Meiosis in Maize: mei Genes and Conception of Genetic Control of Meiosis

MEIOSIS IN MAIZE: mei GENES AND CONCEPTION OF GENETIC CONTROL OF MEIOSIS lnna N. Golubovskaya The N. I. Vavilov All-Union Institute of Plant Industry, Leningrad, 190000, Union of Soviet Socialist Republics

I. Introduction ... ..... .... ... 11. Brief Description of the Maize mei Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. An ameiotic Mutation Controlling the Initiation of Meiosis in Maize.. . B. The afd Mutation Substituting the First Division of Meiosis for Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Blockage of Meiosis after Pachytene: An Effect of the MeiO25 Gene . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . D. rnei Genes of Maize: Impairing the Pairing of Homologous Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Genes Controlling Segregation of Homologous Chromosomes . . . . , . . . . . F. A mei Mutation, elongate (el),Controlling the Second Division of Meiosis.. . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . G. A mei Mutation, uariable (ua),Impairing Cytokinesis .... ... H. mei Mutations ( p a m l and p a d ? ) Causing Nonspecific ....... Multiple Abnormalities of Meiosis. . . . . . . . . . . . I. Precocious Postmeiotic Mitosis: A polymitotic ( P O Gene with a Series of Alleles (ms6 and ms4) . , . . . . . . . . . . . . . . . . . . . . . . . J. Summary 111. Cytogenetic Evidence for the Genetic Control of Meiosis.. . . . . . . . . . . . . . . . . A. The Choice of mei Mutations as Experimental Models B. Independent Action of Genes Controlling C. Consequent Activation of rnei Genes in Meiosis.. . . . . . . . . . . . . . .. . . . . . D. Hierarchy among mei Genes . . . . . . . . . . . . . . . . . IV. Speculation about the Possible Pathways of Geneti V. Conclusion: Theoretical and Applied Aspects of Meiosis Genetics References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . I

149 153 153 158 160 164 167 168 169 169 173 173 174 174 176 177 181 183 185 186

I. Introduction

Knowledge of the genetic control of meiosis may provide a key to understanding the complex and important processes involved in sexual reproduction. In this era of biotechnology and genetic engineering, which provides great possibilities for the construction of new plant and animal genotypes, meiosis should be in the forefront of biological 149 ADVANCES IN GENETICS, Val. 26

English translation copyright 0 1989 by Academic Press, Inc.

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investigations. Creation of new organisms and their fate will depend on whether the gametes can pass through the “sieve” of meiotic selection and can leave viable progeny. In this case, meiosis carries out an important evolutionary function as the barrier in the way of inviable chromosomal or genic combinations (White, 1973). Studies in the field of meiotic biochemistry (Stern and Hotta, 1973, 1974) and ultrastructure of meiotic prophase (Moses, 1956; Bogdanov, 1975; Westergaard and von Wettstein, 1972) stimulated investigations of the genetics of meiosis in the 1970s. These early studies showed that there were enzymes that could take part in meiotic recombination, in DNA repair after damage by UV rays in somatic cells, and in the degree of enzymatic mutagenic sensitivity (Boyce and HowardFlanders, 1964; Kushev, 1972). The necessity for direct study of meiotic genetics stems from cytogenetic investigations of common wheat and its distant hybrids with the subtribe Triticinae. The cytogenetic stability of such hybrids, obtained in vivo and in vitro, appears to depend on genes which control chromosome behavior in meiosis (Riley and Chapman, 1958; Riley et al., 1966; Golubovskaya, 1973; Golubovskaya et al., 1966). Thus, from the general question, whether meiosis is controlled genetically, the following questions derive: the number and identity of the genes involved in meiosis, the genetic mechanisms that occur for the initiation of meiosis, how the main meiotic events (for example, pairing of homologous chromosomes or meiotic recombination) are controlled, and how transition from one event to another is genetically controlled. These and many other questions require answers. Therefore, an essential methodology for investigating such questions was developed. First, mei mutants were collected from Saccharomyces cerevisiae, Neurospora crassa, Drosophila melanogaster, Pisum sativum, and Zea mays. The peculiarities of the genetic control of meiosis in yeast, particularly the question of how the initiation of meiosis is controlled, were investigated with the help of the Saccharornyces and Neurospora mei mutants (Esposito and Esposito, 1969; Klapholz and Esposito, 1980). The mei mutants of D. melanogaster were obtained in order to study the genetic control of meiotic recombination in higher eukaryotes (Baker and Carpenter, 1972; Carpenter and Sandler, 19741,and to examine the relationship of the mei genes to genetic systems controlling DNA repair processes after UV irradiation (Smith, 1973; Smith et al., 1980; Baker et al., 1976a). In addition, the effect of genes controlling meiotic recombination and mutagenesis induced by transposable genetic elements was studied (Green, 1978; Eeken and Sobels, 1981). There exists a n enormous gap between the general concepts relative

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to the genetic control of meiosis and the actual understanding of the intricate peculiarities of such control. Our initial task was to try to understand some of the major peculiarities of the genetic control of meiosis in maize (Golubovskaya, 1975). The basic premise was that meiosis is a universal process for all sexually reproducing organisms. This principle permitted us t o ignore species-specific aspects of meiosis, and allowed us to utilize all available data about all types of mei mutations which have appeared in the scientific literature for classifying mei mutations. To simplify the classification of mei mutations, we compared their effects on separate cytogenetic events of meiosis (i.e., initiation of meiosis, pairing of homologous chromosomes, meiotic recombination, chiasma formation, segregation of chromosomes, the second meiotic division, and cytokinesis). Systematization of mei mutations has revealed that similar types of mei mutations concerning one or several of the above-mentioned meiotic events occur in organisms ranging from unicellular algae and yeast to higher plants, and in animals ranging from nematodes and Drosophila t o humans. A detailed classification of mei mutations was summarized in previous reviews (Golubovskaya, 1975, 197913). In this review the emphasis will be on distinct types of mei mutations, those which show initial cytological effects. On comparing these with each other, we were able to uncover the following main effects they have on the genetic control of meiosis: 1. The seven key cytogenetic steps of meiosis are under strict genetic control, each being controlled by a group of genes acting relatively independently from each other. 2. At least three hierarchical levels of mei genes are noted: there are genes controlling key blocks of meiotic events, those controlling elementary steps in meiosis, and peculiarities of individual chromosomal behavior, including species-specific characteristics of meiosis. 3. The principle of stepwise switching on of mei mutations during meiosis was formulated. Every logical argument demands concrete experimental evidence. For this purpose, it was necessary to have a collection of different rnei mutants affecting the key cytogenetic steps of meiosis. Maize (2.mays L.) was selected for this purpose, because it is well studied genetically and convenient for observing meiotic events (Fig. 1). For the cytogenetic model experiments, nine mei mutants, induced by chemical mutagens, and some mei mutants provided by the Maize Genetics Stock Center (Urbana, Illinois), were used (Table 1).

FIG. 1. The meiotic cytological behavior of chromosomes in normal maize plants. (a-e) Prophase I of meiosis with normal pairing of homologous chromosomes. (a) Zygotene. (b and c) Pachytene. (d) Diplotene. (e) Diakinesis. (f) In metaphase I, 10

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II. Brief Description of the Maize mei Mutants

The mei mutations do not influence vegetative development of plants and do not change their phenotypes. The mei mutations in plants can be revealed only during tassel inflorescence. As a rule, mei mutants display complete or partial male and female sterility, but in some cases they have either male or female sterility only. Special attention to cytological characteristics will be paid throughout the description of maize mez mutants. The genetics of mei mutants are summarized in Tables 2 and 3.

A. AN ameiotic MUTATION CONTROLLING THE INITIATION OF MEIOSISIN MAIZE A recessive ameiotic (am) mutation (chromosome 5 , short arm) was isolated (Rhoades, 1956) and cytologically studied (Palmer, 1971). Homozygous amlam plants exhibit complete male and female sterility. Cytological analyses of homozygotes have revealed that the last premiotic mitosis proceeded regularly, but that the subsequent meiosis did not occur in the meiocytes. Instead of meiosis, there are two to three synchronous mitoses and subsequent degradation of chromatin in the cells. Among the dividing cells of the anther, all stages of the meiotic cell cycle, from interphase to teleophase, are present. The pattern of meiosis in ameiotic mutants grown in Krasnodar (USSR) was similar to that described by Palmer (1971). In my opinion, there was a single exclusion in Palmer’s cytological observation: only the first mitotic division in ameiotic homozygotes was synchronized, and therefore, in the next mitotic cell cycles, only rare cells were seen at different stages of mitosis. To be sure that meiosis was completely omitted in this mei mutant, the prophase of ameiotic mitosis was examined by electron microscopy. Synaptonemal complex (SC) formation was absent in the cells (Golubovskaya and Khristoljubova, 1985). That is, the single mutational action converts the whole meiotic process into mitosis, as shown by the ameiotic mutant. It is reasonable to suppose that the product of the normal allele of the ameiotic gene is responsible for the initiation of meiosis, and that the gene has the highest hierarchical level, i.e., switching on all steps bivalents are seen. ( g ) In anaphase I, 10 chromosomes pass to each opposite pole. (h-n) A second division of meiosis. (h-i) Prometaphase. (j)Metaphase 11. (k-1) In anaphase 11, centromere sister chromatids separate. (m) Telophase 11. (n) Tetrads.

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TABLE 1 List of Maize Meiotic Mutants Cytological effect of mei gene

Symbol for mei gene

Location

References

Comment

Substitution of meiosis by mitosis Substitution of first meiotic division by mitosis

ameiotic (am)

5s

Rhoades (1956); Palmer (1971)

afd W23

6L

Stickiness of chromosomes

sticky (st)

4s

Golubovskaya and Mashnenkov (1975); Golubovskaya (1979a, 1987) Meiotic arrest Beadle (1937) after pachytene Mashnenkov and Golubovskaya (1980) Nelson and Clary (1952) Beadle (1930)

MeiO25

Desynapsis of chromosomes

desynaptic (dy)

asynaptic (as) dsy 1 , dsy 2, dsy 3, dsy 4

Abnormal chromosome segregation

Abnormalities of second meiotic division

1s non-lS, lL, 6L

ms43 A344

non-lS, 5s,5L, 6L, 7L 9s 8L

ms28A344

1s

elongate (el)

8L

diuergent (du)

Golubovskaya and Mashnenkov (1976); Golubovskaya and Urbach (1980) Central spindle Clark (1940) apparatus is not formed Curtis (1983) Golubovskaya and Parallel spindle Sitnicova orientation a t second divi(1980); sion is abGolubovskaya sent and Distanova (1986) Golubovskaya and Delayed Sitnikova depolymerization of (1980); spindle fibers Golubovskaya (1987) Rhoades (1956); Curtis (1983)

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MEIOSIS I N MAIZE

TABLE 1 (Continued) ~~

Cytological effect of mei gene

Symbol for mei gene

Irregular cytokinesis Supernumerary mitoses during pollen grain formation

variable (ua) polymitotic (PO),ms6

Nonspecific abnormalities of meiosis

pan1 A344, pam2 W64A

Location

References

7L

Beadle (1932a)

6s

Beadle (1928,

Comment -

ms6, and ms4 are allelic Precocious Golubovskaya and Urhach postmeiotic mitoses (1980) Golubovskaya and Mapping is Mashnenkov needed to (1976) verify cytology PO,

1933)

ms4 6L

TABLE 2 Inheritance of Eight New Maize mei Mutations as Defined by Pattern of Meiosis and Fertility Segregation in F 2 mei mutant

dsy dsy2 ms43 ms28 ms4

Cross combination Fz(pamlpam x A344) pam2Ii @ afdl+ 0" Fz(afd1afd x W23) dsyl+ @ FZ(dsyl+ X A344) dsy2l+ @ F2(dsy2Idsy2 X A344) ms431+ @ ms43lms43 x ms43/+ F2(ms431ms43 x A344) ms281+ 0" ms28lms28 x m s 2 8 l t Fz(ms28/ms28 X A344) ms4l+ @ F9(ms41ms4 X A3441

' @, Self-pollination.

* Not significant.

Normal fertile

Mutant sterile

Expected ratio

75 43 127 112 132 65 64 36 83 58 102 41 65 18 48 11

22 10 48 39

3:1 3:1 3:l 3:1 3:l 3:1 3:1 3:1 3:l 1:l

42

20 29 5 6 28 21 8 43 5 17 6

3:l 3:1 1:l

3:l 3:1 3:l

x2

value

0.28 1.06 0.55 0.05 0.07 0.09 1.89 3.58 15.82' 10.46' 4.12 1.96 4.48* 0.13 0.15 0.96

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INNA N. GOLUBOVSKAYA

TABLE 3 Allelic Relationships of the mei Mutations as Defined by Pattern of Meiosis and Fertility ~~

Fz segregation

F, segregation Cross combination and meiotic events Homologous pairing dsy2Idsy2 x dsyl+ aslas x d s y l t aslas x a f d l t dsy2Idsy2 x a s i t aslas x dsy2/+ afdli x dsylt Segregation ms43lms43 x ms28l-t ms28lms28 X ms431 t ms43lms43 x d u l t ms28lms28 x d v l t Differentiation pamlpam x p a m 2 I i ualva X p a m l t amlf x afdlt aslas x m s 4 3 l t ms4lms4 x pol+

Normal fertile

Mutant sterile

41 4 24 23

0 0 0 0 0 0

17

40 39

20 27 17

2 8

21 20 8

0 0 0 0 0 0 0 0 2

x2 value Fertile

Sterile

9: 7

-

97 I5 35

1.56

0.33

0.5 5.0

0.25

0.7

107

105 42 20

-

1 :1

-

18

179

141

0.01

4.5

-

-

-

-

-

-

-

-

-

-

-

50

39

0.15

1.4

36

35 116 21 -

0.90

0.01

0.25

2.25 2.28

140 32 -

0.37 -

-

of meiosis simultaneously. Experimental proof for this last statement will be discussed below. If the mutation of a single gene causes conversion of meiosis to mitosis, the principal irreversible product determining the pathway of meiosis must also exist. From this viewpoint, it is interesting to consider the biochemical data, which indicate a meiosis-specific protein, the leptotene protein (L-protein) (Hotta et al., 1984). This 73-kDa protein has been isolated and purified from the nuclear membranes of preleptotene, leptotene, and zygotene cells of lily. The protein has been found to be highly specific in its inhibitory activity, supressing the replication of zygotene DNA (zyg DNA)sequences until the initiation of zygotene. The L-protein has also the capacity to nick the bound DNA in the presence of ATP, and is considered to be responsible for the irreversible commitment of cells to meiosis after entering the premeiotic S phase.

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Initiation of Meiosis and mei Genes in Other Organisms The yeasts S. cereuisiae and Schizosaccharomyces pombe are most convenient for studying the mechanisms of initiation of meiosis. Significant factors for the cells to enter into meiosis in these species, which represent lower eukaryotes, are the heterozygous state of mating sites and nitrogenous starvation. There is a specific point in the GI phase of the mitotic cell cycle, called “start,” which must necessarily be blocked for transition of the cell from mitosis to meiosis. The transition in S. cereuisiae has been shown to be strictly genetically controlled and a function of the normal alleles of the genes of the cell division cycle (cdc genes); namely, cdc’28, cdc+25,cdc+35, tru’3, etc., are needed (Hereford and Hartwell, 1974). The functions of cdcf28 and tra’3 genes were found to be important for entering into meiosis, and the cdc’25 and cdc’35 genes were demonstrated t o be responsible for the choice between meiosis and mitosis and to depend on the components of the nutrient medium. In addition, two genes, CYR 3 and cyr 1, are involved in controlling meiosis and both genes initiate meiosis independent of nutrient components in the medium (Matsumoto et al., 1983; Uno et al., 1982). Biochemical studies of these mutants and their comparison with other cdc genes have allowed the authors to conclude that the initiation of meiosis depends on the repression of CAMPproducts and inactivation of CAMP-dependent protein kinase (Matsumoto et al., 1983). Based on genetic, biochemical, and molecular investigations of cdc mutants in S . cereuisiae, a model of initiation of the cell cycle has been suggested. According to this model, the decreased level of protein kinase enzymatic activity is significant for the beginning of meiosis, and the GI phase of the cell cycle is critical for initiation of meiosis. Recent genetic data obtained using S. pombe have also supported this model (Iino and Yamamoto, 1985a,b). The patl gene is considered to be very important for initiation of meiosis. Investigations of the interaction of the patl gene with a gene defective in the mating system (meil gene), and of the mei2 and mez.3 genes needed for initiation of premeiotic DNA synthesis, have shown that the patl product releases a negative control in meiotic initiation. Additional evidence for the essential role of the loss of protein kinase activity in initiation of meiosis in both yeast species is available from the studies of cdc genes (Shimodo and Uehira, 1985).Recent results of cloning of cdc2 in S.pombe (Hindley and Phear, 1984) have permitted the isolation of the protein product of the cdc2 gene (Simanis and

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INNA N. GOLUBOVSKAYA

Nurse, 1986). It is a 34-kDa phosphoprotein with kinase activity. The protein plays a significant role in entering the “start” point and in initiating the mitotic cycle. When mutant cells are placed on poor nutrient medium, phosphorylation of the cdc2 protein and loss of protein kinase activity occur. They are the basic factors which determine the entry of cells into meiosis (Simanis and Nurse, 1986). The identity and conservation of genetic and molecular mechanisms of cell division initiation in two species of yeast were examined by identifying the protein product encoded by both the cdc’28 gene (5’. cerevisiue) and the cdc+2 gene (5’.pombe) (Lee and Nurse, 1987). The conservation of genetic regulatory mechanisms of the cell division cycle is widespread throughout the evolutionary ladder. A human homolog of the cdc2 gene has been isolated and cloned from a cDNA library of S.pombe. The protein sequence of the human homologue is very similar to that of the cdc2 gene in fission yeast. The CDC2 H s gene encodes a 34-kDa phosphoprotein with kinase activity. The yeast and human proteins have 63% homology in amino acid sequences. These experimental data indicate that the factors by which the cell cycle is controlled are likely to be conserved among yeast and humans (Lee and Nurse, 1987). B. THE ufd MUTATION SUBSTITUTING THE FIRST DIVISIONOF MEIOSISFOR MITOSIS The ufd mutation is another phenomenon in which the entire pathway of meiosis is altered by a single mutation that is induced by a chemical mutagen in maize (Golubovskaya and Mashnenkov, 1975). The recessive ufd gene responsible for the mutant phenotype is located on the long arm of chromosome 6 by B-A translocation stocks (Golubovskaya, 1987). In ufd homozygous plants, typical stages of prophase I, such as leptotene, zygotene, pachytene, and diplotene, are omitted. At a stage which can conditionally be called diakinesis, the 20 chromosomes of maize are represented by univalents, arranged in a disorderly manner in the cells, and a nucleolus is clearly seen. At metaphase I, 20 univalents are lined up in an orderly manner on the plate of spindle. At anaphase I, 20 chromatids move t o the opposite pole as a result of division and separation of centromeres of sister chromatids, as a rule at the first meiotic division instead of the second. Thus, the first division of meiosis in ufd mutants proceeds like mitosis. A t anaphse-telophase of the second division of meiosis, chromatids are distributed irregularly between the two poles. As a result, about 98%

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abnormal tetrads are formed in mutant plants. Homozygotes for the afd gene have male and female sterility. The first meiotic division, being similar t o mitosis, proceeds abnormally. There is pulverization of chromosomes at anaphase I, resulting in 67% abnormal chromosomes. Indirectly, this indicates the defects in the repair process, which normally accompanies meiotic recombination. Meiotic recombination, as experimentally proved (Stern and Hotta, 1974; Hotta et al., 19791, occurs only in the case of intimate pairing of homolgous chromosomes. But the question arises as to how, in the absence of cytologically visible pairing of chromosomes in the afd mutant, events connected with meiotic recombination are possible. Electron microscopic studies of the meiotic chromosomes at prophase I help to understand this contradiction. The short SC fragments were found to appear at the early stage of prophase I, and the morphology is correct in one case and defective in another case. The total length of the SC in the afd mutants is only 12% of that in normal plants (Golubovskaya and Khristoljubova, 1985; Golubovskaya et al., 1980). The formation of the SC is not complete and its short fragments are quickly destroyed and disappear. Whether the afd mutation is due to defects in the pairing of homologous chromosomes or to a systematic mutation induced by changes in the whole meiotic process is not clear. However, in my view the last alternative is more likely, based on a comparison of the afd mutants with asynpatic and ameiotic mutants in maize, as described above.

Comparisons between the afd and the asynaptic and ameiotic Mutants Some asynaptic mei mutants have been described: the c3G gene in D . melanogaster (Gowen, 1933; Gowen and Gowen, 1922; King, 1970; Smith and King, 1968),the asynpatic gene of Triticum durum (Martini and Bozzini, 1966; LaCour and Wells, 19701, the rad6-1 gene of S . cerevisiae (Kundu and Moens, 19821, the syn gene of Secale cereale (Fedotova et al., 1987), and the case of male sterility in man associated with total blocking of the SC formation at prophase I (Vidal et al., 1982). The asynpatic mutations of these different organisms have the same general peculiarities, such as the total blockage of SC formation (in some cases, for example, in rye, durum wheat, and humans, only lateral elements are formed), inhibition of crossing over, and anomalies of chromosomal segregation as a result of the univalent state a t metaphase I. It is necessary to emphasize that meiosis in asynaptic

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INNA N. GOLUBOVSKAYA

mutants proceeds in two divisions and that centromeres of sister chromatids segregate, as a rule, at the second division of meiosis. The difference between the umeiotic mutants and the ufd mutant is that the first division of meiosis of ufd is substituted for mitosis. In the ufd mutants, the second division takes place as in normal plants, but the abnormalities are observed because the function of the second division is carried out in the first division. On the other hand, the existence of the afd mutant indicates that the total reversion of mitosis in the cells irreversibly committed to meiosis (after they enter into the zygotene stage, the ability to form the SC can be considered) is excluded. Also, the behavior of centromeric regions and homologous chromosomal pairing are independent events. An indirect proof for this is the appearance of mei mutants, determining the precocious separation of centromeric regions at the first division of meiosis. Other examples are the pc mutation of Lycopersicon esculentum (Clauberg, 1959) and the ord gene of D . melanoguster (Mason, 1976; Goldstein, 19801, which show common genetic control of disjunction of centromeric regions in chiasmatic meiosis of females and in achiasmatic meiosis of males in D . melanoguster (the ord and the mei-S332a genes) (Sandler et ul., 1968; Davis, 1971; Goldstein, 1980). Hence, it is not surprising that the premature disjunction of sister centromeres can accompany some mutations [the dy mutant in maize (Nelson and Glary, 1952) and the 2982 mutant ofpisumsutiuurn (Klein, 1969)l.It has been suggested by Maguire that it is the dy gene that is responsible for the behavior of centromere regions in maize (Maguire, 1978a,b). OF MEIOSISAFTER PACHYTENE: C. THE BLOCKAGE AN EFFECTOF THE MeiO25 GENE

1 . Inheritance of MeiO25 In 1979, six sterile mutant MeiO25 plants were pollinated with the inbred line of maize W64A (Table 4).Cytological analysis of segregation in the F1 progeny suggested that (1)in general, the segregation pattern of meiosis among F1 hybrids confirmed a 1: 1ratio (26 normal and 30 MeiO25) and the anomalies of meiosis that occur in the first progeny are likely caused by a dominant gene; (2) male sterility is not a reliable criterion for the detection of the mei mutation, as some of the plants of F1 progeny exhibit a Mei025-type meiosis (18 of 30) and are fertile; and (3) a variation of segregation ratio (normal : mutant) ranges from 10: 1 to 3 : 10, indicating that there are differences in

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MEIOSIS IN MAIZE

TABLE 4 Character of Meiosis and Fertility in Maize F1 Progeny from Crossing Sterile Me2025 Mutants with Original Inbred W64A Stock Pattern of meiosis and fertility

Cross (1) 32st x W64A ( 2 ) 32-5st X W64A (3) 3 2 - 7 ~Xt W64A (4) 32-9st X W64A (5) 23st X W64A (6) F1(5) X W64A

Total Normal :Mei

Normal meiosis, fertile

Abnormal meiosis (Mei025) Fertile

Sterile

Total

4

4

3

2 4 5

0 3 2

5 1 1

8 8 10 13 11 6

12

56

4

3 10 2

26 26

0 3 18

30

segregation ratio among individual crosses. It is probable that these differences are due t o the presence of a modifier gene (m factor) which normalizes meiosis in some mutant plants. The m-factor action is dose dependent in such a way that in homozygotes and heterozygoes for m factor, the pattern of meiosis is normalized (Table 4). It has been confirmed cytogenetically that the Mei025 mutation is dominant and has complete expression in homozygous and heterozygous states with respect to meiotic patterns and is incompletely dominant in relation to fertility.

2 . Cytological Analysis of MeiO25 Cytologic analysis of the Me2025 mutants has shown that meiosis proceeds correctly until metaphase I; then the chromosomes lose their contours and ability to move and cling together in a dense cluster. In pycnotic conditions chromatin remains until interkinesis. At this stage, a decondensation of pycnotic chromatin was found, but at metaphase I1 the chromatin in the cells clustered again. As a result of such meiotic anomalies, the pollen grains formed by Me2025 mutants were incapable of fertilization (Golubovskaya, 1979a; see Fig. 4). The occurrence of the modifier genes in the W64A inbred line of maize influences the expression of the Me2025 gene and has led t o the study of the main peculiarities of the latter gene without (or with weaker) action of the m gene. For this purpose mutant plants were crossed with other inbred lines (A344 and W23).Analysis of characteristics of meiosis in the segregating mutants in these crosses has

INNA N. GOLUBOVSKAYA

FIG. 2. The pattern of meiosis in Me2025 mutant segregation in F1 progeny of the individual crosses (Me2025 X A344 inbred line). (a-c) Prophase I. (a) In Pachytene, a regular pairing of homologous chromosomes is seen. (b) Arrest of meiosis after pachytene. (c) At diakinesis. (d and e) A degradation of chromatin is seen in cells. (d) Pycnosis. (el Early stage of lysis. (f 1 Cytokinesis in the cell with pycnotic chromatin. (g) Arrest of meiosis and pulverization are seen. (h) Arrest of meiosis after prophase I. (i) Pollen envelopes are formed around the cell with arrest of meiosis.

demonstrated the same pattern of meiosis as in previous studies, but there seem to be some essential differencies. The observed abnormalities, such as pycnosis of chromatin, were displaced at the pachytene stage, and meiosis in some cells with pycnosis did not proceed further, i.e., meiosis was blocked at the pachytene stage in such cells (Fig. 2).

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This observation suggests that the cytological effect of the MeiO25 gene may be associated with the blockage of meiosis after pachytene. These types of mei mutants are widespread in all eukaryotes examined. 3. mei Mutations Arrest Meiosis after Pachytene in Different Species

The recessive mutation me13 (group I linkage) in N . crassa has been described. It has been found that there is complete arrest of meiosis during early stages (Newmeyer and Galeazzi, 1974; Perkins and Barry, 1977). Two mutations, meil and mei2, were isolated in Podospora anserina (Simonet and Zickler, 1972). Both mutants have the same cytological features: meiosis is blocked at the pachytene stage and chromosomes cling together in dense clusters. The isolation of different alleles of the mei2 gene has permitted study of the influence of this gene regarding the frequency of crossing over. The mei2 gene has been found to cause increased frequency of meiotic recombination close to the centromere region and decreased frequency a t the distal region of the chromosomes (it is demonstrated for the three linkage groups) (Simonet and Zickler, 1972). The m e 8 gene of P. anserina causes chromosomal stickiness at the early displotene stage. In humans, a mei mutation led to the sterility of three sons in one family (Cantu et al., 1981). The reason for the sterility was blockage of meiosis after pachytene. I n the yeast cdc5 mutant (8.cereuisiae), meiosis proceeds correctly at 25"C, but when cultures are transferred to 34"C, meiosis stops at pachytene and remains a t this stage until the yeast culture is returned to 25°C. Electron microscopy of prophase I of meiosis has demonstrated that, in the presence of temperature arrest of meiosis, the SC maintains, and as a result, the frequency of meiotic recombination is increased (Simchen et al., 1981). In the cdc4-3 mutants of yeast, meiosis is also arrested a t pachytene a t 34°C. There is also an increase in meiotic recombination, and reduplicated polar bodies of spindle do not pass to opposite poles but rather lag in the center of the cell (Byers and Goetsch, 1982). The arrest of meiosis at pachytene due to temperature and prolonged retention of the SC in the cells increases the rate of crossing over and nondisjunction of doubled pole structures. The result of these irregularities is that disjunction of homologous chromosomes does not begin (Simchen et al., 1981; Byers and Goetsch, 1982). These facts throw light on the mechanisms of the anomalies in mei mutants, which arrest meiosis at prophase I. Moreover, the data help to interpret these mutations as mutations that act in meiosis inversely in comparison of desynaptics, which lead to the precocious destruction of the SC.

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INNA N. GOLUBOVSKAYA

4. Blockage of Meiosis at Prophase I and the Phenomenon of

Chromosomal Stickiness in sticky Mutants Consideration of the above-mentioned phenomena has led to the conclusion that the sticky mutations described for different species of plants [ Z . mays (Beadle, 1937); S . cereale (Sosnichina, 1970); Allopecurus (Johnson, 1944); and Collznsia tinctoria (Mehra and Rai, 197211 likely act by blocking meiosis at prophase I and that stickiness of chromosomes is the only phenotypic display. Convincing arguments may be as follows: (1) that the meil and mei2 mutations of P. anserina cytologically affect meiosis, causing stickiness of chromosomes after pachytene (and in the case of the me13 mutation, after diakinesislthus, cytologic phenotypes of these mutations are similar to those we observed in plants homozygous for the sticky mutations; and (2) that stickiness of chromosomes, being a primary meiotic abnormality for sticky mutants, is probably due to chromosomal degradation in meiocytes, which is observed cytologically as pycnosis and pulverization of chromatin. The last phenomenon is typical for both sticky and Mei0.25 mutants (Fig. 2). In oogenesis of different animal species, natural arrest of meiosis is observed. Prophase I, metaphase-anaphase I, metaphase 11, and the female pronucleus are the stages in which reversible arrest of meiosis takes place. In mammals, two blockages of meiosis are possible: the first is at prophase I and the second is a t metaphase I1 (Dyban and Baranov, 1978). A study of causes of parthenogenetic development of egg cells in a n inbred line (LTISu) of mouse (Stevens and Varnum, 1974) has demonstrated that natural blockages of meiosis are under genetic control. Use of biochemical markers has shown that parthenogenesis is a n effect of removing the natural blockage of meiosis in some oocytes. Completion of the first and second divisions of maturation following initiation of cleavage division leads to parthenogenesis and the appearance of ovarian teratomas in mice (Stevens and Varnum, 1974; Epping et al., 1977). The genes described in the following section cause reversible blockage of meiosis. Mutations of these genes lead either to irreversible blockage of meiosis, as was previously described for humans (Cantu et al., 1981) or to the reversal of natural blockage of meiosis, as observed in some ovule cells (LTISv). D. mei GENESOF MAIZE:IMPAIRING THE PAIRING OF HOMOLOGOUS CHROMOSOMES One of the first meiotic mutations was a desynaptic mutation (asynaptic or as; chromosome l), isolated and studied by Beadle

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(1930). A second gene, desynapsis (dy),was also isolated (Nelson and Clary, 1952). One important cytogenetic distinction of these mutants associated with their crossing over should be emphasized, albeit without a detailed description of the cytology of the mutants. The as gene increases the percentage of crossing over in close centromeric and distal regions of chromosomes without changing the total length of the genetic chromosomal map (Dempsey, 1959; Rhoades, 1947; Beadle, 1930; Rhoades and Dempsey, 1949). It was found to be connected with redistribution of chiasmata, mostly located in centromeric and distal regions of chromosomes (Miller, 1963). The analysis of crossing over frequency in the intercalary zone of chromosome 3 has demonstrated some decreased recombination frequency, thus confirming Miller’s cytological observations (Nel, 1979). The dy gene does not influence meiotic recombination frequency, and distribution of chiasmata is unaltered. At prophase I, normally developed SC is seen, but it is disturbed a t pachytene (Maguire, 1978a,b). Univalent chromosomes of this mutant undergo the equational segregation in the first meiotic division. The comparison of these two desynaptic mutations in maize allows us to demonstrate the special function of the maintenance of chiasmata independent of crossing over. Possibly the dy gene is responsible for this function. The only question under discussion is whether the function of cohesiveness of sister chromatids in the first division of meiosis should be attributed to the SC (Maguire, 1978a,b, 1979, 1981, 1982). From this position, it is difficult to explain the reductional disjunction of chromatids in the asynuptic mutant of durum wheat (Martini and Bozzini, 1966; LaCour and WelIs, 1970). Mutations of desynuptic genes of maize (dsyl and dsy2) were induced by chemical mutagens (Golubovskaya and Mashnenkov, 1976; Golubovskaya, 1979a; Fig. 3). Two genes, nonallelic and independent of the as gene, were identified, but have not been assigned to a specific choromosome. Ultrastructural analysis of these two mei-mutants was similar, and the normally developed SC was observed at the pachytene stage of meiosis. The total length of SC per cell in desynaptic mutants did not exceed 50% of the SC length in normal plants (Golubovskaya et al., 1980; Golubovskaya and Khristoljubova, 1985). Centromeres of sister chromatids in both mutants divided at anaphase 11. Two additional nonallelic genes of desynapsis (dsy3 and dsy4) were isolated and were shown to be independent of the dsyl and dsyZ genes. Only rare bivalents were observed in cells, and chromosomes were mainly represented by univalents. The pairing of homologous chromosomes is the main event of meiosis. To provide correct pairing, specific for meiotic cells, the SC

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FIG. 3. Comparison of the character of chromosomal disjunction and the shape of the spindle apparatus in normal plants and two mei mutants (dv and ms28) a t the first meiotic division in maize. (a-d) A normal plant: a pattern of chromosome disjunction and spindle shape is observed in pollen mother cells (PMCs) fixed in Wada fluid. (a) In metaphase I, regularly oriented bivalents and a normally formed spindle apparatus are seen. (b and c) Subsequent stages of chromsome segregation a t anaphasetelophase I. Spindle fibers connecting opposite poles to each other and spindle fibers running from each kinetochore toward a pole are well demosntrated. (e-h) A divergent mei mutants, a characteristic of chromosome disjunction observed in PMCs fixed in Newcomber (e and f ) and in Wada (g and h ) fluids. (e) All 10 bivalents lie randomly in the cell; a disorderly orientation of bivalents a t the metaphase plate takes place. ( f ) Disorderly segregation of chromosomes a t anaphase I. (g and h) Chromosomal spindle fibers run radially from cell center to periphery; spindle fibers connecting two poles t o each other are absent. (i-1) A ms28 mei mutant. (i and j ) Incomplete cytokinesis as a result of delaying disassembly of the spindle fibers (k and 1) after the first meiotic division.

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structure is formed. This structure should function in the cell strictly up to a certain time, and the parameters of time are also controlled by genes (normal alleles of desynaptic genes). If premature SC destruction leads to defects in pairing and to the appearance of univalents in cells, then delaying SC destruction will also be observed to cause chromosomal anomalies, for example, arrest of meiosis.

E. GENES CONTROLLING SEGREGATION

OF

HOMOLOGOUS CHROMOSOMES

The three types of mei mutations with segregation and spindle abnormalities (divergent or dv, ms43, and ms28) were studied. A common characteristic of all three genes is that they do not influence the pairing of homologous chromosomes. 1. The divergent (du) mei Mutant The divergent mutation isolated by Clark (1940) was shown to be responsible for determining the divergent shape of the spindle. Attempts to map the du gene with the help of B-A translocations have been made (Curtis, 19831, and the six arms of maize chromosomes lS, 5L, 5S, 6S, 7L, and 9 s are excluded. Analysis of meiotic stages in homozygotes for the dv gene demonstrated the absence of ordered orientation of the homologous chromosomes between opposite poles at metaphase I (Fig. 3e and f ) , perhaps due t o partial or completely blocked metakinesis. The defect in the shape and function of the spindle apparatus has been assumed to be in the du mutants. It seems there is no normal two-pole spindle apparatus in the du mutants: a central spindle apparatus is absent and only chromosomal spindle fibers run radially from the center to the periphery of the cell (Fig. 3; compare a-d and g and h). Thus, the dv gene impairs the spindle structure. 2. T h e ms43 Mutant The ms43 gene, located in the short arm of chromosome 8, is the second mei gene that induces irregular disjunction of homologous chromosomes (Golubovskaya, 1979, see Fig. 5; Golubovskaya and Distanova, 1986). In spite of detailed cytologic analysis of meiosis in this mutant, mechanisms of the irregular segregation of chromosomes remain unclear. Analysis of the spindle morphology in the first meiotic division and analysis of a birefringence of the spindle fibers did not

168

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show distinctive differences in either criteria in the ms43 mutant and in normal plants (Shamina et al., 1981; Shamina and Gruzdev, 1987). Careful analysis of the spindle structure at the first and second divisions of meiosis in the ms43 mutants, fixed in Wada fluid, has shown that the ms43 gene mainly disturbs the orientation of two spindles relative to each other in the second division of meiosis, and the orientation of spindle fibrils within the spindle apparatus. As a result, the two spindles in mutant plants dislocated each other under oblique angles or formed a joint (common) spindle (Fig. 4A and B). A type of mei mutation that changes the orientation of the cell division spindle during meiosis has been described in the potato (parallel spindle or ps gene) (Mok and Peloquin, 1975) and in the sugar beet (Maluta, 1980). In these two cases, the two spindle apparatuses a t the second division of meiosis are situated parallel to each other instead of their normal location a t an angle of 60". In maize, the situation is just the opposite: the normal parallel disposition of the two spindle apparatuses is changed such that the spindles are at different angles.

3. The ms28 mei Mutant The ms28 mei gene is the third gene that effects abnormal segregation of chromosomes in meiosis in maize (Golubovskaya and Sitnikova, 1980); the gene maps to the short arm of chromosome 1(Golubovskaya, 1987). In ms28 homozygous mutants, all anomalies of chromosomal segregation and cytokinesis are dependent upon a delay of spindle fibril depolymerization, which in normal meiosis begins promptly after anaphse I (Fig. 3i-1). In Aspergillus, a recessive mutation, benA33, has been demonstrated to induce hyperstabilization of microtubules in meiotic spindles and results in structural changes of p-tubulin (Oakley and Morris, 1981). In this mutant, similar to the ms28 mutant, the spindle assembly process occurs normally but the disassembly process is either eliminated o r strongly delayed. elongate (el), CONTROLLING THE F. A mei MUTATION,

SECOND DIVISIONOF MEIOSIS

This mutation was obtained and studied by Rhoades (1956). The el gene has been mapped a t the long arm of chromosome 8 (Curtis, 1983). The genetic effect of the mei gene is manifested in the appearance of numerous unreduced egg cells, resulting in presentation of the polyploids in the progeny of the mutant plants. Cytogenetic investigations of microsporogenesis in mutant plants have demonstrated that

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169

the el gene has no influence either on chromosomal pairing or on crossing over (Rhoades and Dempsey, 1966; Nel, 1975). It is probable that the el gene is efficient at the second division of meiosis and causes defective cytokinesis. An additional effect of the gene is an elongated process of chromosome spiralization in meiosis. Alexander (1957) utilized the el gene to obtain polyploids in maize. A recessive mutation with the same cytologic effect (the elongated chromosomes) was obtained in diploid S. cereale (Sosnichina, 1970).

G . A mei MUTATION, variable (ua), IMPAIRING CYTOKINESIS A recessive ua mutation was isolated by Emerson and was investigated and mapped at chromosome 7 by Beadle (1932a). Cytologic analysis of mutant plants has revealed abnormalities in cytokinesis, accompanying both the first and the second divisions. This mutation has different degrees of expression and penetrance of sterility; therefore, portions of the anthers are thrown out and some of the pollen grains can be differentiated. The percentage of sterile pollen grains in different plants ranges from 30 to 90%. In Drosophila a recessive autosomal mutation with the same cytologic and genetic phenotypes was induced by ethyl methanesulfonate (EMS) and was called (ms 2R)(Romrell et al., 19721.

H. mez MUTATIONS ( p a m l AND p a m 2 ) CAUSING NONSPECIFIC MULTIPLE ABNORMALITIES OF MEIOSIS This type of mutation has not been previously discussed in the literature (but see Golubovskaya and Mashnenkov, 1976; Golubovskaya, 1979a). It seems that all events of meiosis are impaired. There are several characteristics of meiosis in the mutant plants: 1. Among uninuclear cells, there are multinuclear microsporocytes (cenocytes) (14%) that contain 2-14 nuclei and divide autonomously and synchronously; there are also polyploid cells (6%). 2. Meiosis, as a rule, begins in all cells synchronously and proceeds asynchronously after pachytene. 3. The degradation of chromatin by pycnosis and lysis is observed in uninuclear and multinuclear cells (19.2%). 4. In different cells of the same anther, meiosis occurs according to its own program: normal cells comprise 39% of the total number; in other cells, either desynapsis is observed, meiosis is substituted for mitosis, or cytokinesis does not occur.

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INNA N. GOLUBOVSKAYA

FIG. 4. The shape of the spindle apparatus a t the first (A) and second (B) meiotic divisions in the ms43 mei mutant (Wada fluid). (A) The first meiotic division. (a-g) Stepwise stages of chromosomal disjunction through metaphase I to interkinesis. Normal spindle apparatus shapes are seen (compare Fig. 3a-d). (B) The second meiotic division (a-i). (a and b) Abnormal segregation of chromosomes in the PMCs without cytokinesis after the first meiotic division. (c) Tripolar spindle in the cell. (d and e) Lack of normal parallel orientation of the two spindle apparatuses a t metaphase 11. (f-h) The two spindle apparatuses approach each other (f and g) and complete fusion of two spindles (h). (i) A two-nucleus cell as a result of meiotic abnormalities.

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171

FIG. 4B.

It is clear that the pam-type mutations concern genetic systems that regulate meiosis. Activation of these systems proceeds in premiotic mitoses, otherwise it is difficult to explain the existence of multinuclear masses of cenocytes at prophase I. The pam mutant looks as if different types of mei genes (ameiotic, desynaptic, ms43, and ua) are joined together in the same plant. This

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173

list could be supplemented with a mu gene, which causes the appearance of cenocytes and was described in barley (Smith, 1942). Only amphidiploids of distant hybrids, for example, Triticale, have the same pattern (but more weak) of meiosis with different types of abnormal meiocytes as p a m mutants. I. PRECOCIOUS POSTMEIOTIC MITOSIS: A polymitotic ( P O ) GENEWITH A SERIESOF ALLELES( m s 6 AND m s 4 ) Beadle (1928) obtained a recessive mutation, termed polymitotic, and located it on chromosome 6. The ms6 gene, allelic to the PO gene, was found among genes that determine nuclear male serility in maize (Beadle, 1928, 1933). Other alleles of the PO gene (rns4)were induced with N-nitrosomethylurea. In mutants homozygous for the PO gene, two postmeiotic mitoses that normally accompany pollen grain formation apparently begin prematurely at the tetrad stage and thus the replication of DNA needed for these process does not occur. It is this event that is responsible for the pattern of anomalies in the meiosis of mutants (Fig. 5 ) and for the formation of sterile pollen grains. Three allelic mutations of the polymitotic gene in maize, isolated in different independent genetic experiments, have indicated that the transition from meiosis to postmeiotic mitosis is controlled by a single gene with a series of alleles. This type of mei mutation is interesting not only in itself but also as a mutation forestalling a sequence of events in meiosis. Probably, a great role in establishing meiosis as a developmental process is played by the mutation providing the precocious events.

J. SUMMARY The types of mei mutants currently known in maize are summarized in Table 1. Similar data for rice mei mutants have been obtained

FIG. 5. A comparison of normal processes of the first postmeiotic mitosis and processes in the polymitotic rnei mutant. (a-d) The first postmeiotic mitosis during the process of maturation of a pollen grain through prophase to telophase in maize. (e-h) The abnormalities caused by the polyrnitotic gene. It is clear that the precocious postmeiotic mitosis induced a t the tetrad stage of meiosis is a primary function of the P O gene. Abnormal segregation of chromosomes during precocious postmeiotic mitosis is a result of a delayed or omitted reduplication of chromosomes. The first postmeiotic mitosis is complete with the formation of octads ( i ) . If the process is incomplete, tetrads separate into definite sporads (i and k).

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INNA N. GOLUBOVSKAYA

(Kitada et al., 1983). Among 281 mutants with nuclear male sterility induced with NMM, 25 appeared to be meiotic; 19 of them influence the pairing of homologs and the other 6 rnei mutations have a defect in cytokinesis and disjunction of homologous chromosomes. The meiotic abnormalities of the MM23 mutant in rice are similar to that of the ms28 maize mutant, and the MM24 mutation in rice causes multiple defects of meiosis like the pam mutation in maize. Let us estimate the percentage of mei mutations among the total number of mutations causing nuclear male sterility. In our experiment the total number of induced visible mutations was 856. Among them, 93 are mutations with nuclear male or both male and female sterility. Of lines with nuclear male sterility, 52 have been studied cytologically, and 9 mutations appeared to be meiotic. If its is considered that half of the mutations of the total number are allelic, the percentage of mutations causing male sterility of the total number of visible mutations is 10.8% (46.6: 428); hence the percentage of the mei mutations among the nuclear ms mutations is 19.3%(9 :46.5) (Mashnenkov and Golubovskaya, 1980). These data are similar to the data obtained for D. melanogaster. Among 600 induced male-sterile mutations, 50 were cytologically analyzed, with 20% consisting of mei mutations (Lifschytz and Meyer, 1977).

Ill. Cytogenetic Evidence for the Genetic Control of Meiosis

A. THE CHOICEOF mei MUTATIONS AS EXPERIMENTAL MODELS The mei-mutants can be utilized to obtain experimental proof that meiosis is under genetic control. Double mei mutants were chosen as an experimental model. To prove that there is independent action of the mei genes that control different cytogenetic events during meiosis, it was necessary to utilize such mutants. Independent expression of the two genes is expected in double mei mutants; i.e., in the progeny of self-pollinated double heterozygotes, the four classes of meiotic patterns were expected in a ratio of 9 :3 :3 : 1.The pattern of last class of plants, comprising 1/ 16 and represented by double homozygotes, was easily revealed cytologically. In conforming with the idea of sequential switching on of genes, meiosis should follow the pathway of the gene switched on first in double homozygous mutants. In genetic terms, this would mean that each mutation is epistatic to the ones aligned next in the ordered series

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175

of the mei mutations. In this case the segregation ratio in Fz progeny will be 9 : 4 : 3 instead of 9 : 3 : 3 : 1,because double mei mutants (1l16) will be added to the class of plants homozygous for the epistatic gene. It is important that the mei genes used should have been located on different chromosomes; in the opposite case, the linkage effect will superimpose on a n interaction of these mei genes. Moreover, mei genes specific for meiosis during microsporogenesis or macrosporogenesis should have been excluded from model experiments. The phenomenon of unisexual male sterility caused by rnei genes (ms28 and ms43, for example) is very mysterious for maize, a monoecious diclinous species. It is important to exclude a specific action of these genes on meiosis in microsporogenesis.

The tassel seed and meiotic Genes The problem of excluding mei genes having specific actions in microsporogenesis can be solved genetically with the help of mutations that determine the sex of the maize flowers. Mutations transforming sex are widely used in genetic analyses of Drosophila to reveal the specific action of lethal genes in males or females (Belote and Lucchesi, 1980). The transformer (tra) and intersex (ix) mutations, which transform genotypic females (XX) into phenotypic males, were used to prove a specific action of the SR (sex ratio) factor on genotypes having Y chromosomes (Miyamoto and Oishi, 1975). For our purposes, the tassel seed mutations, which cause in maize the transformation of male flowers into female flowers, resulting in the development of ovules and silks in the tassel instead of the anthers, are the most efficient. If nongenetic factors are responsible for male sterility without female sterility in some rnei mutants of maize, a n expression of the mez mutations in female meiosis of double homozygotes (tslts meilmez) and the segregation of sterile plants among the tassel seed forms in Fz progeny is expected. If male sterility is determined by a specific action of rnei genes during meiosis in microsporogenesis or of ms genes during gametogenesis after meiosis, all tassel ears of the double mutants will be fertile and the class of tassel seed plants in F2 progeny will be exclusively fertile. In our experiments, the tassel seed (ts2) mutation, the mei afd mutation (responsible for the substitution of the first meiotic division into mitosis and resulting in bisexual sterility), and mutations causing unisexual male sterility were used. The last class is formed by two types of mutations: two mei mutations that induce irregular chromosome disjunction (ms28 and ms43), and the ms2 mutation, which has

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INNA N. GOLUBOVSKAYA

no effect on meiosis but induces abnormalities during the process of pollen grain maturation. The mei ufd mutation and the ms2 mutation were used as controls for ms28 and ms43. Results of genetic analysis for two characteristics-the tassel morphology and sterility in F2 progeny of these combinations of crossesare represented in Table 5. The results of segregation in F2 progeny from crosses between tassel seed mutants and three mei mutants (ufd, ms28, and ms43) correspond with the expected ratio 9 :3 : 3 : 1,and are distinguishable from the pattern of segregation of a cross between ts2 and ms2 mutants. In this last cross, the segregation is 9 :4 : 3, because the tassel seed plants among the F2 progeny are represented by the fertile plants. Thus, the specific action of ms43 and ms28 genes is refuted by these data, and these rnei genes could be used in cytologic experiments despite the fact that both of them caused unisexual male sterility.

B. INDEPENDENT ACTIONOF GENESCONTROLLING DIFFERENT MEIOTICEVENTS For this experiment, we chose two mei genes, the desynuptic gene (us) responsible for the pairing of homologous chromosomes and the TABLE 5 Expression of ms28 and ms43 on the Background of Homoeotic tassel seed Mutation" Fz segregationb

Cross ms43lms43 x ts2lts2 ts2lts2 x ms43lt ms28lms28 x ts2lts2 ts2lts2 x ms281+ ts2Its2 x afdl+ ts2lts2 x afdl+ ms2lms2 x ts2lts2

Genotype of F1 as defined by selfing

a

b

a

b

Total

ts2/+ ms43/+ t s 2 I t ms43/+ ts2l+ t / + ts2/+ ms28/+ ts2/+ ms28/+ ts2I+ +I+ ts2/+ +lafd ts2/+ +I+

61 39 67 64 60 23 72 110

5 3 0 9 21 0 19 0

13 10 23 14 25 5 26

2 4 0 3 5 0 9 0

81 56 90 90 111 28 118 136

ts2/+ +lafd ts2/+ ms2/+

104 181

31 63

25 79

7 0

167 323

Normal

ts2lts2

18

a An experiment with the ms2 gene was made in another year for comparison with the rest of the mei mutants, because the afd mutant was used as a control. a, Fertile plants; b, sterile plants.

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ms43 gene controlling the segregation of chromosomes. A cross was made in the next scheme: P ms43/ms43 x 6 a s / + . In the progeny of double heterozygotes, segregation into four types of patterns of meiosis was obtained: 32 plants had normal meiosis, 7 had the as type, 8 had the ms43 type, and 6 had the as ms43 type, joining the characteristics of the two mei mutations. This ratio corresponds to the expected 9 :3 :3 :1 ratio (x2 = 3.69, df = 3, 0.5 > p > 0.25). The cytology of meiosis in double homozygotes as compared with the original single mutants (Table 6) has shown that the first division of meiosis proceeds like that of the desynaptic (as)mutant, with impaired homologous pairing. The effect of the second mei gene is added and defects of chromosomal segregation and cytokinesis are observed. The independent action of two mei genes responsible for different events of meiosis is proved by the data. This means that the as gene product is not needed for the occurrence of the events controlling the ms43 gene.

C. CONSEQUENTACTIVATION OF rnei GENESIN MEIOSIS The stepwise activation of mei genes, from the initial steps of meiosis through the steps involved in the pairing of homologous chromosomes, has been analyzed. The primary elementary events of this complex process have become clear as a result of investigations and comparisons of different types of mei mutations. These events are shown in

TABLE 6 Meiotic Lesions in Single and Double mei Mutants Affecting Pairing and Segregation of Chromosomes Metaphase I: Number of bivalents (cell %) Genotype aslas +I+

+I+ ms43lms43

aslasms43Jms431

10

9-7

6-4

3-1

0

Total cells

3.8 75.0 0

1.8 24.5 0

16.2 0.5 6.8

49.9 0 27.2

28.3 0 66.0

106 171 132

Meiosis 11: Type of tetrads (cell %) ~

With With Cells at Normal micronucleus macronucleus Polyads M1, A l , A2 aslas +I+

+ I + ms43lms43 aslas ms43lms43

26.5 26.2 9.3

26.5 9.5 22.7

47.0 0 8.0

0 29.9 34.8

0 34.3 25.2

530 695 337

178

,,,

INNA N.GOLUBOVSKAYA

PAIRING OF HOMOLOGOUS CHROMOSOMES

+ Initiation

of meiosis

am, Pam

I

of + Alignment homologs

?

+

as

Initiation of SC formation

+

afd

Completion of SC formation

+

Destruction of formed SC

dSY

FIG. 6 . Elementary steps at the start of meiosis and the rnei genes controlling these steps. This sequence and the genes have been established by investigating different types of mei mutants and by comparing them to each other.

Fig. 6. This logical scheme enables determination of the time of action of the known mei genes controlling the initial events of meiosis: am++ as+ + afd+-+dsy’

If this gene chain is correct, then the ameiotic mutation is epistatic over the asynaptic gene and the others following it, the asynaptic gene is epistatic over the afd gene and the others following it, and the afd gene is epistatic over the dsy gene. Unfortunately, the asynaptic gene completely blocking the SC formation is absent in mei mutation in maize. There are only three types of mei mutations-the ameiotic, which completely blocks meiosis; the afd, which converts the first meiotic division into mitosis; and the desynaptic mutations (dsy and as), which cause premature destruction of the SC and, as a result, defective pairing of homologous chromosomes. Results of epistatic interactions between three pairs of mei genes are demonstrated in Table 7. 1. Interactions between am and afd As expected, four plant genotypes in a ratio of 1 : 1 : 1 : 1 were obtained in the progeny from the first cross shown in Table 7 (A). Only double heterozygotes were of interest to us, because, in their selfing, progeny could be segregated from the double homozygotes. There are three types of meiotic patterns among the progeny: plants with normal meiosis, with the a m type of meiosis, and with meiosis of the afd type in a ratio of 9 : 4 : 3 (x2 = 0.44,df = 2, 0.9 > p > 0.75).

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TABLE 7 Epistatic Relationships of mei Genes Controlling the Sequential Steps of Meiosis

Fz segregation" Cross, genotype, and ratio in the F2 progeny as defined by selfing

Number of family

(A) afdl+ x aml+ 114 +I+ +I+ 1 / 4 a m / + +I+ 114 + I + afdl+ 114 a r n l f afdl+

(B) dsyl+ x afdl+

114 +I+ +I+ 114 +I+ dsyl+ 1 / 4 a f d l + +I+ 114 a f d l f d s y l i

(C) aslas x afdl+ 112 + I + ad+ 112 afdl+ asl+

3 1

Fertility a

b

Type of meiosis C

d

46 0 34 15 52 14 140 116 - 9:7

46 34 52 140

343 0 84 25 346 106 179 141 - 9:7

60 84 64 179

am 0 15 0 64 9:4:3 afd 0 0 23 77

96 33 105 75 - 9:7

26 50

9:4:3 afd 0 24 9:4:3

Total afd 0 0 14 52

46 49 66 256

dSY 0 25 0 64

60 109 89 320

as

7 13

-

33 87

a, Fertile plants; b, sterile plants; c, normal meiosis; d, abnormal meiosis.

This means that the a m gene has recessive epistasis over the afd gene, or, in other words, the presence of the am+ gene product is necessary for initiating the events controlled by the afd+ gene. The am+ and afd+ genes control the same meiotic pathway, although the am+ gene switches on earlier than the ufd' gene during meiosis; i.e., the afd+ gene or its product remains inactive until the product of the am+ gene appears.

2. The Pair afd-dsy The same result has been obtained with other pairs of mei genes [see Table 7 (B)]. Segregation of the meiotic pattern among the progeny of selfed double heterozygotes corresponds well with the expected ratio of 9 : 4 : 3, with epistasis of the afd gene over the dsy gene. To determine whether the interaction of the two last types of mei mutations is a rule and not an exception, the other desynaptic gene (as)

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was paired with the afd gene. In this case [Table 7 (C)], afd is epistatic over as (x2 = 0.5, df = 2 , p > 0.3). These data confirm the previous conclusions concerning the necessity of the afd' product action for realizing the events controlled by desynuptic genes. Analysis of epistatic groups of genes is widely used in genetics for establishing genetic pathways. The common genetic pathway controls complex cellular processes such as sensitivity to radiation and to chemical mutagens. Analysis of double mus mutants in yeast has allowed establishment of three independent genetic pathways for repair of damage caused by UV irradiation and X rays (Haynes, 1978). Genetic analysis of double mutants and isolation of the epistatic groups has shown that there are concrete stages in the genetic regulation of the DNA repair process in yeast (Game and Cox, 1972). The characteristics of interaction of mei mutations with mus mutations in D . melanogaster have been studied (Smith, 1976; Smith et al., 1980). Recombination-defective mutants are known at the X chromosomal loci in Drosophila (mei9 and mei41, for example). In addition, by decreasing the frequency and altering the distribution of exchanges along the chromosome length during meiosis in females, these mutations produce elevated frequencies of nondisjunction of all chromosome pairs. The recombination deficiency and strong mutagen sensitivity of the mei41 and mei9 genes suggest that these loci specify the function essential for both meiotic recombination and the repair of damage caused by mutagens in somatic cells. A demonstration of allelic relationships between mus loci and the mei41 locus suggests the previous conclusion. Analysis of UV sensitivity in the double mutant, with joined mei9 and mei41 loci, has shown that mei9 and mei41 interact synergistically, with increasing UV sensitivity, and suggest that these loci function in alternative pathways for the repair of UV-induced damage. These genetic data have been proved biochemically. It seems that the mei9 locus reduces the rate of both repair replication and pyrimidine dimer excision and that it is defective in a postincision step of excision repair. The mei41 locus is defective in a pathway of postreplication repair (Baker et al., 1976a; Boyd et al., 1976; Smith et al., 1980; Boyd and Setlow, 1976). In the double mutant mei9 m ~ s 2 0 5two ~ , loci interacting epistatically are defective in the process of excision of pyrimidine dimers. The use of double mutants proved to be effective in genetic control of different steps in yeast cell division cycle pathways. Isolation of epistatic groups of cdc genes, genes having additive interactions and independent abilities in S. cereuzsiue, helped to determine the genetic

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code for control of different cell cycle steps (Hartwell, 19781, and to understand the function of cdc genes in the process of chromosomal disjunction (Murray and Szostack, 1987) and the function of centromeric regions (Cumberledge and Carbon, 1987). In our study, double mei mutants were used for investigation of the basic characteristics of genic control during meiosis in higher plants (Golubovskaya, 1979a; Golubovskaya and Urbach, 1980; Golubovskaya et al., 1980). Direct cytogenetic evidence for a postulated chain of rnei genes that activates the initial steps of meiosis and for a conforming, sequential switching off the mei genes has been obtained from studies of double mei mutants in maize. D. HIERARCHY AMONG mei GENES If the highest hiearchical level in mei genes occurs then genic function is required for initiation of several independent meiotic events simultaneously. The occurrence of a gene with such a function in meiosis would require u priori that the locus be involved in both pairing and segregation of homologous chromosomes and also in the control of other meiotic steps. The wild allele of the ameiotic gene, known from the mei genes in maize, could be proposed for this role. The experimental data presented in Table 7 demonstrate that the am+ locus is necessary for initiation and completion of homologous chromosome pairing. To prove that the am+ gene is capable of inducing the disjunction of chromosomal pairs, the double mutants amlam ms43lms43 were used. The ms43 gene is required for disjunction of chromosomes. The disjunction defect in ms43 mutants is manifested as impaired spindle orientation in meiotic cells (Fig. 4B). In addition, it has been demonstrated that the disjunction defect of the ms43 gene shows up independently from the desynuptic gene (as) in double rnei mutants (aslas ms43lms43). If the function of the a m gene is required for meiotic disjunction, it should be expected that a m is epistatic over ms43. In the opposite case, the defect of disjunction induced by ms43 on the background of ameiotic mitosis should be expected, because in ameiotic mitoses the tissue and cell barriers are removed. The results of segregation in F2 progeny in the relative pattern of meiosis seen experimentally clearly demonstrate the epistatic effect of the a m gene over ms43. The segregation of 127 fertile plants and 87 sterile plants corresopnds to the ratio of 9 : 7 (x2 = 0.85). Cytologic analyses of 105 out of 214 plants have indicated that 57 plants have normal meiosis, 27 have meiosis of the a m type, and 19 have meiosis of

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the ms43 type. This segregation is in accordance with the ratio of 9 : 4 :3 (x2 = 0.4). A detailed study of meiosis in ameiotic plants did not reveal any anomalies of chromosome disjunction. All 29 am segregants underwent meiosis typical for ameiotic mutants (Fig. 7). There were no anomalies in the segregation of chromosomes in 574 cells counted at the metaphase-anaphase stages. Therefore, the ms43 mutations are not expressed in ameiotic mitosis. In other words, the am+ gene product is necessary for realizing the events controlled by the ms43 gene. Hence, the wild allele ameiotic function is required for both pairing and disjunction of homologous chromosomes. The use of tusseZ seed mutations and analysis of the nature of the interaction of the mei genes (especially am and ms43) poses a problem

FIG. 7. The nature of cell division of the ameiotic mutants segregated in the Fz progeny in the cross rns43ims43 x a m / + . (a-e) Stages of mitotic cycle in the ameiotic plant, There are neither abnormalities of chromosome segregation nor of spindle shape.

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regarding the specific action of mei genes. Experiments with the tassel seed gene have shown that the mei genes ms28 and ms43 appear to fail to specify function as required for male meiosis. Female sterility occurred only when the male flowers of the tassel were transformed to female (a situation in double mutants tslts mslms).This gives rise to a question about the occurrence of specific genes for meiosis. On the one hand, establishment of the specific meiotic SC cytological structure and the genes controlling formation and function of the SC is most important as evidence for specificity of the genic system in meiosis. On the other hand, in Drosophila and yeast it has been proved that the same recombinant-defective genes are required for both meiotic recombination and DNA repair processes in somatic cells (Baker et al., 1976a; Gatti et al., 1980; Zimmering and Thompson, 1987). Whether the overlapping function exists exclusively for recombination-defective mutations and how the disjunction-defective genes behave are unclear. Till now it was unknown whether the same genes causing defective disjunction of homologous chromosomes could be effective in both meiosis and mitosis if tissue and cell barriers are removed. Double mutants with the am gene can help to answer these questions because the ameiotic gene induces mitotic division in the meiocytes, i.e., the cells are committed to the meiotic process throughout whole pathways of biochemical events. Hence, the ameiotic gene removes specific tissue and cell barriers and there are many possibilities for the expression of mei genes that control disjunction of chromosomes, if the genes have, in part, a common function in meiosis and mitosis. Epistasis of the arneiotic gene over the ms43 gene is evidence that meiotic chromosme disjunction is controlled by specific mei genes that are not effective in mitotic division. In conclusion, one more revision could be accomplished by mutation, i.e., controlling the assembly of the spindle apparatus in meiosis (the divergent gene in maize, for example). IV. Speculation about the Possible Pathways of Genetic Control of Meiosis

It follows from cytological analyses that the general events of meiosis from beginning to end are controlled by a group of epistatic genes, acting in sequence, for example, t o realize the events of the second division of meiosis. An early example supporting this is the dyad gene of Datura stramonzum (Satina and Blakeslee, 1935). Among the genes initiating the key steps of meiosis, the gene that switches on

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the previous steps should be epistatic over the subsequent genes oppressed. This is the principle of cascade regulation of meiosis. A great number of nonallelic cytologically similar mei mutants influencing the same event of meiosis have been identified, indicative of different pathways controlling the same meiotic event. It is interesting t o investigate independent pathways controlling the same meiotic event, for example, pairing of homologous chromosomes. The nullisomic state of two different chromosomes (3B and 5B) in common wheat (Kempanna and Riley, 1962; Riley and Law, 1965) is a case where the 3B chromosome is responsible for chromosomal pairing in such a way that the loss of 3B leads to the desynaptic effect in meiosis. The effect of 5B in meiosis of hexaploid wheat is determined by the Ph gene located on the long arm of this chromosome. The P h gene is responsible for permitting the homoeologous chromosome pairing. The deficiency of the P h locus (in nullisomics, 5B) or the presence of the recessiveph allele in the homozygous state permits the pairing of both homologous and homoeologous chromosomes and, as a result, some multivalents are observed in cells at metaphase I. In double nullisomic plants without 3B and 5B, two types of pollen mother cells are simultaneously present in one anther. There are pollen mother cells (PMCs) with only univalent chromosomes (16%; nullisomic state for 3B is realized) and the PMC with multivalent chromosome configurations (84%; nullisomic effect for 5B chromosome is realized). It is possible that these cytological data can be interpreted as an independent effect of two mei genes controlling the same event in meiosis and acting at the same time during meiosis. At present, it is known that the P h gene does not have any effect on the ability of chromosomes to associate and form the SC. The Ph gene effect could control the rate of pairing (Gillies, 1987). The desynuptic gene, as described above, determines the time of formation of the SC at prophase of meiosis. From studies of double mei mutants it is possible to assign such genes to specific epistatic groups in order to understand the genetic basis of meiotic pathways in higher plants and to indicate the groups of genes controlling the steps of similar pathways in meiotic cells. Initial studies of double mei mutant combinations in maize indicate that the range of interaction observed with cdc yeast mutants and n u s and recombination-deficiency mutants in Drosophila is also present in higher plant system (Hartwell, 1978; Murray, 1987; Smith et al., 1980; Baker et al., 1976b). In direct experiments with double mei mutants, the chain of mei genes a m afd dsy (as) controlling the initial steps of meiosis has

-

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been established. The independent action of the desynaptic (as) and ms43 genes impairing chromosomal segregation and the epistatic interaction between the ameiotic and ms43 genes have also been proved in cytogenetic experiments. This suggests that the as and ms43 genes are involved in parallel chains of rnei genes and, hence, the a m gene simultaneously switches on the two gene chains in meiosis: a m + afd -----+

+ dsy

(as) ms43

Thus, the occurrence of both cascade and fan actions of mei genes in switching on meiosis is likely in effect. V. Conclusion: Theoretical and Applied Aspects of Meiosis Genetics

In addition to “canonical meiosis,” as charcterized for maize and other organisms, variations of meiosis have been observed among virtually all sexually reproducing organisms. There are species with achiasmatic meiosis; species with semimeiosis (Davison, 1984) involving only the first meiotic division and omitting the pairing of homologous chromosomes and crossing over; holocentric species in which the first division of meiosis is equational, i.e., centromeres of sister chromatids divide at the first meiotic division; apomictic plant species in which various transformations from regular meiosis to mitosis take place; and parthenogenetic animal species in which different mechanisms of blockage of chromosome pairing and crossing over occur (see in detail John and Lewis, 1965; Raikov, 1975; Noda, 1975; Oakley and Morris, 1981; Gustafsson, 1946; Rasmussen, 1977; Rasmussen and Holm, 1981). The various types of meiotic processes are interesting in several aspects: (1) in understanding different modes of meiotic division; (2) in the possibilities of several variants of meiosis existing as a result of fixation of meiotic mutations in the evolutionary process; and (3) in the opportunity for predicting the existence of new types of mei mutants. The general peculiarities of mei mutants include the formation of gametes with unreduced diploid chromosome sets, gametes with noncrossover chromosomes, and gametes with aneuploid chromosome sets. All these characters can be used successfully for applied genetic programs. The ability of the elongate mei mutants of maize to produce unreduced egg cells was used to provide tetraploid maize (Alexander, 1957). The same ability of parallel spindle mei mutants in sugar beet and potato was used to obtain polyploids and fertile hybrids in distant crosses (Maluta, 1980; Peloquin, 1983).

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The ability of mei mutants to produce aneuploid gametes can be used to obtain a series of aneuploid stocks in different plant species. Thus, a series of aneuploids in common wheat was obtained by Sears with the help of 3B nullisomics, which have a desynuptic effect in meiosis. The series of aneuploids can be used to obtain addition, substitution, and translocation lines, if they are involved in interpsecies and intergeneric crosses. The mei mutants can be used for genetic construction of apomictic plants. The mei mutants may play a significant role in the creation of a polyploid series of chromosomal sets in the evolution of angiosperms, because they are not only donors of urneduced gametes, but are also testers for the unreduced gametes and they have the ability to give fertile progeny in cases of fertilization of egg cells with unreduced pollen cells.

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