INDUCTION AND INHERITANCE OF MORPHOLOGICAL
MUTATIONS I N COSMARZUM TURPZNZZ BRl3B.l
ROBERT W. KORN
Biology Department, Bellarmine-Ursuline College, Louisville, Kentucky
Received April 24, 1969
ESMIDS are composed of two half cells or semicells which have a particular
symmetry pattern, shape and in some cases, localized spiny outgrowths
(SMITH1950). The ornate morphology of these unicellular algae has attracted
several workers to investigate the morphogenetic basis of cellular patterns in these
organisms. WARIS( 1950) produced several symmetry alterations of Micrasterias
with cold shock, and from the patterns of origin and reversion, postulated a cytofibril mechanism for symmetry determination. KALLIO(1951,1953,1960, 1963)
extended the investigations of Micrasterias and supported WARIS’hypothesis for
symmetry determination, but postulated that the nucleus controls the shape of
the cell.
BRANDHAM
and GODWARD
(1964) found triradiate forms (trilateral symmetry
patterns of semicells) arising from zygotes of a cross between two biradiate
parents of Cosmarium botrytis. Crosses between triradiate cells reared from the
initial products of the zygote (gones) produced the same frequency of triradiate
gones as had the zygotes from the biradiate parents, indicating that the triradiate
state is not inherited.
A study was initiated to produce morphological mutant cells by ultraviolet
radiation in Cosmarium turpinii Brkb. and to determine the pattern of inheritance
of morphologically deviant traits. This eucaryotic alga appears to be a n ideal
organism in which to study several aspects of mutation phenomena, especially
mutation segregation patterns and the parameters of genetic influence on cell
shape. C. turpinii is unicellular, haploid (STARR
1954b) and uninucleate. All cells
of a population are capable of undergoing vegetative reproduction, and so the
system is not complicated by differentiation of various cell types. The size of the
cell (50 EL) permits individual manipulation. Cells can be manually lined up on
agar plates to insure absolute clonal growth, and hence, allow the determination
of the exact generation when a mutation is expressed (Figure 1). A highly
characteristic and stable shape of the cellulose cell wall enables the detection of
morphological deviations due to the mutagenic treatment. Cosmarium has the
additional feature of semicell formation (Figures 2-5) in which each half of the
cell forms a totally new wall after mitosis. Any genotypic change involving cell
wall formation (cell shape) is expressed without the influence of old cell wall
material (from past genotypic states).
1
This investigation was in partlal fulfillment of the Doctor of Philosophy degree, Indiana Univeisity
Genetics 65: 4 1 4 May 1970
ROBERT W. KORN
FIGURI:
1.-A pliriiotypic. 1iig I x i t t c - i i i i i i CosirmYiiiri lurpirtii I h h . T l i e mutant form is
exprcssrd at second division. The two piirrntal srniirells arc wild typr. arid thr two daughter
scmicc4ls cxprrss tlir niutiint trait.
C. turpinii is sexual. and the availability of heterothallic strains affords the
opportunity to determine the heritability of a n y cell alteration. a distinct advantage over the asexual Micrastcrias species studied by WARIS
and KALLIO.
MATERIALS A N D METHODS
-.
Cclls of Cosirinriuin turpinii Br6h.. strains 3 and 24, mating typrs -1- and
rrsprctivrly,
were ohtaincd from tlir Cultun. Collrction of Algnc (Catalogur i t 7 3 3 and 373.1.) at Indiana
Unirrrsity. Stocks and exprrimrntal scrirs were maintained iit 2.5 :t 2°C undrr a rrgime of 16
tin light. 8 hrs dark. and recrivrd white fluorcscmt light at 200 ft-c intcwsity. Cultures wcre
ma inti1inecl in hac te ria-f rrc cord i t ions; stocks w r r r rcarrd in 1iquitl nit4 iuni and cx pr rimrn tal
crlls w r r r plated on 104 agar mrtlium.
The liquid mrdium for stocks was tlir soil-extract nictliuni of Prings!icini (.%an 1M.F).Agar
dishw for U\’-irratliation series containrcl Pringshrim’s medium.
Crlls finm liquid culturrs wcrr ryclrd t w k r through rrntrifugation at 3.000x fi for one niinutr ancl a wash of frcsh medium. Aftrr a third crtitrifugation. t h r rrlls were susprntlrcl in one
nil of fresh medium. One drop of this ccll suspension (ahnut 4.000 crlls) was acldctl to a four (lay
old agar platr. Cells wrrr individually niored into rows and roluniris of 20 x 20 unttrr W X
miiuriifiration. This procrtlurr assurrtl that suhsrquent colonies \vert? clonrs, as wrll as prrinitting
indivitlual examination of crlls to confirm that the rxprriment hcgan with only wild-ty-pc cells.
Ultraviolrt irradiation treatment \vas administrred 1,- rxpnsing plates of 20 x 20 crlls to a
GE grrmicitlal lanip having a niaxinium output at 2537 A for 300 srcontls at a distance of 22 cm.
Platcs were then placrd in tlic tliirk for 12 hrs to prrrent photoreactivation.
Mutant cells wrrr followrd carefully until a clone of four formed within tlir colony clerirrtl
from an original rcll, at which timr t h r crlls were transfcrrcd to anotlirr plate without mutagen.
At each grncration. claughtrr crlls wrn. separatrcl according to a prvrstahlishrtl pattern that R I lo\vrd total reconstruction of cell linmgrs.
Sexual induction and zygotr gerniination procrtlums were those r m p l o y d hy STARR
(I!?%).
and included two nuclrar markrrs: one for mating type (+ and -) and the other for a zygotic
lethal factor (ST.4nn 1954h). In the latter allelic series, zygotes lyse immediately when lrtlial
factor genes arc present in the homozygous ( I / ) state. and survirr when lethal factor genes are in
the heterozygous ( L I ) or homozygous normal (LL)states. Half-tetrad analysis is possible because
the two goncs of the zygote contain non-sistrr nuclei (STARR19541~).Hence. the two gones are
--
MUTATIONS IN COSMARIUM
. _ _._.
12
-
...-
...
43
'3
I
4
F I G W R?-5.--Srqurncr
I:~
of stag<'.; during rrll tlivizion in Cosmnrium. Figurr 3, crll prior to
division; Figurr 3. rarly stage in srmicrll formation; I'igurr 4, latr stage of srmicrll fnrmation:
and Figurr 5 . two dilughtrr crlls upon completion of cell division.
FIGURI:
6 . S c h r m e for spreadinn cells during colony growth to allow reconstruction of crll
linrages. Dark horizonal and vrrtical line indicate cuts in agar for rrfrrence points. Light lines
with numbers indicate dirrction in which dnughtrr cells are moved. and n u m l m inclicntcr genrrntion at which crlls were formed.
44
ROBERT W. KORN
usually different with respect to a pair of alleles, except when crossing over has occurred. Only
clones in which both gones of the zygote survived were scored.
RESULTS
Induction of mutations: A total of 47 different shape mutations were obtained
by UV radiation. One was obtained five times, another twice, and the forty-five
others were obtained only once. A detailed description of these changes will be
given in a later paper. Three hundred seconds of irradiation resulted in 33% survival of plated cells, and a mutation frequemy of 0.04% per treated cell (an
average of one dish in six contained a mutant clone).
The mutation segregation pattern of the UV-induced mutations ranged from
one to four generations following treatment, and involved from 1/8 to all the
cells of the colony (Table 1 ) . Mutations appeared primarily as 1/1,1/2, and 1/4
patterns, but since the mutations mostly appear for the first time in two daughter
cells of the same division, the patterns are more correctly stated as 2/2, 2/4,
and 2/8.
Cell death occurred primarily by the absence of division of a treated cell, or by
lysis of the two daughter cells following the division of a treated cell. Death by
lysis in cells after the first division occurred at a rate of less than one per thousand
divisions, so that no pattern could be ascertained. Cell death did not follow the
same patterns as those for mutations.
Reversion of mutant cells: Some of the UV-induced shape mutations exhibited
patterns of instability leading to symmetry alterations. But in the eight or so
years that the UV-induced morphological mutations have been in culture, no
reversions to wild-type cells have occurred.
Inheritance studies of UV-induced mutants: Seventeen of the 47 different
mutant strains obtained by UV-treatment were checked for sexuality. Of these,
only four were found to have retained mating capacity. Two of these are reported
here.
Bonnet ( b ) cells (Figure 8) differ from the wild-type cell (Figure 7 ) in shape,
but not in symmetry. No reversions to wild-type cells have been observed in this
TABLE 1
Patterns of appearance of UV-induced mutations
Generation appeared
Single*
First
Second
Third
Fourth
Fifth
2
2
1
0
0
5
Total
__
Number of occurrences
Doublef
Quadruplet
13
18
10
0
0
41
~~
* Single = one daughter cell expresses mutant type.
tDouble = both daughter cells express mutant type.
$ Quadruple = both daughter cells of two cells dividing express mutant type.
0
0
2
0
1
__
3
45
.MUTATIONS I N COSMARIUM
9
i
strain. Gaunt ( e ) cells (Figure 9) have both an altered shape and change spontaneously to different symmetry patterns (biradiated. having two lobes; uniradiate. having one lobe; and nullradiate. having no lohrs). FAch of these types
of semicells can produce a daughter semicell like itself. or like either of the other
types. Hence the strain is polymorphic. but contains no phenotypically wild-type
cells.
A cross between crlls of the bonnet strain of matings type (+) without the
zygotic lethal factor [ h(+)L] with wild-type cells of mating type (-) with the
lethal zygotic factor [R(--,l/:] was made and the gone pairs from the zygotes
scored (Table 2 ) . T h e marker results were close to the expected 1:l ratio of the
total products. Recause two gones arise as nonsister nuclei of meiosis, identical
gones (* f.LL. I I ) indicate that crossing over has taken place between
the locus and its centromere (STARR
1954b). T h e morphological traits came out
as 32 bonnct to 64 wild type. in a n unexpected gone-pair ratio of 18BB; 22Rh; 2hh.
T h e reciprocal cross [ . R ( f ) L ]x eh(-)/] resulted in a high frequency of
zygote inviability so that only a few zygotes were analyzed (Table 3). T h a t some
bonpet gones were obtained indicates that the bonnet trait is passed through the
(-) as well as the (f)
mating type.
T h e gaunt trait was studied from the cross [ g ( + ) L ] x [ G ( - ) l ] , and behaved
-.
46
ROBERT W. KORN
TABLE 2
Offspring data from the cross, bonnet (+)L
Total products:
wild type
bonnet
Gone pairs:
bonnet trait
BB
Bb
bb
(+)(+)
B =64
b=32
16
25
2
0
mt(+) =47
mt(-)=44
0
1
0
1
2
0
type (-)I
L=47
1=44
In combination with
(-)(-)
LL
(+)(-)
1
1
x wild
L1
11
Total
18
25
2
0
2
0
18
28
2
in a similar fashion to bonnet in having somewhat unequal classes of the
morphological alteration while the two marker loci followed the expected ratios
(Table 4).
All gaunt-shaped off spring exhibited the symmetry instability that has been
TABLE 3
x
Offspring from the cross, wild type (+)L
Total products:
wildtype
bonnet
Gone paiss:
bonnet trait
BB
Bb
bb
(+)(+>
B = 15
b=ll
(+\(-I
1
8
3
0
0
0
m t ( + ) = 15
mt(-) =11
In combination with
(-)(-)
LL
0
0
2
0
1
0
bonnet (-)1
L = 12
1=14
Ll
11
Total
1
7
3
0
0
0
9
3
1
previously described, and all wild-type cells were completely stable. Hence, shape
and symmetry aberrations of gaunt are inherited as a single trait.
A double mutant cross, [Bg(+)] x [bG(-)] resulted in offspring of both
parental types, and two recombinant types: one morphologically identical to
TABLE 4
Oflspring from the cross, gaunt (+)L
Gone pairs:
gaunttrait
GG
Gg
gg
x
wild type (-)1
Total products:
wild type
G = 35
mt(+) =27
L = 28
gaunt
g=21
m t ( - ) =21
1=28
(+I(+)
0
2
0
(+)(-)
9
10
3
In combination with
(-)(-)
LL
1
3
0
0
2
0
Ll
11
Total
10
12
2
0
1
1
10
15
3
47
M U T A T I O N S IN C O S M A R I U M
TABLE 5
Oflspring datu from the cross, gB (+)x Gb (-)
In combination with
Phenotype
gaunt
bonnet
wild type
gaunt-bonnet
Total
(+)
(-)
4
5
4
4
10
4
13
3
22
25
Total
I:
parental
recombinant
47
wild type, and the other (Figure 10) similar to a nonsexual mutant obtained
from irradiation termed a “nullradiate- 1” (Table 5 ) . The recombinant type
from this cross was found to be asexual. The wild-type gones predominated
(23/47) over either of the parental types (9/47, 8/4T) and the other recombinant type (7/47).
DISCUSSION A N D C O N C L U S I O N
Determinants of cell form: Previous studies on desmid morphology in several
Micrasterias species (WARIS1950) clearly demonstrated that form is the expression of cytoplasmic activities. Later, KALLIO(1 95 1 ) obtained further evidence
of a cytoplasmic system operating in symmetry determination. I n 1963, KALLIO
demonstrated that UV exposure and RNAase treatment produced effects similar
to enucleation, and postulated (1963, see review of WARISand KALLIO1964)
that lobe determination is controlled by a cytoplasmically perpetuated system,
but the nucleus controls the morphological complexity of the cell.
I n this study, the series of mutations induced in the related species Cosmarium,
included both symmetry and shape alterations. The patterns of inheritance for
two selected traits provided information on the mechanism controlling form in
desmids. The altered traits are inherited through both mating types at equal
frequency, can undergo recombination, and are stable through numerous ( >200)
generations.
The transmission patterns of the morphogenetic mutant factors parallel the
behavior of the nuclear markers in all respects except for the frequency of
inheritance. KLEBAHN(1891) observed that the second division of meiosis in
Cosmarium involves two nuclei, one associated with each of the two zygotic
chloroplasts. The second division produces two nuclei at each plastid, and one
of each of these nuclei survives. The obtained ratio of wild-type to mutant clones
can be explained by nuclear competition between sister nuclei of the second
meiotic division in the zygote. Wild-type recombinant nuclei are not formed
until after meiosis, and since they appear in excess over the other nuclear type,
some nuclear phenomenon must occur between the end of meiosis and germination. It is at this time that two of the four meiotic products degenerate.
Competition can occur between sister nuclei when they differ with respect to
a pair of alleles. Mutant effects can go beyond shape alterations. For example,
48
ROBERT W. KORN
bonnet in mating type (-) drastically reduces the zygote viability, indicating
a pleiotropic effect which could extend to fostering nuclear competition.
Significance of mutation-segregation patterns: Observations on the phenotypic
lag patterns for nuclear markers permit an interpretation of the number of
strands in a chromosome. Autoradiographic studies of chromosomes have indicated that eucaryotic cells, such as those found in bean root (TAYLOR
1957) and
procaryotic cells, such as Escherichia coli (FORRO
and WERTHEIMER
1960) have
two longitudinal units, presumably the antiparallel strands of DNA at G, stage
of the mitotic cycle. Reconstructioq models of chromosomes from electron micrographs (SPARVOLI,
GAYand KAUFMANN
1965) suggest, however, that there are
four and eight strands during GI and G, stages, respectively, and electron micrographs of metaphase chromosomes from Vicia (TROSKO
and WOLFF1965) indicate that at least four strands, and probably more are present.
Recent theories of recombination are based on two paired double helices at
prophase I of meiosis (UHL 1965; WHITEHOUSE
1965) and can be incorporated
into various multi-strand models. Hence recombinational analysis has not resolved the problem of the number of strands in a chromosome. Analysis of the
time in generations required for expression of recombinant types is crucial for
the determination of the number of strands in chromosomes at G, and G, stages
(WITKIN1957; CHEN and OLIVE1965). Despite the impressive work done in
this area, conclusions remain indecisive.
The known action of ultraviolet light permits a direct interpretation of the
mutation-segregation patterns. UV-induced mutations appeared primarily as
1/1, 1/2, and 1/4, and almost always occurred simultaneously in two daughter
semicells. Those mutations expressed as a 1/1 fraction can be explained in a twostranded model according to the known action of dimer insertion affecting both
strands of a double helix, or in a four-stranded model as the unlikely alteration
of two separate helices a t exactly the same locus. Further support for the twostranded model can be derived from the 1/2 and 1/4 fractions. The alteration
of a single intact strand at the four-stranded G, stage would produce a 1/4 pattern.
Since it is not known whether the cells were in G, or G, stage during irradiation,
it must be assumed that both were present, and are reflected in the data. Thus,
the UV-induced mutation study supports the ideas put forward by TAYLOR
(1957) for plant cells, KIMBALL(1965) for Paramecium aurelia, and FORRO
and WERTHEIMER
(1960) for bacteria that there is one double helix of DNA in
the chromosome at G, and two double helices at the G, stage of the mitotic cycle.
The results of this study further support the semiconservative replicative pattern
for DNA.
The author wishes to thank Dr. R. C. STARRfor his suggestions during the course of this
study.
SUMMARY
Morphological mutations were produced in Cosmarium turpinii Brkb. with
ultraviolet radiation. Inheritance studies showed that UV-induced mutations are
nuclear. These mutations were studied, particularly with reference to the pat-
MUTATIONS IN COSMARIUM
49
terns in which they appear in clones of treated cells. Irradiated cells gave rise
primarily to clones with 1/1, 1/2, and 1/4 sectors. Considering the known action
of UV-irradiation, and possible multiplicity of chromosomal strands, it is concluded that the chromosome of this eucaryotic cell possesses one double helix
of DNA before, and two double helices of DNA after DNA synthesis.
LITERATURE CITED
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P. E. and M. B. C. GODWARD,
1964 The production and inheritance of haploid
triradiate form of Cosmarium botrytis. Phycologia 4: 75-87.
CHEN,K . 4 . and L. OLIVE,1965 The Genetics of Sordaria breuicollis. 11. Biased segregation due
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F. and S. WERTHEIMER,
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E., H. GAYand B. P. KAUFMANN,
1965 Number and pattern of association of chromonema in the chromosome. Chromosoma 16: 415-435.
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19541, Inheritance of mating types and lethal factor in Cosmarium
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I. H., 1957. The time and mode of duplication of the chromosomes. Am. Naturalist 91:
209-221.
J. F. and S. WOLFF,1965 Strandedness of mitotic chromosomes of Vicia faba as revealed
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H. L. K., I 9 6 Crossing-over. Sci. Prog. London :285-296.
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