Table Of ContentGENETIC EFFECTS OF METHYL BISlffij-DICHLORGETHYL)
ass**1'
MINE IN PARAMECIUM AURELIA. VARIETY 4
by
Robert P. Geckler
asp
Submitted to the Faculty of the
Graduate School in partial fulfillment of
the requirements for the degree
Doctor of Philosophy
in the
'Department of Zoology
Indiana University
1949
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ACKNOWLEDGMENT
The author wishes to acknowledge the many
helpful suggestions and kindly criticisms
offered throughout this investigation by
Dr. T. M. Sonneborn and the group of Para
mecium workers present during the period
of the writerTs graduate study. In addi
tion, the writer is indebted to Dr. R. F.
Kimball and Dr. L. E. Powers for their
valuable suggestions.
TABLE OF CONTENTS
Page
I. Introduction............................... 1
A. The problems....................... 1
B. Effects of mustards in other organisms 2
C. Special features of Paramecium aurelia...... 5
D. Expectations of the action of nitrogen must
ard in Paramecium aurelia. ............... $
II. Materials.......................................16
A. Stocks......... 16
B. The mutagen............................... ..l6
III. Methods.........................................17
A. Exposure of animals to the action of nitrogen
mustard................................ .17
B. Induction of autogamy....................... 19
C. Criteria for reduced viability.............. 21
IV. Evidence for mutagenic activity of nitrogen
mustard on micronuclei..........................23
A. Failure to produce effects which are detect
able during vegetative reproduction......... 23
B. Appearance of reduced viability at autogamy..24
C. Heterogeneous clones; segregation of the
physical basis during vegetative reproduction.30
D. Results of exposing conjugants to nitrogen
mustard.................................... 51
E. Transmission of effects to untreated animals
by conjugation..............................54
F. General discussion............................. 65
V. Results seemingly incompatible with the assumption
of a nuclear basis.................................67
A. Transmission of physical basis through suc
cessive autogamies............................ 67
B. Percentage reduced viability not decreased
in successive autogamies and outcrosses.......... 72
G. Discrepancies from expected equivalance of
mates........................... ............. 76
D. Discussion................ ................... $2
VI. Discussion and Summary..............................$4
VII. Bibliography....... £9
I. INTRODUCTION1
A. The problems. Chemical induction of mutation forms
the basis of one approach to the fundamental biological
problems of the nature of the gene and gene action. In
recent years it has assumed considerable importance in ex
perimental genetics, as evidenced particularly by the in
vestigations of Auerbach and Robson (1944, 1946, 1947a and
1947b). These workers, investigating mustard gases and
related compounds because of their similarity to X-ray in
action on tissues, found them to be potent mutagens in
Drosophila, producing as high as 25 per cent lethal mutations
under some conditions. One of the compounds shown by
Auerbach and Robson (1947b) to be mutagenic was methyl
bis^-dichloroethyl)amine, frequently referred to as ni
trogen mustard. The mutagenic effects of this compound
have been investigated in a number of organisms, including
Neurospora. Escherichia coli and Drosophila (Tatum 1946a,
1946b; Auerbach and Robson 1947b), Nuclear changes induced
by this compound have been investigated extensively in
plants and reviewed by Darlington and Koller (1947). The
results of these investigations indicate that nitrogen mus
tard is effective in producing a multiplicity of changes,
^This investigation was supported by Indiana University and
by Rockefeller Foundation grants in aid of research on Proto
zoan Genetics at Indiana University. The author was a Na
tional Institute of Health Predoctoral Fellow during 1947-49.
both in the genes and chromosomes, irrespective of the or
ganism used.
Such extensive effects, as have been demonstrated on
genes and chromosomes, suggest the possibility of compar
able effects on cytoplasmic genetic factors. Paramecium
aurelia, one of the few animals known to be favorable for
for study of cytoplasmic inheritance, offers opportunities
for investigating the effects of chemicals on both cyto
plasmic and nuclear genetic factors. The inactivating effect
of nitrogen mustard on the cytoplasmic factor, kappa, in
Paramecium aurelia has been investigated and reported else
where (Geckler 1949). The present work reports the effects
of nitrogen mustard on viability in variety 4 of this species,
and makes a start towards discovering the physical bases of
these effects, particularly as to whether they are nuclear
or cytoplasmic or both.
The study of chemical mutagenesis must take into account
the known properties and activities of the compounds being
investigated. In addition, it must be understood how these
effects, known primarily from investigations of other or
ganisms, might be expected to express themselves in Paramecium
aurelia.
B. Effects of mustards on other organisms. In plants
three general classes of effects have been noted by Darling
ton and Koller in their review of the chemical breakage of
chromosomes (1947). The present discussion is based primarily
on their paper and includes only the qualitative effects
-3-
produced by sulfur and nitrogen mustards during mitosis
and meiosis. Observations by Auerbach and Robson, to be
considered later, indicate that similar effects are pro
duced in animals. The types of effects reported in plant
material are as follows.
1. Centromere effects. One or both chromatids of a
chromosome may break at or within the centromere during
either mitosis or meiosis. During meiosis the centromere
may misdivide to give isochromosomes.
2. Nucleic acid effects. These result in errors in
spiralization so that the spiral becomes externally obvious
and at meiosis there is some breakdown of major coiling.
Also, errors in reproduction of the chromosome occur; parts
of the chromosome may fail to divide. If the terminal part
fails to divide, bridges will be formed. If the part which
fails to divide is not at the end, fragments may be formed
as well.
3* Fiber breakage effects. The chromosome thread may
be broken before duplication during the resting stage. This
results in both chromatids exhibiting a break at metaphase.
The broken ends can join to give ring or dicentric chromo
somes. The constituent chromatids may also undergo separate
reunion after reproduction to give inversions and inter
changes. Sometimes at the moment of splitting an error in
the reproduction of the chromosome end may occur to give
chromatid bridges.
-4-
If the thread is broken after duplication of the chromo
some during the resting stage, the break is in only one
chromatid and the foregoing results are correspondingly
altered.
In addition, chromosomes may shatter into minute and
subminute fragments. Interchanges may even produce tri-
radial chromosomes.
Many of the mutations obtained by the use of the var
ious mustards are similar to those obtained by the use of
X-ray (Auerbach, Robson and Carr 1947). However, relatively
fewer translocations are produced. An exposure to mustard
which results in nine per cent lethal production gives only
0.5 percent II-III interchange in Drosophila, while an ex
posure to X-ray giving the same lethal production would
give six percent II-III interchanges (Auerbach, Robson and
Carr 1947). This does not necessarily mean that fewer breaks
are produced since it is possible that restitution could
occur in many cases.
In addition to these effects, exposure to mustard re
sults in production of a large proportion of mosaic progeny.
Thirty to fifty per cent of the mutated progeny are mosaic.
Mosaics produced by mustards have progeny which are mosaic
for the same mutations as the parent, indicating that the
genes or chromosomes are unstable for a considerable period.
-5-
Auerbach and Robson (1947a) found that untreated chromo
somes, introduced into treated oytoplasm were unaffected,
indicating that the chromosomes do not react to mustard
through the cytoplasm. In as much as the tests required
three generations to complete, they do not eliminate the
possibility that most of the immediate action of mustard
may be through the cytoplasm. It does indicate, however,
that the delayed action is not brought about through the
action on the cytoplasm.
Auerbach (194$) also reports that the centromere was
affected in one case from mustard treated material.
Special features of Paramecium aurelia.
!• Multiple macronucleus. The macronucleus,
which controls the phenotype, is inferred to be a compound
structure composed of many units, each containing a full
set of genes (Sonneborn 1947). Radiation resistance during
vegetative reproduction such as reported by Back and Hal-
berstaedter (1945) may be explained by the compound nature
of the macronucleus. In order for mutations in the macro
nucleus to be expressed in all the animals of a clone it
would be necessary to obtain the same mutation many times
because of the many sets of genes present. Thus, mutations
in the macronucleus would not be expressed readily in a
clone as a whole.
2. The micronuclei are diploid and their genes
are expressed after fertilization. In Paramecium aurelia
there are normally two micronuclei, each diploid (Sonneborn
