NOTES
Sporulation in single-spore isolates from amitrole-induced multispored asci of Saccharomyces cerevisiae
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 135.0.78.252 on 11/17/14 For personal use only.
M. ASHRAFA N D J . J . MILLER Deptrrtttrrtrt ttf'Bio1o.q.~.McMtrste, U t i i ~ ~ ~ , : \Htr~triltotr, ity. Otrt., Ctitrocltr L8S 4 K l Accepted August 22. 1978
ASHRAF.M.. and J . J. MILL.ER. 1978. Sporulation in single-spore isolates from amitrole-induced cere~.i.titre.Can. J . Microbiol. 24: 1614-1615. multispored asci of Sticchtrrot,~~ce.c Amitrole treatment causes multispored ascus production by cells of a yeast strain whose asci no~.m>~lly contain two diploid spores. Single spores were isolated from asci containing two to eight spol-es and their ability to gel-minate was determined. Cells in colonies grown from single spores sporulated in the same manner as the parent strain indicating that nmitrole had not induced meiotic division in the developing asci. ASHRAF.M.. et J . J . M I L L E R1978. . Sporulation in single-spol-e isolates from amitrole-induced muitispored asci of Strcchtit~oitr~ces cere\jisicie. Can. J . Microbiol. 24: 1614- 1615. Un traitement par I'amitrole entraine la formation d'une asque i spores multiples chez les cellules cl'une souche de levure qui produit nol-rnz~lernentcles asques contenant deux spores diploides. Des spores ont ete isolees des krsques contenant deux ir huit spores. et nous avons e t ~ ~ dleur i e capacite de germer. Les cellules dans une colonie qui est dCveloppee b partir d'une seule spore sporulent de 121 meme rnaniere que le parent. Ces resultats indiquent queI'~~mitlole n'a pas induit une division meiotique dans I';lsql~een dtveloppement. [Traduit par le journal]
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... .. . . . .. . .. ... .. . .. . . . ..
. . . . . .
.. . . .. . . . . . . . . . .. . .
.
.
.
.
Grewal and Miller (1972) described three strains which normally formed asci conof S. cerei~isi~re taining only two spores, and obtained evidence that the spores were diploid, which would imply that a meiotic nuclear division did not precede ascospore formation. By Giemsa staining they showed that the asci never contained more than two nuclei. We have induced multispored asci in two ofthese yeast strains through addition of amitrole to the presporulation medium (Ashl-af and Miller 1977). In this note, we describe experiments to determine whether the i~ninucleatespores in the multispored asci were haploid or diploid, i.e., whether the amitrole had induced meiosis to occur in the asci, or whether the nuclear divisions that preceded spore formation were equational. Cells of the 19el strain were grown in malt extract - yeast extract - peptone - glucose (MYPG) medium containing 0.1 M amitrole, sporulated in 2% potassium acetate solution, harvested and preserved in a freezer at -20°C, as in our previous study (Ashraf and Miller 1977). Single spores were isolated from asci containing two to eight spores by a micromanipulator technique described elsewhere (Ashraf and Miller 1978). We determined whether the spores were capable of germination on MYPG agar. Colonies produced by spores that germinated were transferred to slants of MYPG agar and
examined for the presence of spores after 2-4 weeks. This yeast strain is capable of sporulation in growth medium. Some single-spore isolates were also tested for sporulation in 2% potassium acetate solution following growth in MYPG liquid medium as in our preceding work (Ashl-af and Miller 1977). Invariably, clones derived from single spores produced asci and each ascus always contained two spores, like the parent strain from which the single spores were derived. Table I shows that the percentage ofspores that germinated after isolation by micromanipulation TABLE1. Germination of ascospores isolated from different types of asci and ability of cells in the cultures grown from single spores to sporulate
Type of ascus -
No. of spores isolated
-
Two-spored Three-spored Four-spored More than 4-s~ored
% of spores that germinated
%of single spore cultures that sporulated
-
56 27 80
55.4 55.6 43.8
100 100 100
40
57.5
100
NOTE: T h e asci were derived from sporulation cultures obtained by adding to 2Z, potassium acetate cells grown in M Y P G containing 0.1 116 amitrole. They were separated from the acetate solution by centrifugation, washed twice, and preserved a t -20°C until needed. The asci were treated with Glusulase, a n d the spores \\,ere separated and germinated o n the surface of a hanging d r o p o f MYPG agar (Grewal and Miller 1972).
NOTES
TABLE2. Viable ascospores within each type of ascus of asci with: Ascus type
No. of asci
N o viable spores
one viable spore
two viable spores
Two-spored Three-spored Four-soored
28 9 20
28.6 22.2 25.0
32.1 22.2 20.0
39.3
-
--.77 7
33.3 15.0
-
25.0
three viable spores
-four viable spores -
15.0
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 135.0.78.252 on 11/17/14 For personal use only.
NOTE:Six asci with five to eight spores were also studied. Their germination frequencies were as follows:
416.517.4/8, 518. (Numerator
number o f spores that germinated; denominator
=
115, 415.
total number o f spores per ascus).
FIG.I. Ge~minatingxscospores which remain closely attached to each other. Magnification x 2400. F I G . 2. Germinating ascospores which have almost separated from each other. Magnification x 2400.
did not cot-relate with the number per ascus. From Table 2 it is apparent that among asci o f a given kind (i.e., containing two, three, or four spores) there was no consistent tendency for viabre or non-viable spores to occur together in a single ascus. Since the single spores derived from amitroleinduced multispored asci resembled those from two-spored asci regarding ability to sporulate, it may be concluded that the nuclear divisions in the multispored asci did not yield haploid spores, that is, the ability to complete meiotic nuclear division had not been restored by amitrole. Thus the effect of amitrole was apparently to induce the occurrence of a succession of equational nuclear divisions in asci of our yeast strain. As suggested in our preceding paper (Ashl-af and Miller 1977), this may be related to the effect of amitrole on cytokinin levels in the cells. An observation of interest during the germination studies was that development of buds on ascospores in non-dissected asci could occur either
before the pair of spores showed evidence of separation from each other or afterwards (Figs. 1 and 2). Regardless of the number of spores per ascus, we did not observe conjugations at germination. (With multispored asci, however, it was difficult to be certain that no fusions occurred.) The apparent absence of fusions is in accord with the presumed diploid nature of the spores which was indicated by the ability of all single-spore cultures to sporulate. Acknowledgement This work was supported by a grant from the National Research Council of Canada. ASHRAF,M.. and J . J . M I L L E R 1977. . Induction of multispored asci in two-spored strains of Scrcchtrrot?~ycescerevisicre by amitrole. Can. J . Microbiol. 23: 690-694. 1978. A simple, inexpensive micromanipulator for isolation of single spores from yeast asci. Microscope, 26: 35-40. GREWAL.N. S., and J . J . M I L L E R1972. . Formation of asci with two diploid spores by diploid cells ofSncc11cironlyces. Can. J . Microbiol. 18: 1897- 1905.
Sporulation in single-spore isolates from amitrole-induced multispored asci of Saccharomyces cerevisiae
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 135.0.78.252 on 11/17/14 For personal use only.
M. ASHRAFA N D J . J . MILLER Deptrrtttrrtrt ttf'Bio1o.q.~.McMtrste, U t i i ~ ~ ~ , : \Htr~triltotr, ity. Otrt., Ctitrocltr L8S 4 K l Accepted August 22. 1978
ASHRAF.M.. and J . J. MILL.ER. 1978. Sporulation in single-spore isolates from amitrole-induced cere~.i.titre.Can. J . Microbiol. 24: 1614-1615. multispored asci of Sticchtrrot,~~ce.c Amitrole treatment causes multispored ascus production by cells of a yeast strain whose asci no~.m>~lly contain two diploid spores. Single spores were isolated from asci containing two to eight spol-es and their ability to gel-minate was determined. Cells in colonies grown from single spores sporulated in the same manner as the parent strain indicating that nmitrole had not induced meiotic division in the developing asci. ASHRAF.M.. et J . J . M I L L E R1978. . Sporulation in single-spol-e isolates from amitrole-induced muitispored asci of Strcchtit~oitr~ces cere\jisicie. Can. J . Microbiol. 24: 1614- 1615. Un traitement par I'amitrole entraine la formation d'une asque i spores multiples chez les cellules cl'une souche de levure qui produit nol-rnz~lernentcles asques contenant deux spores diploides. Des spores ont ete isolees des krsques contenant deux ir huit spores. et nous avons e t ~ ~ dleur i e capacite de germer. Les cellules dans une colonie qui est dCveloppee b partir d'une seule spore sporulent de 121 meme rnaniere que le parent. Ces resultats indiquent queI'~~mitlole n'a pas induit une division meiotique dans I';lsql~een dtveloppement. [Traduit par le journal]
.
... .. . . . .. . .. ... .. . .. . . . ..
. . . . . .
.. . . .. . . . . . . . . . .. . .
.
.
.
.
Grewal and Miller (1972) described three strains which normally formed asci conof S. cerei~isi~re taining only two spores, and obtained evidence that the spores were diploid, which would imply that a meiotic nuclear division did not precede ascospore formation. By Giemsa staining they showed that the asci never contained more than two nuclei. We have induced multispored asci in two ofthese yeast strains through addition of amitrole to the presporulation medium (Ashl-af and Miller 1977). In this note, we describe experiments to determine whether the i~ninucleatespores in the multispored asci were haploid or diploid, i.e., whether the amitrole had induced meiosis to occur in the asci, or whether the nuclear divisions that preceded spore formation were equational. Cells of the 19el strain were grown in malt extract - yeast extract - peptone - glucose (MYPG) medium containing 0.1 M amitrole, sporulated in 2% potassium acetate solution, harvested and preserved in a freezer at -20°C, as in our previous study (Ashraf and Miller 1977). Single spores were isolated from asci containing two to eight spores by a micromanipulator technique described elsewhere (Ashraf and Miller 1978). We determined whether the spores were capable of germination on MYPG agar. Colonies produced by spores that germinated were transferred to slants of MYPG agar and
examined for the presence of spores after 2-4 weeks. This yeast strain is capable of sporulation in growth medium. Some single-spore isolates were also tested for sporulation in 2% potassium acetate solution following growth in MYPG liquid medium as in our preceding work (Ashl-af and Miller 1977). Invariably, clones derived from single spores produced asci and each ascus always contained two spores, like the parent strain from which the single spores were derived. Table I shows that the percentage ofspores that germinated after isolation by micromanipulation TABLE1. Germination of ascospores isolated from different types of asci and ability of cells in the cultures grown from single spores to sporulate
Type of ascus -
No. of spores isolated
-
Two-spored Three-spored Four-spored More than 4-s~ored
% of spores that germinated
%of single spore cultures that sporulated
-
56 27 80
55.4 55.6 43.8
100 100 100
40
57.5
100
NOTE: T h e asci were derived from sporulation cultures obtained by adding to 2Z, potassium acetate cells grown in M Y P G containing 0.1 116 amitrole. They were separated from the acetate solution by centrifugation, washed twice, and preserved a t -20°C until needed. The asci were treated with Glusulase, a n d the spores \\,ere separated and germinated o n the surface of a hanging d r o p o f MYPG agar (Grewal and Miller 1972).
NOTES
TABLE2. Viable ascospores within each type of ascus of asci with: Ascus type
No. of asci
N o viable spores
one viable spore
two viable spores
Two-spored Three-spored Four-soored
28 9 20
28.6 22.2 25.0
32.1 22.2 20.0
39.3
-
--.77 7
33.3 15.0
-
25.0
three viable spores
-four viable spores -
15.0
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 135.0.78.252 on 11/17/14 For personal use only.
NOTE:Six asci with five to eight spores were also studied. Their germination frequencies were as follows:
416.517.4/8, 518. (Numerator
number o f spores that germinated; denominator
=
115, 415.
total number o f spores per ascus).
FIG.I. Ge~minatingxscospores which remain closely attached to each other. Magnification x 2400. F I G . 2. Germinating ascospores which have almost separated from each other. Magnification x 2400.
did not cot-relate with the number per ascus. From Table 2 it is apparent that among asci o f a given kind (i.e., containing two, three, or four spores) there was no consistent tendency for viabre or non-viable spores to occur together in a single ascus. Since the single spores derived from amitroleinduced multispored asci resembled those from two-spored asci regarding ability to sporulate, it may be concluded that the nuclear divisions in the multispored asci did not yield haploid spores, that is, the ability to complete meiotic nuclear division had not been restored by amitrole. Thus the effect of amitrole was apparently to induce the occurrence of a succession of equational nuclear divisions in asci of our yeast strain. As suggested in our preceding paper (Ashl-af and Miller 1977), this may be related to the effect of amitrole on cytokinin levels in the cells. An observation of interest during the germination studies was that development of buds on ascospores in non-dissected asci could occur either
before the pair of spores showed evidence of separation from each other or afterwards (Figs. 1 and 2). Regardless of the number of spores per ascus, we did not observe conjugations at germination. (With multispored asci, however, it was difficult to be certain that no fusions occurred.) The apparent absence of fusions is in accord with the presumed diploid nature of the spores which was indicated by the ability of all single-spore cultures to sporulate. Acknowledgement This work was supported by a grant from the National Research Council of Canada. ASHRAF,M.. and J . J . M I L L E R 1977. . Induction of multispored asci in two-spored strains of Scrcchtrrot?~ycescerevisicre by amitrole. Can. J . Microbiol. 23: 690-694. 1978. A simple, inexpensive micromanipulator for isolation of single spores from yeast asci. Microscope, 26: 35-40. GREWAL.N. S., and J . J . M I L L E R1972. . Formation of asci with two diploid spores by diploid cells ofSncc11cironlyces. Can. J . Microbiol. 18: 1897- 1905.
