ROBERT W. KORN. Biology Department, Bellarmine-Ursuline College, Louisville, Kentucky. Received April 24, 1969. ESMIDS are composed of two half cells or ...
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
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
BRANDHAM, 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 to spindle overlap. Genetics 51 : 761-766.
FORRO, F. and S. WERTHEIMER, 1960 The organization and replication of desoxyribonucleic acid in thymine deficient strains of Escherichia coli. Biochim. Biophys. Acta 40 : 9-21. KALLIO,P., 1951 The significance of nuclear quantity in the genus Micrasterias. Ann. Bot. SOC. Zool. Bot. Fenn. “Vanamo” 24: 122 pp. -, 1953 On the morphogenesis of Desmids. Bull. Torrey Bot. Club 80: 247-263. -, 1960 On the morphogenesis of Micrasterias 1963 Effects of ultra-violet ainericana in clone culture. Nature 187: 164-166. -, light on cell division in Micrasterias ihomasiana. Ann, Acad. Sci. Fenn. Series A 4: 70-75.
KIMBALL,R. F., 1965 The indication of reparable prernutational damage in Paramecium aurelia by the alkylating agent triethylene amine melamine. Mut. Res. 2: 413-425.
H., 1891 Studien ueber Zygoten I. Die Keimung von Closterium and Cosmarium. KLEBAHN, Pringsheim’s Jahrb. Wiss. Bot. 22: 415-41.3. SMITH,G. M., 1950 T h e Freshwater Algae of the United States. McGraw-Hill, New York. SPARVOLI, E., H. GAYand B. P. KAUFMANN, 1965 Number and pattern of association of chromonema in the chromosome. Chromosoma 16: 415-435. STARR,R. C., 1954a Heterothallism in Cosmarium botrytis var. subiimidium. Am. J. Botany 42: 477-481. -, 19541, Inheritance of mating types and lethal factor in Cosmarium botrytis var. subtimidium. Proc. Natl. Acad. Sci. U.S. 40: 1060-1063. -, 1964 The culture collection of algae at Indiana University. Am. J. Botany 51 : 1013-1044. TAYLOR, 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 TROSKO, by enzyme digestion studies. J. Cell Biol. 26: 125-136. UHL,C. H., 1965 Chromosome structure and crossing over. Genetics 51: 191-207. WARIS,H., 1950 Cytophysiological studies i n Micrasterias. IT. The cytoplasmic framework and its mutation. Physiol. Plantarum. 3: 326346. WARIS,H. and P. KALLIO.1964 Morphogenesis in Micrasterias. Advan. Morphogenesis 4: 4580. WHITEHOUSE, H. L. K., I 9 6 Crossing-over. Sci. Prog. London :285-296. WITKIN, E. M., 1956 Time, temperature, and protein synthesis: A study of ultraviolet-induced mutation in bacteria. Cold Spring Harbor Symp. Quant. Biol. 21 : 123-140.
