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ENZYME
STUDIES
ON
CHROMOSOMES
DANIEL MAZIA
The title of this paper is purposely ambiguous.
In part, it deals with the action of enzymes on
chromosome structure. The other part concerns the
enzymatic activity of chromosomes. In no case, it is
hoped, will chromosome chemistry automatically
be equated with gene chemistry. If future discoveries justify such an equation, the situation will
be a happy one. For the present, our crude notions
of the chemistry of the chromosome may be seriously
restrictive in our thinking about the genes. The
problems of chromosome mechanics are important
enough in themselves, and the chromosome, as will
be shown later, may represent fax~orable material
for the study of the problems of the gene reproduction.
CHEMICAL STRUCTUREOF CHROMOSOMES
Proteins. The facts concerning the protein analysis of the nucleus have been adequately reviewed
(Gulick, 1938 et seq.; Mayer, 1938). The highly
basic proteins, protamines and histones, have received a major part of the attention. It has often
been stated that these are too simple to possess
the versatility of gene material, but thymus nucleohistone, for instance, has been reported to have a
molecular weight of over 2,000,000 (Carter and
Hall, 1939). However, even the meager analytical
data available point to the presence of other proteins in the nucleus. Huiscamp in 1905 obtained
from thymus a "nucleoprotein X," which he believed to come from the cytoplasm. I have prepared this material and found, first, that it is
strongly positive with the Feulgen reaction and,
second, that the combination with thymonucleic
acid is very stable. It seems likely that this is a
protein of nuclear origin. Mayer has isolated from
purified thymus nuclei a sulphur containing protein
with an isoelectric point of about five.
This is all that chemical analysis has produced.
The work does not even allocate the substances
found to chromosomes, much less to parts of chromosomes. For finer study we turn to optical methods
and histochemical methods. This paper represents
the results of a combination of digestion methods
with staining and ultraviolet microscopy.
The use of enzymes in histochemistry goes back to
Schleiden (1839). In the study of chromosomes,
nuclease was used by van Herwerden (1913) and
trypsin by Caspersson (1936). With our present
knowledge of enzyme specificity we can do much
more than test for the presence or absence of particular kinds of substances. Since enzymes are now
known to be specific for particular linkages (and
even for particular linkages only when in specific
relationship to other linkages) it is possible to
apply enzymes to the study of structures rather
than substances. By observing the effect of specific
enzymes on chromosome structure, we may evaluate the role of particular linkages in maintaining
that structure. We have here potentially the finest
of instruments for chemical microdissection.
Methods. The methods used are simple. Whole
cells may be immersed in the enzyme solutions. Recently, I have found it possible to apply enzyme solutions to isolated single Sciara salivary chromosomes. After incubation, the chromosomes are observed with appropriate techniques. For nucleic
acid studies, we used the Feulgen method and the
ultraviolet absorption technique. Since there is no
good color test for protein, although we have used
the ninhydrin reaction, we have worked most extensively on material in which chromosomes may
be most easily observed without staining. Sciara,
Drosophila and Chironomus have been used for
most kinds of experiments.
It has proved necessary to work at pH 5 or below
even with enzymes with an alkaline pH optimum.
In moderately alkaline solutions, the salivary chromosomes disperse into a fibrous mass. In strongly
alkaline solutions, the dispersion takes the form
described by Calvin, Kodani and Goldschmidt,
(1940).
Effects oJ proteolytic enzymes. Trypsin. Trypsin
completely digests and dissolves salivary chromosomes, as originally reported by Caspersson. The
same effect has been observed with onion root-tip
chromosomes and Tradescantia pollen tube chromosomes by my student, Miss Katharine Maneval.
Pepsin. Preliminary observations of the effect
of pepsin on Drosophila salivary chromosomes were
reported by Mazia and Jaeger (1939). The facts are
1) that the continuity of the chromosomes is not
destroyed by the digestion and 2) that the chromosomes are drastically reduced in volume after digestion. The shrinkage takes place very rapidly in a
0.1 percent solution of commercial pepsin in 0.2 percent HC1. There is no effect apparent on the Feulgen
staining of the chromosomes, except that it becomes
more compact. Ultraviolet photographs have not
yet been taken. The effect may be observed particularly well in isolated chromosomes held stretched
in an agar gel. Here one observes a great lateral
shrinkage, the longitudinal contraction being prevented. The shrinkage is greatest in achromatic
bands.
I have in the past interpreted this experiment as
indicating the presence in the chromosome of a
matrix, composed of a different protein from the
structural protein of the chromosome itself. This
matrix protein is digestible by pepsin whereas the
"skeletal" protein is not. The matrix protein occupies a considerable part of the volume of the
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E N Z Y M E STUDIES ON CHROMOSOMES
chromosome, and may account in part for the large
size of salivary chromosomes. This interpretation is
now confirmed by the ultraviolet absorption measurements of Caspersson (1940) who finds in the
achromatic bands a concentration of a "protein of a
globulin type," characterized by absorption around
wave length 2800 A. The pepsin indigestible "structural" protein would then be assumed to be a histone-like protein. The further evidence for this
will be given below.
If the salivary chromosome is characterized by a
high concentration of the "matrix" protein, it
might be expected that the action of pepsin on active
chromosomes might be smaller or absent. Miss Maneval has observed the effect of pepsin on onion roottip chromosomes. A considerable shrinkage of the
chromosome mass is evident, though details are difficult to make out in these preparations. Evidently
even here there is some matrix though less than
in the case of the salivaries.
Intracellular proteases. Papain. At the Gene Conference of 1940, the question was raised: why are
not chromosomes digested when exposed to the
enzymes of the cytoplasm? This question, of course,
applies to any cell structure. The answers have so
far not been satisfactory. In view of our growing
knowledge of intracellular proteases, especially due
to the work of the Bergmann laboratory, it is possible to attack this question.
In experiments with papain commercial papain
was used, with both cysteine and HCN as activators.
The conditions were those defined by Bergmann ~
and Fraenkel-Conrat (1937). Sciara chromosomes
only were used. No visible digestion was obtained
either with activated or inactivated solutions at
pH 5 for periods as long as 3 6 hours.
Tissue enzymes. Various tissues have been extracted and their proteolytic activity against chromosomes tested. The conditions were those defined
by Bergmann and Fraenkel-Conrat (1937). Cysteine was used as activator. The following tissues
were used: 1) beef liver, 2) pig liver, 3 ) beef
spleen, 4) frog liver, 5) frog kidney, 6) frog testis,
7) autolyzing yeast. The results were almost uniformly negative. In only one tissue was an enzyme
found which affected the protein of the Sciara chromosomes, and that was frog liver. The extract had
to be activated by cysteine. The fact that this activation is required indicates that we are dealing with
a typical intracellular protease, but why only the
enzyme of frog liver was effective will be explained
only when its activity against artificial substrates
will have been studied. This information might give
an important clue concerning the nature of the
linkages responsible for the chromosome structure.
Tumor enzyme. Since dividing cells should be actively carrying on synthesis and breakdown of
chromosome proteins (Caspersson, 1940), it seems
likely that one would find in such cells a protease
which affects chromosomes. Fruton, Irving, and
Borgmann (1940) have found active proteases in
41
tumor tissues. I tried a sample of Jensen rat sarcoma, which was extracted with phosphate buffer
at pH 4, activated with cysteine, and applied to
Sciara salivary chromosomes. It produced no striking effects on the chromosomes; the tumor enzyme
behaved as did the majority of intracellular enzymes.
Autolysis. Finally, an attempt was made to determine whether the salivary gland cells themselves
possessed a protease to which the chromosomes were
susceptible. Glands were placed under toluol for
seven days, then stained by the Feulgen method.
No digestion of chromosomes was observed to have
taken place. It will be interesting to see whether
glands of young larvae may not behave differently.
Also, we should expect to find an effective protease
in the pupa.
Protaminase. Well (1935) has described in some
detail a pancreatic enzyme which will split certain
linkages in protamines, but which cannot digest true
proteins. Since the presence of protamines in the nucleus is suspected, this enzyme has been applied to
chromosomes. Actually, I used a crude preparation
in which tryptic activity was inhibited by albumin
and phosphatase activity by means of phosphate.
After incubation, it was observed that the chromosomes were still present, but would not take a Feulgen stain. The effect is not due to phosphatase, nor
to ribonuclease, since the latter is heat stable, while
the active enzyme in my preparations is not. It
seems probable that the effect is due to protaminase
or to some unknown enzyme. Tests with protamine
substrate indicated considerable protaminase activity in the preparation.
If we are dealing with protaminase in this experiment, it would seem that protamine-like polypeptides are present in the chromosome and that the
nucleic acid component of the chromosome is attached to the protein through these polypeptides.
Not much can be made of this experiment until we
use a pure enzyme of known specificity. It is interesting, however, to find that an enzyme which apparently is not a nuclease can remove nucleic acid
from the chromosome without affecting the fundamental structure of the chromosome.
Interpretation and summary. In interpreting these
experiments, we must first inquire into the meaning
of "digestion." In a solution, digestion has taken
place when one chemical linkage has been split.
The "digestion" of a structure means something
else. It means that enough of the architecturally
important linkages have been split so the structure
is either radically altered or dissolved. If we are
interested in the molecular architecture of the system, our digestion method becomes a method for
evaluating the responsibility of particular bonds,
recognized by their susceptibility to enzymes of
known specificity, in the maintenance of the structure. For instance, it may be true, though it probably is not, that papain splits as many peptide
linkages in the chromosome as trypsin. But the
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42
DANIEL MAZ1A
bonds for which trypsin is specific may be those
which hold the structure together. Such a possibility
can be tested on a model system.
Since the simplest structural model for a chromosome is a nucleoprotein fiber, I have undertaken to
compare the digestion of chromosomes with the
digestion of a variety of artificial protein fibers. A
very simple technique, based on the findings of Derivichian (1939) and Langmuir was used to prepare
the fibers. Derivichian had found that a good surface
film of almost any protein could be made by dropping on the surface an aqueous solution containing
added surface-active material, such as amyl alcohol.
Langmuir has shown that a completely compressed
protein film becomes an elastic fiber or bundle of
fibers, which is very easy to handle.
All attempts to prepare a fiber of protamine failed,
as predicted by Wrinch (1936). Thymus histone and
thymus nucleohistone formed excellent fibers. Thymus "nucleoprotein X" formed good fibers. These
were compared with fibers of albumin and casein.
All of the fibers were digestible by trypsin. All but
histone and nucleohistone were digestible by pepsin. The behavior of histone mixed with other proteins, especially "nucleoprotein X" was studied.
The molecules were mixed in solution. When pepsin
was applied to the mixture, part of the material
of the fibers went into solution immediately, and
the rest contracted. In a few cases the contraction
threw the residual fibers into spiral form. The contraction following digestion of one constituent is,
of course, characteristic of the behavior of the
chromosome. But the perplexing problem is the origin of the continuity of the indigestible portion. One
possible interpretation, applicable to the chromosome, is that in the mixture we actually have a
chemical combination of the basic histone and the
more acidic protein. The film formed is, then, a
film of the histone "salt," and the fiber is a histone
fiber much inflated by the other protein. When the
latter is digested, the fiber contracts.
All of the other proteolytic enzymes used on
chromosomes have been applied to these fibers. It
was found, astoundingly, that no enzyme tried, other
than pepsin and trypsin, has any visible effect on
any of the artificial protein fibers. It would seem,
on the one hand, that fibrous organization alone
would explain the resistance to digestion of the important structures of the cell. It would seem, on the
other hand, that the enzymes of the digestive system represent a remarkable biochemical adaptation
to the needs of the organism.
If information on enzyme specificity may be extrapolated from artificial substrates to fibers and
chromosomes, the data may be interpreted. Since
trypsin digests chromosomes and all the fibers, and
since this splits peptide linkages neighboring free
basic groups, such linkages must be vital in holding
all protein fibers together. In the case of a basic
protein like histone, trypsin should be most effec-
tive, as it is. Pepsin, on the other hand, is active
against linkages between certain amino acids in the
neighborhood of free acidic groups. It would not be
expected to be effective against bistone, and it is
not. This is more direct evidence for the histone nature of the continuous structural protein of the
chromosome.
The inactivity of the intracellular proteases raises
interesting questions. Certainly these split many
bonds in a solution of a protein like albumin. Is
there some physical condition which prevents their
attack on the same bonds in a fiber? Or are these
bonds so placed in the fiber that the fiber continues
as such even though many peptide linkages are
opened? Are these conditions which do not seem
to favor digestion favorable to synthesis? This is
the most interesting question of all. If the enzyme
simply cannot attack the fiber, then, so far as the
enzyme is concerned, the concentration of protein
is always zero, and this, of course, would tend to
shift its action in favor of protein synthesis.
However, as Bergrnann (1939) points out, not
only the physical conditions, but also a slight difference in chemical configuration may determine
that an intraceltular protease shall act synthetically.
It might be that the linkages in the protein fiber
possess such characteristics, but in examining the
published data I have not found any substrates
which are synthesized by intracellular proteases
and digested by trypsin or pepsin.
The evidence for a continuous histone-like fibrous
structure in the chromosome seems fairly complete,
except for the unexplained case of the frog liver
intracellular enzyme which digests salivary chromosomes but not fibers. The existence of the matrix
of more acidic protein seems also probable. The
picture of the interrelations of these fits very well
the picture of Caspersson (1940). The evidence for
protamine-like molecules associated with the nuclei
acid is less convincing.
Chromosome proteins in plants. In the experiments on the digestion of plant chromosomes now
being carried on by Miss Maneval, the results obtained generally parallel our data on salivary chromosomes. It has, however, often been stated that
"nuclear" proteins, protamines and histones, have
not been isolated from plants. The nucleoproteins of
plants have, in fact, been neglected. Kiesel and
Belozerski (1934) found evidence for the presence
of both ribonucleoprotein and desoxyribonucleoprotein in bean seedlings, but do not classify the
protein. Belozerski (1939) has found that the onion
bulb contains no ribonucleic acid, but only desoxyribonucleic acid. If this is the case, this is suitable
material in which to seek nuclear nucleoproteins.
I have begun such an investigation, and, following
the techniques (Kossel, 1928) used on animal material have obtained a small amount of nudeoprotein. The material has not been analyzed quantitatively, but its qualitative reactions are as follows:
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E N Z Y M E STUDIES ON CHROMOSOMES
it gives a strong Feulgen reaction, a positive ninhydrin reaction, a positive Sakaguchi reaction for arginine, a weak Millon reaction.
Fibers have been prepared and subjected to digestion. Their behavior is histone-like; they are
digested by trypsin but unaffected by pepsin. The
ultraviolet absorption spectrum of a solution in
0.01 N NaOH has been determined. The expected
peak at 2650 A is absent, indicating a relatively
low concentration of nucleic acid in the protein. The
nucleic acid content may well have been reduced
in the process of purification by repeated precipitation in acid and resolution in alkali. The protein
absorption is more pronounced, and is shifted toward wave lengths longer than 2800 A. This shift
is found by Caspersson to be characteristic of proteins of the histone type. In fact, the curve for
the onion protein resembles closely some of Caspersson's curves for nucleohistone preparations.
There seems, therefore, no good reason for assuming
any great difference between the chromosome proteins of animals and plants.
Nucleic acid in chromosome structure. Since thymonucleic acid may form large elongated molecules (Signer, Caspersson, and Hammarsten, 1938),
nucleic acid has been assigned various roles in
maintaining the continuity and the apparent fibrous
structure of chromosomes (Wrinch, 1936; Gulick,
1938; Astbury and Bell, 1938). If chromosome
structure were in any way dependent on the nucleic
acid component, the structure should be destroyed
when the nucleic acid is removed. Miss Jaeger and
I succeeded in removing it by application of beef
spleen "nuclease" (van Herwerden, 1913). In
Drosophila, we found, after digestion, that the carmine staining material had been removed, but the
protein part of the chromosome, demonstrable by
the ninhydrin reaction, was still present in its characteristic form.
It has since been found that Sciara is much
more satisfactory material, since it is easy to observe the chromosomes even after they will no
longer stain. Some evidence of banding is preserved, indicating the expected difference in protein distribution in the different bands. Very recently, Mr. Roman and I have taken ultraviolet photographs of isolated chromosomes after nuclease digestion. There is no doubt that the form of the
chromosome is independent of its staining constituent.
Since the Feulgen reaction is a test only for the
desoxyribose constituent of thymonucleic acid, one
is not certain whether the rest of the molecule might
not remain. This can be tested by observations of
ultraviolet absorption. Mr. Hayashi and I have
taken photographs of Sciara material at wavelengths
2650 and 4380. In the visible, the chromosomes are
clearly made out both before and after nuclease
treatment. But in the ultraviolet, after nuclease
treatment, the chromosomes absorb no more strong-
43
ly than the cytoplasm and are hardly discernible.
It is clear that "nuclease" removes both the sugar
and the basic components of nucleic acid from the
chromosomes.
It is now possible to be more precise as to the
nature of the nuclease action. Nucleases are of three
classes: 1) polynucleotidases, which break down
highly polymerized polynucleotides to smaller units;
2) nucleotidases, which are phosphomonoesterases,
and split phosphoric acid from nucleotides; and 3)
nucleotidases, which separate the carbohydrate and
the basic components of nucleotides.
Since our "nuclease" removes both the base and
the sugar the point of action must be either the
linkage of sugar to phosphoric acid, or else some
linkage which connects the whole nucleotide to the
protein, which linkage might be either through
the phosphoric acid or through the base at the other
end. The former possibility may be tested, since
the properties of phosphatases are well known, and
enzymes like intestinal phosphatase actively dephosphorylate nucleotides. It is well known that phosphatase action is very reversible and is, therefore,
inhibited by inorganic phosphate. In most of our
experiments, we have used intestinal phosphatase
(beef) and studied its action in the presence and
absence of phosphate. Phosphate very effectively inhibits the splitting of the nucleic acid from the chromosomes. Therefore, it may be concluded that the
"nuclease" action we have been describing is a
phosphatase action, and that the nucleic acid is attached to the rest of the chromosome through its
phosphoric acid.
The study of phosphatase action on chromosomes
also gives some insight into the state of aggregation
of the nucleic acid in the chromosomes. Levene and
co-workers (1939) have found that the rate of dephosphorylation of thymonucleic acid by intestinal
phosphatase decreased as the molecular weight increased. They also found in the intestinal extract
an enzyme which they called a nucleodepolymerase,
which reduces large polynucleotide molecules to
smaller ones preparatory to dephosphorylation. The
depolymerase may be separated from the phosphatase by adsorption on aluminum hydroxide.
I have prepared intestinal phosphatase presumably free from the depolymerase in this way, and
find that it is still effective in removing nucleic acid
from salivary chromosomes. Although these experiments have not yet received adequate chemical control, the conclusion would be that the nucleic acid
is not present in the form of very high molecular
weight units.
Caspersson has been working on the same question, using dichroism in the ultraviolet as a measure
of the extent of orientation of nucleic acid molecules along the chromosome axis. He takes issue with
the conclusion of Schmidt (1939), and others, that
the birefringence observed in the visible is adequate
evidence of orientation in the living chromosome.
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DANIEL MAZIA
44
Caspersson considers this birefringence to be distinctly an artefact. ,Caspersson also points out that
his findings do not exclude the orienting effect of
nucleotide chains on polypeptide chains postulated
by Astbury and others, since units as small as molecular weight 20,000 would be adequate for this.
THE
ENZYMES
OF C H R O M O S O M E S
The nucleus has often been postulated to be the
center of the metabolic activity of the cell. So
far as energy turnover is concerned, all the evidence
on this point has been negative (Brachet, 1938).
The nucleus seems to be occupied with the process
of reproduction. We should like to study the reproduction of "phenotypic" genes, but this we
cannot do. However, everything in the chromosome is reproduced including such easily identifiable constituents as nucleic acid. The reproduction of nucleic acid, in the process by which a
chromosome makes more chromosome, may be studied by means at o u r disposal.
Where the reproduction is rapid, the problem of
raw material is important. Koltzoff (1938), Painter
(1940), and Brachet (1940) have suggested, in the
case of the egg, that the raw materials are formed
during oogenesis and stored in the cytosome as ribonucleic acid or its constituents. Caspersson and
Schultz (1938), Brachet (1936) have found evidence of such storage not only in eggs, but also in
the cytoplasm of other rapidly dividing tissues.
Recently, Dr. Ballantine and I have begun to
study the enzymes that may be involved in the
synthesis of new chromosome nucleic acid. The
material I am about to report represents only a
brief summer of exploratory work, and necessarily
has many gaps.
We have studied two of the enzymes that might
by involved in the synthesis of nucleic acid in the
chromosome. First, v~e have studied phosphatase or
nucleotidase. Secondly, we have studied polynucleotidase activity.
Our material was the eggs of Arbacia. For preparation of nucleated and enucleated fragments we
used the centrifuge technique (Harvey, 1940). It
TABLE i. EF~FECTOF FERTILIZATION ON ACTIVITY 01~
ARBACIA PIIOSPHATASE
rag. P/cc. eggs/hr.
Experiment
6/23
6/16
pH
7.0
4.3
Unfertilized
Fertil~ed
0.625
1.27
0.635
1.01
was found that whole Arbacia eggs possessed a high
phosphomonoesterase activity, dephosphorylating
both glycerophosphate and depolymerized nucleic
acid. The phosphata~e is remarkable for its very
acid pH optimum. In fact, we did not go low
enough on the pH scale to find an optimum.
TABLE 2. I~HOSPHATASE ACTIVITY OF ARBACIA
EGO FRAGMENTS
Substrate: 0.5 percent sodium glycerophosphate
pH: 5.3
Temperature: 25~
Cell
nag. P/cc. eggs/hr.
Whole
Nucleated half
Enucleated half
1.93
2.10
3.30
The problem of localization was attacked in two
ways. First, fertilized eggs (having a diploid chromosome number) were compared with unfertilized.
Secondly, the phosphatase activity of nucleated
and enucleated egg fragments was compared. Tables
1 and 2 summarize these two experiments. Fertilized
and unfertilized eggs possess the same activity, and
there is no indication that nucleated fragments contain a store of the enzyme. If nucleotides are synthesized or broken down in the nucleic acid metabolism
of these cells, the process takes place in the cytopIasm.
If the chromosome contains nucleic acid of any
degree of polymerization, there should be present
in the cell a polynucleotidase. The presence of such
an enzyme may be measured in two ways. First,
indirectly, by its effect on the rate of dephosphorylation of highly polymerized nucleic acid. We have
found that Arbacia material acts on nucleic acid
which has not previously been depolymerized, indicating the presence of the depolymerase. Secondly, depolymerization leads to a decrease in the
viscosity of nucleic acid solutions. Such experiments
require careful control since the viscosity of these
solutions is sensitive to external factors. We used the
far from ideal Hess method of viscosity determination, in order to work with small amounts of
material. Since care was taken to maintain a constant pressure head, thixotrophy was not a variable.
It was first found that the whole Arbacia egg does
contain an active depolymeryzing enzyme. This is
not a phosphatase since it is not affected by phosphate and works at alkaline pH values at which
Arbacia phosphatase is inactive. We have not yet
determined the complete activity-pH curve of the
polynucleotidase.
Table 3 shows the effect of fertilization on the
polynueleotidase activity of Arbacia eggs. It is clear
that increase in chromosome number is accompanied by increase in enzyme activity, though the
experiment does not establish a causal relation. A
more conclusive experiment is given in Table 4,
in which the activities of nucleated and non-nucleated fragments are compared. The answer here is
very clear cut, since the non-nucleated fragments
show no activity whatsoever, while the nucleated
fragments are active.
In order specifically to attribute the activity to
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E N Z Y M E STUDIES ON CHROMOSOMES
the chromosomes, we plan to separate the fragments
after the nuclear membrane has broken down, following E. B. Harvey (1940). That the activity is
in the chromosomes is strongly suggested by the
effect of fertilization, since here little "nuclear
TABLE 3. EFFECT OlVFERTILIZATIONON"ACTIVITYOF .
ARBACIA POLYNUCLEOTIDASE
Substrate: 5 percent sodium thymonucteate
pH: 9.0
Temperature: 25 ~
Viscosity change. Centipoise/cc/hr.
Experiment
Unfertil~ed
Fertil~ed
0.29
0.26
0.31
0.73
0.69
0.70
8/15
8/21
8/23
TABLE 4. POLYNUCLEOTIDASEACTIVITYOF ARBACIA
EGG FRAGMENTS
Substrate: 5 percent sodium thymonucleate
pH: 9.0
Temperature: 25~
Viscosity change, centipoise/cc./hr.
45
does the amount of thymonucleic acid (Brachet,
1933). Since our polynucleotidase is in the chromosomes, it must presumably reproduce, unless it is
stored in sufficient quantity at the beginning of development. With such ideal material as sea urchin
eggs, with which one may obtain any amount of
cells all approximately in the same phase of mitosis,
one may follow the exact course of the presumed
reproduction. I have carried out some experiments
along this line, but the results have not been uniform. In a number of cases there has seemed to be a
sharp increase in enzyme activity during early
prophase. However, certain problems of technique
must be solved in carrying this work further. It is
not going to be easy to assure adequate contact between such a localized enzyme and an added substrate. The rise during prophase might merely indicate the greater ease of contact between chromosome and nucleic acid after breakdown of the nuclear membrane. I mention this project at some
length only because it seems to promise such a
direct attack on a fundamental problem.
SUMMARY
Experiment
Whole
egg
Enucleated
half
Nucleated
half
8/13
(unfertilized)
--0.37
0.00
--0.84
8/17
(unfertilized)
--0.19
0.00
--0.476
8/19
-0.51
-0.14
-1.79
sap" is probably introduced with the sperm cell.
However, activity in the nuclear membrane certainly
can not be excluded.
Control experiments were carried out with heated
egg extracts. The enzyme is heat-labile, which
would distinguish it from the ribonuclease of Dubos
(1938) and Kunitz (1940).
This experiment seems strongly to support the
ideas of those who consider that much of the preparatory work in the formation of chromosomal nucleic acid takes place outside the chromosome. In
fact, it would seem that the chromosome itself, if
we can assume the reversibility of the polynucleotidase, begins with fully prepared nucleotides or
even small polynucleotides, and organizes them into
the more complex and presumably more specific
polynucleotides that seem so important in gene
reproduction.
An enzyme which is localized in a chromosome,
and which is active in the process of chromosome
reproduction, must itself reproduce! In a system
like the Arbacia embryo the mitotic rate does not
fall off during early development, and the amount
of chromosome increases at an accelerating rate as
No attempt has been made to draw a diagram
of the molecular organization of a chromosome.
Possibly such a diagram will be a contribution of
this Symposium. But the following facts have been
brought out.
1) The salivary chromosome, and, very likely, the
plant chromosome, seems to be composed of a continuous framework and a matrix which occupies a
considerable volume.
2) The matrix seems to be composed of protein
containing many acidic groups.
3) The continuous skeleton seems to be composed of a histone-like protein.
4) It is possible that the chromatic bands also
contain a protamine-like substance to which the nucleic acid is attached.
S) The behavior of the chromosome toward enzymes parallels the behavior of artificial nucleoprotein fibers, suggesting a fibrous organization for the
chromosome "skeleton."
6) Proteins organized into fibres are not visibly
digested by intracellular proteases. It is suggested
that the system is favorable to protein synthesis.
7) A nucleoprotein whose properties resemble
those of histone has been separated from plant material.
8) Nucleic acid is attached to the protein part of
the chromosome through it s phosphoric acid residues,
9) Removal of nucleic acid does not affect the
continuity of the chromosome.
10) The nucleic acid of the salivary chromosome
is probably not in a highly polymerized form.
11) In Arbacia eggs, an active phosphatase (nu-
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46
DANIEL MAZIA
cleotidase) has been found. It is not localized in the
nucleus.
12) Arbacia polynucleotidase is strictly localized
in the nucleus. The sperm nucleus brings a portion
of this enzyme with it when fertilization takes place.
13) An investigation of the reproduction of the
polynucleotidase has been begun.
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DISCUSSION
GREENSTEIN: The resistance of native protein
fibers to enzymes is illustrated by experiments on
wool.
MAZlA: IS this case not usually attributed to the
abundance of sulphur as disulphide?
MELAMPY: Wool fiber is digested by the clothes
moth larva.
HOLLAENDER: Does what Mazia has said about
the low polymerization of nucleic acid apply only
to the salivaries?
MAZIA: Yes, I have worked only with the sailvaries and could not generalize.
SCHULTZ: I wonder whether Caspersson's argument for birefringence as an artefact could not
apply even to sperm, since in sperm there is so much
nucleic acid in so little space.
GREENSTEIN: The degree of polymerization in nucleic acid depends on the solvent and is at a maximum in water. In sperm, a lower concentration of
protein is present, and hence a higher degree of
polymerization may be assumed to be present than
in chromosomes where the proportion of protein
is greater.
CHILD: Have you measured the polynucleotidase
activity of sperm?
MAZIA: Not successfully. The problem of breaking down sperm was too difficult.
MIRSKY: I was interested to hear that the fibers
formed by rolling up films of nucleohistone and egg
albumin are not digested by cathepsin. Some native
proteins are either not digested at all or digested
very slowly by certain proteolytic enzymes whereas
the same proteins are rapidly digested when they
are denatured. It has been suggested that denaturation renders a protein digestible because, as a result
of the unfolding process t h a t takes place during
denaturation, some peptide bonds which were inaccessible to the enzyme in the folded (or native) state
become accessible. When a protein film is folded up
to form a fiber, certain peptide bonds may become inaccessible to cathepsin much as they are in
a native protein. This possibility can be examined
experimentally by removing a portion of the nucleohistone film with a wire loop and placing it while
still unfolded in a solution of cathepsin.
MAZlA: Histones when prepared as fibers are not
soluble, but it is difficult to separate the nucleic acid
entirely and therefore to be sure that we have
unconjugated histone. No protein is soluble if prepared as fibers in the way I described.
SCHULTZ: The rate of cell division decreases as
division proceeds; this can be interpreted through
Brachet's data as related to using up the ribose nucleic acids in the cytoplasm.
MAZIA: If the decrease in the rate of division
meant that the enzyme were "diluted" in the course
of development, this would invalidate the assumption that it reproduces itself.
SCHULTZ: More likely the substrate is being used
up.
MULLER: I doubt that sperm bodily brings in the
enzyme but suggest that it brings in the possibility
of enzyme production.
MAZlA: As said above, I have not been able to
study sperm directly. Only by doing this could one
test Muller's suggestion directly.
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ENZYME STUDIES ON CHROMOSOMES
Daniel Mazia
Cold Spring Harb Symp Quant Biol 1941 9: 40-46
Access the most recent version at doi:10.1101/SQB.1941.009.01.006
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