T contained in a sac and usually originate from the reduction of a single

Transcription

1 ASCI IN SACCHAROMYCES WITH MORE THAN FOUR SPORES CARL C. LINDEGREN AND GERTRUDE LINDEGREN Southern Illinois University, Carbondale, IIlinois Received March HE ascomycetes are characterized by the production of spores which are T contained in a sac and usually originate from the reduction of a single diploid zygote nucleus. In Saccharomyces, meiosis produces 4 haploid nuclei, each of which is usually converted into a spore ; the standard ascus is 4-spored. In most of the higher ascomycetes the standard number of spores in an ascus is 8, although an ascus may contain fewer than four, or as many as a thousand or more. An 8-spored ascus is achieved by a third division, followed by the production of a spore wall arqund each of the 8 nuclei. In Neurospora, this process is followed by a fourth division inside the spore, producing binucleate spores. LINDEGREN and SCOTT (1937) showed that some strains of Neurospora may produce as many as 20 spores in an ascus, but only eight of the spores contain nuclei. The spore wall is produced by an autonomous process which may enclose a mass of cytoplasm (without a nucleus) in a spore wall. DODGE and APPEL (1944) found strains of Neurospora which produced only a single ascospore; the entire ascus wall was transformed into a spore wall. In Neurospora tetrasperwza 4 (instead of 8) spores are usually produced, each containing 4 (instead of 2) nuclei. There is no evidence that more than 16 nuclei are present in any single ascus of Neurospora, although the spore number may varyfrom 1 to niore than 20. More than 8 spores containing nuclei have not been observed in any single ascus of Neurospora. Although two supernumerary mitoses always occur, there is no evidence that more than two ever occur. In other genera, notably Dipodascus and Thelebolus (BESSEY 1935), the number of spores may vary from about 100 to 1000 per ascus. In these genera, it seems probable that all the spores are descended from 4 haploid nuclei produced by meiosis from a single nucleus. Repeated mitotic divisions of the 4 nuclei after reduction are not uncommon in other ascomycetes, resulting in asci containing 16, 32, 64 or more nuclei before spore formation begins and producing asci which contain a corresponding number of spores in each ascus. In most ascomycetes 8 nuclei are originally formed and asci containing fewer than 8 spores usually result from the degeneration of some nuclei, rather than the inclusion of a large number of nuclei within a single spore wall. Among the yeasts, Schizosaccharomyces produces either 4 or 8 spores in a single ascus ; presumably the 8-spored asci are produced by a third division of the 4 nuclei resulting from reduction. In Schizosaccharomyces it is very un- 1 This work was supported by research grants from the Southern Illinois University, The American Cancer Society of the National Institutes of Health, Public Health Service and Anheuser-Busch, Inc. GENETICS 38: 73 January 1053.

2 74 CARL C. LINDEGREN AND GERTRUDE LINDEGREN usual to find asci containing other than either 4 or 8 spores. In Saccharomyces, on the contrary, asci containing fewer than 4 spores are usually more abundant than the standard 4-spored asci, presumably resulting from the degeneration of one or more nuclei. LINDEGREN (1949) proposed that some 1-spored asci in Saccharomyces were produced by the formation of a spore wall around an unreduced diploid nucleus, but this may be a relatively rare event. In contrast to the high frequency in Saccharomyces of asci with fewer than 4 spores, asci with more than 4 spores are extraordinarily rare. The method of their formation has become a matter of considerable interest since WINGE and ROBERTS ( 1950) have suggested that irregular segregations may result from the mitotic division of the nuclei produced by reduction, resulting in S (or more) nuclei in a single ascus, followed by the degeneration of all but 4. If the zygote were heterozygous for a Single pair of genes and qnly 4 of S nuclei survived, 3 of which were recessive and 1 of which was dominant, an actual 1 : 1 ratio would be transformed to a 3 : 1 ratio. MUXDKCR (1950) has shown, however, that WINGE and ROBERTS hypothesis is incapable of explaining all hybrid asci that have been analyzed. The present paper reports the direct genetical analysis of hybrid asci which contained more than 4 spores. The asci with more than four spores were obtained from an inbred stock which produced them together with many 4-spored asci (as well as many asci with fewer than 4 spores) ; the many-spored asci were in the minority even in this selected stock. Table 1 shows the pedigree in which the selected stock w-as developed. Eight-spored ascus No. 1 was found among the asci obtained from a mating of by This hybrid was marked, however, for only 2 good characters (a/a and ME/nte) and therefore analysis of the ascus did not ---- TABLE 1 Pedigree showing the development of breeding stocks capable o/ producing a high lrequency of asci with more than four spores X x 8308 amemg x amemg amemgp X a MEmgP No spored No spored x x a GAmgp x agamgp a GAmgp x agamgp No spored No spored No spored No spored No spored No spored No spored No spored No spored Abbreviations: The symbols GA, ME, MG indicate genes controlling the ability to ferment the sugars galactose, melibiose and alpha-methyl glucoside; corresponding symbols in small letters indicate inability to ferment these carbohydrates. The symbols, a and a indicate mating type. The symbols, P and p indicate pink and white respectively; white cultures are adenine-independent while pink cultures are adenine-dependent. Ascus No. 1 contained eight spores numbered in order from to 12255, thus culture 12251, which was a parent of the succeeding generation, arose from ascus No. l.

3 ASCI WITH MORE THAN FOUR SPORES 75 contribute to the question at hand. The other asci, excepting No. 5, were segregating for 4 markers. When 4 markers are used a maximum of 16 classes of offspring is expected. These are all listed in the second column of table 2. In the first column is a number used to designate each of the classes. To analyze the data it is necessary to know the kinds of asci which could he produced by a hybrid heterozygous for four gene pairs. These are shown in table 3. Previous data had TABLE 2 Types of offspring found in 9 multispored asci. The second column shows the 16 possible genotypes obtainable from a hybrid heterozygous for a/a, GA/ga, MG/mg, and P/p. The numbers in the first column are used in tables 3 and 4 to indicate these different genotypes. It tuill assist in the analysis to obserue that pairs I and 16, 2 and 15, etc. form a series which are complementary for all four loci. Class Types of Ascus number No- Offspring 2l Total 1 agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp agamgp Number of viable spores Number of WDeS Numbr of original spores Heterozygous for ME/me instead of GA/ga. a Not diagnosed for a/a, may be 3 or 11. Not diagnosed for a/a, may be 8 or 16. shown that the four markers were not linked and this is confirmed by the finding in the last column of table 2, showing that each of the 16 classes occurs with approximately equal frequency. Table 3 is constructed by expanding the tables of WHITEHOUSE (1942) (for 4-gene hybrids) and collecting all the unordered asci under similar headings. Sixty types of unordered asci are possible. Corrections for the linkage of a and GA to their respective centromeres did not seem necessary for the purposes of the present analysis.

4 76 CARL, C. IJNDEGREN AND GERTRUDE LISUEGREN TABLE 3 The 60 different possible types of unordered asci following segregation in a hybrid heterozygous for 4 genes. The different types of asci are indicated by using their class numbers (table 2) to show the genotypes of the spores. There are 8 possible ditype asci (e.g., , 28 complementary tetratypes (e.g., ) and 24 non-complementary tetratypes (e.g., ). If each of the factors is assumed to be independent of its centlomere the tetratypes are each of equal frequency and 4 times as frequent as the ditypes I Table 4 shows the types of spores isolated from these asci together with the reconstruction of the asci. Eight of ten asci contained more than four kinds of spores. This fact obviously excluded supernumerary mitoses, and suggests that asci Nos. 2, 3, 4, 6, 7, 9, 10, and 11 originated by the fusion of two or more diploid cells before reduction or by the fusion of the asci after reduction. Ascus No. 10 contains four identical pairs, but the reconstructions suggest that five different asci are involved. Sporulation often occurs in a row of cells connected end to end, and if the ends were to fuse one long ascus would be produced. Ascus No. 8 suggests the possibility that supernumerary mitoses had occurred. However, the calculation of its probability if 2 asci had fused by PAPAZIAN (1952) has shown that an ascus of this type may be expected to occur about once in 55 times if 2 asci had fused and, since 10 asci were sampled thete is at least one chance in five that an ascus of this type would be found. It may be concluded, therefore, that one ascus of this type is not unexpected. Ascus No. 5 was produced in a mating in which regular segregations for both MG/wzg and P/p occurred in all the other asci. An excess of the types MG and p appeared. This could be the result of the fusion of two cells in a strand of triploid or tetraploid cells. The high frequency of duplicate types in asci with more than four spores suggested the probability that a restricted type of supernumerary mitosis might occur involving the division of a single nucleus. A calculation by DR. A. M. MARK (personal communication) of the expectation of none, 1, 2, and 3 duplicate pairs in an 8-spored ascus if 16 types are available is given below : P (0) = 0.23 P (1) = 0.52 P (2) = 0.1s P (3) = 0.07 where P (n) is the probability of n pairs. These figures reveal that pairs of identical genotypes might occur in approximately half of the asci. The observed

5 ASCI WITH MORE THAN FOUR SPORES 77 ' TABLE4 Classification of spores in multispored asci with reconstruction of probable asci produced. The asci are reconstructed by rearranging the types of suwiuing spores into tetrads corresponding to those found in table 3. An expected spore which may have failed to survive is indicated in italics. Ascus No. No. of spores Types of surviving spores Reconstructed asci isolated or 11, or , 8or 16, or or ' or frequency of duplicate pairs conforms approximately to this expectation. Although these calculations were made without consideration of the restrictions imposed on expectation by the limitation in possible types of asci, it did not seem necessary to carry out the elaborate calculations that this correction would involve to show that a high frequency of duplicate pairs can be expected. SUM MARY It is concluded that supernumerary mitoses do not occur in the majority of asci of Saccharomyces which contain more than four spores. Data are presented which suggest that asci with more than four spores usually arise through the fusion of adjacent sporulating cells. LITERATURE CITED BESSEY, E. A., 1935 Textbook of Mycology. Blakiston, Philadelphia. 495 pp. DODGE, B. O., and APPEL, A., 1944 Breeding Neurospora from aborted asci. Bull. Torrey Bot. Club 71:

6 78 CARL C. LINDEGREN AND GERTRUDE LINDEGREN LINDEGREN, CARL C., 1949 The Yeast Cell, its Genetics and Cytology. Educational Publishers, Inc., St. Louis. 384 pp. LINDEGREP;, CARL C., and SCOTT, MARY ALLAN, 1937 Formation of the ascospore wall in Neurospora. La Cellule 65: MUNDKUR, B. D., 1950 Irregular segregations in yeast hybrids. Current Science 19: PAPAZIAN, H., 1952 Unordered tetrads. Amer. Nat. 86: WHITEHOUSE, H. L. K., 1942 Crossing-over in Neurospora. New Phytol. 61: WIXGE, O., and ROBERTS, C., 1950 Non-Mendelian segregation from heterozygotic yeasts and asci. Nature 165: 157. Corrigenda : Asci 2 and 11 originally contained 6 and 11 spores, respectively, instead of 8 and 14 spores, as printed in tables 1, 2 and 4.