Mol Gen Genet (1981) 184:434-439 © Springer-Verlag 1981

Intragenic Location of an Unstable Insertion Element within Gene b5 of the Fungus Ascobolus immersus B. Decaris Laboratoire de G6n~tique I e t Laboratoire associ6 n°86 au C.N.R.S., Universit6 Paris-Sud, F-91405 Orsay Cedex, France

Summary. A fine structure map of gene b5 has been established in Ascobolus immersus and the unstable mutant site b5-301 (phenotype: ascospore coloration) has been found to map within this gene. This map was constructed using seven b5 mutants induced by ICR 170 and is based on the additivity of recombinant frequencies and confirmed by three point tests. The unstable site 301 is located between the induced sites. In particular, mutant 249 is located to the left of site 301, whereas sites 601 and 754 are located to the right. Previous studies showed that the inducing gene of mutant b5-301 reversions are either closely linked to the b5 locus or within it in certain strains. The study of asci resulting from reciprocal recombination between unstable mutant site and several induced mutant sites showed that neither crossovers located on the left nor the right of site 301, separate the unstable site from the inducing gene. Thus, the inducing gene was found to map within gene b5 as did the inducible site. These results constitute a genetic argument showing the presence of an insertion element. In this case, the insertion structure contains at least the integration site (inducible site) and the inducing gene which allows the excision.

In Ascobolus, it is possible to distinguish unambiguously wildtype reverted meiotic products from the wild-type products resulting from recombination. This fact enabled us to show genetically the existence of an insertion in the gene and to demonstrate that this insertion bears one gene conferring the inducing property. This demonstration first requires the establishment of a very precise map of one gene, and secondly it requires the location of both the unstable site and the inducing gene in this map. Finally, we have to show that the inducing gene and the inducible site are not separable by genetic recombination. This principle is simple, but its realization is only possible if several conditions are met. A reliable genetic map must be established. Wild recombinants resulting from crossovers must be distinguished from those resulting from gene conversion. Moreover, the inducing or non-inducing and the inducible or non-inducible characters of each meiotic product have to be characterized.

Material and Methods Strains

Introduction The study of unstable ascospore color mutants of the fungus Ascobolus immersus enabled us to describe recently a new genetic instability system (Rizet et al. 1979, Decaris et al. 1981). It submits to the similar general functional system described in maize and studied originally by McClintock (see for review Fincham and Sastry 1974). Several unstable mutants affect ascospore color, and revert to wild-type with very high frequencies of revertant asci. These reversions are indistinguishable from back mutations. They occur in the presence of a gene known to induce reversion. In the case of mutant b5-301 (Decaris et al. 1978) we showed that instability results from the interaction of two kinds of genetic elements: 1) The inducible site governs the reversion ability, and 2) the inducing gene is involved in the reversion induction of the inducible site. Several inducing genes which map in different chromosomes (Decaris et al. 1979), are known to act in trans. We observed three different situations: the inducing gene and unstable mutant b5-301 in certain strains may be located on different chromosomes or they may be on the same chromosome in two different positions either, loosely linked or very closely linked to this gene. In the latter case, it has not been possible to separate them by recombination.

0026-8925/81/0184/0434/$01.20

All the strains used in this study belong to the stock 50 of Ascobolus immersus. The general techniques of culture, germination, crossing and spermatization have been described (Rizet et al. 1960 ; Lissouba et al. 1962 ; Lewis and Decaris 1973 ; Decaris et al. 1978). Genetic Markers Used Unstable mutant b5-301 map on the first linkage group (Nicolas et al. 1981) about 20 map units from the centromere. The matingtype locus is closely linked to this centromere. Gene rdl (round spores) is located between b5 and the centromere, about 5 units from b5. Recovery of Induced Mutants in Gene b5 The mutagen ICR170, an acridine, was used to induce mutations (Leblon 1972). ICR170- induced mutants were produced in the following way. The wild-type strains 2 7 1 . 5 - and 271.21 + were selected because the spontaneous mutation frequency among their offspring is exceptionally low (8.3 x 10 -4 estimated on 3 x 104 asci). Mycelia were treated in 30 p~g/ml ICR170 for 130 min at 22 ° C. Each treated mycelium was crossed with wild-type strain. Mutant tetrad frequencies (4wt:4 m) asci were estimated at 1.9 x 10 3 (on 4.2 x 104 observed asci). Among 166 harvested

435 mutants, seven were found to belong to gene b5. (mutants 198, 249, 337, 609, 707, 754 and 782).

Tests Revealing the Presence of [nduc&g Gene Reversions of b5-301 occur only during development of fruitbodies, after fertilization and before the onset of the dikaryotic phase (Decaris et al. 1978). Theis period corresponds to a heterokariotic structure where nuclei from male and female origin are present in the same cytoplasm. The inducing character is only expressed by the female strain. So, in order to know the inducing property of a given strain, it must be used as the female parent. If it is an unstable strain (i.e. both inducing and inducible) it induces reversions of its own nuclei. If this female also induces the male reversions (trans acting action) it means that the male is an inducible strain. The origin of reverted nuclei is revealed by the marker rdl. Results

between two mutant strains bearing a different allele of gene rdl ( m l r d l x m 2 r d l + ) , both types of recombinant asci 2wtrd:2mrd:4mrd ÷ and 2wtrd ÷ :2mrd + :4mrd are obtained. Numbers of these two types are not equal (Table 3). In particular, it is the case in 249rd×609rd + and 249rd+ x 609rd crosses or 249rdx754rd ÷ and 249rd ÷ x754rd crosses. So, disparity between both recombinant asci is not due to a property of any alleles but the consequence of the relative order among both mutants and external marker. Moreover, there is no polarity of the B,F.C. (Table 1). Thus, the disparity is not due to differences in the conversion frequencies of the mutant site. Hence, these results indicate that the major class of recombinant asci resulted from two kinds of events, conversion and reciprocal recombination between both mutant sites, whereas the minor class of recombinant asci was due to conversion alone. Thus, the comparison between the two classes led to the relative gene order: rd-249~509 (or rd-249-754). When all the results are taken into account, no discrepancy appears, confirming the map established on the basis of the additivity of recmnbination frequencies.

Map of Gene b5 Certain mutants are known to be able to modify both the type and frequency of intragenic recombination events and therefore, with such mutants, it is possible to construct an intragenic map. Leblon et al. (1981) have already established that ICR170-induced mutants can be used to establish a reliable map of gene b2 based on the criterion of the additivity of recombinant frequencies. We have used seven ICR170-induced mutants of gene b5 to establish an intragenic map. The conversion spectrum of these mutants was obtained by crossing the mutant strains by the wild-type parents (Table 1). Conversions are only of meiotic type : we observed only 2wt: 6m (2 wild-type spores: 6 white spores) or 6wt:2m asci. The basic frequencies of conversion (B.F.C.) are homogenous and are between 10°/0o and 20°/oo. The dissymmetry coefficient (D.C.) which are the ratio between asci of wild-type conversion and those of mutant type conversion, are higher than 1 for all of these mutants. These particulars confirm that these mutants are really induced by ICR170 since Leblon (1972) showed that these B.F.C. and D.C. are specific to this mutant type. Offsprings of mutant x mutant crosses are shown in Table 2. The frequencies of recombinant asci were used to establish the map showed in Fig. 1. The relative order of sites is confirmed by the following three-point tests: in the offspring of crosses

Table 2. Offspring of crosses between 2 mutants of gene b5. Frequencies of recombinant asci Mutant

249

337

198

7,820 a 6b

249

609

4wt:4m

2wt:6m

6wt:2m

3wt:5m

754

782

5,880 2

9,170 0.9

3 , 0 0 0 11,590 0.3 0.8

4,440 0

7,610 2

25,080 6.7

4,920 30,840 6.3 7.9

9,760 3.8

12,110 2.6

2,980 2.3

3,430 2

337

609

5,120 2.4

16,730 5 6 , 2 5 0 0.1 0.1

707

19,840 0

754

3,200 0.9 7,770 0.5 12,240 0.5

a number of observed asci b frequency of recombinant asci 2wt:6m x 103

Table 1. Conversion spectrum of ICR170 induced mutants of gene b5 Mutants

707

5wt:3m

other asci

BFC ×

10 3

C.D.

198

5,217

68

20

0

i

-

i7 (13-20)

3.4

249

2,723

26

10

0

0

-

13 (9-18)

2.6

337

3,200

34

29

3

1

6 0wt:8m l iwt :7m

19 (15-25)

1.2

609

1,784

16

7

0

0

1 0wt:8m

13 (8-19)

2.3

707

1,280

12

3

0

0

-

754 782

2,006

14

5

0

0

-

12 (6-19) 9 (6-15)

4 2.8

2,706

25

10

0

1

-

13 (9 18)

2.5

B.F.C. : Basic frequency of conversion D.C. : Dissymmetry coefficient: ratio between asci showing conversion toward mutant type on those showing conversion toward wild-type

436 2wt: 6m recornbinanf I~SC/ RL R# 0.73 0.04

- - " --

1.1 0.86 1.17

•

0.25 0.51

17 56 1 2 11 113 7 103 6 18 I 9 6 27

301 I I

2/+9

q '~'J

•I

1~

,I

II

II

~1 I I

t I)

337

707 6.7

30

27 163 7 56 11 66

6.3 3.8

6

23 115 26

2.6 2.42.3

• +

25

31

7

48

2 J

~--

0.9 0.8

~

I

C

3

11

~ 0 . 3 ~ ~'~-" 0.9 ~

5

8

~-- 0.5 ~

6

13

~0.5~

Fig. 1. Location of unstable site 301 on the map of gene bY. Average frequencies × 103 of 2wt:6m asci obtained in the offspring of crosses between 2 mutants. Values of major recombinant asci RR concern asci having the same association between wild recombinant and rdl marker as that of the right mutant. Minor values RL correspond to asci showing the same association between wild recombinant and external marker as that of the left mutant

Table 3. Type and number of observed recombinant asci in offspring of mutant x mutant crosses Crosses 198rd + x 249rd 198rd + x 609rd 198rd + x 754rd 249rd x 337rd + 249rd x 609rd + 249rd + x 609rd 249rd x 707rd ÷ 249rd x 754rd + 249rd ÷ x 754rd 249rd x 782rd ÷ 337rd ÷ x 609rd 337rd* x 754rd 609rd x 782rd + 754rd x782rd ÷

2wtrd: 2mrd: 4mrd + 23 4 11 15 12 53 7 8 117 11 31 48 8 13

249 337 198 782 707 754 609

Observed asci after projection

35,710 36,080 33,200 23,810 20,520 31,580 53,890

Frequencies of 2wt: 6m 103

0.73 0.05 0.51 0.25 1.17 0.88 1.1

(0.5-0.11) (0.3-0.8) (0.09-0.6) (0.7 1.7) (0.6 1,3) (0.8-1,4)

Observed recombinant asci 2wtrd:6m

2wtrd + :6m

56 2 6 1 6 7 11

17 1 27 9 18 103 113

119

14-

--

Mutant

609

7.9

~ 2

Table 4. Observed asci in the offspring of ~ induced mutant rd + x o~ unstable 301 rd crosses and frequencies of observed asci 2wt:6m

2wtrd + : 2mrd + : 4mrd 115 1 3 26 66 18 56 46 20 66 25 7 5 6

Location of Unstable Mutant Site 301 To map unstable genetic sites, it is very important to be able to distinguish wild-type recombinants from wild-type revertants. In Aseobolus, it is easy to distinguish recombinants which appear

as 2wt:6m asci from revertants which appear as 4wt:4m asci. Moreover, it is possible to prevent unstable mutants from reversing by using unstable strains as male with a non-inducing female strain. In this case, the only wild-type products result from recombination. Unstable strains b5-301 rdl were crossed by induced mutant b5-rd ÷ strains which are non-inducing to analyse the recombination frequencies in three-point crosses. Recombination frequencies are given in Table 4. The frequency of 2wt: 6m asci is almost negligible in crosses with mutant 337. This result suggests that mutant 301 site is located near 337 induced mutant site. In all other cases, frequencies of 2wt : 6m asci are higher and the numbers o f both types of 2wt:6m asci are strongly different. For mutant 249, 2wt:6m frequencies are higher than the others. This observation is related to the fact that 249 is the only mutant site localized on the left of 337. Conversely, all mutants located to the right o f mutant 337 yield the major class asci of the type 2wtrd + :6m. All these observations suggest that 301 site is located within gene b5 (Fig. 1). Moreover, it is possible to carry out complementation tests by using double spores which have two different nuclei, and observations of this sort indicated that no complementation exists between unstable mutant 301 and all of the induced b5 mutants (Decaris 1981). In Tables 3 and 2 concerning mutant 337, it is instructive to compare the frequency of recombinant asci among the offspring of unstable 301 x induced mutant crosses and 337 x induced mutant crosses. In all cases, values obtained from mutant 301 are lower than those obtained from mutant 337. Thus, unstable mutant 301 appears to reduce recombination frequencies in crosses involving mutants m a p p e d on either side. A n another aspect of this modification of recombination frequencies is shown by the conversion spectrum o f mutant 301 obtained from crosses between 19 unstable 301 strains and wildtype parents (Table 5). All unstable 301 strains were used as female, allowing the observation o f 8wt:0m asci which reveal reversion events. Post-meiotic segregations (3wt: 5m or 5wt: 3m) were very rare and could correspond to mixed asci. The f e w 0wt:8m segregations obtained in only three crosses probably were either new mutations or projections of immature asci. Moreover, frequencies of 2wt:6m and 6wt:2m asci were low, about 2.10 -3, and the D.C. is close to 1, These values can be compared to those obtained with induced mutants (Table 1). The conversion frequencies of unstable mutant were 10 times lower than those of induced mutants, and their D.C. was also much lower with the exception of mutant 337.

437 Table 5. Conversion spectrum of unstable m u t a n t b5-301 : asci obtained in crosses unstable m u t a n t x wild strain ( 2 7 1 . 5 - or 271.21 + ) Strains

4wt :4m

2wt:6m

6wt:2m

3wt:5m

5wt : 3m

0wt : 8m

8wt:0m

61114+ 1619+ 21+ 28+ 31+ 3437+ 40+ 445062+ 686976+ 77+

1,118 1,990 2,204 832 1,426 1,910 2,116 1,926 2,584 1,428 863 2,628 472 1,640 580 1,584 1,436 3,244 1,464

0 4 0 0 8 2 1 2 2 2 2 4 1 2 0 2 3 0 0

3 4 3 1 1 0 2 4 0 3 2 2 0 0 0 0 0 2 0

0 0 2 0 0 0 0 0 2 0 0 0 0 1 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0 0 0 4 0 0 2 0 0 0 0 0 0 0 0 0 0 0

11 11 9 2 6 17 5 35 1 4 5 43 3 1i 3 1 5 0 1

Total

31,445

35

27

5

0

7

173

2 m

B F C x 10 ~

C.D

3 (0.5-0.8) 4 (2-8)

2 (1 5) 1 (0.03-7) 6 (3 12) 1 (0.1 4)

1 (0.3 4) 3 (I-7) 2 (0.4~4) 3 (I-5) 5 (1 12)

2 (l 5) 2 (0.05-12) 2 (0.4~5) 0 1 (0.1-5) 2 (0.4-6)

1 (0.08-2) 0 2.1 (1.6-2.7)

1.3

Table 6. Analyses of m u t a n t strains belonging to 2wt:6m recombinant asci obtained in 9 induced m u t a n t (non-inducing)x ~ unstable 301 (inducing and inducible) Crosses

Type of the tested m u t a n t strain 1

2

3

4

5

6

7

8

2 tested strain x 4 inducer inducible 301

4wt:4m a 4wt:4m b 2wt:6m c

+ + 0

0 0 0

0 + 0

0 0 0

0 + +

0 0 +

0 + 0

0 0 0

6' tested strain x inducer inducible 301

4wt:4m" 4wt:4m u 2wt: 6m °

+ + 0

+ + 0

+ 0 0

+ 0 0

+ 0 +

+ 0 +

+ 0 0

+ 0 0

4wt: 4m" 4wt: 4m b 2wt : 6m °

0 0 +

0 0 +

0 0 +

0 0 +

0 0 0

0 0 0

0 0 0

0 0 0

301 inducer inducible

301 non inducer inducible

301 non inducer inducible

301 non inducer non inducible

induced mutant inducer

induced mutant non inducer

double mutant inducer

double mutant non inducer

tested strains × induced m u t a n t non inducer Genotype of the tested strain

a 4wt : 4m asci revealing reversions of the female nuciei b 4wt: 4m asci revealing reversions of the male nuclei ° 2wt : 6m asci revealing recombination events between the two m u t a n t sites + and 0 mean that the type of asci are present or not

Location of the Inducing Gene By u s i n g s t r a i n s w h e r e t h e i n d u c i n g g e n e is closely l i n k e d to or w i t h i n gene b5 ( D e c a r i s et al. 1979) it is p o s s i b l e to try to s e p a r a t e t h e i n d u c i n g gene f r o m t h e u n s t a b l e site by genetic r e c o m b i n a t i o n . F o r this p u r p o s e , it is n e c e s s a r y to select crossovers l o c a t e d as close as p o s s i b l e to m u t a n t 301 site by u s i n g i n d u c e d m u t a n t sites as m a r k e r s . T h e r e f o r e , t h e n a t u r e o f t h e r e c o m b i n a t i o n a l e v e n t ( c o n v e r s i o n v.s. c r o s s i n g - o v e r ) u n d e r l y i n g e a c h r e c o m b i n a n t a s c u s m u s t be d e t e r m i n e d .

M u t a n t s 249, 609 a n d 754 were c h o s e n b e c a u s e their sites c o r r e s p o n d e d to t h e very e n d s o f t h e m a r k e d p a r t o f gene b5, a n d b e c a u s e t h e y p r o d u c e t h e h i g h e s t r e c o m b i n a t i o n frequencies. On the other hand, the two unstable strains used are 165.106+rd a n d 1 6 5 . 1 2 9 - r d b e c a u s e we d e m o n s t r a t e d p r e v i o u s l y t h a t their i n d u c i n g gene w a s closely linked to or w i t h i n gene b5. T h a t is to say, all r e c o m b i n a n t asci o r i g i n a t e d f r o m c r o s s e s b e t w e e n a n i n d u c e d m u t a n t strain, w h i c h itself is n e i t h e r i n d u c i n g n o r inducible, a n d t h e u n s t a b l e m u t a n t 301 s t r a i n s ( i n d u c i n g a n d inducible) b e a r i n g m a r k e r rd+. R e c o m b i n a n t asci a r e expected

438 Table 7. Analyses of recombinant asci obtained in crosses rd301m + x rd+301 +m

Mutant

Recombinant asci

Number of asci harvested

Number of asci analysed

Types of asci conversion of 301

conversion of induced mutant

reciprocal exchange

249

2wtrd:2mrd:4mrd + 2wtrd + :2mrd + :4mrd

10 4

6 4

2 (1) 1 (1)

2 (2) 3 (1)

2 0

609

2wtrd :2mrd: 4mrd + 2wtrd + :2mrd ÷ :4mrd

1 10

1 5

0 0

1 (1) 2

0 3

754

2wtrd : 2mrd: 4mrd ÷ 2wtrd + :2mrd + :4mrd

1 18

1 8

1 1 (1)

0 2 (1)

0 5

Numbers in parentheses indicate asci that are the result of other recombination events associated with detected conversion to yield 2 wild-type spores and 6 mutant spores. These latter may be either 301, or induced mutant or double mutant. All of them may or may not be inducing, and may or may not be inducible. In order to reveal mutant ascospore characteristics, each mutant strain was successively used in three different crosses: 1) used as female, it was crossed with unstable 301 male strain (inducing and inducible), 2) used as a male, it was crossed with a 301 inducing and inducible female, and 3) still used as male, it was crossed with an induced mutant strain (non inducible and non inducing). The eight types of possible mutant strains and the expected results in these different crosses are summarized in Table 6. One can see that the results from the three different crosses allow the 8 types of mutant strains to be distinguished without ambiguity. The results of this analysis are given in Table 7. Fifteen recombinant asci result from mutant 301 conversions or induced mutant conversions. Eight of these asci also show reciprocal associated recombinations either between mating-type and rdl loci, or between rdl and b5 loci, or in gene b5 itself between both mutant sites. Ten other asci show one double mutant meiotic product. They belong to the major class of recombinant asci and no associated crossing~over is observed. With regard to the inducing character, all 301 mutant and all double mutant strains are inducing while none induced mutant strains are inducing. The intragenic reciprocal exchange detected in these 10 tetrads really corresponds to crossing-over events. These results allow to map the inducing gene within gene bS. Discussion

Theoretically, two different interpretations can explain the occurrence of the asci listed in Table 7. They may result either from intragenic crossing over or from conversion assuming symmetrical hybrid D N A i,e. on two chromatids. In fact, only the first hypothesis is in agreement with the results for four reasons: a) The disparity between both types of recombinants, associated with the lack of conversion polarity, implies the existence of crossing over in gene b5 (see above), b) The low dissymmetry of conversions in this gene supposes that the major part of hybrid D N A is asymmetric (Rossignol et al. 1978). c) There is another difference between both types of asci: no ascus with a double mutant product is associated with crossing over out of the gene and this is quite understandable if these asci do not result from conversions, d) Finally, in spite of the low number of asci analysed, those with double mutant product are observed as expected in the major class of recombinant asci. Therefore, one can conclude that the events leading to these

asci involved crossovers. We saw that all double mutant products were inducing. That means that the inducing gene was never dissociated from the unstable mutant site whether the crossing over is located to the left (i.e. between 249 and 301) or to the right (i.e. between 301 and 609 or 754). These conclusions are compatible with the idea that the inducing gene and the unstable site 301 are located within gene bS. As a result of the proximity of the inducing and unstable sites, no recombination was observed between these two elements. Moreover, a previous study showed that, in some strains, reversions are most often accompanied by the lost of the inducing factor situated at locus b5 (Decaris et al. 1979). These results constitute the most direct argument for the existence of an insertion element in the unstable site. Hence, the insertion element is at least composed of an integration site (inducible site) and an inducing gene which control the excisions. Such a structure explains the simultaneous loss of both the inducing gene and the reversion site (if the reversions are the consequence of excisions). In other strains, the inducing gene of the reversions of mutant 301 is not always lost simultaneously and in these strains the inducing gene is not located near the unstable site. In these instances, the reversions do not remove the inducing property (Decaris et al. 1979). A comparison between this Ascobolus system and the maize system of McClintock has been made in previous papers (loc. sit.). The inducible site and the inducing gene are respectively analogous to receptors and regulators in maize. From the beginning, McClintock (1951, 1956) supposed the existence of a transposable element. In the case of unstable mutants in maize, the insertion would correspond to the mutant and its excision would be revealed by the phenotypic reversion. The same hypothesis was proposed as a basis for genetic instability in Drosophila (Green 1967, 1977). With the recent development and sophisticated biochemical techniques, insertion elements have been physically documented and chemically characterized in pr0caryotes and in eucaryotes (see for review, Calos and Miller 1980). In particular, in yeast, sequence analyses of unstable mutants 912 and 917 of gene His4 showed the existence of an insertion and its molecular structure (Chaleff and Fink 1980; Roeder and Fink 1980). The results reported in this paper emphasize several different properties of this element. 1) They show that this element may be located between induced mutant sites of gene bS. A similar situation is known in Maize (Nelson 1968) and in Drosophila (Green 1969).

439 2) The effect of unstable m u t a n t b5 301 on recombination reinforces the insertion hypothesis. Similar results were obtained with the unstable m u t a n t s of gene b l and b2 (Decaris et al. 1980). Low frequencies of meiotic conversions a n d low D.C. were also obtained in the case of large deletions (Girard a n d Rossignol 1974; Fogel et al. 1978). The possible existence of a correction system specific to large insertions or deletions (Radding 1978) may explained the type of conversion spectrum. Moreover, the decrease of recombination frequencies may be due to a modification by the insertion of the propagation of meiotic hybrid D N A (Rossignol et al. 1978). 3) The element integrates in gene b5 whether or not the inducing property is expressed. In one case, the inducing gene is m a p p e d in gene b5 and in other cases, it may m a p elsewhere. Does this situation m e a n that this element would be divisible i.e. the inducing gene may be separated from the inducible site? Such a property is u n k n o w n in bacterial elements. A similar situation was found by McClintock in maize (1965). F o r example, the Spm system at least implies two functions which act in trans: a suppressive function of the expression of the structural genes a n d a m u t a t o r function which allows the excision of the receptor element. McClintock's observations imply that these genes may or may not be a part of the Spm element. In yeast, b o t h properties exist but they are not coded by the element located at the unstable m u t a n t site (Roeder et al. 1980). 4) This element codes for a function inducing the reversions. In bacteria, transposons Tn3 (Heffron et al. 1979) Tn5 (Egner a n d Berg 1981) and T n l 0 (Foster et al. 1981) code for one transposase. But for Tn5 a n d T n l 0 at least, transpositions and excisions result from independent events. In Ascobolus, does the inducing function of reversion correspond to an excisase? Do excisions and transpositions arise from independent events? In Ascobolus, these questions c a n n o t presently be answered, but in maize, reversions and transpositions are closely connected in the case of the instability of pvv allele induced by element M p (Greenblatt and Brink 1962; Greenblatt 1968). Therefore, this situation underlines a n i m p o r t a n t difference between bacterial systems and one eucaryotic system. Acknowledgments. This work has been realized in the laboratory of Prof. G. Rizet. I thank him for useful discussions during the course of this study. I wish to thank Dr. T. Leonard and Dr. J.M. Simonet for critical reading of the manuscript. I also thank Mrs A. Gregoire et Mrs. M. Dahuron for their technical assistance. This work was supported by grants from the Universit~ Paris-Sud, from the Centre National de la Recherche Scientifique (L.A. no. 86 and ATP microbiologie 79) and from the Institut National de la Sant6 et de la Recherche M6dicale (ATP no. 78-79-110).

References Calos MP, Miller JH (I980) Transposable elements. Cell 20:579 595 Chaleff DT, Fink GR (1980) Genetic events associated with an insertion mutation in yeast. Cell 21:227 237 Decaris B (1981) Les mutants instables d'Ascobolus immersus: le r61e dans l'instabilit6, de l'insertion et de l'excision d'~16ments transposables. Thesis, University of Paris XI, Orsay Decaris B, Francou F, Kouassi A, Lefort C, Rizet G (1980) Genetic instability in Ascobolus immersus: Modalities of back-mutations, intragenic mapping of unstable sites and unstable insertion. Preliminary biochemical data. Cold Spring Harbor Syrup Quant Biol 45:509 517 Decaris B, Francou F, Lefort C, Rizet G (1978) Unstable ascospore color mutants of Ascobolus immersus. I. Temporal occurrence and modalities of back-mutations. Mol Gen Genet 162:69 8i Decaris B, Lefort C, Francou F, Rizet G (1979). Unstable ascospore color mutants of Ascobolus immersus. II. Determinism and possible mechanisms of gene instability. Mol Gen Genet 170:191 202

Egner C, Berg DE (1981) Excision of transposon Tn5 is dependent on the inverted repeats but not on the transposase function of Tn5. Proc Natl Acad Sci USA 78:459-463 Fincham JRS, Sastry GRK (1974) Controlling elements in maize. Annu Rev Genet 8 : 15-50 Fogel S, Mortimer R, Lusnak K, Tavares F (1978) Meiotic gene conversion. A signal of the basic recombination event in yeast. Cold Spring Harbor Symp Quant Biol 43:I325-1341 Foster TJ, Lundblad V, Hanley-Way S, Halling SM, Kleckner N (1981) Three T n l 0 - Associated excision events: relationship to transposition and role of direct and inverted repeats. Cell 23:215217 Girard J, Rossignol J-L. (1974) The suppression of gene conversion and intragenic crossing-over in Ascobolus immersus. Evidence for modifiers acting in the heterozygous state. Genetics 76:221 242 Green MM (1967) The genetics of a mutable gene at the white locus of Drosophila melanogaster. Genetics 56 : 467-482 Green MM (1969) Mapping a Drosophila melanogaster "controlling element" by interalielic crossing-over. Genetics 61:423 428 Green MM (1977) The case for DNA insertion mutations in Drosophila. In: Burkhari AI et al. (eds) DNA insertion elements, plasmids and episomes. Cold Spring Harbor Laboratory, New York, p 437 Greenblatt IM, Brink RA (1962) Twin mutations in medium variegated pericarp in maize. Genetics 47:489-501 Greenblatt IM (I968) The mechanism of modulator transposition in maize. Genetics 58 : 585 597 Heffron F, McCarthy BJ, Ohtsubo H, Ohtsubo E (1979) DNA sequences analysis of the transposon Tn3: Three genes and three sites involved in transposition of Tn3. Cell 18:1153-1163 Leblon G (1972) Mechanism of gene conversion in Ascobolus irnrnersus. I. Existence of a correlation between the origin of mutants induced by different mutagens and their conversion spectrum. Mol Gen Genet 115 : 36-48 Leblon G, Haedens V, Kalogeropoulos A, Paquette N, Rossignol J-L (1982) Aberrant segregation patterns and gene mappability in Ascobolus immersus. Genet Res Camb (in press) Lewis LA, Decaris B (1974) The induction of apothecial formation in Ascobolus immersus by a spermatization technique. Trans Br Mycol Soc 63 : I97-199 Lissouba P, Mousseau J, Rizet G, Rossignol J-L (1962) Fine structure of genes in the ascomycete Ascobolus immersus. Adv Genet 11 : 343380 McClintock B (1951) Chromosome organization and gene expression Cold Spring Harbor Symp Quant Bioi 16:13-47 McCIintock B (1956) Controlling elements and the gene. Cold Spring Harbor Syrup Quant Biol 21 : 197 216 McClintock B (1965) The control of gene action in maize. Brookhaven Symp Biol 18 : 162-184 Nelson OE (1968) The waxy locus in maize II. The location of the controlling element alleles. Genetics 60:507-524 Nicolas A, Arnaise S, Haedens V, Rossignol J-L (1981) Ascospore mutants and genetic map of Ascobolus immersus stock 28. Gen J Microbiol i25:257-272 Radding CM (1978) The mechanism of conversion of deletions and insertions. Cold Spring Harbor Symp Quant Biol 43:1315-1316 Rizet G, Engelman N, Lefort C, Lissouba P, Mousseau J (1960) Sur un ascomyc6te int6ressant pour l'6tude de certains aspects du probl+me de Ia structure du g6ne. CR Acad Sci Paris 250:2050 2052 Rizet G, Lefort C, Decaris B, Francou F, Kouassi A (1979) Unstable ascospore color mutants of Ascobolus irnmersus. III. Mapping, Temporal occurrences and modalities of back-mutations of 34 new unstable mutants. Mol Gen Genet 173 : 293-303 Roeder GS, Fink GR (1980) DNA rearrangements associated with a transposable element in yeast. Cell 21:239 249 Rossignol J-L, Paquette N, Nicolas A (1978) Aberrant 4:4 asci, disparity in the direction of conversion and frequencies of conversion in Ascobolus immersus. Cold Spring Harbor Syrup Quant Biol 43:1343-1352 C o m m u n i c a t e d by W. Gajewski Received July 25/September 28, 1981