate the template abilities of the resulting chromatin.
Before reviewing the above literature it would be instructive to note incidentally that the picture in prokaryotic RNAregulation is far from simple
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THE EUKARYOTIC
CHROMOSOME
165
or complete. Indeed the simplicity of the classic lactose operon is misleading.
In the lactose operon one specific repressor (71) is made and has a single
binding site on the DNA(72) that inhibits further progress of RNApolymerase in transcribing RNAfrom the genome (73). By contrast the product
of the arabinose gene C (74) is a repressor with a repressor binding site but
is converted to an inducer by binding arabinose. The operon is presumably
only inducible if the protein-arabin0se complex is bound to a specific region
of the DNAwhich is distinct from the repressor site. Evidence for positive
control is also found in the rhamnose (75) and maltose (76) operons.
In operons involved in synthesis of some amino acids, tRNAsseem to be
implicated in repression of the operons (77-79) Temperature-sensitive synthetase mutants have been isolated and it can be shown that at elevated temperatures the operon can not be repressed (79, 80) This has implicated the
charged tRNAin repression (81, 82). If efficient binding of the repressor
the operator gene requires the protein to bind the charged tRNAas a cofactot, the above temperature dependence is explained. An even simpler model
has the charged tRNAas the repressor. At the present there is no evidence,
however, that the tRNAis involved in repression at the level of transcription.
The amino acid synthesis operons are of special concern because RNA
is involved in their control. Several authors have evidence for the importance
of RNAin the regulation of RNAsynthesis in higher organisms (83, 84).
is likely in fact that evolution has made full use of several forms of negative
and positive control as will be discussed below.
Finally the discovery of the sigma factor (85) further complicates RNA
regulation in bacteria, as it seems that different factors mayinfluence specificity of RNApolymerase recognition of DNAsites (86, 189). With sigma attached to polymerase the polymerase transcribes
in vitro only early T4
genes. Travers & Burgess (87) claim that sigma or similar factors may
necessary for all natural initiation.
Implications for higher organisms where
large blocks of genes must be regulated (as in mitotic regulation) are obvious.
The references to the bacterial systems are given to establish a minimal
range of complexity. In fact recent genetic and biochemical evidence of eukaryote systems makes the "operon hypothesis" (88) highly unlikely. Formally operons constitute a clustering of genes of related functions into one
unit of control on the DNA.Coordinate repression and induction is explained by the fact that one mRNAmolecule contains the information for
translation of several genes. In Salmonella (a prokaryote) the histidine
operon is a cluster of ten enzymes under one control. In Neurospora crassa
(a eukaryote), which has seven linkage groups, the histidine genes are
spread to four different chromosomes (89). Though smaller clusters are
seen in the four chromosomes, it seems that they have several properties
that may declassify them as operons. The products of the clustered genes
form an aggregate enzyme (90). The clustering may therefore be necessary
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166
HEARST & BOTCHAN
for the aggregation, not control (91). On the other hand, the aggregrate
enzyme may be one polypeptide chain.
One predicts from the operon hypothesis that the more close linked the
functions of a pathway the more likely is clustering of the genes. However
the ~ and fl subunits of hemoglobin are unllnked (92). Another example
comes from interpretations
of lactic acid dehydrogenase (LDH) patterns
mouse-humanhybrid cells. In tissue culture as the chromosomes are selectively lost from the hybrid cells, it appears as if the A and B chains of LDH
are unlinked (93). Without belaboring the point it is clear that significant
evidence for operons has not been discovered in higher organisms (93a).
fact from the above it seems as if clustering has been selected against, in
the evolution of eukaryotes, at least in these cases. We must then ask what
the selective advantage against clustering is, since the operon has several
distinct advantages, including efficient usage of DNAspace, and protection
against separation of functions resulting from crossover events. Perhaps
having individual control over each enzyme at the level of RNAas opposed
to having coordinate control at this level enables the cell to be more flexible.
This would be of particular
advantage in a biochemical pathway that is
coordinated at times with another, through one or a few enzymes. Relative
concentrations of these enzymes could be controlled independently. Another
advantage to individual control as opposed to the operon could be a protection against frameshift and nonsense mutations. These provide reasons for
having individual control over genes, but in fact, do not provide any insight
into why these genes are found on different chromosomes.
CO~,aSE CO~TaOL
Workers are in general agreement about the inactivity
in situ of the
metaphase chromosome. The general agreement is also extended to an explanation of this shutoff. In the process of coiling and condensation the once
exposed DNAbecomes inaccessible
to RNApolymerase. A priori other
methods for a complete halt in RNAsynthesis are conceivable. Amongthese
perhaps the most elegant is the removal of RNApolymerase activity from
the condensing material.
However, Hotta & Stern (94) have shown that
there is no difference between RNApolymerase levels in crude metaphase
chromosome preparations
and in metabolically active meiotic chromosome
preparations. It should not be assumed, however, that RNApolymerase levels are always constant, in fact, recent evidence by Kohl et al. (95) shows
that amounts of polymerase vary at different stages of Rana pipiens development. Clearly other studies on polymerase levels in other systems are
called for, before any general statement on the cause of the biological inactivity of metaphase chromosomesis accepted.
There seems to be high correlation
between highly condensed chromosomal material and genetic inactivity in cells other than those in metaphase.
During development it is common to see increasing
amounts of clumped
"heterochromatin"
material in interphase cells [for a review see Brown
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THE EUKAR¥OTIC
CHROMOSOME
167
(96)]. Several authors (68-70, 97-100) have correlated this physically
densed material with genetic inactivity. Autoradiographic studies sfiow that
the condensed material does not incorporate RNAprecursors. The isolated
heterochromatin does not support in vitro synthesis while enchromatin does
(97). The fact that the repression in the condensed chromatln can be removed by treating with trypsin (98) demonstrates that some repressors are
involved with proteinaceous material. The DNAin heterochromatin generally melts at higher temperatures than in euchromatin (97) which is consistent with the notion that proteins strongly bound to DNAalter its physical
state in some way to prohibit transcription. Muchattention has been focused
on histories as repressors since the initial observation of Huang & Bonner
(99) that histones annealed to DNAcan repress transcription
in vitro.
When the very lysine-rich histones are selectively
extracted from native
chromatin, large increases in template responses to RNApolyrnerase have
been measured (101). As mentioned earlier,
extraction of these histones
from chromatin has no measured effect upon the secondary structure
of
DNA(45), though increases in solubility
(102) and changes in particle
sizes are observed (44). Apparently histones can repress synthesis
merely precipitating
chromatin (103). This may be an important in vivo
phenomenon, though it has been shown (104) that soluble chromatin can
also be repressed and that polyrnerase molecules have access to individual
DNA molecules in chromatin comparable to pure DNAas measured by the
apparent K,~ (105). Therefore, in some cases histones must affect either the
initiation of RNAchain growth or its elongation, and not the actual binding
of the enzymeto the template.
Heterochromatization can be either "constitutive,"
occurring in both maternal and paternal chromosomesin the same locus in interphase, or "facultative," that is one condensed and the other open. Perhaps the most striking
example of heterochromatization
is that which occurs to the X chromosomes in mammals(106, 107). Here one whole chromosome stays in its condensed metaphase-like configuration. Probably all genes associated with this
condensed body are not expressed and if one has a heterozygote allele, mosaics
occur, as a result of the random selection of the X chromosomewhich stays
inert from cell to cell (108, 109).
Though the cited literature serves as strong evidence for gross inactivations of genetic material due to condensation and coiling of the chromosome, one must remain cautious. It is not dear that condensation is the
cause of the inactivity. It may be a reflection of it. Without any rigid criteria of nativeness, such as ultrastructure morphology, the fact that in vitro
euchromatin has a higher response than heterochromatin or chromosomes
can only be taken as consistent with and not proof of this hypothesis. In
vitro systems have been shown to produce RNAsindistinguishable
from in
vivo-produced RNAsin a number of laboratories
(83, 84). However, these
workers did not attempt to fractionate
euchromatln from heterochromatin.
Recently DeBellis et al. (110) have isolated euchromatic and heterochro-
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HEARST
& BOTCHAN
marie chromatin from rat liver and Novikoff hepatoma, and have found that
after one adds excess E. coli RNApolymerase to either, no difference in
template response is found. However, their incubation mixture contained .25
N NaC1which did have a stimulatory effect on the heterochromatin.
This
high salt concentration
may have extracted proteins from the condensed
material. It would be interesting
to see if in fact the RNAsproduced in
their system were simil~r to native RNAas measured by hybridization
competition.
Puffing in the giant dipteran chromosomesseems to be a reflection of active RNAsynthesis rather than the initiation of it through the uncoiling of
the DNA.Treatment of the puffs with actinomycin causes a reduction in the
puff size (111). This establishes a direct relationship between puffing and
RNAsynthesis. RNArelease from the puffs is energy dependent and if one
stops this process of turnover by treating with 2,4-dinltrophenol (a mitochrondrlal poison), the puff is maintained even if RNAsynthesis is stopped
with actinomycin. 2,4-Dinitrophenol alone has no effect on the puff size.
Lezzi (112) has found that protein synthesis occurs in the puffs and has described the content of the puff as RNAchains which are coated with ribosomes engaged in protein synthesis. A dependence upon RNAsynthesis and
loop structure in lampbrush chromosomes has also been reported (113).
Despite the above reservations
it seems more productive to accept the
hypothesis that condensation is important in genetic regulation as well as. in
formation of metaphase chromosomes. If molecules can specifically
condense or uncoil regions of a specific chromosometheir effect upon template
activity can be determined. Pogo et al. (114) have shown that ionic strength
conditions can control amounts of heterochromatin in isolated nuclei, and
therefore levels of RNAsynthesis. Regulation of ionic conditions within the
cell nucleus may be very important in increasing total activity; however,
specificity for the variable response of local regions of the chromosometo
changes in ionic strength must originate from chromosome composition and
structure and not the salt per se (115).
It is our view that a hierarchy of genetic regulation exists governing
RNAtranscription.
The first level is the control of chromosome uncoiling.
That is to say if a region stays heterochromatic or chromosome-like it will
not be active even if inducers are present that would at a second level give
transcription.
Alternately before mitosis a region of a chromosome can become condensed. The condensed X chromosome provides an example, for
levels of control. Consider that there is genetic homology between the condensed chromosome and the extended one. Inducers must promote transcription on one chromosomeand have no effect on the other.
Position effects (116-118) provide another line of evidence for this first
level of control. Whena euchromati¢ section of a higher organism chromosome is translocated
by crossing-over to a region of another chromosome
that remains heterochromatic at interphase, the translocated region becomes
heterochromatic itself. These translocations are probably large enough to in-
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THE EUKARYOTIC
CHROMOSOME
169
sure that at least one control gene has been translocated, yet at its new position, this control gene is not expressed.
A reasonable approach to studying control at this level would include the
comparison of the composition and. structure
of metaphase chromosomes
with that of partially unfolded chromosomes (heterochromatin) and active
euchromatin. Similarity in basic fiber structure between chromosomes and
chromatin has been observed by several authors (67, 119). The basic fibers
of the chromosome (see Figure 2) have the same appearance as those
chromatin (120). It is tempting from this observation to postulate that additional molecules are added to the chromatln fiber at discrete spots and serve
as glue for chromosomal folding (121). Though more nonhistone acid-soluble proteins and acid-insoluble proteins are found in isolated chromosomes
than in chromatin, this is not the complete story.
Numerous workers (14, 97, 122, 123) have proposed that histones of extended and condensed chromosomes are similar. Actually these authors did
not look at specific hlstones but total histone content. The use of fast green
stains in measuring histone content is suspect since it is known that sidechain modification of histones can alter the effect (124). Comings(125)
looked for qualitative differences with polyaerilamide gel eleetrophoresis
and could find no variation in histone types in metaphase chromosomes and
interphase chromatin. Sadgopal (18) has carefully compared both the qualitative and quantitative aspects of histones in chromosomesof HeLa to HeLa
ehromatin. Significantly the amount of histone extractable is constant; however, a larger amount of the arginine-rich histones and a smaller amount of
the lysine-rich histones are observed in metaphase chromosomesthan in interphase chromatin. The differences are small and the significance of this
phenomenon is obscure since the very lysine-rich histones are known to be
of structural importance in metaphase chromosomes (61). It will be interesting to compare these results with quantitative
data on the relative
amounts of histones in heterochromatin and euchromatin. One must emphasize the importance of doing amino acid analysis in doing quantitative comparisons in histones since it is known that contamination is likely even in
what appear to be chromatographically pure fractions (126).
Other studies also indicate that more than a simple addition of nonhistone molecules to the basic chromatln fiber is responsible for chromosome
folding and coarse control of RNAtranscription.
It is the general result
that histone synthesis is dependent upon the mitotic state of the cell (1, 2)
and that indeed the rates of synthesis of the histone classes are similar
(127), although Stellwagen & Cole (128) have recently reported that
arginine-rich and lysine-rich histories are synthesized twice as fast as other
histones in mammaryglands. Turnover studies are perhaps more important
in studying the importance of histones in the control of the cell cycle since
it cannot be assumed that all histones are synthesized for the same length of
time. Gurley et al. (129) have found that the very lysine-rich histones turn
over in Chinese hamster cells in tissue culture whereas the other histones
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170
HEARST & BOTCHAN
seem to be stable, showing no loss of prelabel. This indicates that the very
lysine-rich histones do not play a passive role and are degraded between divisions.
Important quantitative differences in relative histone concentration in
cells in different stages of development have been measured. Fambrough et
al. (130) show that as the pea seed develops, a steady increase in the
amount of very lysine-rieh histones relative to total histone occurs. This is
consistent with the view that the lysine-rieh histones are important in heterochromatization. Other authors have also reported the increase in the very
lysine-rich histories in developing organisms (131, 132).
The very lysine-rlch hlstones are not a simple class of molecules. Bustin
& Cole (133) have been able to fraetionate this class of histones into several subclasses, and shoxv that different tissues have different chromatographic elution profiles and amino acid composition. Kinkade reports that
the relative amounts of the very lysine-rieh histone in different tissues vary
(134). Kinkade believes that these relative qnantitative differences bring
about qualitative
differences in genomie repression.
He postulates that
aggregates of the different histones make different repressors. It seems unlikely that there is enough information in the histories alone to give hundreds of thousands of specific aggregates. It is not necessary, however, for
these histories to have complete specificity at the level of individual genes. It
will be interesting to see if different very lysine-rlch hlstones are associated
with large specific parts of chromosomes. The involvement of the very lysine-rlch hlstones in genetic regulation is implicated by the work of Hobman & Cole (135). These authors show that in mammary gland organ explants, the very lyslne-rich hlstones can be fractioned into the established
five subgroups. In the presence of insulin and hydroxycortisone the cells undergo rapid DNAsynthesis. If prolactin is added to the media, casein synthesis is induced. Correlated with this synthesis is a specific reduction in one
of the very lysine-rlch histone fractions and an increase in another fraction.
The above implicates the lyslne-rich histones in a genetic regulatory sense
as well as in metaphasccoiling.
It is not surprising to find that each major class of hlstones binds equally
well with homologous and heterologous DNAs(104) since thcse h{stones
are probably fonnd in all eukaryotes. However each class may have specificities relative to each other, which could be important in coarse regulation.
Evidence from binding studies indicates that different hlstones bind with
different binding constants (136). More convincing evidence that is consistent with the involvement of bistones in a coarse level of regulation has
been provided by Hurwitz et al. (I37). They have found that DNAbound
with nonsaturating amounts of histone I serves as a template for G~C rich
RNA, whereas DNA-hlstone III-IV complexes are primers for A-T rich
RNAs.Johns & Butler (138) have looked for the relative specificities
of the
different histone classes by analyzing base composition in calf thymus DNA
precipitated by histones. These authors observe a change in G-C content in
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THE EUKARYOTIC
CHROMOSOME
171
the unprecipitated fraction of DNAfrom 45.6% to 49.4% upon precipitating
80% of the DNAwith the very lysine-rich
histone. They interpret
this
change as insignificant,
but from physical studies of Sueoka (190) on the
composition heterogeneity of DNAisolated according to usual methods, the
distribution
in the G-C content of calf thymus DNAhas a standard deviation of only 5%. From this number the maximumshift in G-C content expected in the unprecipitated
DNAupon precipitation
of the 80% of the
DNAhavng the lowest G-C content would be 7.5%. This 7.5% is surely an
over estimate of that expected because the spread in G-C content was determined from band width in a density gradient. Such an analysis is now
known to require a virial correction (191) which would make the measured
density heterogeneity smaller. We must conclude that the results of Johns
and Butler show a significant specificity in the precipitation of DNAby the
very’ lysine-rich histones.
The results of Hurwitz et al. are consistent with the work of Leng &
Felsenfeld (139, 140) who have shown that poly-L-lysine is somewhat specific for binding to poly-A-T. Work done in this laboratory (141) indicates
that the binding of poly-L-lysine to DNAis probably not a good model for the
observed level of specificity in the binding of the histones and that this specificity must involve other interactions as well as the side chains of lysine.
Olins (142) has found that the very lysine-rich histones do not compete for
binding to DNAwith actinomycin D. He has also shown that the binding of
the very lysine-rich
histones to DNAinhibits glucosylation of T2 phage
DNA.This evidence is consistent with a large-groove binding site for these
histones although it does not exclude some sort of external binding assymetric with respect to the grooves. Carroll & Botchan (141) have shown that
penta-L-lysine does compete for binding with actinomycin well before pentaL-lysine begins to form aggregates that would necessarily exclude some sites
for actinomycin binding. The competition between penta-L-lysine and actinomycin for DNAbinding sites seems to be unaffected by the presence of glucose in the large groove of DNA.These results are consistent with a smallgroove or external binding of penta-L-lysine. Circular dichroism data (140,
142) also indicate differences between polylysine binding and that of the
very lysine-rich histones.
Other candidates for a role in coarse regulation include the specific modification of side chains of chromosomal proteins. Modifications including
pho’.sphorylation (143), acetylation (144), and methylation (145) have
proposed as methods for introducing heterogeneity into the basic hlstone
frac.tions. At least in the case of acetylation it seems unlikely that the modification will increase significantly the information content of histones. Acetylation of the e-amino group of lysine occurs in the arginine-rich histones
and the complete sequence of histone IV has been reported (146). Only one
residue is methylated and only one lysine side chain is acetylated. Pogo et al.
(147) looking at synchronously diving cells in regenerating liver show that
at least 50%of the total acetylation occurs only in the very argini~e-rich
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172
HEARST & BOTCHAN
histones (hlstone IV). From the sequence work of DeLang eet al. (146)
appears that the nuclear enzymes for methylation and acetylation are specific to two particular side chains in histone IV. This limited modification
does not significantly increase the information content of this class of histones ; however, the relationship between these enzymeactivities and the mitotic cycle is of muchinterest. Tidwell et al. (148) show that methylation
histone IV occurs after the first peak in DNAsynthesis in regenerating
liver cells, preceding mitosis. Acetylation however is an early event in the
regenerating liver system (147), preceding the first peak in RNAsynthesis.
The degree of methylation of histones in metaphase chromosomes relative
to dispersed chromatin has not been investigated. Indeed it would be interesting to knowif in fact the ratio of e-acetyl lysine to free lyslne side chain
in residue ~16 of histone IV is proportional to the ratio of the number of
cells in G1 + S to the number in G2 and mitosis. Investigations on the activities of nuclear phosphatases (149) and kinases (150) specific to chromosomal proteins are also of interest in relation to the mitotic cycle.
A frequent observation with metaphase chromosomes has been that mercaptoethanol disperses the material (151, 152). Sadgopal (18) has observed
that when histones are extracted ~rom metaphase chromosomes a large portion stay at the origin in polyacrilamlde gel electrophoresls. This does not happen in the interphase extraction of histones. Whenthis material is treated
with mercaptoethanol the large polymerized proteins dissociate and band at
the arginine-rich histone spot. It is the arginine-rich histone UI that contains the total cysteine of the histone fraction. He observed that the nonhistone proteins also are highly crosslinked by S-S bonds. He observes that
this increase in S-S aggregation at metaphase is independent of the isolation procedure (isolating
chromosomes at acid pH and at neutral pH),
which provides some evidence that this is not an artifact of oxidation. Ord
& Stocken, (132, 153) have also found a low ratio of thlol to thiol plus disulfide in histones from condensed chromatin and a high ratio from dispersed chromatin in both sea urchin and calf thymus. In inactive unfertilized sea urchin eggs the ratio is 20%, in active precleavage eggs the ratio is
60%. The importance of these observations relative to chromosomal folding
and consequent coarse regulation of synthesis is obvious.
There is a class of nuclear nonchromosomal proteins with a total thiol
content which is large relative to the content of chromosomes. These thlols
were first studied by Rapkine (154) and later by Sakai & Dan (155).
nonchromosomal thiols are mostly reduced at cell division and oxidized at
interphase and thus have properties opposite to those of the chromosome. It
is possible that transfer of oxidation state between the two classes of thiols
occurs during the cell cycle. The states of the thiols in the chromosomeand
mitotic apparatus are of obvious structural importance; however, it is also
possible that hlgh-energy sulfur esters are important as an energy source
during mitosis. This thought is given credence by the fact that the dividing
cell seems to have its energy source "built into" the mitotic apparatus and is
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THE EUKARYOTIC
CHROMOSOME
173
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indifferent to metabolic poisons during mitosis (156) ; however, mercaptoethanol can be an arresting agent if applied before metaphase and at metaphase is without effect (151).
The nonhistone protein content of metaphase chromosomes as was previously mentioned is considerably higher than in interphase material. However, no information other than this exists either about their composition or
function.
What is clear is that there are a large number of chemical modifications
of chromosomal components which may have importance in the condensation of interphase chromosomesand which are at present poorly understood.
FINE CONTROL
Many workers have been led to look for molecules other than histones
that are important in the specific regulation of the chromosomeat the level
of the gene. They have been motivated by the fact that there are a small number of molecular types of histone, the apparent qualitative similarities
in
histones from tissue to tissue (130, 134), from organism to organism (2,
157-159), and the inability to find specificity of binding to DNAin vitro.
There are four major classes of histones as resolved by the chromatographic system of Rassmussen, Murray & Luck (156) and the most heterogeneity seems to be found in the very lyslne-rleh hlstone class where there
are perhaps 6 to 12 resolvable subgroups (133, 134). The one gene-one histone model is clearly impossible. Although as mentioned earlier it still seems
logically possible to have different permutations and combinations of these
relatively few histones to give more diversity, it seems highly unlikely that
there could be such a controlled aggregation without other informational
molecules.
Two models of regulation have been proposed. Neither accounts for the
quantitative
changes in the histone complement of a developing organism
(130, 132) or for the quantitative
changes in the histone complement
seen in response to a hormone (135), nor do they explain differences
turnover of histones (128, 129) and the significant metabolism of hlstones
at the polymer level (143-145). However, neither precludes these phenomena
from being significant at another level of regulation. The first hypothesis
most clearly stated by Huang & Bonner (160) and by the same group again
in their review article (161) can most aptly be called the director hypothesis. Here a complex of histones is given sequence information by the presence of another director molecule.
Huang & Bonner (160) have discovered a dihydrouridine-rich
class
RNAmolecules that are covalently linked to a nonhistone protein (162).
They have discussed an aggregation of 10-20 histone molecules per one
RNA-protein molecule and considered this complex as the specific repressor.
This class of small (35) I~NAmolecules has now been found in Ehrlich rat
ascites tumor (163), chick embryos (84), pea bud, and calf thymus (164).
It is methylated to a degree similar to rRNA(165), although it has a base
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174
HEARST & BOTCHAN
composition different from that of rRNAand is not rapidly labeled though
no information exists as to its turnover.
Several groups have been able to dissociate chromatin with high salt and
urea, and reconstituted the product by slow removal of the salt by dialysis,
followed by removal of the urea by dialysis (83, 84, 166).
Bekhor et al. (83) and Huang & Huang (84) have been able to isolate
chromatin from pea cotyledons and chick embryos, respectively,
and show
that by dissociating
chromatin, destroying RNAwith either Zn(NOa)~
RNase, and then reconstituting
the chromatin as described above, they can
alter the template response of the chromatin so that the RNAsproduced
with exogenous polymerase in their in vitro system cannot compete effectively with RNAextracted from the in vivo source. The RNAs produced
from chromatin reconstituted
without treatment with RNase or Zn (N’Oa)2
cannot be distinguished from native RNAby the hybridization competition
techniques of Gillespie & Spiegleman. Several different kinds of RNAare
present in this chromatin and it is not clear from this experiment if only the
special 3S piece is involved.
Plausibility
for the involvement of RNAin gene regulation comes from
the results of Shearer & McCarthy (167), Kijima & Wilt (168), and Attardi
et al. (169) who show that a large fraction of RNAthat is transcribed
never leaves the nucleus. This rapidly turning-over RNAcould be involved
in some way with regulation at the level of DNAby being a precursor to a
more long-lived species, that is produced by specific cleavages and methylation. Weinberg & Penman (170) have reported monodisperse small molecular
weight RNAthat is long-lived and is present in the nucleus. However this
RNAis different
from protein-RNA in molecular weight, and base composition.
Clearly there are several possibilities for those nuclear RNAsthat do not
get to cytoplasmic polysomes. Kijima & Wilt (168) have suggested that the
transfer of RN’Ato the cytoplasm is nonconservative and that some regulation system involving specific cleavages of RNAis operative. It does seem
energetically
extravagant for the cell to make RNAonly to degrade it;
however, some observations consistent ~vith this hypothesis have been made.
Birnstiel et al. (171) have shown that the transfer of rRNAto the cytoplasm is nonconservative and Sarmarina et al. (172) have seen a protein
structure
associated with RNAin the nucleus which may be important in
the transfer of RNAto the cytoplasm. It should be obvious, however, that
these observations are not at odds with the involvement of these nuclear
RNAsin regulation.
Belabor et al. (83), and Huang & Huang (84) have shown that chromosomal RNA(cRNA) is important in regulation of the template responses
their chromatin; however, it is clear that other molecules are also important. Huang & Huang show that when the cRNAis destroyed, the level of
in vitro synthesized RNAhybridization
with DNAincreases from about
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THE EUKARYOTIC
CHROMOSOME
175
2~% to 5% by weight of single-stranded
DNA. RNAprimed from pure
DNAhybridizes
with 50% of the DNA(173). If the protein-RNA were
involved in all forms of repression, the repressive histones would, upon removal of the RNA, reconstitute
in a random fashion, and all possible RNA
sequences would be transcribed. The hybridization level would thus be comparable to that obtained with pure DNA. The discrepancy between the 5%
and 50% can be rationalized
if one assumes that a major portion of the
genome is repressed speeifically
by molecules other than protein-RNA and
that a small percentage of the repression involves proteln-P,N’A. This
amount of the genome may be involved in amino acid biosynthesis.
The involvement of RNAin the control of amino acid synthesis in bacteria has
already been mentioned. The loss of specific control in this piece of the reconstituted
chromatin exposes enough new sequences of DNAto account
for the poor hybridization
competition between the RNAs made in vitro
with and without destruction of cRNA.
The evidence published by Bekhor et al. and Huang & Huang is not consistent with the view that the RNAis an inducer (174, 175) since an increase in the level of hybridization is seen after RNase treatment of ehromatin.
It is clear that other molecules aside from RNAcould form specific
complexes with hlstones to repress regions of the genome and thus serve as
directors.
Another general model for gene level control is the masking hypothesis
of Paul & Gilmour (166, 17’6). In this model genes are nonspecifically repressed by histones, a view given plausibility by the fact that histories by
themselves can repress all RNApolymerase template capabilities
of DNA.
In this model genes are specifically induced by the interactions of histories
with other nonhistone proteins (see also 177, 178). A variation of this
model is that histones are actually displaced from DNAby these nonhistone
molecules.
Paul & Gilmour can also isolate chromatln that shows template activity
similar to the in vivo activity. Their system is organ dependent, that is, a
chromatin from an organ will’produce RNAssimilar to in vivo ]iN’As from
the same organ, but recognizably different from in vivo RNAsof a different
organ. They find that when histories are annealed with DNA,no RNAscan
be produced. Whenthe total nonhistone fraction of proteins is added to the
separated mixture of DIq’A and histones, an induction of synthesis occurs
producing RNAsthat compete effectively
with native in vivo synthesized
RNA. However, the degree of competition deviates significantly
from
competition expected if both RNAsamples were identical.
This indicates
that some sequences are made in vitro that are not made in vivo.
The degree of competition is also significantly less than that measured by
Huang & Huang" (84) and Bekhor et al. (83). It is hard t6 compare these
papers since Huang & Huang and Bekhor et al. nse different materials and
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176
HEARST & BOTCHAN
different isolation procedures from Paul & Gilmour. One possible source of
difference could be the presence of cRNA, whose presence or absence cannot be easily detected in the template response if one merely looks for hybridization saturation values, as opposed to competition type experiments.
Preliminary evidence from Paul (179) indicates that the RNAcontent
his nonhistone protein fraction is very low (in some cases less than 0.2%).
The experiments reported by Paul & Gilmour (176) are consistent with the
masking hypothesis though they are also consistent with the director hypothesis assuming the directors are nonhistone proteins.
Paul & Gilmour also find that not all repression depends solely upon the
histone. Uponextracting histones from chromatin there still remain significant differences between the level of hybridization of the RNAmade from
dehistonized
chromatin and from pure DNA. This repression can be
moved from the DNAby extracting
the nonhistone proteins and then restored to give the same level of hybridization as before, implying specificity.
Not all of the nonhistone chromosomal proteins are operating in this function as some must be interacting with histones. Marushuge et al. (180) have
extracted one type of nonhistone protein and have shown that no repression
ability exists in this class. A negative result, however, must be viewed with
caution since denaturation is possible when extracting with detergent as
these authors did. The result of Paul & Gilmour implies at least two different kinds of specific regulation, one involving histone and other interacting
molecules, and one involving solely nonhistone protein. Taken together with
Bonners’ group, at least three different sorts of regulation at the level of
transcription are operative; perhaps some of these specific controls are involved with large blocks of genes or even specific chromosomal coiling, perhaps some with the regulation of individual cistrons.
Without genetic analysis the task of unraveling these control mechanisms in higher organisms is perhaps hopeless. On the other hand, the genetic complexity of a gene that is under nonspecific positive control at the
coarse level of regulation and perhaps under both positive and negative control at the fine control level is enormous. Furthermore, in view of the evolutionary constancy of the primary sequence of histone IV (158) and the constancy of peptide maps of all the other histone fractions (2), it appears that
mutations in these histones are strongly selected against. This will make the
selection of mutants difficult.
The lethal anucleolate mutant in Xenopus laev/s is extremely interesting.
Berlowitz & Birnstiel (181) have found that
this mutant, which is devoid in rRNAsynthesis, the histone fractions I and
II are missing. Further work on mitotic and developmental abnormalities in
this organism should provide valuable information as to the role of these
histones.
Despite the complexity introduced by different levels of genetic control,
if proper criteria can be established to classify the different levels experimentally, they can each be understood. A combination of genetic and biochemical informaton is likely to provide these criteria.
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THE EUKARYOTIC
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BASE SEQUENCE REDUNDANCY IN
CHROMOSOME
THE EUKARYOTIC
177
CHROI~IOSOME
It is generally believed that the amount of DNAin higher organisms is
much greater than is needed for the production of the required mRNA
(182). It is not clear why there is so much more DNAin unicellular eukaryotes than in bacteria with similar metabolic pathways. One logical hypothesis is that this additional DNAis associated with the control of the more
sophisticated structure and division cycle of the higher organism. The process of cell differentiation
must require a still more complex control system
and therefore more DNA.
A special class of DNAhas been observed in eukaryotes.
Britten &
Kohne (183) have observed that when the DNAof these organisms is heat
denatured, a certain fraction of it renatures rapidly under appropriate conditions. They argue that this phenomenonresults from the fact tibet some
DNAsequences are repeated many times in the total genome. The collection
of DNAsequences which are able to renature under prescribed conditions is
called a family. These families have been observed to contain 10a to 10s repeated elements. Such redundant families are found in organisms ranging
from protozoa to humans. At present no knowledge is available concerning
the degree of identity two sequences must have to be members of the same
family; nor is there good evidence for how long these sequences are, although they are estimated to average about 400 nucleotides long. This
length is a factor of 25 greater than the length predicted for such families
on a random basis. Given the amount of sequence homology found in proteins and the degeneracy of the code, there is at present no obvious eonneetlon between the redundancy of these DNAsequences and known protein
sequences.
Britten & Davidson (174) have presented an elegant theory for gene
regulation in higher cells. The theory implicates the redundant sequences of
DNAas control sites for RNAtranscription.
One argument that seems to
be very powerful in involving some redundant sequences in control is that if
many "producer genes" (analogous to "structural
gene" in bacteria)
are
under the control of a single external signal, the internal message (e.g., repressor, inducer) must utilize redundancy at some level (184). However,
it seems unlikely that a control molecule would need a sequence of 400 nucleotides to effect transcriptional
control. The linear dimension of such a
sequence is 30 times greater than the diameter of most globular protein
molecules. Such redundant sequences may be linear arrays of sites for many
control molecules controlling one producer gene as suggested by Britten &
Davidson. The probability of significant redundancy between the linear arrays of control sites should be high because manyproducer genes are likely
to respond to the same control molecules.
There is another class of redundant DNAwhich is not transcribed by
the cell. Large amounts (10% of the genome) of an A-T rich DNAhave
been observed in mice (185). The function of this DNAis not clear but
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178
HEARST
& BOTCHAN
this DNAis probably not transcribed
in vivo (186). It is known to be uniformly distributed
among all chromosomes (7). It is possible that these sequences are important to the structure
of the chromosomes. Unlike the majority
of the DNAsequences in the mouse cell, these do not release their
chromosomal proteins
in 2 M NaCI (7). It is likely that such untranscribed
DNAsequences exist in other organisms as well, although they do not have
unique G-C contents.
It would be interesting
with respect to both types of redundancy to see if
there are families unique to individual
chromosomes. Of particular
interest
would be the Y chromosome, because this chromosome is only transcribed
during spermatogenesis
(187), in the male gonad.
Such redundant regions could influence
structure
both by their binding
characteristics
for proteins
and by self-interaction
of complementary regions of the same DNAstrand.
Such a looping capability
in DNA has been
demonstrated
by Flamm et al. (186) using mouse satellite
DNA.
Redundant DNA can also contain
producer genes. For example there are
1400 copies of rDNA per genome in Xenopus laevis
(188). This third class
of redundant DNA, which is believed to result
from amplification
of important producer genes, involves families
containing
a smaller number of repetitions than those usually associated with the other two classes.
LITERATURE
CITED AND ANNOTATIONS
1. Stellwagen, R. H., Cole, R. D., Ann.
Rev. Biochem., 38, 951 (1969)
2. Hniliea, L. S., Proof. Nucl. Acid Res.
Mol. Biol., 7, 25 (1967)
3. Bonnet, J., Dahmus, M. E., Fambrough, D., Huang, R. C., Marushige,
K., Tuan, D. Y. H.,
Science, 159, 47 (1968)
4. DuPraw, E. ~., Ceil and Molecular
Biology, 514-90 (Academic Press.
New York. 1968)
5. Wolfe, S. L., Biological Basis of
Medlelne, 4, 23-42 (]~ittar, ]~. t~..
Ed., Academic, 1969)
6. Ris, H., Proc. Roy. Soc., Set. B,
164, 246 (1964)
7. Maio,J. J’., Sehildkraut, C. L., )’. Mol.
Biot., 40, 203 (1969)
S. Huberman,J’. A., Attardi, G., .L Mol.
Biol., 29, 487 (1967)
9. Mendelsohn, J., Moore, D. E., Salzman, N. P., L Mol. Biol., 32, 101
(1968)
I0. Franeeschini, P., Giacomoni, D., Atti
Assoc. Genet. Ital., 12~ 248 (1967)
11. Reglmbal, T., Mel, H., Biophys..t.,
9, A-254 (1969)
12. Salzman, N. P., Moore, D. E., Mendelsohn, J., Proc. Natl. Acad. Sci.
U.S., 56, 1449 (1966)
13. Huberman,~I. A., Attardi, G., .L Cell
Biol., 31, 95 (1966)
14. Maio, I. I., Schildkraut, C. L., ].
Mol. Biol., 24, 29 (1967)
15. Epstein, W., Beckwith, J’. R., Ann.
Rev. Biochem., 377 411 (1968)
16. Chorazy, M., Bendich, A., Borenfreund, E., Hutchison, D. J., )’.
Cell Biol., 19, 59 (1963)
17. Cantor, K. P., Hearst, J. E., Proc.
Natl. Acad. Sd. U.S., 5~, 642
(1966)
18. Sadgopal, A., Studies on Chromosomal
Proteins o[ HeLa Cells Durin9 the
Ceil Division Cycle (Doctoral
thesis, California Inst. Teeh.,
Pasadena, Calif., 1968)
19. Cantor, K. P., Hearst, I. E., .L MoL
BioL (In press, 1970)~ Cantor,
K. P., Isolation and Characterigatlon of Metaphase Chromosomes
of a Mouse Ascites Tumor (Doctoral thesis, Univ. California, Berkeley, 1969)
20. Murray, K., .r. Mol. Biol., $~, 125
(1969)
21. Tissleres, A., Watson, J’. D., Schlessinger, D., Hollingworth, B. R.,
L Mol. Biol., 1, 221 (1959)
22. Schmid, C. W., Botehan, M. (Unpublished data)
23. Kaufmann, B. P., Botan. Rev., 14,
57 (1948)
24. Mazia, D., The Cell, 3~ 77-412
Annual Reviews
www.annualreviews.org/aronline
Annu. Rev. Biochem. 1970.39:151-182. Downloaded from arjournals.annualreviews.org
by UC Berkeley on 08/16/05. For personal use only.
THE
EUKARYOTIC
(Brachet, J., Mirsky, A. E., Eds.,
Academic, New York, 1961)
25. Abuelo, J. G., Moore, D. E., I. Cell
Biol., 41, 73 (1969)
26. Wolfe, S. L., I. Ultrastruct. Res., 12,
104 (1965)
9.7. Ts’o, P. O. P., Microsomal Particles
in Protein Synthesis,
156-68
(Roberts, R. B., Ed.,. Pergamon,
New York, 1961)
28. Walker, I. 0., J. Mol. Biol., 14, 381
(1965)
29. Taylor, J. H., Molecular Genetics,
Part I, 62-111 (Academic, New
York, 1963)
30. Cairns, J., J. Mol. Biol., 15, 372
(1966)
31. Huberman,J. A., Riggs, A. D., Proc.
Natl. ,4cad. Sci. U.S., 55, 599
(1966)
32. Sasaki, M. S., Norman, A., Exptl.
Cell Res., 44, 642 (1966)
33. DuPraw, E. J., Nature, 206, 338
(1965)
34. DuPraw, E. J., Rae, P. M. M.,
Nature, 212, 598 (1966)
35. Ris, H., J. Biophys. Biochcm. Cytol.,
Suppl., 2, 385 (1956))
36. Gay, H., ]. Biophys. Biochem. Cytol.,
Suppl., 2, 407 (1956)
37. DuPraw, E. J., Proc. Natl. Acad.
Sci. U.S., 53, 161 (1965)
38. Ris, H., Chandler, B. L., Cold Spring
Harbor Syrup. Quant. Biol., 28,
1 (1963)
39. Pardon, J. F., Wilklns, M. I-I. F.,
Riehards, B. M., Nature, 215, 508
(1967)
40. Ris, H., Regulation of Nucleic Acid
and Protein Biosynthesis,
11-21
(Koningsberger, V. V., Bosch, L.,
Eds., Elsevier, Amsterdam, 1967)
41. Wilklns, M. H. F., Zubay, G., Wilson,
H. R., 1. Mol. Biol., 1, 179 (1959)
42. Pardon, J. F., Wilkins, M. H. F.,
Richards, B. M., Nature, 215, 508
(1967)
43. Ohba, Y., Biochim. Biophys. ~lcta,
123, 76 (1966)
44. Chalkey, R., Jensen, R. H., Biochemistry, 7, 4380, 4388 (1968)
45. Tuan, D. Y. H., Bonnet, 5., ]. Mol.
Biol. (In press)
46. Mirsky, A. E., Burdick, C. J., Davidson, E. H., Littau, V. C., Proc.
Natl. Acad. Sci. U.S., 61, 592
(1968)
47. Littau, V. C., Burdick, C. L, Allfrey,
V. G., Mirsky, A. E., Biochemistry,
54, 1204 (1965)
48. Taylor, J. H., ]. Mol. Biol., $1, 579
(1968)
CHROMOSOME
179
49. Nagata, T., Proc. Natl. Acad. ScL
U.S., 49, 551 (1963)
50. Wolf, B., Newman,A., Glaser, D. A.,
J. Mol. Biol., 32, 611 (1968)
51. Pritchard, R., Lark, K. G., ]. Mol.
Biol., 9, 288 (1964)
52. Yoshikawa, H., Sueoka, N., Proc.
Natl. Acad. Sci. U.S., 49, 559
(1963)
53. Yoshikawa, H., Sueoka, N., Proc.
Natl. Acad. Scl. U.S., 49, 806
(1963)
54. Ryter, A., Jacob, F., Compt. Rend.,
257, 3060 (1963)
55. Ryter, A., Jacob, F., Ann. Inst.
Pasteur, 107, 384 (1964)
56. Ryter, A., Jacob, F., Ann. Inst.
Pasteur, 110, 801 (1966)
57. Cuzin, F., Jacob, F., Compt. Rend.
Acad. Sci., 260, 5411 (1965)
58. Ganesan, A. T., Lederberg, T.,
Biochem. Biophys. Res. Commun.,
18, 824 (1965)
59. Sueoka, N., Quinn, W., Cold Sprino
Harbor Syrup. Quant. Biol., 33,
695 (1968)
60. Moses, M. J., Coleman, J. R., Role
Chromosomes in Development, 1149 (Locke, M., Ed., Academic,
New York, 1964)
61. Comings, D. E., Kake{uda, T.,
Mol. Biol., 33, 225 (1968)
62. Pawlowski, P. J., Berlowitz, L.,
Exptl. Cell Res., 56, 154 (1969)
63. Taylor, J. H., Ann. N.Y. Acad. Sci.,
90, 409 (1960)
64. Prescott,
D. M., Bender, M. A.,
Exptl. Cell Res., 26, 260 (1962)
65. Konrad, C. G., ]. Cell Biol., 19, 267
(1963)
66. Baserga, R., 1. Cell Biol., 12, 633
(1962)
67. Hsu, T. C., Exptl. Celt Res., 27, 332
(1962)
68. Frenster, J. H., Allfrey, V. G.,
Mirsl~’, A. E., Proc. U.S. Natl.
Acad. Sci., 50, 1026 (1963)
69. Littau, V. C., Allfrey, V. G., Frenster, J. H., Mirsky, A. E., Proc.
U.S. Natl. ~tcad. ScL, 52, 93
(1964)
70. Brown, S. W., Wur, U., Science,
145, 130 (1964)
71. Gilbert, W., Muller-Hill, B., Proc.
U.S. Natl. ~Icad. Sci., 56, 1891
(1966)
72. Gilbert, W., Muller-Hill, B., Proc.
U.S. Natl. ~cad. Sci., 58, 2415
(1967)
73. Resnlkoff, W. S., Miller, J. H.,
Scalfe, J. G., Beckwith, ~. R., J.
Mol. Biol., 43, 201 (1969)
Annual Reviews
www.annualreviews.org/aronline
Annu. Rev. Biochem. 1970.39:151-182. Downloaded from arjournals.annualreviews.org
by UC Berkeley on 08/16/05. For personal use only.
180
HEARST
& BOTCHAN
74. Englesberg, E., Sheppard, D. E.,
tional effects, and did statistical
Squires, C., Meronk, F., Jr., J.
analysis to see if any clustering
could be detected. They came to
Mol. BioL 43~ 281 (1969)
the conclusion that at the level to
75. Power, J., Genetics, 55~ 557 (1967)
which the genetic map had been
76. Schwartz, M., Ann. Inst. Pasteur,
investigated, functionally related
118, (1967)
genes can be regarded as occurring
77. Sehlesinger, S., Magasanik, B.,
randomly along the chromosome.
Mol. Biol., 9~ 670 (1964)
94. Hotta, Y., Stern, A., Nature, 210,
78. Eidlic, L., Neidhardt, F. C., Proc.
Natl. Acad. Sci. U.S. 53, $39
1043 (1966)
(1965)
95. Kohl, D. M., Greene, R. F., Flickinger, R. A., Biochim. BiophXs.
79. Freundlich, M., Science, 157, 823
(1967)
Acta, 179, 28 (1969)
80. Roth, J. R., Ames, B., J. MoLBiol.,
96. Brown, S. W., Science, 151, 417
(1966)
22, 325 (1966)
81. Silbert, D. R., Fink, G. R., Ames, B.,
97. Frenster, J. H., Nature, 206, 680
J. Mol. Biol., 29, 335 (1966);
(1965)
Fink, G. R., Roth, J. R., .r. Mol.
98. Allfrey, V. G., Littau, V. C., Mirsky,
Biol., 88, 547 (1968)
A. E., Proc. Natl. Acad. Sci. U.S.,
82. It is possible that an uncharged tRNA
49, 414 (1963)
99. Huang, R. C. C., Bonnet, J., Proc.
is important in rRNA repression
as well; see Nierlich,
D. P.,
Natl. Acad. Sci. U.S., 54, 960
Proc. Natl. Acad. Sci. U.S., 60,
(1962)
1345 (1968)
100. Granboulan, N., Granboulan, P.,
83. Bekhor, I., Kung, G., Bonnet,
ExPtl. Cell Res., 88, 604 (1965)
101. Georgiev, G. P., Ananleva, L. N.,
1. Mol. Biol., 89, 351 (1969)
84. Huang, R. C. C., Huanff, P. C.,
Kozlov, J. V., J. Mol. Biol., 22,
Mol. Biol., 39, 365 (1969)
365 (1966)
85. Burgess, R. R., Travers, A. A., Dunn, 102. Ohlenbusch, H., Olivera, B., Tuan,
J. J., Bautz, E. K., Nature, 221~
D., Davidson, N., J. Mol. Biol.,
43 (1969)
25, 299 (1967)
103. Sonnenberg, B. P., Zubay, G., Proc.
86. Travers, A. A., Nature, 2~, 1107
Natl. Acad. S¢i. U.S., 5% 415
(1969)
R. R.,
87. Travers, A. A., Burgess,
(1965)
Nature, 229, 537 (1969)
104. Huang, R. C. C., Bonner, J., Murray,
K., J. Mol. Biol., 8, 54 (1964)
88. Stent, G. S., Science, 144, 816 (1964)
105. Marushige, K., Bonnet, J., 3". Mol.
89. Ahmed, A., Case, M. E., Giles, N.,
Brookhaven Natl. Lab. Syrup., 17,
Biol., 15, 160 (1966) ; Shih, T. Y.,
Subunit Structure of Protgins :
Bonnet, J., J’. Mol. Biol. (In press)
Biochemical and Genetic Aspects,
106. Lyon, M. F., Nature, 190, 372 (1961)
53 (1964)
107. Mittwoeh, V., I. Med. Genet., 1, 50
90. Giles, N. H., Case, M. E., Partridge,
(1964)
108. Russell, L. B., Trans. N.Y. Acad.
C. W. H., Ahmed, S. I., Proc.
Sci., 26, 726 (1964)
Natl. ~lcad. Scl. U.S., 58, 1453
109. Davidson, R. G., Mnn. N.Y..dcad.
(1967)
ScL, 151, 157 (1968)
91. Fink, G. R., Genetics, 58, 445 (1966),
110. DeBellis, R. H., Benjamin, W., Gellhas found a similar situation in
the histidine genes of yeast. Exhorn, A., Biochfin. Biophys. Res.
tensive purification in one duster
Commun., 86, 166 (1969)
has failed to separate components 111. Beermann, W., Cell Differentiation
and Morphogenesis, 24-54 (Northof the aggregate enzyme. Rytka,
J., Fink, G. R. (Manuscript in
Holland, Amsterdam, 1966)
preparation)
112. Lezzi, M., Chromosoma,21, 82 (1967)
92. Epstein, C. ~’., Motulsky,A. G., ProtTr.
113. Izawa, M., Allfrey, V. G., Mirsky,
Med. Genet., 4, 97 (1965)
A. E., Proc. Natl. ~tcad. Scl. U.S.,
93. Nabholz, M., Migglano, V., Bodmer,
49, 544 (1963)
114. Pogo, A., Littau, V. C., Allfrey,
W., Nature, 22~, 358 (1969)
V. G., Mirsky, A., Proc. Natl.
93a. Elston, R. C., Glassman, E., Genet.
Res., 9, 141 (1967) These authors
Acad. Sci. U.S., 515, 550 (1966)
have classified
D. Melanogaster
115. Kroeger, H., Lezzi, M., Ann. Rev.
mutants according to their tuneEntomol., 11, I (1966)
Annual Reviews
www.annualreviews.org/aronline
THE
EUKARYOTIC
116. Lewis, E. B., Advan. Genet., 3~ 73
(195o)
117. Hannah, A., Advan. Genet.,
Annu. Rev. Biochem. 1970.39:151-182. Downloaded from arjournals.annualreviews.org
by UC Berkeley on 08/16/05. For personal use only.
(1951)
4, 87
118. Becket, H. J., ZooI. Anzeig. Suppl.,
24, 283 (1961)
119. Davies, H. G., Small, J. V., Nature,
217, 1129, (1968).
120. The substructure nature of the "9,00
A" fiber in chromatin is in dispute
as is the "200 A" fiber in metaphase chromosomes. A 100 A fiber
is seen in active euehromatin
(Frenster,
J. H., Nature, 205,
1341, 1965), which may undergo
further coiling to give the 200
fiber or mayin fact aggregate with
other fibers.
121. Cole, A., Nature, 19~, 211 (1962)
122. Dingman, C. W., Sporn, M. B.,
Biol. Chem., 239, 3483 (1964)
123. Loewus, M., Nature, 218, 474 (1968)
124. Berlowitz, L., Proe. Natl. Acad. Sci.
U.S., 54~ 476 (1965)
125. Comings, D. E., )’. Cell Biol.,
699 (1967)
126. Stellwagen, R. H., Cole, R. D.,
Biol. Chem., 243, 4452 (1968)
127. Gurley, L. R., Hardin, 5. M., ~lrch.
Biochem. Biophys.,
128, 9.85
(1968)
128. Stellwagen, R. H., Cole, R. D.,
Biol. Chem., 244, 4878 (1969)
129. Gurley, L. R., Hardin, J. IV[., Longham, W. H., Fed. Proe., 27, 797
(1968)
130. Fambrough, D. M., Fujlmura, F.,
Bonner, J., Biochemistry, 7, 575
(1968)
131. Backstrom, S., Acta EmbroyoL Morphol. E~rptl., 8, 20 (1965)
132. Ord, M. G., Stoeken, L. A., Bioehem.
J’.~ 107~ 403 (1968)
133. Bustin, M., Cole, R. D., ~’. B~ol.
Chem., 24~, 4500 (1968)
134. Kinkade, 5. M-, Jr., or. Biol. Chem.,
244, 3375 (1969)
135. Hohman, P., Cole, R. D., Na~rz,
223, 1064 (1969)
136. Aklnrimisl,
E., Ts’o, P. O. P.,
Bonnet, J., L Mol. Biol., 11, 128
(1965)
137. Hurwitz, J., Evans, C., Babinet, C.,
Skalka, A., Cold Sprln# Harbor
Syrup. Quant. Biol., 28, 59 (1963)
138. 5ohns, E. W., Butler, 5. A. V.,
Nature, 204, 853 (1964)
139. Leng, M., Felsen~eld, G., Proc. Natl.
Acad. S¢i. U.S., 56, 1325 (1966)
140. Shapiro, J. T., Leng, IV[., Felsenfeld,
G., Biochemistry, g, ~219 (1969)
CHROMOSOME
181
141. Carroll, D., Botchan, M. (Manuscript
in preparation)
142. Olins, D., J. Mol. Biol., 4$, 439
(1969)
143, Klein-Smith, L. J., Allfrey, V. G.,
Mirsky, A. E., Proc. Natl. Acad.
Sci. U.S., 55, 1182 (1966)
144. Stevely, W. S., Stocken, L. A.,
Biochem. ]., 100, 20 (1966)
145. Allfrey, V. G., Faulkner, R., Mirsky,
A. E., Proc. Natl. Acad. Sci. U.S.,
51, 786 (1964)
146. DeLange, R. J., Fambrough, D. M.,
Smith, E. L., Bonner, J., ]. Biol.
Chem., 244, 319 (1969)
147. Pogo, B. G., Pogo, A. O., Allfrey,
V.
G., Sci.
Mirsky,
Natl.
Acad.
U.S., A.,
59, Proc.
1337 (1968)
148. Tidwell, T., Allfrey, V. G., Mirsky,
A. E., ]. Biol. Chem., 243, 707
(1968)
r149.
.
Meisler, M. H., Langan, T. A.. o
Cell Biol., 35, 91A(1967)
150. Langan, T. A., Science, 162~ 579
(1968)
151. Mazia, D., Zimmerman,A. M., Exptl.
Cell Res., 15, 138 (1958)
152. Bodenfreund, E., Fitt, E., Bendich,
A., Nature, 191, 1375 (1961)
153. Ord, M. G., Stocken, L. A., Biochem.
Y., 98, 888 (1966)
154. Rapklne, L., ,4nn. Physiol. Physlochim. Biol., 7, 382 (1931)
155. Sakai, H., Dan, K., Exptl. Cell Res.,
16, 24 (1959)
156. Rassmussen, P., Murray, K., Luck,
J., Biochem. Y., 1, 79 (1962)
157. DeLange, R. ~., Fambrough, D. M.,
Smith, F. J., Bonnet, .l., or. Biol.
Chem., 243, 5906 (1968)
158. Smith, E. L., DeLange,
Bonnet, J. (Submitted to Fed.
Proc. )
159. Fambrough, D., Bonnet, 5., Biochem.
or., 5, 2563 (1966)
160. Huang,
C., Bonner,
Proc.
Na~l.
Acad.R.Sci.
U.S., 54,5, 860
(1965)
161. Bonnet, 5, Dahmus, M. E., Fambrough, D., I-Iuang,
R. C.,
Marushige, K., Tuan, D. ¥.,
Science, 159, 47 (1968)
162. Huang, R. C., Fed. Proc., 26, 1933
(1967)
163. Dahmus, M. E., MeConnell, D.
Biochem. Y., 8, 1524 (1969)
164. Shih, T. Y., Bonnet, 5., Biochim.
Biophys. Acta, 182, 30 (1969)
165. Dahmus, M., Studies on Chromosomal
RNA and Effect of Hydrocortisone on the Template Activity of
Liver Chromatin (Doctoral thesis,
Annual Reviews
www.annualreviews.org/aronline
182
166.
167.
168.
169.
Annu. Rev. Biochem. 1970.39:151-182. Downloaded from arjournals.annualreviews.org
by UC Berkeley on 08/16/05. For personal use only.
170.
171.
172.
173.
174.
HEARST
& BOTCHAN
California Inst. Teeh., Pasadena.
Calif., 1969)
Gilmour,R. S., Paul, J., .r. Mol. Biol.,
40, 137 (1969)
Shearer, R., McCarthy, B. J., Biochem. J., 6, 283 (1967)
Kijima, S., Wilt, F. H., 3.. Mol. Biol.,
40, 235 (1969)
Attardi, G., Parnas, H., Hwang, M..
Attardi, B., J. MoLBiol., 20, 145
(1966)
Weinberg, R. A., Penman, S., ]. Mol.
Biol., 88, 289 (1968)
Birnstiel, M., Speirs, J., Purdom,I.,
Jones, K., Loenlng, U. E., Nature,
219, 454 (1968)
Samarina, O. P., Lukanidin, E. M.,
Molnar, J., Georgieu, G. P., J.
Mol. Biol., 39, 251 (1968)
A quantitative understanding of hybridization is not yet available, so
interpretational difficulties exist
with such experiments. For example, a 12-met (depending upon
G-Ccontent) is stable at hybridization temperatures.
Given the
large size of the enkaryote genome,
each 12-met appears 100 times in
a random array. Secondly, from
the klnetle data of Brltten (manuscript in preparation) it appears
that ordinarily hybridization takes
place only with the redundant
sequences, which are only a fraction of the total genome.
Britten, R. J., Davldson, E. H.,
Science, 165, 349 (1969)
175. Frenster, J. H., Nature, 206, 1269
(1965)
176. Paul, J., Gilmour, R. S., .L Mol. Biol.,
~4, 305 (1968)
177. Dingman, C. W., Sporn, M. B., J.
Biol. Chem., $39, 3483 (1964)
178. Wang, T. Y., Johns, E. W., Arch.
Biochem. Biophys., 124, 176 (1968)
179. Paul, J. (Personal communication)
180. Marushige, K., Brutlag, D,, Bonner.
J., Bioehem..~., 7, 3149 (1968)
181. Berlowitz, L., Birnsteil, M., Science,
156, 78 (1967)
182. Discussion following Goodwin,]3. C.,
Histories (de Reuck, A. V. S., Ed.,
Little, Brown, Boston, 76-77 pp.,
1966)
183. Britten, R. J., Kohne, D. E., Scie~rce,
llil, 529 (1968)
184. Britten, R. J, (Personal eommunicat/on)
185. Flamm, W. G., Bond, I~. E., Burr,
H. E., Biochim. Biophys. Acta,
123, 652 (1966)
186. Flamm, W. G., Walker, P. M. B.,
MeCallum, M., 3". Mol. Biol., 40,
2123 (1969)
187. Hennig, W., J. Mol. Biol., 38, 227
(1968)
188. Birnstiel, M. L., Cell Differentiation,
178-95 (de Reuck, A. V. S.,
Knight, J., Eds., J. & A. Churchill,
London, 1967)
189. Travers, A. A., Nature, 223, 1107
(1969)
190. Sueoka, N., 3". Mol. Biol., 9, 31 (1961)
191. Sehmld, C. W., Hearst, J. E., 3". Mol.
Biol., 44, 143 (1969)
Annu. Rev. Biochem. 1970.39:151-182. Downloaded from arjournals.annualreviews.org
by UC Berkeley on 08/16/05. For personal use only.
Annu. Rev. Biochem. 1970.39:151-182. Downloaded from arjournals.annualreviews.org
by UC Berkeley on 08/16/05. For personal use only.