VOL.

53, 1965 GENETICS: BEISSON AND SONNEBORN 275

Y1,Y2,. . . be a sequence of independent two-valued random variables, Yn+i = -s

or f3 + ns with probabilities (1 - W) and W, where s is a small positive number
and W is then determined by the condition -E(Y.) = aV(Yn). Verify that
the probability that for some n, - 3 + Y, + . . . + Yn > 0 converges to 1/(1 +
ad) as s -. 0, and let X,, = Yn - E(Yn). This completes the proof.
The theorem can be extended to say that for each y > 0, if rT is the least n if
any for which (Xi + . . . + Xn) . -yT +GI + . . . + An) + .(V + * * .Vn).
* +
then the probability that there is some n < rz for which (1) holds is less than
(,y/(( + 3))(1/(1 + ac)); and this bound is sharp.
The material of this note, including proofs of Lemmas 1 and 2, will appear as
part of our forthcoming book, How to Gamble If You Must (New York: McGraw-
Hill), Theorems 2.12.1 and 9.4.1, and an illustrative application of the theorem
will appear in the forthcoming article, "A sharper form of the Borel-Cantelli lemma
and the strong law" by L. E. Dubins and D. A. Freedman.
* Partially supported by NSF grant GP-2593 (to L. E. Dubins).
Doob, J. L., Stochastic Processes (New York: John Wiley and Sons, Inc., 1953).

CYTOPLASMIC INHERITANCE OF THE ORGANIZATION OF THE

CELL CORTEX IN PARAMECIUM A URELIA*
BY JANINE BEISSONt AND T. M. SONNEBORN
DEPARTMENT OF ZOOLOGY, INDIANA UNIVERSITY

Communicated December 14, 1964

The cortex of Paramecium aurelia exhibits a high degree of structural differentia-
tion (Fig. 1). At each level of organization observable within the limits of resolu-
tion of the optical microscope, the cortical pattern is remarkably constant and re-
produces faithfully through a regular cycle of changes during growth and fissions.
However, this highly stable organization can be experimentally modified. Sonne-
born' obtained several variants of the normal pattern by fusion of mates after con-
jugation, by partial loss of supernumerary structures, and by "cortical picking," an
accident of separation after conjugation in which one of the mates acquires a piece
of its partner. Sonneborn' showed, by all available methods of genetic analysis,
that these abnormal patterns behaved like "cortical mutations." Each variation
proved to be hereditary although the variants were genotypically identical to wild
type, as though the existing pattern of cortical organization itself determined the
pattern that arose during subsequent reproduction. This ordering of new by old
cell structures, which has been called "macrocrystallinity"2 or "cytotaxis,"3 might
be an important, although still little recognized, mechanism in cell heredity. We
therefore undertook further experiments and extended the analysis to more ele-
mentary levels of structural organization.
The analysis exploited various grafts of a piece of one cell on a whole cell, using
stock 51 (syngen 4) of P. aurelia. All of these arose from conjugating pairs which
remained united by a cytoplasmic bridge instead of separating immediately after
conjugation. Some pairs eventually separated spontaneously in such a way that a
276 GENETICS: BEISSON AND SONNEBORN PROC. N. A. S.

part of one conjugant went to the other. In other pairs most of one conjugant was
cut off with a micromanipulator, the residual part persisting as a graft on the other.
The progeny of animals bearing such spontaneous and experimentally produced
grafts were observed from fission to fission in order to follow the development of the
grafted piece. The methods of culture and handling of the organism have been
described elsewhere.4 For detailed study of the cortical geography, specimens were
prepared by a modification of the silver nitrate impregnation technique' and pieces
of isolated cortex by a simplification of the digitonin technique.6 Each method
revealed details of organization not shown by the other.
Determination at the Level of the Repeating Unit of Cortical Structure.-Origin and
nature of the mutant "twisty": This mutant was derived from an exconjugant that
received a piece of its mate during spontaneous breakage of the conjugation bridge.
The piece formed a little tail near the posterior end on the ventral side. Because of
its posterior position, it passed to successive opisthes (posterior products of trans-
verse fission), but became less and less obvious, disappearing by the fourth fission.
Nevertheless, the clone derived from the fourth successive opisthe displayed ab-
normal swimming, designated as "twisty." This gives the impression of marked
twisting during progression forward, but is merely an exaggeration of normal spiral
progression.
Twisty swimming has a structural basis which appears from a comparison of
normal (Fig. 1) with twisty (Fig. 2) cells. Unlike the regular spacing of the rows of
cortical granules in normal cells (Fig. 1A,B), cells of the twisty clone (Fig. 2) show
a patch of four rows bounded on one side by an unusually wide space and, on the
other side, by an unusually narrow space. The intervening rows consist of cortical
units with reversed polarities: instead of the normal polarity described in the
Figure 1, legend the kinetosomes lie to the left (instead of the right) of the unit mid-
line, the parasomal sacs are to the left (instead of the right) of the kinetosomes, and
the kinetodesmal fibers (see Figs. 1C and 3) emerge to the left (instead of the right)
of the kinetosomes and extend posteriorly (instead of anteriorly). This reversal
of both antero-posterior and right-left polarities will be symbolized henceforth by
RP; it is equivalent to 1800 rotation in the plane of the body surface. Such rows
of RP units constitute the first reported exception to the rule of desmodexy,7 the
rule that kinetodesmal fibers in Ciliates always lie to the right of the kinetosomes.
This exception explains the abnormally wide and narrow spaces at the edges of the
patch of RP rows in the twisty clone (Fig. 2, rpr). When a row of normal units has
a row of RP units on its left, the larger clear areas of units in both rows are adjacent
and the space therefore appears unusually wide. Conversely, when an RP row has
a normal row on its left, the visible structures of both rows are brought into juxta-
position, leaving hardly any clear space between them.
Experimental production of cells with rows of RP cortical units: The simplest
hypothesis as to the origin of the twisty clone is that the tail of the ancestral excon-
jugant included a piece of cortex of its mate which became assimilated in reversed
orientation. This assumption is reasonable, for exconjugant pairs held together
by a cytoplasmic bridge often twist into a settled heteropolar position (Fig. 4A).
Moreover, it can be verified experimentally. By cutting off most of one cell from
a heteropolar pair of exconjugants, one can observe that the remaining fragment
becomes grafted to the intact cell and that the progeny include twisty sublines of
 VOL. 53, 1965 GENETICS: BEISSON AND SONNEBORN 277

descent. But the easiest way to obtain clones of cells with complete rows of RP
cortical units was the following.
Pairs of conjugants united in heteropolar position (Fig. 4A) were isolated. Such
pairs grow and divide and at each fission (Fig. 4B, C, D) give off a proter from each
end while, in the central heteropolar double opisthe, the zone of union extends
progressively. By about the third fission (Fig. 4D) the opisthes are fused up to or
beyond the vestibular region, so that the fission furrows cut through a mixed cortex.
At this stage each newly arising proter has most of its cortex derived from its "own"
parent cell, but contains a little heteropolar bit in its posterior end. If such a cell
is cultured, the normal process of cortical growth associated with fission brings
about, in successive opisthes, the extension forward of the "grafted" rows. In the
third opisthe they extend all the way to the anterior pole. All the clones derived
from isolation of the third opisthe of third proters given off by a heteropolar pair
displayed the twisty swimming behavior. Each clone was characterized by a dis-
tinctive number (up to 12) of rows of RP cortical units inserted as a single patch or
as two or three patches separated by a few normal rows.8
Inheritance of rows of RP cortical units: The original twisty, as well as the many
twisty clones obtained by the procedure just described, reproduced true to type
through hundreds of fissions, dozens of autogamies, and conjugations with normals,
with or without endoplasmic exchanges. Occasionally one or more RP rows could
be lost from a particular subline of descent.
This persistence of rows of RP cortical units can be understood from Dippell's9
analysis of production of new cortical units. She has shown that new structures
(kinetosomes, parasomal sacs, and kinetodesmal fibers) arise within each "old"
unit in locations and with polarities that are fixed in relation to the "old" com-
ponents of the unit. For example, new kinetodesmal fibers grow out from kineto-
somes on the same side and in the same direction as the old ones, and comparable
definite spatial relationships are shown by new and old kinetosomes and parasomal
sacs. Then, longitudinal growth of cortical membranes and appearance of trans-
verse partitions yield identically organized units from each old one. In this way
the units of a row are traceable to a common ancestral unit, the orientation of which
is always perpetuated by the same mechanism, regardless of the orientation of the
unit and of the polarity of the cell.
Determination at the Level of Differentiated Fields of Cortical Rows.-The question
may now be asked: what determines the path followed by a row of cortical units
and (or) what determines the differentiation of groups of rows into characteristic
patterns (Fig. 1A, B): circumoral and vestibular fields around the ingestatory
apparatus, right fields of rows running from anterior suture to posterior pole, left
fields running from anterior to posterior sutures, and dorsal field extending from
pole to pole?
First, as became evident from further observation of the twisty clones, the paths
followed by the rows are not intrinsic properties of any of them. All RP rows were
initially located close to the suture line on the right side. In the course of hundreds
of fissions, they were observed to be located progressively further to the right,
eventually reaching the left side of the cell. The mechanism of this shift relative
to the oral meridian is not yet understood, but it is clearly not peculiar to RP rows,
for the normal rows interspersed among them accompany them. As such groups
278 GENETICS: REISSON AND SONNEBORN Puoc. N. A. S.

r~~~~~~~~

A
ts*X f DcXr
rxy aHXJpr Pk r
v B b 4 A

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~.,'.mr
~ ~ ~ . * Fe

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A-~ ~ ~
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~~~i-
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at
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DO D / pr3l

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oNo
*s * C1 clone o
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oonost
3l incnpom
F te
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i;
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SZ
e sdor rblet
 VOL. 53, 1965 GENETICS: BEISSON AND SONNEBORN 279

of normal and RP rows are followed into different fields, they fit the path char-
acteristic of the region in which they come to lie. These paths are therefore some-
how imposed on the rows by their location or their "cortical environment." This
will now be demonstrated by a second type of observation.
In a pair of exconjugants united by a cytoplasmic bridge, one exconjugant was
cut (Fig. 5A, B) so as to remove roughly the anterior 2/3 of the cell including the
whole ingestatory apparatus (vestibule, mouth, and gullet). What remains of the
posterior left field (Fig. 5B, 12) of the amputated cell (represented in black) is fused
to the posterior right field (ri) of the host. Conversely, 1800 away (on the side not
visible in the figure), what remains of the posterior right field of the amputated cell
is fused to the host's posterior left field. The host's vestibule lies anterior to the
graft and is surrounded on both sides by only host circumoral material. Figure
5C, D, E shows the development of the graft during the first three fissions. In the
first two successive opisthes (ol, 2), the cortical growth accompanying fission en-
larged the graft forward to the vestibular region. Then by the third fission (Fig.
5E) the "daughter" vestibule, which goes to the opisthe, came to lie at the juncture
of the right field (ri) of the host with the left field (12) of the graft. At the other
juncture (1800 away) between host and graft cortex, the whole ingestatory appara-
tus being absent, no circumoral or vestibular fields developed; but at both junctures
the other features (anterior and posterior sutures and cytopyge, see Fig. iB) of the
FIG. 1.-Normal cortical geography of Paramecium aurelia. (A) Dorsal view, and (B) ventral
view, of a silver nitrate impregnated cell. Magnification: X 416. A cell's right and left sides
and anterior and posterior ends are defined in relation to the major landmarks of the ventral
aspect. "To the right" means clockwise around the body from the anterior (as) and posterior
(ps) sutures in (B) when facing the anterior end (top of fig.); the right and left sides [r and t in
(A) and (B)] are to the animal's right and left of the sutures, respectively. (C) Diagrammatic
enlargement of the unit of cortical structure, including one or two kinetosomes (k) or ciliary basal
bodies, slightly to the right of the unit midline, one parasomal sac (psc) to the right of the kineto-
some and anterior to it (or to the posterior kinetosome in units possessing two), and one kineto-
desmal fiber (kf) emerging to the right and extending anteriorly (a) from the kinetosome (or from
the posterior one when two are present). The 1 or 2 kinetosomes and parasomal sac of one unit,
lying close together, appear as a single granule (silver deposit) in photographs (A) and (B), but
they are partially resolvable (see Fig. 2). Kinetodesmal fibers show only in digitonin preparations
(see FIG. 3).
a, anterior. as, anterior suture. c, cytopyge or cell anus. cvp, pores of the contractile vacuoles. k,
kinetosome. kf, kinetodesmal fibre. 1, left. p, posterior. ps, posterior suture. psc, parasomal sac. r,
right. v, vestibule leading inward to mouth and gullet.
FIG. 2.-Part of dorsal surface of a silver impregnated typical cell of the twisty clone after about
300 cell generations. Magnification: X 1800. In the four rows with reversed polarities (rpr),
the compound granules "point" to the observer's right, while in the normal rows (nr) they point
to the observer's left (i.e., animal's right).
FIG. 3.-Digitonin preparation of a cell from a clone possessing a single row with reversed
polarities (rpr). The kinetodesmal fibers in this row, unlike those in the normal rows (nr), emerge
to the animal's left (1) and extend posteriorly (toward bottom of figure). Magnification: X
1800.
FIG. 4.-Origin of cells possessing some rows of cortical units with reversed polarities. (A)
Pair of exconjugants united in heteropolar position; (B), (C), and (D), first, second, and third
fissions of the heteronolar doublet cell. prl and prl', pr2 and pr2', pr3 and pr3', are the three pairs of
proters (anterior fission products) given off from the two anterior ends (a) of the heteropolar
doublet at its first three fissions. Small ovals are the vestibules leading inward to the mouth and
gullet. In the diagrams [(A), (B), and (D)], one row of cortical units on either side of the juncture
between the two fused cells is represented by a row of triangles pointing in opposite directions.
This shows the opposite polarity of the units, the points of the triangles representing the position
of the parasomal sacs (see Figs. 1C and 2). The photograph (C) shows that all rows beyond the
juncture are oriented like the row nearest the juncture. Note that at the third fission (D), the
proters (pr3 and pr3') receive a mixed cortex including oppositely oriented rows on both sides of the
juncture line.
FIG. 5.-Origin of clone of incomplete doublets (lacking one ingestatory apparatus) by excision
of part of one conjugant of a homopolar fused pair. For description, see text.
280 GENETICS: BEISSON AND SONNEBORN PROC. N. A. S.

midventral meridian were present. Between the two midventral meridians, on

both sides, was a "back" with its two contractile vacuole pores (as in Fig. 1A).
This incompletely double organization (lacking one ingestatory apparatus), which
constitutes another type of cortical variation and has been previously described,'
characterized the clone descended from the third posterior fission product (Fig. 5,
o3) of the grafted cell.
The significant features of this development are (1) that the piece of grafted left
field (12) has acquired the vestibular and circumoral differentiations when it be-
came associated with the host's ingestatory apparatus, and (2) that the left field of
the host, originally possessing the circumoral and vestibular patterns, lost these
differentiations when it developed 1800 away in the absence of an ingestatory
apparatus, along the right field of the graft.
In more general terms, the presence or absence of a given cortical organization
(ingestatory apparatus in this case) determines whether other characteristic pat-
terns (vestibular and circumoral fields in this case) will differentiate during growth
of adjacent or nearby regions. This confirms the conclusion already drawn from
the behavior of rows of RP cortical units: the cortical environment determines the
path or form the rows take. Comparable observations, not reported here, on the
differentiation of the cytopyge and the ingestatory apparatus show that changes in
the surrounding cortical organization result in production of abnormal structures
or in failure to form them altogether. In agreement, definite cortical requirements
for oral differentiation have also been reported for Stentor'0 and other Ciliates.
Discussion.-The major conclusion from our results is that, at each studied level,
the organization, i.e., presence, orientation, and location, of newly formed cortical
structures is determined by the cortical environment existing at the time of their
development. This raises the question of the mechanisms of perpetuation of both
normal and variant cortical organizations and of the role of genes in this perpetua-
tion.
The variations reported here are perpetuated during sexual and asexual reproduc-
tion in the direct cortical line of descent. As set forth previously, the variations
persist in the absence of any other known differences between "mutant" and normal
cells. The absence of genic and nuclear differences has been demonstrated by the
genic and nuclear identities of host and graft and by breeding experiments. The
absence of endoplasmic differences has been shown again by endoplasmic identity
of host cell and donor cell and, in some cases, by exchange of endoplasm between
normal and variants during conjugation. This, of course, does not mean that genes
do not play any role in the cortical organization. They are, in all probability,
necessary for production of the specific molecules that make the structures, and
gene mutations are known which result in altered cortical organization. Never-
theless, the coexistence, on the same cell, of a normal oral meridian and of a deficient
one lacking circumoral differentiations, and of normal and RP rows of cortical
units, could hardly be accounted for by any noncortical difference, even by dif-
ferences in local concentrations of genic products since the RP rows, for instance,
persist as such as they "move" around the cell.
The mechanism of perpetuation of cortical variations is less reliable than genic
mechanisms. Irreversible losses of parts and eventually of all of the variant pat-
tern may occur with a higher order of frequency than mutations or losses of a spe-
VOL. 53, 1965 GENETICS: BEISSON AND SONNEBORN 281

cific gene. Nevertheless, certain variants, such as complete doublets and cells with
rows of RP cortical units, can apparently be maintained indefinitely with periodic
selection. Even those which revert 100 per cent to normal and do so rapidly (for
example, incomplete doublets that revert after 30-40 cell generations) transmit the
variant organization to a remarkably large number of progeny cells (109 to 1012 in
the example cited). Hence, even imperfections of transmission do not greatly
lessen the problem of accounting for the impressive degree of transmission that
does occur in the less persistent types.
How then can the hereditary maintenance of cortical variations in the absence
of relevant nuclear or endoplasmic differences be understood? Would the assump-
tion that the cortical structures themselves, or any components of them, replicate
resolve the problem? The question is most pertinent in regard to the kinetosomes
which have been thought to be both self-reproducing and directive or instrumental
in morphogenesis." Recent evidences, at the electron microscope level, still leave
the matter unsolved. However, even if they reproduced themselves or even if they
contained DNA, this would not explain the hereditary maintenance of our cortical
variations. These variations are not changes in the kinetosomes or associated
structures per se. They are variations in spatial relationships among elementary
structures, which themselves appear to be structurally unaltered.
The maintenance of normal or variant patterns of organization can be understood
simply in terms of existing structures and the restraints they impose on the develop-
ment and positioning of new structures. For example, grafted segments of rows of
cortical units can be integrated only by being sandwiched between the existing
rows. They are forced to grow parallel to them and their only degree of freedom
is to have normal or reversed polarities. Once a row is integrated, no degree of
freedom is left: as Dippell has shown,9 new parts, as they arise, are positioned
within, and in definite relations to, those of the existing unit.
We have no evidence concerning the molecular nature of such structural con-
straints or interactions between structures or groups of structures, but it may be
worth calling attention to the absence of any necessary theoretical conflict between
our results and the implications of the "self-assembly" hypothesis which ascribes
the formation of organelles and subcellular structures solely to the properties of
their constituent molecules. Our results merely underline a missing and probably
decisive component in the system of molecular interactions which should have been
recognized a priori, namely, the existing organization that constitutes the milieu
where other subcellular organelles and structures are assembled. Newly formed
molecules do not enter a vacuum, but a structured cell, the molecules of which are
an essential part of the determinism for locating, orienting, and patterning new
molecular formations. In this perspective it is conceivable that any subcellular
structure might be affected, in the course of its formation, by changes in physical,
chemical, or organizational properties of the surrounding intracellular milieu.
Some changes may not allow the new structure to be formed at all; but with some
probability they may induce a structural alteration in it. A modified structure
might in turn, by itself constituting a change in the milieu, no longer permit the
formation of the normally patterned structure, but permit reproduction of only the
modified form. Such interactions at the molecular level might then result in hered-
itary extragenic variations.
282 GENETICS: ALEXANDER AND GLANGES PROC. N. A. S.

Whatever the case may be concerning the possible mechanisms of cytoplasmic

mutations, our observations on the role of existing structural patterns in the deter-
mination of new ones in the cortex of P. aurelia should at least focus attention on
the informational potential of existing structures and stimulate explorations, at
every level, of the developmental and genetic roles of cytoplasmic organization.
Summary.--Pieces of cortex of Paramecium can be grafted onto a whole cell and
become integrated, yielding a modified cortical pattern which is maintained through
both sexual and asexual reproduction. Two types of modified cortical patterns
were studied: presence of some rows of cortical units with reversed polarities and
presence or absence of whole fields of rows of cortical units. An analysis of the
development and maintenance of the modified patterns shows that, both at the
level of fields of rows and at the level of the cortical unit, the organization (presence,
location, orientation, and shape) of newly formed structures is determined by the
cortical environment existing at the time of their development.
*
Contribution 756 from the Department of Zoology, Indiana University. Aided by grants
from the Centre National de la Recherche Scientifique and from the Delegation Gendrale A la
Recherche Scientifique et Technique, and by grant COO-235-13 of the Atomic Energy Commis-
sion to T. M. Sonneborn. The authors are very much indebted to Dr. Ruth V. Dippell for sharing
much of the experimental work with the twisty cultures and for invaluable discussions of every
aspect of the work.
t Present address: Laboratoire de Biologie Experimentale, Faculte des Sciences, Orsay (Seine-
et-Oise) France.
I Sonneborn, T. M., The Nature of Biological Diversity, ed. J. M. Allen (New York: McGraw-

Hill Book Co., 1963), p. 165.

2 Weiss, P., The Molecular Control of Cellular Activity, ed. J. M. Allen (New York: McGraw-
Hill Book Co., 1962), p. 1.
3Sonneborn, T. M., these PROCEEDINGS, 51, 915 (1964).
4 Sonneborn, T. M., J. Exptl. Zool., 113, 87 (1950).
5 Chatton, E., and A. Lwoff, Compt. Rend. Soc. Biol., 104, 834 (1930).
6 Child, F., and D. Mazia, Experientia, 12, 161 (1956).
7 Chatton, E., and A. Lwoff, op. cit., 118, 1068 (1935).
8 The possession of rows of RP cortical units is doubtless the basis of the twisty swimming be-
havior, but the relation of the orientation of cortical units to the direction of the ciliary beat is still
obscure.
9 Dippell, Ruth V., Excerpta Medica, Intern. Congr. Ser. 77, 16 (1964).
10 Tartar, V., J. Exptl. Zool., 144, 187 (1960).
1 Lwoff, A., Problems of Morphogenesis in Ciliates (New York: John Wiley & Sons, Inc., 1950).

GENETIC DAMAGE INDUCED BY ETHYLENIMINE*

BY MARY L. ALEXANDER AND EVALEA GLANGESt
THE GENETICS FOUNDATION, DEPARTMENT OF ZOOLOGY, UNIVERSITY OF TEXAS
Communicated by Wilson S. Stone, December 15, 1964
The induction of genetic changes by chemical agents was established by Auerbach
and Robson' using mustard gas treatment of Drosophila. Before that time, in-
dications of the mutagenic potentialities of chemicals had been obtained in other
biological forms, especially plant material.2 Further testing with mustard gas re-