intragenic suppression at the locus in ascobolusintragenic suppression at the b2 locus in ascobolus...

INTRAGENIC SUPPRESSION AT THE b2 LOCUS IN ASCOBOLUS ZMMERSUS. I. IDENTIFICATION OF THREE

DISTINCT GROUPS OF SUPPRESSION’

G. LEBLON AND N. PAQUETTE2

Laboratoire de Gbnne’tique, Centre &Orsay, Umuersitb Paris X I 91405, Orsay, France

Manuscript received July 18, 1977 Revised copy received April 17, 1978

ABSTRACT

A reversion study of EMS- or ICRl70-induced ascospore color mutants in Ascobolus immersus is reported. Twenty-three new intragenic suppressors were isolated within the b2 locus. These are localized within three distinct groups on a genetic fine-structure map containing 21 identifiable sites. The pattern of reversion is discussed according t o the known specificity of EMS and ICR170 in Ascobolus immersus.

INTRAGENIC suppression of a primary mutation occurs when a second mutation, appearing in the gene at a site distinct from the original mutation site,

completely or partially restores the function that has been lost in the original mutant. Genetic and biochemical studies have revealed the existence of various types of intragenic suppression.

The primary and secondary mutations may be base substitution missense mutations as observed by HELINSKI and YANOFSKY (1963) in the E. coli tryptophan synthetase A protein. The active polypeptide chain in the suppressed mutants appears to differ from the wild type by two amino acid residues. The suppression is thought to result from restoration of some tertiary configuration that alters the activity of the enzyme’s catalytic site. In effect, the deleterious effects of the primary mutational site are somehow compensated by the second substitution mutation.

By studying reversions of a nonsense mutant of E . coli alkaline phosphatase, WEIGERT and GAREN (1965) have shown that intragenic suppression involving two substitution mutations can also occur within a single codon. In this case, the primary nonsense codon is changed to a missense codon following a base-pair substitution at one of the two remaining sites of the codon. Extensive data are available concerning intracodon suppression from studies on the tryptophan synthetase A protein of E. coli (YAKOFSKY, ITO and HORN 1966) and on the structural gene for bacteriophage T4 head protein (STRETTON, &LAN and BRENNER 1966). This type of suppression is particularly well documented in

Supported in part by a grant f” the “Laboratoire associe n”86 du Centre National de la Recherche Scieutifique. * Present address, Department de Biologie, Facult6 des Sciences et de GBnie, Universite Laval, Quebec GIK 7P4,

Prov. Quebec, Canada.

Genetics 90: 475-488 November, 1978.

476 G. LEBLON AND N. PAQUETTE

yeast where intracodon suppression of missense and nonsense mutants at the iso-I -cytochrome C gene has been confirmed by amino acid sequence analysis (SHERMAN and STEWART 1971).

Intragenic suppression of a mutant in the initiating codon has also been observed in yeast; biochemical analysis indicates that reverse mutations in this case can introduce new initiator sites (SHERMAN and STEWART 1971).

The primary and secondary mutations can be addition/deletion frameshift mutations. These mutations shift the reading frame by adding (+ phase-shift mutants) or deleting (- phase-shift mutants) one base pair in the nucleotide sequence of the gene, thus generating a completely new amino acid sequence in the protein beyond the mutated site. Combinations of (+) and (-) frameshift mutations can lead to suppression by restoration of the correct reading frame, except for the region between the two mutation sites. This was extensively studied in the rZZ B gene of bacteriophage T4 with proflavin-induced mutants (CRICK et al. 1961 ) , and subsequently confirmed by biochemical analysis in the T4 lysozyme (STREISINGER et al. 1966) and in the tryptophan synthetase A gene of E . coli (BRAMMAR, BERGER and YANOFSKY 1967). Yeast frameshift mutants were identified by sequence changes in iso-l-cytochrome C (STEWART and SHERMAN 1974).

Intragenic suppression of a frameshift mutation may rarely occur by a base- pair substitution at a second site in the gene when the second mutational lesion affects signals for chain initiation o r chain termination (SARABHAI and BRENNER 1967; RIYASATY and ATKINS 1968).

Complete evidence for intragenic suppression in eukaryotes is very difficult to obtain. Since biochemical studies such as the few cited above cannot usually be performed in higher organisms, evidence must be obtained by genetic means. The suppressor mutation obtained in reversion experiments must be isolated and then reassociated with the original mutation to confirm intragenic suppression. This requires a resolving power sufficient to detect recombination between the suppressor and the primary mutational sites.

Evidence for intragenic suppression at the mtr locus of Neurospora crassa has been obtained, but selective methods for the recovery of mutant phenotype from backcrosses of revertants to wild type have failed to yield the suppressor (BRINK, KARIYA and STADLER 1969).

At present a few definitive reports of intragenic suppression in an eukaryote, including relief of polarity by internal suppressors and inactivation by secondary mutation of the suppressor activity of a mutant tRNA, have been published (SHERMAN and STEWART 1971 ; HAWTHORNE and LEUPOLD 1974; CULBERTSON et a1 1977).

Evidence for intragenic suppression has been obtained at the b2 ascospore pigmentation locus of the ascomycete Ascobolus immersus (LEBLON 1972b). We were able to perform a genetic test for second-site reversion by virtue of an efficient system for the selection of rare mutant apigmented spores among many nonmutant pigmented spores, and the high frequency of recombination at the b2

GROUPS OF INTRAGENIC SUPPRESSION 477

locus. Five secondary intragenic mu tations were recovered from revertants with restored pigmentation. All of the secondary mutations are in close proximity to the primary lesion that they suppress and produce by themselves spores devoid of pigment like the primary mutation.

The ascospore color system in Ascobolus is also useful for the study of intragenic recombination (RIZET et al. 1960), e.g., the gene conversion spectrum, defined by the frequencies of each kind of conversion event (2+6m, 6+2m, 3+5m, 5+3m), can be investigated by crossing the mutant with wild type and determining the frequencies of spore color segregations other than 4:4.

This series of papers intends to present all data now available on intragenic suppression in the b2 ascospore pigmentation locus, to report some conclusive experiments regarding this kind of suppression and to discuss questions raised by findings on the conversion spectrum of the intragenic suppressors. In this first paper, the search for new intragenic suppressors and their isolation are reported, together with an attempt to localize them with respect to each other in the b2 locus.

MATERIALS A N D METHODS

Media: Culture, germinating and crossing media have been described by RIZET et al. (19601, LISSOUBA et a2. (1962) and Yu SUN (1964).

Mutants used: Strains of each mating type carrying the primary mutations, AO, EO and PO (previously designated ICRb2 A38, EMSb2 47E, and ICRb2 A20, respectively) were employed in this study. These mutations abolish spore pigmentation. Secondary intragenic mutations were recovered from revertants with restored spore pigmentation by appropriate crosses. The secondary mutations used in these experiments were A i , A2, A3 and A4, which suppress the AO mutation, and E l , which suppresses the EO mutation. Tertiary mutations that suppress the secondary mutations were also selected. These partially or fully restore spore pigmentation in the presence of the secondary lesion.

Reversion experiments and isolation of revertants: The procedures have been derived from those described by STADLER, TOWE and ROSSIGNOL (1970) and LEBLON (1972a). Mutagenesis was carried out a t room temperature. Mycelia growing on the surface of sheets of cellophane were treated in 0.1 M phosphate buffer (Na,HPO,, KH,PO,) a t pH7 with the chemical mutagens 2 methoxy-6 chloro-9- [3- (ethyl-2-chloroethyl) amino propylaminol acridine dihydrochloride (ICRI 70), N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS). Treatment time was 130 min for ICR170, 15 min for NTG and 300 min for EMS. The ICR170 treatment was carried out in darkness.

Previous results (LEBLON 1972b) concerning the reversion of ICR170- and EMS-induced mutants were considered in choosing the mutagens used in this study. ICR170 was used to induce suppressors of the EMS-induced secondary mutant A2 and EMS to induce suppressors of the ICRl70-induced primary mutant FO and secondary mutant El . An attempt was made to induce tertiary mutations of AO with NTG.

After treatment, the material was rinsed once in buffer and placed on a fresh crossing plate. At the same time, untreated mycelia from a strain of opposite mating type carrying the same mutation was inoculated at the edge of the plate. The plates were then incubated at 23" until maturation. Ascospore color revertants were detected by scanning collecting plates of asci for octads containing four colored spores (revertant asci). Single spores from revertant asci were isolated from independent collecting plates, i.e., corresponding to different crossing plates, germi- nated and subsequently analyzed.

478 G . LEBLON AND N. PAQUETTE

Genetic analysis and screening of revertants: Some basic principles for the analysis of revertant asci have already been published (LEBLON 197213). In the present study, the following screening methods were used:

(1) Revertants with a phenotype distinct from the wild type: Revertants with an ascospore phenotype distinct from the dark-brown coloration of ascospores produced by the wild-type strain are suspected of originating from a suppression event. Therefore, they were crossed to the wild- type strain to confirm their phenotype and to determine whether or not the suppressor mutation occurred at the original locus.

(2) Revertants with the wild-type phenotype: Revertants showing an ascospore coloration indistinguishable from the dark-brown pigmentation of wild type spores might originate from back-mutation or suppression. On the former hypothesis the conversion spectrum, e.g., the frequencies of spore color segregations other than 4:4 observed in the progeny of the backcross, is expected to be identical to the ones established in the cross of the primary mutant to wild type.

On the latter hypothesis, the conversion spectrum characteristic of the secondary mutation is detected (Figure 1). Therefore, any significant difference between the backcross and the control cross is an indication of either intragenic or external suppression. If the two crosses give the same result, revertants are crossed to wild type in order to discriminate. Obviously, intragenic

ASCUS Ascus Ascus

g e n o t y p e p h e n o t y p e g e n o t y p e

su+m su+m

su m su m

s u m su m

su m s u m

2m:6m+ 2 w : 6 c 6su:2su+

m m 0 0 0 0

m+ m+ - 0 m+ m+ 0 -

m+ m+

m m

in m

m m

m+ m+

6m:2m+

0 0 0 0 0 0 0 .

6w:2c

su+m su+m

su+m su+m

su+m su+m

s u m su m

2su: 6 su+

BACK MUTATION INTRAGENIC SUPPRESSION

FIGURE 1 .-Identification of an intragenic suppressor (su) by determining the frequencies of segregations other than 4:4 in the progeny of the backcross of revertant x original mutant (m).

In the case of a back mutation, this cross produces the Same result as in the control cross (wild type x original mutant). In the case of an intragenic suppression, the frequencies of segregations other than 4:4 depend on the conversion spectrum of su. Obviously if the actual conversion spectrum of su is the inverse conversion spectrum of m, the cross wiU produce the same result as in the control cross and we cannot discriminate. w = white; c = colored.


suppressors displaying the primary mutation inverted conversion spectrum cannot be discrimi- nated in this way (Figure 1). However, they may be screened by a third method as follows.

( 3 ) Modified conuersion spectrum of nearby type C mutant crossed to the reuertants: It has been shown previously (LEBLON and ROSSIGNOL 1973; ROSSIGNOL and HAEDENS 1978) that type A or B mutation (mutation yielding only 6:2 conversions with either an excess of 6+:2m or 2+:6m) can modify the conversion spectrum of a nearby type C mutation (mutation produc- ing postmeiotic segregation, PMS, that is 5+:3m and 3+:5m together with 6:2) in such a way that PMS become much less frequent; one can use this property of type A and B mutations by crossing revertants to available nearby type C mutants in order to screen those originating from intragenic suppression and showing a wild-type phenotype. Crossing a revertant arising from intragenic suppression to a type C mutant brings together three heterozygous sites, e.g., A su f -- , the primary mutation (A or B) in cis array with the suppressor and the type C mutant + + C in repulsion. One may expect that at least the primary A or B mutation will modify the conversion spectrum of the type C mutation and particularly diminish PMS segregation frequency. In the case of a back mutation, crossing the revertant to the type C mutant amounts to the same thing as crossing the latter to the wild-type strain (control cross). In such crosses the normal conversion spectrum of the type C tester mutant is detected. Therefore, any change observed in the frequencies of segregations other than 4:4 was ascribed to the presence of a suppressor mutation in the revertant. Back mutation was inferred from the observation of the normal type C conversion spectrum.

The isolation of secondary and tertiary mutations: When a revertant was suspected of originating from intragenic suppression, the primary mutant was sought among the progeny of the cross of revertant by wild type among rare asci showing one or two white spores. These white-spored strains were backcrossed to the primary mutant to determine whether they contained a mutation at the same site. When this was observed as was the case for some of them, the secondary mutation was sought among the others that appeared to recombine infrequently with the primary mutant. These two kinds of white-spored strains are expected to originate by recombination between the primary and the secondary mutation sites.

The suspected secondary mutant strains were characterized as true secondary mutant strains as follows: (1) In the case of revertants with a phenotype distinct from the wild type, pigmented spores from the cross of the suspected secondary mutant and the primary mutant were crossed to wild type. Characterization of the secondary mutation was obtained when four spores of the revertant phenotype were recovered in each ascus besides four spores of the wild-type phenotype. (2 ) In the case of revertants with the wild-type phenotype, suspected secondary mutants were crossed to each other. Absence of recombination was considered a sufficient test for characterization of the secondary mutation. The same method applied to the characterization of tertiary mutations.

RESULTS

Isolating the secondary and tertiary mutants Strains carrying either the primary mutation EO or the secondary mutations

E l , A2, A3 or A4 of the b2 locus were treated either with EMS, ICRl70 or NTG. Ascospore color revertants were detected by scanning collecting plates of asci for octads containing four colored spores. The frequency of revertant asci was determined. Unmutagenized control crosses were run in parallel with these treatments to determine the frequency of spontaneous revertants. For each treatment, a sample of independent revertants was isolated and analysed to determine whether they had resulted from back mutation or suppression.


TABLE 1

Reversion of El, A2, A3, A4 and FO mutations of the b2 locus

Inde- Revcant Inde- pendent Reversions Reversions

Mutation concentration X10-5 asci' l o 5 asci reversion+ tested phenotype phenotype Mutagen and Asci Revertant asci per pendent reversions wild not wild

E l Spontaneous EMS 0.05 M

EMS 0.1 M

EMS 0.15 M

A2 Spontaneous ICR170 20pg ICRl70 40 pg ICR170 60 pg

A3 Spontaneous A4 Spontaneous

NTG 10,20,40 pg FO Spontaneous

EMS 0.05 M

EMS 0.10 M

EMS 0.15 M

- - 1.48 0 1 0 2.34 20 8.55 9 9 3 6 1.53 41) 26.14 24 18 4 14 1.09 9 8.26 3 3 1 2 4.69 8 1.71 4 4 0 4 1.92 11 5.73 9 4 0 4 I .99 12 6.09 6 6 1 5 1.73 21 12.14 7 7 0 7 2.40 10 4.17 5 5 1 4 3.72 14 3.76 11 2 0 2 2.00 11 5.50 7 7 0 7 3.0 0 1 0

50.9 250 4.9 54 38 19 19 36.0 270 7.5 58 26 16 10 23.0 406 17.6 41 28 10 18

-

- - -

* Incomplete revertant asci containing at least three colored spores are included. -/- Independent reversion events are estimated by the number of independent collecting plates

containing revertant asci. Independent collecting plates containing only an incomplete ascus with one, two or three colored spores are included.

The results are given in Table 1. The concentrations used and the corresponding frequencies of revertant asci observed are indicated for each treatment.

Clearly the secondary mutation E l was strongly induced to revert by EMS; among 30 independent revertants analysed, eight proved to be back mutations and 22 originated from tertiary mutations resulting in intragenic suppression; in the latter, the revertant phenotype is characterized by the presence of a broad belt of pigment at the spore equator, while the rest of the spore is faintly pigmented (belted phenotype). One of the tertiary mutations was recovered independent of the secondary mutation site. This tertiary mutation was called E2. The secondary mutation A2 reverted spontaneously. However, the frequency of revertant asci was increased about seven-fold by the mutagen ICRI 70. All the revertants obtained were brown spored. Among the 21 independent revertants analysed, one proved to originate from a back mutation and the others from tertiary mutation. Two tertiary mutations from revertants of spontaneous origin (respectively called A7 and A8) were recovered independent of the secondary site.

The secondary mutation A3 also reverted spontaneously. The revertants showed a brown-spore phenotype. Five independent reversion events were studied. One proved to involve a back mutation, while the others implicated a tertiary mutation.

The secondary mutation A4 reverted spontaneously. The frequency of revertants obtained from NTG-treated mycelia did not increase as a function of the mutagen concentrations used. Accordingly the results of the various mutagenic

GROUPS OF INTRAGENIC SUPPRESSION 48 1

treatments were grouped. The observed frequency of revertants obtained after exposure to NTG (5.5 X is similar to the frequency of spontaneous revertants (3.8 X The nine revertants analysed are pink spored. They all originated from tertiary mutations resulting in intragenic suppression. Two tertiary mutations were recovered independent of the secondary mutation site (A5 and A&).

Reversion of the primary mutation FO was strongly induced by EMS. Various revertant phenotypes were readily distinguishable in the progeny of the treated cross: some revertants showed the dark-brown phenotype of the wild-type spores, others showed the pink phenotype of revertants of A0 and still other revertant asci contained four very faintly pigmented spores that sometimes displayed a somewhat more pigmented area at the equator. Ninety-two revertants obtained from distinct EMS-treated mycelia were analysed, and 47 of these revertants correspond to secondary mutations that restore a spore pigmentation clearly different from that of wild-type spores. Twelve of these secondary mutations were separately recovered (F2, F3, F4, F5, F6, F7, F8, F9, FIO, Fll, F12 and Fl3). The 45 other revertants show a wild-type phenotype. A more elaborate study of a sample of 24 of these wild-type revertants showed that 11 reversion events correspond to back mutations, while 13 involve a secondary mutation. Six of these secondary mutations were separately recovered (Fly Fl4, Fl5, FI6, F17 and Fl8).

Table 2 summarizes the analysis of spontaneous or mutagen-induced revertants of mutations of the b2 locus. Previous results concerning primary mutations A0 and EO (LEBLON 1972b, 1974) are also included in this Table. The following facts must be pointed out: (1) The four secondary mutations (A2, A3, A4 and El), as well as the three primary mutations (AO, EO and PO), can revert by intragenic suppression. (2) Of these at least six can revert by an apparent reversal of the parental mutation (back mutation). ( 3 ) External suppression was not observed. (4) EMS did not induce reversion of the EMS-induced mutation (EO), but it did induce reversion of the three ICR-induced mutations (AO, E l

TABLE 2

Intragenic suppression and back mutation of b2 mutations

b2 mutation A0 A2 A3 A4 EO E l FO

Origin (ICR)' (SP) (EMS) (EMS) (EMS) (ICR) (ICR)

- - 0 22 71 2 - - - 0 8 21 + 8

1 ICRI 70 su 0 16 + 0 1 - 15

0 0 NTG su 0 3. 2 - 0 0

SP su 2 4 4 9 + 6 0 1 0

- EMS 4-

- - - - - - - - - - - - - -

- - - - - -

* The origin of the mutation is indicated in parentheses. t su : Intragenic suppression; + : back mutation.


and FO) . ( 5 ) ICR did not induce reversion of the ICR-induced mutation AO, but it induced reversion of the EMS-induced mutation EO and the spontaneous secondary mutation A2. (6) NTG did not induce reversion of the two EMS-induced mutations. It did induce reversion of the ICK-induced mutation AO.

Table 3 summarizes the main peculiarities of the 28 mutations recovered from revertants of b2 primary or secondary mutations. Based on the three prinlary mutations used (AO, EO and FO), three intragenic groups of mutations capable of intragenic suppression may be recognized (group “A”, group “E” and group ‘‘I?’’).

TABLE 3

Main features of the primary, secondary and tertiary mutations at the b2 locus

Mutation -___-- A0 A1 A2 A3 A4 A5 A6 A7 A8

EO E1 E2

FO F1 F2 F3 F4 P5 F6 F7 F8 F9 FIO FII F12 F13 F14 F15 F16 F17 F18

Origin Parent

Phenotype of the double mutant

parent-suppressor

ICR SP SP EMS EMS SP SP SP SP

EMS ICR EMS

ICR EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS

- A0 A0 A0 A0 A4 A4 A2 A2

- EO E1

- BO PO PO BO FO PO PO PO FO PO FO FO FO FO FO FO PO PO

brown brown brown pink pink pink brown brown

- pink-belted brown-belted

- dark- brown

pink pink pink

pink or belted pink Pink pink Pink pink

pink or belted pink or belted pink or belted dark-brown dark-brown dark-brown dark-brown dark-brown

GROUPS O F INTRAGENIC SUPPRESSION 483

Mapping the mutations The mutations within each group were mapped with respect to each other,

based on the frequencies of recombinant asci observed in the progeny of two- factor crosses (Figures 2 , 3 and 4). The existence of six, three and twelve distinct mutation sites was shown. As one can see, the frequencies of recombinant asci in crosses involving mutations capable of intragenic suppression within a given group are generally lower than those obtained from crosses involving mutations in distinct groups (Table 4). This indicates that the three genetic maps probably do not overlap and that the three groups are localized in distinct regions of the b2 locus. This was partly confirmed by the use of two large deletions that divide the b2 locus into three segments (GIRARD and ROSSIGNOL 1974). Figure 5 shows that groups “F” and “Ey are located within region I, while group “A” is located within region 111. When tested against the reference deletion 138, mutations of group “F” produced frequencies of recombinant asci ten times higher than those obtained with mutations of group “E’. This may suggest that group “E” is closer than group “F” to region 11. -

A8 A7 A5

A4 A2 A3 AI A0 A6

1 .05 (1.74) 0.16 , 0.44 0.46 0.02 > < 3

> < 1.07 < 2.03 (3.86)

< 0.85 0.58 > < >

4 2.61 (6.10) > 4 0.87 D

< 1.51 D

< 2.76 (5.53) *

< FIGURE 2.-Genetic map of group “A”. Crosses between mutants grouped under the same

bracket give no recombinant asci. Numbers below the map are the mean numbers of 6 white: 2 colored asci per thousand asci observed in the progeny of two-factor crosses involving mutants A4, A2, A3, AI, A0 and Ab. The standard errors (x 103) are as follows: A4 x A2 0.43; A4 x A3 0.61; A4 x AI 0.53; A4 x A0 0.47; A4 x A6 1.10; A2 x A3 0.03; A2 x AI 0.24; A2 x A0 0.09; A2 x A6 0.38; A3 x AI 0.08; A3 x A0 0.14; A3 x A6 0.46; AI XAO 0.12; AI x A6 0.18; A0 x A6 0.01.

Mean numbers of 7 white: I colored per thousand asci are given in parentheses. The standard errors (X 103) are as follows: A4 x A2 0.55; A4 x A3 0.84; A4 x AI 0.82; A4 x A0 0.66; A4 x A6 1.40.

3.04 (4.97)


EO E2 El

* > e 0.21 0.82 s

4 1.69

FIGURE 3.-Genetic map of group “E”. Numbers below the map are the mean numbers of 6 white:2 colored asci per thousand asci observed in the progeny of two factor crosses. The standard errors ( x 103) are as follows: EO x E2 0.10; EO x E1 0.19; E2 x E1 0.25.

>

- - - F12

c___ - FI1 F9 F IS F8 F17

F4 F6 F14 F15 F1 FO F 16 ~3 n o ~2 ~ 1 3 ~5

0.08 0.06 ~ ~ 0.59 ~ ~ 0.49 ~ 2 .3 2.11 > < . f < v - - - - - < > < D < 0.39 2 .02 13.92

4 > <

< > <

1.39 8.17 b

2 . 5 0 9.52 >

B ~

8.43 < >

r

< 14.52

- < 28.62

FIGURE 4.-Genetic map of group “F’. Numbers below the map are the mean numbers of 6 white:2 colored asci per 1000 asci observed in the progeny OI two-factor crosses involving mutants F4, F6, F15, FO, F16, F1U and F5. The standard errors (x 103) are as follows: F4 X F6 0.03; F4 x F15 0.15; F4 x FG 0.51; F4 x F16 1.00; F4 x Fi‘O 2.30; F4 x F5 2.59; F6 X F15 0.04; F6 x FO 0.37; F6 x F16 0.72; F6 x F1O 1.32; F6 x F5 7.28; F15 x FO 0.24; F15 X F16 0.67; F15 x F10 0.82; F15 x F5 1.49; FO x Fi‘6 0.22; FO x FZO 1.07; FO X F5 0.97; F16 X FfO 0.94; F16 x F5 0.14; F1O x F5 0.51.

Crosses between mutants grouped under the same bracket give very few or no recombinant asci; in these cases, their relative order has not been determined.


TABLE 4

Results of crosses involving nutations in different groups*

Cross 8w:Oc

FO X El 2079 2002

FO X A0

E l X A0

1924 2000

2777 2098

Mean number Ascus type of 6w:Zc

6w:Zc 4w:4c Zw:6c per I O 3 asci _ _ _ _ _ ~ ~ ____ 175 2 1 80 181 2 0

el 3 0 0 89 171 4 0

65 0 0 22 43 0 0

* For each couple of mutations, two different strains have been used. Data from J.-L. ROSSIGNOL (unpublished results).

As one can see by comparing Table 3 with Figures 2 ,3 and 4 that the intensity of pigmentation of spores produced by double-mutant strains decreases as the relative distance between the two mutated sites increases.

DISCUSSION

Twenty-three new b2 mutations were isolated from ICRl70-or EMS-induced b2 primary or secondary mutations. These mutations were localized within three distinct groups (“A”, “E” and “F”’) on a genetic fine-structure map containing 21 identifiable sites. These groups were localized in distinct regions of the b2 locus. Several facts strongly suggest that the intragenic suppression reported in this study involves frameshift mutations: (1) the spores produced by the intragenic suppressors as single mutants are completely devoid of pig-

I I I I

R E G I O N I \ R E G I O N 1 1 \ R E G I O N 1 1 1 I I

10 , I I

I I I I I

t I

I 188 I I I

I

group gy+ F I

1 I

j grow I A

~

80 2 2 -i >

r

FIGURE 5.-Position of groups “A”, “E” and “F” in the bZ locus as indicated by crosses to deletion mutants 10 or 138. The mean numbers of 6 white:2 colored asci per 1000 asci observed in the progeny of two-factor crosses are reported below the map.

486 G. LEBLON A N D N. PAQUETTE

ment. (2) The sites of suppression map very close to the site of the suppressed mutation in terms of recombinational frequencies. ( 3 ) Second-site mutations can revert as a consequence of compensatory tertiary mutations. (4) The intensity of spore pigmentation of parent suppressor double mutants decreases as the distance between the sites increases. A confirmation of this hypothesis will be reported in the next paper of this series.

The results of the mutagenic treatments performed in the present study are compatible with the specificity and complementarity of action of EMS and ICR170 suspected in Ascobolus immersus (LEBLON 1972b, 1974) in that: (1) EMS induced reversion of ICR-induced suppressors (three mutants) , but did not induce reversion of EMS-induced ones (one mutant), and (2) ICR170 induced reversion of an EMS-induced mutant (one mutant) , but did not induce reversion of an ICR-induced one.

Our interpretation of these observations is that EMS and ICRI 70 each induce a single type of injury to DNA, leading to frameshift mutation and the two types are complementary (for example, addition or deletion of a single base pair) ( LEBLON 1972b).

Although ICR170 is known to induce small additions or deletions in various organisms (for review, see DRAKE 1970), such a specificity has seldom been reported for alkylating agents such as EMS (MALIJNG and DE SERRES 1968). STREISINGER et al. (1966) suggested that frameshift mutations may be the consequence of mispairing errors during the repair of single-strand interruptions in a double-stranded DNA molecule. It is worthwhile to point out with regard to this hypothesis that EMS is known to induce such single-strand breaks directly in DNA (LOVELESS 1966; LAWLEY 1966), probably indirectly like MMS in consequence of repair of alkylated sites (FRIEDBERG and GOLDTHWAIT, 1969; LINDAHL 1976), and now to produce mutations of the addition/deletion type in Ascobolus immersus.

The availability of numerous intragenic suppressor mutations in different regions of a given locus is a particularly advantageous situation for studying genetic processes such as gene conversion. I t can provide a genetic system to investigate the role of map position of a mutation within the locus on the conversion spectrum as well as other factors e.g., the nature of the mutational change. Moreover in crosses such as primary-secondary double mutant to wild-type, the interaction between mutant sites during recombination can be precisely studied.

We are much indebted to J.-L. ROSSIGNOL for helpful discussions and communication of unpublished results. We thank R. E. ESPOSITO for critical reading of the manuscript. Technical assistance by S. LEBILCOT and V. HAEDENS is gratefully acknowledged.

LITERATURE CITED

BRAMMAR, W. J., H. BERGER and C. YANOPSKY, 1967 Altered amino-acid sequences produced by reversion of frameshift mutants of tryptophan synthetase A gene of E. coli. Proc. Natl. Acad. Sci. US. 58: 1499-1506.

GROUPS O F INTRAGENIC SUPPRESSION 48 7

The detection of reverse mutations at the mtr locus in Neurospora and evidence for possible intragenic (second site) suppressor mutations. Genetics 43: 281-290.

CRICK, F. H. C., L. BARNETT, S. BRENNER and R. J. WATTS-TOBIN, 1961 General nature of the genetic code for proteins. Nature (Lond.) 192: 1227-1232.

CULBERTSON, M. R., L. CHARNAS, M. T. JOHNSON and G. R. FINK, 1977 Frameshifts and frameshift suppressors in Sacchrrromyces cereuisicle. Genetics 86 : 745-764.

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