GENETICS OF SORDARZA FZ-MICOLA. VII. GENE CONVERSION

GENETICS OF SORDARZA FZ-MICOLA. VII. GENE CONVERSION AT THE G LOCUS IN INTERALLELIC CROSSES

...... ......

Y. KITAN1 AND L. S. OLIVE

Department of Botany, University of North Carolina, Chapel Hill, N . C. 27514

Received October 9,1968

1 1

HE preceding paper in this series (KITANI and OLIVE 1967) dealt with gene Tconversion in crosses between g heteroalleles and wild type. The investigation of conversion at this locus is continued in the present study of crosses between the various heteroallelic mutants.

MATERIALS A N D M E T H O D S

The following heteroallelic mutants of the g locus were studied: g, (gray-spored), the original hyaline-spored isolates h,, h,, and h,, and the derivative hyaline-spored mutants h,, and hZb. The origin, nature, and complementation relationships of these mutants have been described (KITANI and OLIVE 1967). The linked morphological markers have also been described elsewhere (EL-ANI, OLIVE and KITANI 1961), as has their behavior in relationship to conversion at the g locus (KITANI, OLIVE and EL-ANI 1962; KITANI 1963; KITANI and OLIVE 1967). Linkage relationships of the morphological markers to the g locus are shown in Figure 1. An unlinked marker i, which imparts a slight indigo color to the spore wall, was used in most crosses to distinguish certain types of cmversion asci from those having spindle overlap (“spore slippage”). + mat h +

sp + g , cor s p + h, cor

+ mat g, +’ The crosses studied were: -, where h represents h,, h,,, h,, or hlb,

+ mat g,h, cor + mat g,h, cor , and

S P + + t SP + g, +‘ Aberrant asci were detected by direct microscopic examination, after which they were dis-

sected to determine the genotypes of all germinable spcres. Normal appearing 4 gray:4 hyaline asci resulting from conversion are visually indistinguishable from the background population of 4:4 asci and are therefore excluded from the dissections. This includes normal appearing 4:4 conversion asci containing one or two double mutant (g,h) spores which are visually indistinguishable from normal 4:4 asci because of the epistasis of all h alleles to g,. In addition, a certain combination of conversion events can lead to the production of 4:4 asci that are indistinguishable, even by dissection, from those normally produced.

mat glocus cor 1 1 I

1 Supported by grants AI-04425 and 2TlGM216 from the Public Health Service and GB4998 from the Naitonal Science Foundation. A major part of this research was performed at Columbia University, New York City. Detailed data on all asci analyzed genetically have been deposited with the Editor of Genetics. The following mutants have been deposited with the American Type Culture Collection: sp , mat, cor, mat g,, g, cor.

Genetics 62 : 23-66 May 1969

24 Y. KITANI A N D L. S. OLIVE

A B

FIGURE 2.-Pigmentation test for genotypes of hyaline spores. using tester culture p , ! , A. Dark zones ohtained with p l + h genotype. B. Dark zones lacking with glh genotype.

Segregation of p , can be determined by direct observation of progeny lacking an h allele. hut hyaline progeny suspected of carrying the g, allele must he subjected to further analysis. Two tests were found useful in distinguishing double mutant hyaline spores. the first involving my- celial pigmentation and the second, crosses with tester cultures. The pigmentation test required the hyaline progeny to he paired with a test mycelium of genotype g , t l , r 5 ( I , , , tan-spored X- ray mutant obtained hy KITANI at Kyoto University; r5, restricted growth mutant obtained by treatment with UV and 5-bromouracil, hy Mr. SEYMOUR LEWIS at Columbia University; no linkage among the three mutants). A special testing agar, described in the next paragraph, was used. If the hyaline progeny was gl+h, a darkly pigmented zone appeared between a hetero- karyotic zone formed and the tester mycelium; whereas, no such zone of color appeared when the progeny was a double color mutant (Figure 2). In the crossing test, used on randomly selected hyaline isolates to test the validity of the above method, the hyaline progeny were mated with gray-spored (g,) and wild-type cultures. In the cross with the gray-spored culture, gl+h progeny yielded a small percentage of wild-type spores, while g,h progeny failed to do so. In the cross with wild type, g lh yielded a small percentage of gray spores, while g l + h did not. Both methods were found to be dependable and in agreement with each other.

Modified culture and dissection media described in a previous paper (KITANI and OLIVE 1967) were also used in the present investigation. To prevent bursting of hyaline spores, the following hypertonic mounting solution was used: 10 g sucrose, 5 g glucose, 0.7 g sodium acetate, 100 CC

water. The medium for determining genotypes of hyaline progeny by the pigmentation test (de- vised by MARJORIE C. POLLICE) is composed of the following: 17 g Difco cornmeal agar, 15 g sucrose, 12 g glucose, 2 g yeast extract, 0.1 g crystalline KH2P0,, and 1 1 water.

RESULTS

I. Types of conriersion asci: From the various crosses between g, and the heteroallelic hyaline mutants, 17 visually different ascus types have been obtained, and an eighteenth type was found in a confirming back cross. Likewise, 16 types have been observed in crosses of the double mutant glh, with wild type. The combined total of aberrant ascus types observed in these crosses is 20 (see Figure 3 and Table 1 ) . It was first proposed by WHITEHOUSE (1964) and experimentally supported by KITANI and OLIVE (1967) and EMERSON (1969) that the aberrant ascus types from mutant x wild-type crosses could be explained by various com-

CONVERSION IN INTERALLELIC CROSSES 25

K

a 1 8

*

e

0 I I m n o k p q l r s r t U

FIGURE 3.-Expected ascus phenotypes in interallelic crosses. A-U, from g, x h (repulsion) crosses; a-u, from g,h, x wild type (coupling) cross. (Frequencies in Table 1 .)

o : o . o . o . o .

h n

0 : o . o . o . - E

. . . .

CONVERSION I N INTERALLELIC CROSSES 27

binations of three modes of base correction-mutant to wild type, no correction, or wild type to mutant-in the hybrid DNA of the two involved chromatids (Figure 4a). If one applies to interallelic crosses the concept that base correction events occur independently at the two mutant sites after simultaneous hybrid DNL4 formation in both chromatids, then the variety of aberrant ascus types possible in these crosses could be predicted from the various possible combinations of events shown in Figure 4a. The genotypic results of these combinations of events are shown in Figure 4b. Since the three modes of base correction apply to two different sites (gl and h) in these crosses, the maximum number that will allow for all possible types of conversion asci is ( 3 2 ) 2 = 81, but since the pattern is symmetrical with respect to the two involved chromatids the maximum num-

= 45 (see footnote ber of genotypically different ascus types must be

2). Since a glh double mutant spore is not distinguishable from an h single mutant spore, visually distinguishable ascus types are expected to number only 21 (see Figures 3 and 4b for gl x h crosses and Figures 3 and 4c for g,h x wild type crosses). The only missing ascus type in the actual observations was H (or h), which is expected to be one of the rarest, since the combination of 3 4- :5g and 6 4- : 2h types required to produce H are both rare in mutant x wild type crosses. Furthermore, no visually distinguishable ascus types other than those predicted in Figures 4b and 4c were found. Of the 45 ascus genotypes predicted, 41 have been found.

The results suggest that gene conversion is a two-step process involving (1) hybrid DNA formation between two of four chromatids of a bivalent (for rare exceptions see EMERSON 1969), presumably involving both mutant sites simultaneously (die question of simultaneous participstion of both sites will be discussed later), and (2) nonsimultaneous but interrelated choices in mode of base correction at both sites in a hybrid DNA segment.

11. Total frequencies of aberrant asci: The total frequencies of aberrant asci in crosses between the various g heteroalleles and wild type did not differ significantly from each other (KITANI and OLIVE 1967), regardless of the relative locations of mutant sites in the cistron (refer to pages 44 and Figure 11 for the arrangement of mutant sites and their relative positions). Also, in the crosses between gl and its hyaline heteroalleles in the present study, no significant differences in total frequencies of aberrant asci have been observed (Table 2), nor have there been any significant deviations from the frequencies found in single mutant x wild type crosses. The frequencies observed for the interallelic crosses are as

(392 + 32 2

2 Formulae for all the possible cmversion ascus types and the maximum number of genotypically different ascus types are as follows: (1) ( m n l ) n 2 = all possible conversion ascus types. (2) (mni)n? + ,nz

= maximum number of genotypically different ascus types. m = number of 2

choicas in the m d e of base correction (fixed at 3: mutant + wild type, no correction, wild type + mutant). nl = number of chromatids involved (fixed at 2; formula would be void if more than 2). n2 = number of sites employed in the cross (unlimited, but 2 in these studies). In both formulae. the region of hybrid DNA formation in the locus is fixed at 1; otherwise the formulae would be void.

\ C h r o m a - I A

B correction

t i d 3 ( + ) \ I Substitution

Substitution

*+-m 6f: 2 m

ct -m

Substitution

Restoration

5+ :3m

N o r m a I 4+:4m

Substitution

B

no correct i on

5 t : 3 m

Res tor at i on

Aber ran f

4 + : 4 m

3 -t :5m

Sub st i t u t i o n

c l Re +-m st or a t i on I Normal

4+: 4 m Res tor a t i on

3 $:5m

Restoration

2 t: 6 m

h 5+:5a(r)l 2+:6a . . . , .

3+:5g(r) i 2+: 69

A f r )

S

Q

! -

6 t : 29

t : 3 h ( r )

R

L 5+:3g(s) N.4t:49(

5+:3g(r) A. 4t:4g 3+:5g(s) 5+:3g(r) A.4+:4g 3+:5g(s)

J D E

6 t : 2 g 5+:3g(s) N.4+4g(s) 6+:2g 5+:3g(s) N.4t:4~(s)

K F C 5+:3g(r) A.4t:4g 3-k:5g(s) 5+:3q(r) A.4+:49 3+:5g(s) I I 6+: 2 g 5t:3g(s) N,4t:4g(s) 6 t : 2 g 5+:3g(s) N.4+4g(s) I C In

N.4t:+(r)I 3 t :Sd r ) ZC: 6 g 1N+t:4q(r) 3+:5g(r) 2+:6g


I + I A f r l

I

I

I I I I 6 + : 2 h

6+:2g 5+:3g(r) N4t*g(rl 6+ :2g 5t:3g(r1

5+:3g(s) A. 4+:44 3+:5g(r) 5+:3g(s) A.4t:4g

2+: 6 9 t(4t:4g(s) 3+:5g(s A.4C: 4

t p h q l

n h b jd N.4+:4g(s) 3+:5g(s)

N.4t:4g(rl

3+:5g(r)

2+:6g

I

d

fk -

N4+Wrl

5+:3g(s) A.W.49 3+:5g(r) 5+3g(s) A..&49 3+:5g(r)

r I d fk f

5f:3g(s) A,4t;4g 3+:5g(r) 5+:3g(s) A.W.49 3t:5g(r)

\1.W.4g(s) 3+:5g(s) 2+: 6 9 M.44g(s) 3+:5g(s) 2+:69

A B C (SI f r )

m

i

e

gc

N . 4 + : 4 h ( r )

6 t : 29 5+:3g(r) N4+:4g(r)

5+:3g(s) A.4+4g 3+:5g(r)

N,4+4g(s) 3+:5g(s) 2+ : 6 g 3+:5 h ( r )

6 t : 2 9 5+:3g(r) N.4$.4g(r)

5+:3g(s) A.4$:49 3+:5g(r)

C N.&.4a(s) 3+:5a(s) 2+:6a

2+:6 h

6+: 2 g 5+:3g(r) N.WAg(r)

5+:3g(s) A.4+:49 3t:5g(r)

FIGURE 4.-Ascus genotypes resulting from various combinations of base correction modes. (For Figures 4b and 4c, refer to Figure 3 for ascus phenotypes). Figure 4a. Mutant x wild type crosses (6+:2m and 2+: 6m types are substitution-restoration and restoration-substitution combinations, respectively). Figure 4b. Interallelic crosses: repulsion. Figure 4c. Interallelic cross: coupling.

follows: gl X h,, 1/577; g1 x h,, 1/470; g , x h,, 1/395, and g, x h4b, 11487. In these crosses conversion ascus type E, which has a normal appearing segregation of 4 gray: 4 hyaline spores, could not be included among the aberrant asci, since it is visually indistinguishable from phenotypically similar, normally produced asci. Therefore, the real frequencies for all conversion asci must be somewhat greater than the values given. Using as an index the e-type:i-type ratio (the m- type is unrecognizable here) from the cross glh, x wild type (the nature of which is not essentially different from that of mutant x mutant crosses, but in which e is equal to E in the combination of fundamental ascus types for gl and h and is visually distinguishable; see footnote 3), one may conclude from the observed proportion of e-type asci that the absence of this group, and therefore of E asci, from the data would not drastically affect the results (not more than 26% in the most affected case; see Table 4 and footnote 3).

3 In the combination of restoration base correction, no correction, and substitution base correction at the g , site of both chromatids 2 and 3, e type is equivalent to M type (as arranged in Table 5 ) , and the ratio of e:i is considered equivalent to M:I. Therefore, the frequency of E could not be


TABLE 2

Total frequencies of conversion asci in various crosses and proportions of normal 4 f : 4h types among these*

Sum of all ascus types Number of aberrant other than asci obtained

normal N o m ” Normal Normal Frequency of conversion 4+ : 4 h 4 f : 411 4+ ; 4h 4+ ; 4h asci, one in total of:

Crass percent percent excluded included Observed Estimated

h2 h x + 91.9 8.1 96 104.5-f 478 439 h x g, 90.8 9.2 99 109 577 <577

h x g, 95.2 +.8 100 105 . . . h,,, h x + 96.4 3.6 97 1oo.q 458 441

. .

h:, h x + 95.2 4.8 100 105.lt 434 41 3 h x g , 41.1 58.9 99 241 470 <470

h X + 93.8 6.2 53 56.5t 43 8 41 1 h, S,h x g, 84.0 16.0 43 57.w 529 444

h x g, 40.5 59.5 62 153 395 <395 g,h x + 40.6 59.4 41 101 . . . . . .

h x g, h4b h x + 82.5 17.5 47 57.0t 463 382

52.6 47.4 80 153 487 <487

* Real values in interallelic crosses are higher because E asci are indistinguishable from normal

t Estimated. asci.

The relatively small variation in frequencies of aberrant asci among interallelic crosses and the insignificant deviations of these frequencies from those observed in mutant x wild type crosses suggest that hybrid DNA formation at the g locus in both involved chromatids is a single event, even when two mutant sites are involved in the crosses. This might be extended to consider gene conversion as involving a single event of hybrid DNA formation for the whole cistron rather than an additive combination of individual events for the various sites.

directly correlated with the frequency of e. However, the M:E ratio could be estimated inter- mediately by means of a computation. The M: (I fE) ratio in the normal 4+:4h group is not observable because of the loss of E, but the equivalents of the ratio in other ascus types are available (e.g., G:C in 3+:5h, Figure 4b). Also, the actual M:I ratio (1:0.7, Table 4) is available, thus enabling one to obtain the M:E equivalents from the M:(I+E) equivalents. Two available M:(I+E) equivalents are the G:C ratio (1:2.1) among 3+:5h asci, and the ratio of the sum of all ascus types that correspond to M, and the sum of all ascus types that correspond to I and E (1:l.l) in the pool of all fundamental ascus types for the h site, exclusive of normal 4+:4h and 2+:6h. The M:E equivalents expected from the above two M:(I+E) equivalents are 1:1.4 (i.e., 2.1-0.7 = 1.4) and 1:0.4 (i.e., 1.1-0.7 = 0.4), respectively. Since the latter sample is larger and less specific than the former, the real M:E ratio is probably closer to the latter. Figure 4c could be reorganized to correspond with Figure 4b with regard to restoration-substitution relationships at the g1 site, but at the expense of equality in arrangement of fundamental ascus types for the g, site. The process of reorganization includes the exchange in positions of A(s) with C(r) and A(r) with C ( s ) and the relocation of all ascus types to match the altered locations of the fundamental ascus types for the g, site.


This idea will be subsequently discussed in reference to the polaron hypothesis of the RIZET group.

111. Asymmetrical distribution of Ra and Rp classes among aberrant ascus types with respect to base correction behauior of the involved chromatids: All aberrant asci were relegated to two classes with regard to the absence (Ra) or presence (Rp) of recombination of closely linked markers flanking the g locus, the marker mat being proximal to the g locus and the marker cor distal to it. All asci belong- ing to 5+:3h, normal 4+:4h and 3+:5h groups, relative to the hyaline site only, are subdivided into two subclasses each, namely, Ra-1 with crossing over absent in the marked region, Ra-2 with apparent double crossing over flanking the g locus, Rp-1 with a single crossover proximal to the g locus, and Rp-2 with a single crossover distal to g (see KITANI and OLIVE 1967, Figure 2). The relationships of recombination classes and subclasses to ascus types and the origin of these ascus types with respect to mode of base correction in the two involved chromatids are shown in Figure 5 .

All aberrant asci were placed in three groups with regard to combinations of modes of base correction in the two participating chromatids (refer to Figure 5 ) . Group 1 is called the restoration group, in which the h site on chromatid 2 has undergone the restoration type of 'base correction (i.e., to its original s t a t e i n this case to hyaline) or failed to correct, while the h site on chromatid 3 also

(SI

A or a (SI

Ra-2 i Ra-2 i

Rp-l Rp-l *-

+-m

6 + : 2 m m a l g Lows cor

Ro

B or b no cor rec t ion

I

5+:3 m ( r ) mat g locus t o r

Ra- I

5 +: 3m (S I 1 Aberrant 4+:4m

R a

I Normal 4 + : 4 m ( s ) C or c

C or c ( r )

+-m

N o r m o l 4 + : 4 m ( r )

Ra-l mat g locus cor

R P 2 :I:

Ra-l , ,

RP-2 :I; 2 + : 6 m

Ra . Rp

FIGURE 5.-Origin of recombination classes and subclasses. (Capital letters for mode of base correction for g1 site; small letters for h site.)

32 Y. KITANI AND L. S. OLIVE

TABLE 3

Asymmetries between classes Ra and R p (recombination of outside markers absent or present, respectively) and their subclasses*

TABLE 3a

Restoration group+

P-value Cross Total Ra Rp Ra:Rp 1:l 1.4:l 1.8:l

X + 43 25 18 1.4:1 >.e0 >.95 >.30

with normal 4+:4h 37 22 15 1.5:l >.20 >.SO >.50 x g1withoutnormal4+:4h 31 17 14 1.2:l >.50 >.50 >.20

h*

normal 4+ :4h alone 6 5 1 5.O:l . . . . . .

X + 25 18 7 2.6:l >.02 >.IO >.50

h,, with normal 4+:4h 35 24 11 2.2:l >.02 >.20 >.50 x g, withoutnormal4+:4h 32 22 10 2.2:l >.02 >.20 >.50

normal 4+:4h alone 3 2 1 2.0:l . . . . . .

X + 64 46 18 ' 2.6:l < . O l >.02 >.e0

with normal 4+:4h 133 107 26 4.1:l g.01 Q.01 g.01 x glwithoutnorma14+:4h 55 38 17 2.2:l r . 0 1 >.IO >.30

normal 4+:4h alone 78 69 9 7.7:l g.01 g.01 g.01

h,

X + 32 19 13 1.5:l >.20 >.90 >.50

h4 with normal 4+:4h 75 65 10 6.5:l g.01 g.01 g.01 x g, without normal 4+:4h 28 23 5 4.6:l <.Ol =.Ol r . 0 5

normal 4+:4h alone 47 42 5 8.4:l g.01 g.01 g.01

45 29 16 1.8:l >.05 >.50 >.95

g1h4 with normal 4+:4h 49 43 6 7.2:l g.01 g.01 g.01 x + without normal 4+:4h 19 13 6 2.2:l >.IO >.30 r . 7 0

x g1

normal 4+:4h alone 30 30 0 W : l . . . . . .

X + 20 12 8 1.5:l >.30 >.90 >.lo

h4b with normal 4+:4h 77 62 15 4.1:l g.01 g.01 g.01 x g1withoutnormal4+:4h 38 26 12 2.2:l r . 0 2 >.20 >.50

normal 4+:4h alone 39 36 3 12.O:l . . . . . .

h x + and g,h, x g, crosses pooled 229 149 80 1.86:l <.Ol >.02 =.SO 66 42 24 1.8:l >.02 >.30 >.go g, x +

underwent the restoration type of base correction (to wild type in this case) or failed to correct. (Aberrant 4:4 asci with no correction on either chromatid are placed in group 3 . ) Thus group 1 is comprised of 5+:3h, normal 4+:4h and 3+: 5h asci in which one or both chromatids underwent restoration base correc-

CONVERSION I N I N T E R A L L E L I C CROSSES 33 TABLE 3b

Substitution group+ ~~ ~~

P-value Cross Total Ra Rp Ra:Rp 1:1 1:1.4

X + 15 10 5 2.0:l r . 2 0 r . 0 5

with normal 4+:4h 29 20 9 2.2:l >.20 >.02

normal 4+:4h alone 4 2 2 1:l.O . . . .

X + 11 4 7 1:1.8 . . . .

h2

x g , without normal 4+:4h 25 18 7 2.7:l >.02 <.01

with normal 4+:4h 29 11 18 1:1.6 >.02 r . 2 0 x g , without normal 4+ :4h 27 10 17 1:1.7 >.IO >.50

h,,

normal 4f :4h alone 2 1 1 1:l.O . . . .

X + 19 10 9 1.1:l >.80 >.50

with normal 4+:4h 76 15 61 1:4.1 g.01 g.01

normal 4+:4h alone 64 9 55 1:6.1 g.01 g.01

h3

x g, without normal 4+:4h 12 6 6 1:l.O . . . .

X + 15 5 10 1:2.0 ~ . 0 5 ~ . 2 0

h* with normal 4+:4h 54 5 49 1:9.8 g.01 g.01 x g, without normal 4+:4h 10 5 5 1:l.O

normal 4+:4h alone 44 0 44 l : W

x g1 17 9 8 1.1:l >.80 >.50

g1h4 with normal 4+:4h 33 2 31 1:15.5 g.01 g.01 x + without normal 4+:4h 3 1 2 1:2.0

normal 4+ :4h alone 30 1 29 1:29.0

X + 10 5 5 1:l.O . . . .

with normal 4+ :4h 52 9 43 1:4.8 g.01 g.01 x g, without normal 4+:4h 19 8 11 1:1.4 >.30 >.95

h4b

normal 4+ :4h alone 33 1 32 1:32.0 . . . .

h x + and g,h, x g, crosses pooled 87 43 44 1:1.03 >.30 >.IO g1 x + 52 23 29 1:1.3 >.30 >.70

tion. This group corresponds to the upper right three squares of Figure 4b-c. Group 2 is referred to as the substitution group, in which the h site on chromatid 2 underwent the substitution type of base correction (to wild type on this chromatid) or did not correct, and the h site on chromatid 3 underwent substitution base correction (to hyaline on this chromatid) or did not correct, but with correction occurring on at least one chromatid. In other words, group 2 involves 5+: 3h, normal 4+: 4h and 3+: 5h asci in which either or both chromatids underwent substitution base correction. The group corresponds to the lower left three squares of Figure 4b-c. Group 3, the like mode group, comprises those asci in


TABLE 3c

Like-mode group+

Cross P-value

Total Ra Rp Ra:Rp 1:l 1.4:1

X + 39 25 14 1.8:l >.05 >.30

with normal 4+:4h . . . . . . . . . . .

normal 4+:4h alone . . . . . . . . . . . .

h*

x g, without normal 4+:4h 43 27 16 1.7:l =.lo >.50

X + 53 30 23 1.3:l >.30 r . 8 0

. . . . . . . with normal 4+:4h . . . . . .

normal 4+:4h alone . . . . . . . . .

h,,

x g, without normal 4+:4h 41 28 13 2.2:l =.02 -.20 . . . .

X + 63 29 34 1:1.2 >.50 g.05

. . . . . . . . with normal 4+: :h . . . . .

normal 4+:4h alone . . . . . . . . . . . . .

h3

x g, without normal 4+:4h 32 19 13 1.5:l >.e0 >.90

X + 27 16 11 1.4:l >.30 >.go

. . . . with normal 4+:4h . . . . . . . . .

normal 4+:4h alone . . . . . . . . . . . . .

h4

x g, without normal 4+:4h 24 16 8 2.0:l >.IO >.30

h4b

x g1 38 17 15 1.1:l >.70 >.50

. . . . with normal 4+:lh . . . . . . . . .

normal 4+:lh alone . . . . . . . . . . . . . x + without normal 4- t :4h 19 13 6 2.2:l >.IO >.30

X + 17 4 13 1:3.5

. . . . . . . . . with normal 4+: ! h . . . .

normal 4+:4h alone . . . . . . . . . . . . . x g, without normal 4,+:4h 24 15 9 1.4:l >.20 >.50

which the h site of both chromatids underwent the same mode of base correction; that is, to hyaline (restoration for chromatid 2 but substitution for chromatid 3) or to wild type (substitution for chromatid 2 but restoration for chromatid 3), or no correction. Group 3 includes 6+:2h, aberrant 4+:4h and 2+:6h asci, and these are contained in the remaining three squares of Figure 4b-c.

The asymmetrical distribution of classes Ra and Rp among these asci was very interesting, as shown in Table 3a-d. In Table 3a, which represents group 1, the restoration group, there is in all interallelic crosses, except h, x g,, a very sig-


TABLE 3d

All asci pooled+

P-value Cross Total Ra Rp Ra.Rp 1:l 1.1:4

X + 97 60 37 1.6:l z . 0 2 >.30

with normal 4+:4h 109 69 4.0 1.7:l <.Ol >.20 x g, without normal 4+:4/z 99 62 37 1.7:1 > . O l >.30

h,

normal 4+:4h alone 10 7 3 2.3:l . . . .

X + 89 52 37 1.4:1 >.05 >.98

with normal 4+:+h 105 63 42 1.5:l >.02 >.70 x g1 without normal 4+:4h 100 60 40 1.5:l >.02 >.70

nxmal4+:4h alone 5 3 2 1.5:l

h,,

X + 145 85 61 1.4:l >.02 >.95

with normal 4+:4h 241 141 100 1.4:1 < . O l >.95 x g, without normal 4+:4h 99 63 36 1.8:l <.Ol >.20

normal 4+:4h alone 142 78 64 1.2:l >.20 >.30

h3

X + 74 40 34 1.2:l >.30 >.30

h4 with normal 4+:4h 153 86 67 1.3:1 >.lo >.50 x g, without normal 4 + :4h 62 44 18 2.4:l g.01 >.02

normal 4+:4h alone 91 42 49 1:1.2 >.30 >.Ol

94 55 39 1.4:l >.05 >.98

g1h4 with normal 4+:4h 101 58 43 1.4:l >.IO >.80 x +without normal 4+:4h 41 27 14 1.9:l >.05 >.30

normal 4+ :4h alone 60 31 29 1.1:l >.80 >.30

x g1

47 21 26 1:1.2 >.30 >.05

h4b with normal 4+:4h 153 84 67 1.3:l >.IO >.50 x g, without normal 4+:4h 80 47 33 1.4:l >.IO >.go

normal 4+ :4h alone 72 37 35 1.1:l >.50 >.20 h x + and g,h, x g, crosses pooled 547 313 234 1.34:l e.01 >.50

215 130 85 1.5:l <.01 >.50

X +

8 1 x + nificant deviation of the Ra:Rp ratio from the 1: 1 ratio expected if there is no factor which causes asymmetry. The asymmetry in this group was always in favor of Ra over Rp. A similar asymmetry, but reverse in direction, appears in Table 3b, which represents group 2 or the substitution group. The asymmetry observed in these two tables is very similar to that observed by FREESE (1957a, b) , MURRAY ( 1963) , and FOGEL and HURST ( 1967), although these authors have not distinguished restoration, substitution and like mode groups. No significant deviation from the 1 : 1 ratio of Ra: Rp asci was observed in the like mode group (Table 3c) or when all three ascus groups were pooled together (Table 3d). In the like

TABLE 3e

Proportions of subclasse4

Cross Total Ra-l Ra-2 Ra-1:Ra-2: Total Rp-2 Rp-1 Rp-2:Rp-1

X + 35 25 10 2.5:l 23 18 5 3.6:l

with normal 4+:4h 42 22 20 1.1:l 24 15 9 1.7:l X g, withoutnormal4+:4h 35 17 18 1:l.l 21 14 7 2.0:l

normal 4+:4h alone 7 5 2 2.5:l 3 1 2 1:2.0

h2

X + 22 18 4 4.5:l 14 7 7 1.O:l

with normal 4+:4h 35 24 11 2.1:l 29 11 18 1:1.6 x g, withoutnorma14+:4h 32 22 10 2.2:l 27 10 17 1:1.7

ha

normal 4+:4h alone 3 2 1 2.0:l 2 1 1 1:l.O

X + 56 46 10 4.6:l 27 18 9 2.0:l

with normal 4+:4h 122 107 15 7.1:l 87 26 61 1:2.3 x g, withoutnormal4+:4h 44 38 6 6.3:l 23 17 6 2.8:l

normal 4+ :4h alone 78 69 9 7.7:l 64 9 55 1:6.1

h3

24 19 5 3.8:l 23 13 10 1.3:l

with normal 4+:4h 70 65 5 13.0:l 59 10 49 1:4.9 x g, withoutnormal4+:4h 28 23 5 4.6:l 10 5 5 1.O:l

normal 4+:4h alone 42 42 0 0o:l 49 5 4 4 1:S.S

- X +

h4

x g1 38 29 9 3.2:l 24 16 8 2.0:l

g1h4 with normal 4+:4h 45 43 2 21.5:l 37 6 31 1:5.2 x +withoutnormal4+:4h 14 13 1 13.0:l 8 6 2 3.0:l

normal 4+:4h alone 31 30 1 30.0:l 29 0 29 l : W

X + 17 12 5 2.4:l 13 8 5 2.6:l

h4b with normal 4+ :4h 71 62 9 6.9:l 56 15 43 1:2.9 x g1withoutnormal4+:4h 34 26 8 3.3:l 23 12 11 1.1:l

normal 4+:4h alone 37 36 1 36.0:l 35 3 32 1:10.7

h x + andg,h4 x g, crossespooled 192 149 43 3.4:l 124 80 44 1.82:l g, x + 65 42 23 1.8:l 53 29 1:1.2

* Ra-I, no crossing over in g region; Ra-2, apparent double crossing over flanking g; Rp-I, single crossover proximal to g ; Rp-2, single crossover distal to g. t Table 3a represents upper-right three squares of Figure 4b; the origins of these ascus types

are: no correction on chromatid 2 and correction to wild type (restoration) on chromatid 3 (5+:3h), correction to hyaline (restoration) on chromatid 2 and correction to wild type (restoration) on chromatid 3 (normal 4+:4h), and correction to hyaline (restoration) on chromatid 2 and no correction on chromatid 3 (3+:5h).

Table 3b is the counterpart of Table 3a, representing lower-left three squares; the origins of these ascus types are: correction to wild type (substitution) on chromatid 2 and no correction on chromatid 3 (5+:3h), correction to wild type (substitution) on chromatid 2 and correction to hyaline (substitution) on chromatid 3 (normal 4+:4h), and no correction on chromatid 2 and correction to hyaline (substitution) on chromatid 3 (3+:5h).

Table 3c represents upper-left, centre and lower-right three squares; the origins of these ascus types are from the combination of like-mode base correction on both chromatids, i.e., correction to wild type (6+:2h), no correction (aberrant 4f:4h) and correction to hyaline (2+:6h).

Table 3d represents all asci obtained in each cross. Table 3e represents all asci shown in Tables 3a and 3b.

$ Ra-I and Rp-2 are from Table 3a; Ra-2 and Rp-I are from Table 3b.


mode group and pooled groups the P-values for the hypothetical ratio 1.4: 1 (a ratio obtained by pooling all crosses) gives a better fit than does a 1: 1 ratio for Ra:Rp.

The examples of extreme asymmetry between classes Ra and Rp in interallelic crosses (Tables 3a, 3b) appear to result exclusively from the presence of a large proportion of asci having a normal segregation of 4+:4h (for the origin and frequency estimation of this type, refer to WHITEHOUSE 1964; EMERSON 1966; and KITANI and OLIVE 1967). If the latter are removed from the totals (noted in the tables as “without normal 4+: 4h”), the P-values show fit to ratios from 1 : 1 to 1.8: 1 in Table 3a and 1 : 1 to 1 : 1.4 in Table 3b. This seems to correspond to the minor asymmetry observed in mutant X wild type crosses (Table 3, the first cross in each set of crosses involving a single hyaline mutant).

The data presented in Table 3a-d indicate that: (1) the asymmetrical tendencies in the Ra:Rp ratio show a reverse relationship in restoration and substitution groups, and there is no significant asymmetry in the like mode group; (2) the asymmetrical Ra: Rp ratio is almost exclusively caused by the presence of normal 4+:4h type conversion asci, and (3) regardless of the great asymmetries in restoration and substitution groups, the pool of all three groups (restoration, substitution and like mode) shows only a relatively minor deviation (1.4: 1) in favor of Ra over Rp (Table 3d), a deviation that had already been observed in single mutant x wild type crosses.

Asymmetry was observed, not only in the Ra: Rp ratio, but also in Ra-1 : Ra-2 and Rp-2:Rp-1 ratios, as shown in Table 3e (ratios ordered on the basis that Ra-1 and Rp-2 are the recombination subclasses for the restoration group and Ra-2 and Rp-1 for the substitution group).

In single mutant x wild type crosses the Ra-1:Ra-2 ratios were in favor of Ra-1 and showed relatively moderate variation among the crosses (ranging from 1.8: 1 in gl X wild type to 4.6: 1 in h, X wild type), and Rp-2: Rp-1 ratios were in favor of Rp-2 (except in gl x wild type). In mutant x mutant crosses the same tendencies were observed in both Ra-1: Ra-2 and Rp-2:Rp-1 ratios when normal 4f: 4h type conversion asci were excluded (exceptions: Ra-1 : Ra-2 ratio in h, x gl and Rp-2: Rp-1 ratio in h,, X gl) . However, when the normal 4+: 4h conversion type was included, a marked modification of the Ra-I : Ra-2 ratio in favor of Ra-1 appeared in crosses h, x gl, glh, x wild type, and h4,, x gl. But inclusion of the normal 4+:4h group reversed the relationship of Rp-2:Rp-1 in favor of Rp-1 in all mutant x mutant crosses except h, x gl.

When the left-hand members of ratios Ra-1:Ra-2 and Rp-2:Rp-1 are conspicu- ously greater than the right-hand in value, this means that the restoration group is larger than the substitution group in a cross, whether single mutant X wild type or interallelic. The tendency of the normal 4+:4h conversion ascus type to increase more or less the proportion of Ra-1 (restoration) asci and to increase Rp-1 (substitution) rather than Rp-2 (restoration) asci is of considerable interest and must be examined in the light of the striking modification of the Ra: Rp ratios in the normal 4+:4h conversion group alone or when this group is included in the totals.

Y. K I T A N I A N D L. S . OLIVE 38

Ascu - type

r (U .. + U)

c In

+ ts)

r CO

+ (U

-

lutan - 12

12a

h3 h 4

h4b JI h4

- n2

h2a h3 h4 JI h4 h4b

hz h2a h3 h4 ai h4 h4 b

hz hza h3 h4 aihd

h4b

-

-

- hz hza h3 h4 ai hr h4b

hz h2a h3 h 4

-

gI h, h4b -

#=+ - v w

J .... I .... I .... I .... 1 .... I . . . . I . ) . . . . I . . . . I . . . . l . . . . l . . . . l . . . . ) . . . . I - 7 * - - -

.I .... I . . . . I . . . . I , . . . . I . . . . I .... I .... I .... I .... I .... I .... I .... I .... I .... I -L

I 1

I I I - - I I 1

- t i & 7

. I . . . ..I. .. . . I . . . . I . . . . I . . . . I . . . . I . . . .I . * . . I . , . . I , . . . I . . . . I . . . . I , . . . I . . . . I

I I I - ~ . . . . I....1....1....1....1....1....1....1....1....1....1....1....1....*

-1 .... I .... I .... I .... I . . . . I . . . . I . . - - - - * - l . . . . I . . . . I . . . . l . . . . I . . . . I . . . . I .

... l . . . . 1 . . . . 1 . . . . I . . . . l . . . . 1 . . . . 1 . . . . I

CROSS HETEROZYGOUS FOR ONLY O N E SITE

m CROSS HETEROZYGOUS FOR B O T H S ITES

.. I . . . . I . . . . l . . . . I . . . . I . . . . I . . . . , . . . . ,

FIGURE 6.-Modification of ascus type proportions for h sites by the g , site in interallelic crosses. The intermediate vertical bar indicates observed frequency (in percent), and the terminal ones give the 95% confidence limits.


ZV. Variations in proportions of ascus types in different crosses and the modification of these proportions for the h sites by the presence of gl in the cross: As shown in Table 1, proportions of the 21 visually distinguishable aberrant types of asci vary among mutant x mutant crosses, just as the proportions of the five fundamental aberrant ascus types (normal 4+: 4h conversion asci not being distinguishable) varied among mutant x wild type crosses (KITANI and OLIVE 1967). If the 21 ascus types from interallelic crosses are divided into six groups (the normal 4+:4h type being recognizable here) with regard to the fundamental aberrant ascus types for the h site and the data compared with those from the corresponding h x wild type crosses, the proportions of the various fundamental types in interallelic crosses are quite different from the corresponding ones in single mutant x wild type crosses (Figure 6 ) . A statistically significant decrease in proportion of aberrant 4+: 4h asci from that found in the corresponding h X wild type cross is a general occurrence in h x gl crosses (except for the small overlap of confidence limits in crosses glh, x wild type and hb x g,, for which the single mutant X wild type controls were small in number). In addition, there were decreased frequencies of 3+:5h asci in crosses h, X g l , h, X g , and glh4 X wild type. Seemingly complemental to the previously noted decreased proportions of aberrant 4+: 4h asci in interallelic crosses was the significant increase in 2-k: 6h in h, X g, (with confidence limits only narrowly overlapping), 3+: 5h in h,, X g l , and normal 4+:4h conversion asci in all other crosses. In spite of these apparent complemental modifications, three such ascus types in each cross escaped the modification, and only one additional type in each cross showed moderate to significant modification.

There are several notable features in the above-mentioned modifications of ascus types. First, the ascus type which always shows a reduced frequency in these interallelic crosses is the aberrant 4+:4h type, which results from a combination of no-correction events on both chromatids. Whenever some other ascus type is reduced in frequency, it is one that has had a combination of no correction on one chromatid and a correction of wild type to mutant on the other (3+:5h) . In cross h, x g l ascus type 2+:6h, which may be a reduced type, has resulted from a combination of wild type to h corrections in both chromatids. These results may mean that hybrid DNA at the h site is more ‘likelv to be corrected in h X gl crosses than in h x wild type.

It is also obvious that the ascus types which showed increased frequencies in crosses h, X gl and h,, x gl differ from those of other h X gl crosses. The 2+:6h type appears to have increased in frequency in these two crosses (confidence limits slightly overlapping in both cases), and the 3+: 5h type is significantly increased in h, X g,, while the normal 4+:4h conversion type (types M, I, and E, Figure 4b) has increased to a number amounting to half or more of the total in all other h x gl crosses as well as in the cross glh, x wild type. This could mean that hybrid DNA which would have remained uncorrected in h x wild-type crosses is, in h x gl crosses, induced to undergo correction in a manner characteristic of each hyaline heteroallele. Reference to Figures 3 and 4b will show that M and I types of normal 4+:4m conversion asci are phenotypically distinguish-


able, whereas E type is not. On the other hand, Figure 4a shows that all normal appearing 4 f : 4m conversion asci are phenotypically indistinguishable from non-conversion asci. Later, we shall discuss the relationship of the above results to the generally accepted concept that hybrid DNA formation may not always include both mutant sites in interallelic crosses (the frequency of exclusion of the second site presumably being correlated positively with its distance from the first). Such an occurrence would result in normal 4: 4 segregation of the excluded site.

V. Modification of proportions of aberrant ascus types with regard to the g1 site by the presence of various h alleles in gl x h crosses: Data on the gl site in interallelic crosses are less complete because of the tendency of many hyaline spores to burst before germination. Thus a number of hyaline double mutant spores ( g l h ) are lost before they can be analyzed for the presence of gl. To reduce this tendency, hypertonic mounting solution and dissecting agar have been found effective (MATERIALS AND METHODS). Experimental results have shown that there is no difference in the bursting frequencies or survival rates of h and glh spores.

The double color mutants, containing both gl and h (h3, h,, or h 4 b ) , were detected by the two-test method described in MATERIALS and METHODS. Thus far, no double color mutant has been found in crosses h, X gl and h,a X gl. A total of 83 selected hyaline spores from the former and 30 from the latter cross were tested. These spores were selected for position and outside marker alignment in aberrant asci" which, in crosses between gl and h, or h, (in which all germinable spores were analyzed), had been found to be characterized by relatively high frequencies of double mutants. Since none of these hyaline spores proved to be a double mutant, it seems reasonable to conclude that the frequency of the glh double mutant where h, alleles are concerned is negligible (estimated to be less than 4% of tested hyaline spores, 95% confidence). Therefore, all hyaline spores from aberrant asci obtained in crosses of gl with h,, h,,, or h2b5 were classified as h single mutant spores.

For crosses gl x h,, h,, and h4b and the cross glh4 x wild type, all aberrant asci were reclassified, insofar as possible, with respect to fundamental ascus types for the gl site (Figure 7). Because of the epistatic nature of h over gl and the loss of many hyaline spores, it is necessary to consider minimal and maximal percentages for each fundamental ascus type, the minimal based on experimentally determined genotypes and the maximal on the largest number possible for a fundamental ascus type. For example, the cross gl x h, was relatively inefficient for the recovery of the double mutant glh3. Therefore, comparatively low minimal values

4 Hyaline spores selected for further testing were: ( 1 ) the hyaline spore from the gray-hyaline spore pair in ascus types R, K and C; ( 2 ) any hyaline spore pair recombinant for outside markers in ascus types C, G, M, and I; and (3) any two hyaline spore pairs that show outside marker recombination in type A asci.

5 The hnb allele is a derivative of h,, having appeared in a hyaline-wild-type spore pair in an aberrant 4+:4h ascus from the cross h,, x wild type. In crosses with wild type, h,, spores have a slightly more bluish tint than those of either h, or A,,. All crosses among the h, group of alleles fail to yield wild-type spores.

CONVERSION IN I N T E R A L L E L I C CROSSES 41

lross 0 5 IO 15 20 25 30 35 40 45% - ' ................................................. - S I X + h2 xg I hzaxgi hzbx gl

.

U U)

i N

-. . . . . . . -.-

Crass 0 5 I O 15 20 25 30 -. ............................... gi x + hzxg i h2axgi hzbx g i h3xgi h4x gi gih4 x+ h4bxgl

gi x + h z x g i hzaxgi hZbXgl h3 x g i h 4 x g i gihsx+ h4bxgi

g1 x+ h2xgi h2oxg1 hebxgl h 3 x g i h4x gi gth4x+ h4bxgi

-

g l x + h z x g i hzaxgf hzbxgl h 3 x g 1 h 4 x gi grh4x+ h4bxgl - - Cross; gi x +

k-1 Cross, group1 hyaline mutant x gl ------U Cross, group2 hyaline mutant x g l t -----I Confidence limits, 9 5 % probability *--------a Minimal and maximal percentages

bounding allowance for actual frequency

g l x + h z x g l 1-4 hzaxgl ,-I

h2bXgl + h3xg1 ~ _ _ _ _ - _ _ _ _ _ - h4xg1 ----U

g i h 4 ~ + -------U

h4bXgI ------U - .................................. FIGURE 7.--~odification of ascus type proportions for g, site by h sites in interallelic crosses.

(Confidence limits given for data where minimal and maximal percentages are shown, the lower limit based on the minimal percentage and the higher on the maximal percentage.)

were obtained for the various fundamental ascus types for gl: 6+:2g1, 7.9%; F;+:7gl, 10%; normal 4:4, 15.4%; aberrant 4:4, 2.9%; 3+:5gl, 1.6%; 2+:6gl, 0.4%; a sum of 38%. Equivalent totals of minimal percentages for other crosses were as follows: gr X h,, 54%; gl X hhb, 45%; and g,h, X wild type, 73%, the last cross being the most efficient for analysis of the double mutant.

On the other hand, if the same asci are reconsidered from the standpoint of all possibilities for classification of undetermined asci (Figure 7), it is apparent that the latter could fall into from one to several types. Therefore, the sums of the hypothetical maximal percentages for the various ascus types in any cross will exceed 100%. For example, in the cross gl x h, a maximal total percentage of 195% was obtained (6+:2g1, 37.8%; 5-+:3g1, 65.5%; normal 4:4,46.9%; aber-


rant 4:4, 24.5%, 3f:5gl, 18.2%; 2+:6gl, 2.5%). Other maximal percentages were as follows: gl X h,, 166%; g, X h4b, 198%; and glh, X wild type, 143%. Confidence limits in Figure 7 are based on the minimal and maximal percentages obtained as described above.

In contrast to the sharp modification in behavior of h sites in the presence of gl in crosses (Figure 6), the behavior of the gl site was generally only moderately modified by the presence of an h allele (Figure 7). However, some significant modifications did occur in some ascus types: (1) There was a reduction of the 6+: 2g1 type in the cross glh4 X wild type, and similar tendencies are suspected in crosses gl x h, and g, x h,. (2) A significant increase occurred in the normal 4+:4g, conversion type (limited to the restoration group) in the cross g,h, x wild type, and 'the same was suspected in crosses gl x h, and g, x h,. (3) A significant reduction in aberrant 4+:4gl asci occurred in crosses g, x h, and gl X h?, (to a lesser degree in gl X h z b ) , but the range between maximal and minimal frequencies in the other crosses is too great to permit further meaningful conclusions.

The tendencies towards reduction in frequencies of the aberrant 4f : 4h type and an increase in frequencies of the normal 4+:4h conversion type were very conspicuous and widespread in all gl x h crosses, as already demonstrated, and these tendencies have also been found characteristic of the gl site in at least some gl x h crosses. If these observations on the modification of proportions of fundamental ascus types with regard to both the g, and h sites are combined, two entirely different explanations seem possible. One explanation might be that when two mutants with sites relatively far apart in the cistron are crossed, hybrid DNA formation is less likely to include both sites simultaneously, and this would leave the excluded site in a normal condition in both chromatids, thus permitting it to segregate normally in a 4+:4m ratio while the other site segregates aberrantly. The second explanation would contend that when any two heteroallelic mutants are crossed, hybrid DNA formation occurs as a single event throughout the cistron, but the mode of base correction of each mutant site is modified by the presence of the other, as compared to the behavior of each mutant site in mutant x wild type crosses. Since the differences in these two concepts are of fundamental importqnce to an understanding of gene conversion, they will be further considered in the discussion.

VI. Comparison of gl and h sites with respect to asymmetrical proportions of restoration and substitution groups: Asymmetries in Ra-1 : Ra-2 and Rp-2: Rp-1 ratios have been previously demonstrated for various h sites, with or without the involvement of gl (Table 3e), and it was explained that this asymmetry was equivalent to the asymmetrical ratio between restoration and substitution groups. Figure 8 contains a detailed analysis of the pertinent data from several view- points. Group 1 in this figure represents the pooled data from crosses between gl and the h, alleles, and group 2 represents the pooled data from crosses between g, and h,, h, (in both coupling and repulsion crosses), and hlb (refer to Figure 11 for the relative locations of these two groups of mutant sites).

Among 5 f : 3m ascus types (both 5+: 3h and 5+:3g,) , a clear-cut restoration- substitution asymmetry appeared, with respect to h sites, in h x wild type crosses

CONVERSION IN I N T E R A L L E L I C CROSSES 43

Ascus type and Cross

i + w gr.2 gi

A. ASCUS proportion for h s i tes

0 5 I O 15 20 25 30 ................................. 35 40 ........

. .

B. ASCUS proportion for gl site Ascus type X O 5 IO 15 20 25 30 35 40 and Cross ........................................ , - g t x + zz - g I x g r . l +

-._-------_---------..U g l xgr.2 UJ

N I . . . . . . . . .

I . . . . . . . . .

. . . . . . . . . . . . . . . .

+ + + + gr.2 x

g l ......................................... + crosses,mutant x +. 1-1 crosses,glwpl

zz v)

91 x gr. I .. m

r g l x g r . 2 ~ _ _ _ - _ _ _ _ b ".--_-___U . . . . . . . . . U g i x + w

* g i x gr. I + W

i *--U g i x gr.2

xgi. 4 crosses,grwp2hxg!. -----A see Figure7. ........................................

FIGURE 8.-Relationship between restoration and substitution base correction among the various ascus types in all crosses; differences in behavior of group 1 and group 2 hyaline mutants. (Group 1 = h,, h,, and hzb; group 2 = h,, h, and &.)

of group 1 and in h x gl crosses of group 2, and, with respect to the gl site, in gl x h crosses of group 1. In each case, the asymmetry observed in one set of crosses in a group did not appear in the alternate set in that group. In the normal 4+: 4m conversion type no significant asymmetry was observed for any h site, whereas a statistically significant asymmetry for the gl site was found in h x gl crosses of group 1 and a possibly significant asymmetry (with minor overlap of confidence limits) in group 2. The differential behavior of h and gl sites with respect to restoration and substitution asymmetry, especially for the normal 4:4 type of conversion ascus, must be a key element in any discussion of the involvement of one or both sites in an interallelic cross in hybrid DNA formation.

Among 3 f : 5m asci of h x wild type crosses, both group 1 and group 2 showed


significant asymmetry for h sites, while in h X gl crosses only group 2 showed asymmetry for the h sites and none in any of these crosses showed asymmetry for the gl site.

At this point the question arises as to why there should be asymmetry between restoration and substitution events in aberrant asci and why the conversion behavior of one heteroallele is markedly affected by the presence of a second in a cross. The results might be explicable on the basis that restorational base correction is accomplished with greater facility than substitutional correction in a chromatid (no reversal of this asymmetry has been observed). The tendency towards a greater proportion of restoration events in interallelic crosses is en- hanced or decreased in accordance with the particular heteroalleles involved (Figure 8). The data correlate less well with the concept that a significant number of conversion events may involve only one of two paired chromatids in hybrid DNA formation at one or both sites.

VIZ. The effects of gl and h, upon each other in heterozygous and homozygous states and in repulsion and coupling alignment: The subject of the interaction of two heteroalleles in the conversion process was introduced in the foregoing section. If this effect of gl on the h heteroalleles resulted only from the presence of gl in the cross, then the behavior of h, in the cross g,h, X gl should be the same as in the cross h, x gl (repulsion), as well as in the cross glh, x wild type (COU-

pling). If this effect were the result of a kind of enzymatic interaction between repair enzymes for gl and h, alleles, repulsion-coupling differences would be expected.

Comparisons of a pair of crosses homozygous for the gl site with a pair heterozygous for the site showed statistically significant differences in three ascus types, as already described in section IV. These results indicate that heterozygosity at the gl site rather than the mere presence of the gl allele in the cross was the key factor in altering proportions of ascus types. The fact that coupling-repulsion differences in alignment of two heteroallelic mutant sites did not give significantly different proportions of any ascus type fails to support the idea of an effective interaction of repair enzymes involving gl and h, sites.

The effect of h, on conversion at the gl site was somewhat similar to the effect of gl upon h,, but the ascus types affected were different. The 6+:2gl type was reduced in the cross glh, x wild type as was the 2+: 6gl type in the cross gl X h,, rather than the aberrant 4 f : 4 g l type (in contrast to the reduction of aberrant 4+: 4h asci in all gl x h crosses) , and the normal 4+: 4gl type is significantly increased in the cross glh, x wild type and possibly in the cross gl x h,. The normal 4+:4gl conversion type was also increased in crosses of gl with h,.

V I I I . Differences in conuersion behavior of the original hyaline alleles and their modified derivatives and the problem of mapping: Some differences in proportions of aberrant ascus types in wild type x original h and wild type x derivative h crosses were reported in the foregoing paper of this series (KITANI and OLIVE 1967). The present studies show similar differences between original and derivative h mutants in crosses with gl. As shown in Table 2 and Figure 6, in the cross hlb x gl the percentage of normal 4-k: 4h conversion asci is lower than in the


Ascus type Cross 5 IO 15 20 25 30 35 40 45 50 55 60 65 70 . . . . . . . . . . . . . .

h4 x + RI g l h4 x g l

h 4 x g l g i h4 x +

a h 4 x + .- ro g i h 4 x g I U) + h 4 x g i

g i h 4 x +

U ) .

+

rt---J - rc--l - . . . . . . . . . . . . . . . -

I I - - -c . . . . . . . . . . . . . . .

h 4 x + ;; T. g i h 4 x g1 L 4 h 4 x g l

a g i h 4 x + h 4 x + 7 g i h 4 x gI

o 7 h 4 x g l g i h 4 x +

U) E

1 I

I - - - . . . . . . . . . . . . . . . r I -

r I . . . . . . . . . . . . . . . 2 h 4 x + I J

h . . . . . . . . . . . . . . . m g l X + -

; ;T .

-2 g1 x + v ;;t g 4 g l x h4 , * - _ _ - - _ _ _ _ - - - _ _ - - - - _ - k 2 g l h 4 x + ,-___-_--_-_- - $ a g l x + r3-1

:2 ' f !

m

r -k g l x h4 ---__--__----U g1h4 y + ----_______--U

L . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . U)

+ g1 x h4 ,-,---------U

g i h4 x + __-__---------- -- . . . . . + cross heterozygous for both si tes

#-----A see Figure 7

m g 1 x + + $ g l x h4 p W

g l h4 x + -----U . . . . . FIGURE 9.--Effects of homozygosity and heterozygosity at g, site on h, site, and comparison

of repulsion and coupling alignment of heteroalleles.


Ascus types compared 0 5 10 15 20 25 30 35 40 45 5 0 , 55 60

I . . . . , . . . . , . . . . , . . . . , . . . . , . . . . I . . . . , . . . . , . . . . , . . . . I . ' . . . * , . . . I - M only - C . G B F I I

pooled b

I 1

All types except M,C, G 8F poobd

h 4 X g l ,-, h 4 b X g l

I . ... ~ . . . . I . . . . ~ . . . . I . . . . I . . . . I . . . . I . . . . I . . . . , . . . . , . . . . I . . . . I

FIGURE 10.-Comparison of h, and h,, in crosses with g, with respect to proportion of M type asci and the negative correlation in frequency with certain other ascus types.

cross h, x g,, especially with regard to the M ascus type, while the percentage of C type is larger than in the latter cross. Figure 10 contains an analysis of the types of aberrant asci (M, C, G, F) that clearly show these differences between original and derivative mutants. When M asci alone are considered, the confidence limits for the cross involving h4, do not overlap with the higher range of values for h,. By contrast, pooled asci of C, G, and F types showed a reversed relationship of these values, with confidence limits overlapping only slightly. When all other types of aberrant asci are pooled, no differences in conversion behavior of h, and h4b are apparent.

An analysis of the data shows that the decrease in M type asci in the cross h4b X g,, as compared with h, X gl, is complemented by the increase in C, G, and F types. With respect to the h site, C and G asci are both of the 3+: 5h type, and the F asci are aberrant 4+: 4h. Therefore, the differences observed between h, and hi, in crosses with gl have to do with normal 4+:4h, 3+: 5h, and aberrant 4+:4h conversion types. There is no apparent explanation why only M and not I asci (both being of the normal 4+:4h type) are affected in these crosses. Whatever the mechanism responsible, the significantly different proportions of M asci (with one pair of wild-type spores) and C, G, and 1' asci (with one wild type spore in G and none in C and F) in these two crosses probably have an important mean- ing.

The frequency of wild-type recombinant (prototrophic) progeny (especially in M type asci which at least visually resemble tetratype asci) is commonly considered as an index to the distance between sites, and fine structure mapping in fungi has been based primarily upon this concept. In the present studies, however, conflicting evidence has been obtained with two sites, h, and h4,,, which appar- ently occupy essentially similar positions in the cistron.6 In particular, the frequency of M asci €ound in the cross h, x gl has differed significantly from that obtained in the cross htb X gl. These observations suggest that the frequency of wild- type recombinant spores is not necessarily an exact index to the distance between heteroalleles.

The fact that no spontaneous mutants for the g locus have been obtained and that complementation characteristics of derivative alleles are similar to those of the originals indicates that the

CONVERSION IN I N T E R A L L E L I C CROSSES

a Ascus proportion s Spore proportion c 4 x s

a : c (c/a %I

47

8.7 I .4 5.6

1.55:i (64.4)

h 4 p h 2 h 4b hs .

a Ascus proportion s Spore proportion c 4x s

a : c (c/a %)

21. I 15.4 4. I 2.3

16.4 9.2 1.2s:i (77.7) 1.67:i(ss.7)

1 1 . 1 I .5 6.0

I .85:1 (54.

<o. I <o. I - -

a Ascus proportion s Spore. proportion c 4x s

a ; c (c/a %I

a Ascus proportion s Spore proportion c 4 x s

a : c (c/a %)

23.6 3.8 15.2

1.55:l ( 6 4 . 4 )

18.5 3.6 14.4

1.28:l (77.8)

FIGURE 11.-Comparison of methods of gene mapping at the g locus. (a = number of asci with any number of wild-type spores/lO4 asci; s = number of wild-type spores/l04 spores; in second set of data, numbers in bold face type are for h4b, while ordinary type for the same interval represents h4,)

A map of the g locus, based primarily on frequencies of wild-type spores recovered from interallelic crosses, is shown in Figure 11. Earlier complementation studies (KITANI. and OLIVE 1967, Table 2) have also contributed to the ordering of sites. In this map, distances between heteroalleles can be considered from the standpoint of two different sets of values-the first based on the proportion of asci containing one or more wild-type spores, and the second based on the proportion of wild-type spores. For fine structure mapping of the cistron only the second method has been commonly used.

If, as was generally assumed at one time, a recombinant prototroph picked up in random analysis represented one of a pair of prototrophs from a tetratype ascus, then the two sets of values above could be directly correlated, the value

derivative mutants have arisen by some modification of the original mutant site rather than by additional mutation within the cistron. The possibility of a modifier site in the cistron cannot be ruled out at this time. Further studies on the nature of the derivative mutants are being made.


from random spore analysis being one-fourth that for ascus analysis. As demonstrated by the present data, however, no such direct correlation can be made, since asci containing wild-type spores are not of one but many types (Figure 3 ) and may contain from one to four wild-type spores. The data assembled in Figure 11 further demonstrate the lack of good correlation between the first method of mapping (based on proportion of asci containing any number of wild-type spores) and the second (based on proportion of wild-type spores). The ratios (a: c ) of values obtained by these two methods varied from cross to cross, depending upon the heteroalleles used, from 1.28: 1 (in cross gI X h,) to 1.85: 1 (in cross h, X h,). (The c/a percentages vary from 54.1'% for h, x h, to 77.8% for gl x h ? ) . Thus it is clear that established methods of fine structure mapping in fungi can not be accepted without some reservations. This subject will be further discussed in the following section.

I X . Data selected for detailed examination in eunluating specific concepts: (1) Significance of the M:I ratio and the proportions of M and I asci with two, one, or no double mutant spores. If the drastic increase in normal 4+:4h conversion asci in crosses between group 2 hyaline mutants (h3, h,, hrb) and gl over the proportions found in the corresponding h X wild type crosses (see section IV) were attributable to intracistron (interallelic) crossing over between the ,yl and h sites, these normal 4+: 4h types would be exclusively M type and genotypically tetratype with a pair of double mutant spores. However, the actual results fell far short of these conditions. I type asci, containing only one wild-type spore, were not much less abundant than M type (Table 4 ) , and the proportion of asci containing two double mutant spores was low among both M and I type asci (Table 5 ) . Especially in the cross h, x gl, the sum of actually determined M asci with one or no double mutant spores (actual number = 17, the minimal iiumber in the allowance discussed in section V) was greater than the estimated maximal number for asci with two double mutant spores (estimated number = 15, the highest number in the allowance in section V) . The results show that the actual number of tetratype asci-the only ones that could possibly be considered as products of intracistron recombination by crossing over-did not constitute the majority of M

TABLE 4

M : Z and i : e ratios

Pooled data Cross Group 1 h' Group 2 h'

Type of ratio h2X g, h,, X gl h, X gl h, X g, g,h, X -!- h, X gl mutants mutants

rt 3 : 3 2 : l 39 :38 2 9 : 1 8 . _ . . 2 0 : 1 9 5 : 4 88 :75 M : I si- 4 : O 2:O 45:20 31 :13 . . . . 16:18 6 : O 9 2 : 5 1

r . . . . . _ . _ . _ _ _ . _ . . 21 : 9 _ _ . . _ _ . _ _ . . _ i:e s _ _ . _ , . , . . _ , _ _ _ _ _ 13:17 . . . _ _ _ _ _ , , . _

pooled . . . . . . . . . . . . . . . . 3 4 : 2 6 . . . . . . . . . . . .

pooled 7 : 3 4 : l 84 :58 60:31 . . . , 36:37 11 : 4 180:126

* Group 1 h = h, and hza; Group 2 h = h,, h, and h4,,. t r = restoration base correction; s = substitution base correction.


TABLE 5

Proportions of asci containing no, one or two double mutant spores in ascus types M and I , and proportions of asci containing no, one or two single mutant spores in ascus types e and i'

Number of gl h Repulsion crosses Repulsion Coupling CMSS

Ascus k,+ h,) h,Xg, h4 x gl h4b x gl crows p l e d gl h,X + trpe spores r s Pooled r s Pooled r s Pooled r s Pooled r s Pooled

no

M one (e)

two

no

I one (3

two

* Numbers in parentheses are maximum expected, genotype and ascus t y p e s in parentheses are for coupling cross.

type but comprised only a small segment of the total population of normal 4f: 4h type, including M, I, and E asci. Thus any attempt to explain the great increase of the normal 4+:4h type in interallelic crosses over those occurring in mutant x wild type crohsses on the basis of crossing over between heteroalleles is ruled out.

On the other hand, if it is proposed that hybrid DNA does not always cover the whole cistron and as a consequence frequently excludes one site from the hybrid DNA segment, then the latter would segregate normally and the included site would generally segregate aberrantly. Thus the large proportions of I asci, which clearly show aberrant segregation at the gl site but normal segregation at the h site (Table 4) could be accounted for, as could the small number of M asci containing a pair of double mutant spores as well as those with a pair of single and double mutant hyaline spores (Table 5). It should be noted that the presence of only one or no double mutant spores in M asci also signifies aberrant segregation at the gl site.

(2) Absence of the wild type-g,h spore pair in repulsion crosses and the g1 single mutant-h single mutant spore pair in a coupling cross. Among the 21 conversion ascus types from repulsion crosses (upper part of Figure 3 ) , there are some (G, J, K, Q, S) which contain a wild-type-hyaline spore pair, and one ascus type (0) contains two such spore pairs. Among the 21 conversion types for coupling crosses (lower part of Figure 3 ) , there are some with a gray-hyaline spore pair (c, d, fk, 1, r) and one type ( f ) with two such spore pairs. The presence or absence of the g,h double mutant among the hyaline spores of wild type-hyaline spore pairs in repulsion crosses and the presence or absence of the h single mutant condition among the hyaline spores of gray-hyaline spore pairs in the coupling cross were studied in detail. As shown in Table 6, only about half of these spore


TABLE 6

Frequency of non-parental genotype in hyaline spore of odd spore pair' ~~

Ascus type involving Repulsion cross odd spore pair h , X g l h , X g , h , X g ,

Coupling cross Grand h P b X g l Total Ascus type g, h , X + total

G 12 12 7 J 13 21 9

K 15 9 6 ot 0 16 10 Q 0 1 2 s 0 1 2

spore pairs 40 60 36 Total number of odd

Number of hyaline spores germinated 20 23 19

Number of parental hyaline spores identified 20 23 19

hyaline spores identified 0 0 0

Number of non-parental

12 C 3 8 d 0 4 ft 2

10 fk 6 4 1 1 3 r 6

41 177 . . 18 195

21 83 . . 9 92

21 83 . . 9 92

0 0 . . 0 0

* The odd spore pair is the wild type-hyaline spore pair in repulsion crosses and the gray-

-t Numbers for these ascus types are doubled, since they contain two odd spore pairs. hyaline spore pair in coupling cross.

pairs were analyzable because of the loss of hyaline spores. However, none of the 83 tested spores from these particular spore pairs in repulsion crosses showed a double mutant genotype (Table 6). Similarly, all of the nine tested hyaline spores from the coupling cross were double mutants ( g l h ) . Combining these, none of the 92 tested hyaline spores were different from the parental hyaline genotypes used in the crosses. This indicates that the hyaline spores from wild type-hyaline spore pairs in repulsion crosses and gray-hyaline spore pairs in the coupling cross probably consistently carry the genotype of the parental hyaline mutant in the cross ( h single mutant in the repulsion crosses and glh double mutant in the coupling cross). The statistical probability of these spores being glh double mutant in wild type-hyaline pairs or h single mutant in gray-hyaline pairs is less than 4% (95 % confidence limits).

The above results are expected in hypotheses favoring hybrid DNA formation by an exchange of single DNA strands between homologous chromatids. How- ever, if a re-annealing of once hybridized DNA within the original chromatid may occur, then non-parental hyaline genotypes would be expected among the above described odd spore pairs. In this connection it should be noted that re- annealing of base-corrected strands to the original half-chromatids is an essential feature of the HOLLIDAY (1964) model, but the above odd spore pairs are avoided in HOLLIDAY'S modification of the model to exclude one of the mutant sites from hybrid DNA formation. On the other hand, these particular odd spore pairs are not expected in the WHITEHOUSE-HASTINGS (1 964) model or in the modification

CONVERSION IN INTERALLELIC CROSSES

A.4+:4 m

+-m Y '

3 + : 5 m ( r ) no correction

IO-(y1+ y 2 1 [ '0 - ( XI +

(IO- (yl +y2 )]

I y 2

x2 [IO - ( y l +y2 5

+*" X I

3 + : 5 m ( s )

[ l o - ( x I + x 2 ~ y 2

6 + : 2 m

X I y l

2 - t : 6 m

x 2 y 2

5 + : 3 m ( S I x I [I 0 - ( y I + y 2 I]

[ lo - (x I+x2) ]y I I x 2 yl

51

FIGURE 12.-Degrees of preference in the two involved chromatids in modes of base correction.

of it by WHITEHOUSE (1965), in which localized DNA synthesis is required to replace disintegrated segments in the region of dissociation.

( 3 ) Preference in mode of base correction in the two chromatids involved in the conversion event. It is clear that the following tests become meaningless if the frequencies of hybrid DNA formation in the two involved chromatids are different, specifically with regard to whether a chromatid carries the wild-type or mutant allele (in Figure 4a, chromatid 2 originally carries the mutant allele and chromatid 3 the wild type). However, there have been no reports that clearly demonstrate such a situation. An attempt was first made to determine for each of the two chromatids the degree of preference for the three modes of base correction (mutant to wild type, non-correction, and wild type to mutant). As shown in Figure 12, z1 and zz represent the degrees of base correction from mutant to wild type and wild type to mutant, respectively, and 10 - (z, -I- z,) the degree of non-correction on chromatid 2 (the sum of the three degrees being I O ) . Simi- larly, yl, yz , and IO - (yl + y.) represent the corresponding values for chromatid 3. Calculations to obtain numerical values for zl, zz, yl, and y z were made as follows:

(a) zl value; [IO- (y1+yz)Iz1=1Oz1 - Xlyl - 5 1 yz [obs percent of 5 f 3m (s)] [obs. percent [obs. west.

normal 4+,4m (s)]

of 6+ .em] percent of

(b) zz value; 110- (y1+yz)lz,=1022 - zzy1 - xz yz [obs percent of 3+ 5m (r)] [obs. or est. [obs. percent

percent of of 2 f 6ml normal 4+.4m ( r ) ]

Y. KITANI A N D L. S. OLIVE

yl value; [lo - (x1-t x,)] y1= 10 yl - xlyl - 5 2 y1 [obs. percent of 5+:3m (r)] [obs. percent [obs. or est.

nonnal 4+:h (r)]

of 6+:2m] percent of

y z value; [ l o - ( ~ 1 + ~ 2 ) l y , = 1 O y z - sly2 - XZ y2 [obs. percent of 3+:5m (s) ] [obs. or est. [obs. percent

percent of of 2f:Gml normal 4+ :h (s)]

In these computations, percentages for normal 4+:4m conversion asci in mutant x wild type crosses were estimated (KITANI and OLIVE 1967, Table 3 ) and shared evenly between restoration and substitution groups. Some error may be expected in this method but should not be great in view of the small proportions of this type estimated to be present.

TABLE 7

Degrees of preference in mode of base correction. The sites graded in the table are those in bold type in the first column*

+ +-m No correction + - m Chromatid Chromatid Chromatid Chromatid Chromatid Chromatid

2 3 2 (r) 3 (SI . ,. X f 4.20

x h . . .

X + 0.99

x g1 1.87

X + I .49

x g1 2.40

X + 0.47

x g1 3.15

X + 0.36

x g1 3.67

x g1 1.24

X + 4.02

X + 1.39

x 81 3.04

5.20 3.25

. . . . . .

3.29 6.1 1

2.54 4.23

2.72 7.12

2.88 4.43

1.18 4.97

4.33 1.70

0.98 4.41

4.17 1.57

3.04 3.45

4.75 1.53

2.88 3.83

3.65 2.10

3.71 2.55

. . . . . .

5.67 2.90

3.87 3.89

7.02 1.36

4.16 3.17

7.22 4.57

2.19 5.15

5.78 5.23

2.01. 4.76

4.46 5.31

1.19 4.4.5

4.53 . 4.77

2.82 4.86

1.09

. . .

1.04

3.59

0.27

2.96

1.61

3.48

3.25

3.79

2.50

4.02

2.59

3.52

* Estimation is based on formulae listed in text. + (r) = restoration base correction. (s) = substitution base correction.


The estimated degrees of preference in modes of base correction shown by chromatids 2 and 3, based on the current observations, are presented in Table 7. Two points of particular interest are apparent in this table: (a) within the like mode base correction category, restoration (+ -+ m in chromatid 2; + + m in chromatid 3) was, without exception, more frequent than substitution correction in both mutant X wild type and mutant x mutant crosses in both chromatids, and (b) degrees of preference varied between mutant x wild type and mutant X mutant crosses with regard to the same hyaline mutant.

AS to why restoration is always preferred over substitution in both chromatids, two possible explanations come to mind, both of which require a model such as that proposed by HOLLIDAY (1964) or by WHITEHOUSE and HASTINGS (1965). First, the single DNA strand in each participating chromatid that remains unbroken in the dissociation process leading to hybrid DNA formation, or the one that retains its proximal connection in the event of an exchange, might more often have the initiative in base correction than would the other strand (allowing also for some individual differences among heteroalleles) . Alternatively, if hybrid DNA formation at a mutant site may involve one chromatid and not the other (but with random occurrence in the two chromatids), this would give the other chromatid an apparent preference in restoration correction, whereas, there would have been no actual correction in that chromatid at all.

The fact that in h X wild type and h X g, crosses differences in degrees of preference were found for the same hyaline allele is another aspect of the observation in section IV that the proportions of aberrant ascus types for one mutant site are modified by the heterozygous presence of another heteroallelic mutant site in the cross.

In Table 8 the values given in Table 7 for degrees of preference in mode of base correction in the two chromatids are subjected to the x2 test. In this table the frequencies of all nine ascus groups (6+:2m, 5-k: 3m(r), 5+:3m(s), normal 4+:4m(r), normal 4+:4m(s), aberrant 4+:4m, 3+:5m(r), 3+:5m(s), and 2+:6m) were computed from the degrees of preference (Figure 12) and compared with the observed frequencies of these ascus groups. For mutant X wild type crosses, both restoration (r) and substitution (s) groups of normal 4+:4m conversion types were pooled, since the “observed value” for this ascus type in these crosses was not the actually observed value but that estimated for all normal 4+:4m conversion asci. All mutant x wild type crosses, including glh4 X gl, and crosses of h, and h,, (group 1) with gl showed good to fair fit except for crosses h, x wild type (P < .Ol) and h, x wild type (P > .05). On the other hand, crosses of h,, h,, and hlb (group 2) with gl and the coupling cross glh4 X wild type did not show fit. These results tend to confirm the idea that there is a preference in mode of base correction in the involved chromatids f a v o h g restoration, but within certain limits imposed by the particular hetemallele. The different combinations of various degrees of preference shown in Figure 12 determine the frequencies of the nine ascus groups. No mechanism other than the modification in degrees of preference by the gl allele is needed to explain the results of crosses h, X gl and h,, X gl, but some additional explanation is needed for the alteration

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37

13.2

1 9.

38

12.3

8 7.

34

4.07

17.

92

10.7

9 8.

90

8.53

5.

41

7.08

17.

01

4.67

18.

04

12.0

8 8.

69

10.4

8 6.

71

8.19

13.

28

3.65

17.

89

9.99

7.

92

10.3

5 4.

62

5.26

12.

35

5.41

17.

11

9.05

11.

84

8.43

10.

78

>.50

> .

80

<.O

l - e .2

0

>.05

g

.01

< .01

>.

02

>.IO

g

.01

z.3

0

-

>.30

>.go

g

.01

<.Ol

<.

Ol

>.50

< .

01

E.2

0 -

>.50

* Obs

erve

d va

lues

for

mut

ant

x f c

ross

es (

and

g,h4

x g

, cr

oss)

are

bas

ed o

n es

timat

ions

app

lied

in t

he p

revi

ous

pape

r (K

ITA

NI a

nd O

LIV

E, 19

67, T

able

t Deg

rees

of

free

dom

are

7 in

all

test

s fo

r mut

ant x

+ cro

sses

(an

d g,

h, x

g,

cros

s), 8

in a

ll u

nadj

uste

d te

sts

for

inte

rall

elic

cro

sses

, 6 w

hen

only

nor

mal

$ (1

) =

Prop

ortio

n of

nor

mal

4+

:4h

asci

adj

uste

d to

the

lev

el i

n h

x f c

ross

es,

(2) =

Pro

port

ions

of

both

nor

mal

4+:

4h

and

aber

rant

4+:

4h a

sci

3) ;

(r)

and

(s)

grou

ping

s ar

e no

t use

d in

thes

e cr

osse

s.

4*+:

4h ty

pe is

adj

uste

d, a

nd 5

whe

n bo

th 4

+:4h

ty

pes

are

adju

sted

.

adju

sted

to th

e le

vels

in h

x +

cross

es.


by gl of the expected proportions of the nine ascus types resulting from crosses between group 2 hyaline mutants and gl.

In an attempt to determine the cause of the above stated lack of fit among proportions of the nine ascus groups (with regard to h sites) and among the estimated proportions in all crosses between group 2 hyaline mutants and gl, two tests were applied. In the first, a large majoritj. of normal 4+:4h conversion asci were removed from the total in crosses between group 2 hyaline mutants and gl, and the remnant of normal 4+:4h conversion asci left in the total was adjusted to the proportion of this group estimated as being present in the corresponding h X wild type crosses. Then the degree of preference for each chromatid was estimated on the basis of the adjusted proportions. Finally, the observed proportions of seven ascus groups (the two groups of normal 4+:4h type being artificial and therefore untestable) were tested against the computed proportions.

In the second test, a majority of normal 4+:4h conversion asci were removed from the total in the same manner as in the first test, and a number of aberrant 4+:4h asci were added to the total to make the proportion of this type equal to that in each corresponding h x wild type cross. Then the same procedure applied to the first test was followed (the number of tested ascus groups being six in this test, since both the aberrant 4+:4h and the normal 4+:4h conversion types were artificial).

The first test was to determine whether the lack of fit in crosses of group 2 hyaline mutants with gl to the general x2 test (previously described in Table 8) was due to the excess of normal 4+:4h type, as indicated by much of the foregoing data. The second test was to determine whether the decreased proportion of the aberrant 4+: 4h type, as well as the excess of the normal 4+: 4h conversion type, was part of the reason for lack of fit to the general x2 test.

The results of these two tests are shown in Table 8. Both tests showed improved fit in all crosses, especially in crosses h, x gl and hab X gl. Furthermore, the fit was even better for the second test in all crosses except glh, X wild type (P < .01 in both cases). These results suggest that a more suitable test can be made by excluding from the total a majority of normal 4+:4h conversion asci rather than considering that all such asci have the same origin. The results also show that a better fit can be obtained by excluding both normal 4+:4h conversion asci and aberrant 4+:4h asci from the total conversion group rather than by excluding normal 4+: 4h conversion asci alone.

In summing up all tests applied to all crosses available, several interesting points may be listed. First, the two chromatids involved in a conversion event show different degrees of preference in mode of base correction. Second, one heteroallele alters the degrees of preference at the other heteroallele in mutant X rlutant crosses. Third, the proportions of all ascus types and the proportions of restoration and substitution groups within an ascus type are determined by the combination of degrees of preference in both chromatids in all crosses (mutant X wild type and interallelic) except group 2 h X gl crosses. Fourth, it was found more suitable to segregate a majority of normal 4+:4h conversion asci from the total conversion asci in group 2 h x gl crosses and consider their origin separately.


Fifth, an even better fit was obtained by considering the origin of normal 4+:4h conversion asci in relation to the fluctuating proportions of aberrant LE+: 4h asci.

DISCUSSION

Since the gene conversion models of HOLLIDAY (1946) and WHITEHOUSE and HASTINGS (1965) include several steps in which variations are possible, considerable variety may be expected among the end products of meiosis. Therefore, existing data (including the present results) can neither confirm nor deny these models with any degree of certainty. On the other hand, there is a limitation to the variety of genotypes among aberrant 8-spored asci which is imposed by the nature of the models, including the fact that only half an octad is involved (since only two chromatids participate in conversion at any one time). Several basic features of these models are now favored by many investigators, including our- selves, since these models come closer than others to explaining the varied results of conversion.

The above two models embody several common features: (1) The linearly arranged DNA molecule contains at intervals, possibly flanking the cistrons, dis- continuities variously termed “linkage structures”, “linkage points”, etc. ( 2 ) Hybrid DNA formation between two homologous chromatids occurs in short segments. (3) Base correction tends to take place among incorrectly paired bases in the hybrid DNA; failure to correct leads to postmeiotic segregation. (4) A relationship exists between hybrid DNA formation and the excess of reciprocal exchanges observed in the vicinity.

There are several distinct differences between the HOLLIDAY and WHITEHOUSE- HASTINGS models, the most conspicuous being the disintegration and resynthesis of short segments of single-stranded DNA and the greater number of reciprocal chromatid exchanges required by the latter model. Before any attempt was made to test the present data against either model, it seemed advisable to isolate all recognizable types of aberrant octads and classify them in all possible categories, thereby adding an additional dimension to the bulk of gene conversion studies that have been based primarily on random spore analysis (selected prototrophs), unordered octad analysis, and tetrad or octad analysis limited by the absence of spore markers.

In the present paper, as in the foregoing (KITANI and OLIVE 1967), the data are analyzed in terms of somewhat simplified models (Figure 13) that are based on features of both HOLLIDAY and WHITEHOUSE-HASTINGS versions. The following steps are involved in the revised models. At linkage points in two paired homologous chromatids, dissociation of single DNA strands in each double helix occurs in a closely paired region (the breaks presumably occurring at 5’ positions). If initiated at identical linkage points, hybrid DNA is reciprocally produced by half-chromatid exchange without outside marker recombination (Model I) ; if initiated at opposite ends of the cistron in the two chromatids, then hybrid DNA is reciprocally formed in the region of an exchange (Model 11). In the latter event, towards the conclusion of reannealment of the two detached strands with

s t e p

I n t a c t c h roma t i d s

of involved bivalent

Dissociation of

DNA single strands

Competition between dissociated and intoct strands for atachment

Rejoining o f ,DN,A single strands with those o f homologs

Dissociation a t opposite e n d o f cistron

Rejoining of DNA single strands with those o f homologs

Base c o r r e c t i o n


M o d e l I M o d e l E

( s e e F i g u r e s 4 a and 5 ) FIGURE 13.-Hypothetical models of gene conversion, Model I involving no exchange of out-

side markers, Model I1 leading to recombination of outside markers.

TA

BL

E 9

Com

pari

sons

of p

redi

ctio

ns f

rom

unm

odif

ied

mod

els

in F

igur

e 13

wit

h ac

tual

obs

erva

tions

Rep

orts

of

othe

r an

thos

Pr

edic

tion

Dat

a of

KIT

ANI a

nd OLIVE

In a

gree

men

t with

pre

dict

ion

Not

in a

gree

men

t with

pre

dict

ion

(1 )

Tot

al c

onve

rsio

n fr

eque

ncie

s eq

ual i

n m

utan

t x

wild

-typ

e cr

oss

(2)

Tot

al c

onve

rsio

n fr

eque

ncie

s eq

ual i

n in

tera

llelic

cro

ss (

also

eq

ual t

o th

ose

of m

utan

t x

wild

type

)

(3)

Ra:

Rp

rati

o =

1:l

(4)

Ra-

1:R

a-2 =

1:l

R

p-2:

Rp-

I =

1 :I

(5)

Max

imum

num

ber

of c

on-

vers

ion

ascu

s typ

es;

5 (v

isua

lly a

berr

ant)

+

1 (n

orm

al se

greg

atio

n)

(5’)

Max

imum

num

ber o

f co

n ve

rsio

n as

cus t

ypes

; 45

in in

tera

llelic

cro

ss

(6)

No

corr

elat

ion

betw

een

inte

ralle

lic d

ista

nces

and

fre-

qu

enci

es o

f w

ild-t

ype

spor

es

Fit

(K

ITA

NI an

d O

LIV

E, 19

67)

Fit

(Thi

s rep

ort,

Sect

ion

11)

Obs

erve

d ra

tio

was

1.

4:l

(Sec

tion

111)

Res

tora

tion

and

subs

titut

ion

grou

ps d

evia

te in

opp

osite

di

rect

ions

; nor

mal

4:1

. typ

e di

ffer

ent

from

oth

er ty

pes

5 (a

berr

ant)

+ 1 (

norm

al)

41 a

ctua

lly

obta

ined

Par

tial

cor

rela

tion

EM

ER

SON

an

d Y

u-SU

N (

1967

),

RO

SSIG

NO

L

(196

1.),

EM

ER

SON

F

IEL

DS

(u

npub

lishe

d)

(196

6) a

nd K

RU

SZE

WSK

A

and

GA

JEW

SKI (1

967)

(Uns

elec

ted

octa

d an

alys

is h

as n

ot y

et b

een

esta

blis

hed)

MIT

CH

EL

L (1

955)

, MU

RR

AY

F

OG

EL

an

d H

UR

ST

(196

7)

( 196

3) a

nd F

INC

HA

M

( 196

7)

Non

e M

UR

RA

Y (196

3), F

INC

HA

M

(196

7),

and

FO

GE

L

and

HU

RS

T

(196

7)

5 (a

berr

ant)

+ 1 (

norm

al):

2

(abe

rran

t) : R

OM

AN

(1 96

3)

EM

ER

SON

( 1

966)

5

(abe

rran

t) :

CA

SE an

d G

ILE

S (19

64)

and

FO

GE

L

and

HU

RS

T

(1 96

7)

Non

e 54

pre

dict

ed b

y E

ME

RSO

N

(196

9)

Non

e M

ajor

ity

of r

epor

ts, b

ut w

ith

exce

ptio

ns i

n m

ost


each other, the two intact strands are forced from their linkage points, after which they pair with each other. In either event 'base correction may or may not occw in unmatched parts of the hybrid DNA, leading to the three modes of base correction behavior: mutant + wild type, no correction, and wild type + mutant.

With regard to the revised models, several predictions might be made: ( 1 ) The entire cistron being simultaneously involved in hybrid DNA formation, frequencies of conversion asci should be equal for all sites in the cistron in all mutant X wild type crosses. (2) The same equality should apply to interallelic crosses, and frequencies of conversion asci should be the same in both types of crosses. (3) If Models I and I1 applied with equal frequency, recombination classes Ra and Rp should show a 1:l ratio. (4) Ratios of recombination subclasses Ra-1 and Ra-2 and subclasses Rp-2 and Rp-1 should be 1 :l; hence the ratio Ra-1: Ra-2: Rp-2: Rp-1 should be 1 : 1 : 1 : 1. (5) The maximum number of conversion genotypes among the asci of mutant X wild type crosses should be six (one of which appears as normal 4: 4), and the maximum number in an interallelic cross should be 45 (one of which appears as normal 4ml: 4m2), but epistasis of one allele over the other (i.e., h over gl) would lead to 21 visually distinguishable conversion phenotypes (one with normal 4mI:4m, segregation), as in the present studies (only H type missing). (6) No correlation between frequency of wild type recombinants and distances between alleles would be inherent in the models.

In Table 9, the above stated expectations are compared with our results and those of others. The six points listed above and in the table are further discussed below:

(1) Total conversion frequencies in mutant X wild type crosses. In S. fimicola all mutant alleles in crosses with wild type gave similar conversion frequencies (KITANI and OLIVE 1967). This was also true in the study of Ascobolus immersus by EMERSON and YU-SUN (1967). It is considered that these results indicate the involvement of the whole cistron in hybrid DNA formation in mutant x wild type crosscs.

(2) Total conversion frequencies in mutant x mutant crosses. If involvement of the whole cistron in hybrid DNA formation is a general phenomenon, the total conversion frequencies should be the same in all crosses between pairs of alleles in the same cistron and equal to the frequencies for all crosses between these alleles and wild type. This expectation appears to be met in the present data, as shown in section I1 of the RESULTS. No equivalent data on this subject are available, since techniques for unselected octad analysis have not been perfected for other species.

(3) Ratio of recombination classes. The expected ratio between classes Ra and Rp, based, respectively, on the absence or presence of outside marker recombination, is 1 : 1 as shown in Table 9. Previous data on mutant x wild type crosses (KITANI and OLIVE 1967) showed a generally good fit to the expectation, and the present data irom interallelic crosses show fair fit to this ratio for each of the fundamental ascus types as well as for the totals. However, there was a still better fit of Ra:Rp to the ratio 1.4: 1 for all conversion types in all crosses except for those conversion asci which show normal 4i-:4m segregation at one of the two

60 Y. KITANI A N D L. S . OLIVE

mutant sites in several interallelic crosses (Table 3d and section I11 of RESULTS). It was suggested earlier (KITANI and OLIVE 1967) that this minor deviation may have resulted from the switch-back of the strands in hybrid DNA after base correction. However, the absence of g,h-wild type spore pairs in interallelic crosses makes this idea untenable. An alternative explanation is that the rate at which Model I applies is higher than that for Model 11, possibly because of the absence of competition for attachment of free ends of the DNA strands in the former. (4) Ratios of recombination subclasses. The expected 1: 1 ratios of Ra-1:Ra-2

and Rp-2:Rp-1 (Section 111, RESULTS) were not realized. Our data show that the Ra-1 subclass is 2.4-13 times greater than Ra-2 in mutant x wild type and interallelic crosses (excluding all normal 4 f : 4 m type for a particular allele), except in the cross gl x h,. A similar but weaker asymmetry was found in the Rp-2:Rp-1 ratio, with subclass Rp-2 ranging from equality with Rp-1 to three times as large in all crosses (again with the exclusion of normal 4 f : 4m conversion asci for the allele being analyzed, Rp-1 being larger than Rp-2 in this type). The larger subclasses Ra-1 and Rp-2 represent asci in which restoration base correction has occurred, while the smaller subclasses Ra-2 and Rp-1 pertain to asci in which substitution correction has occurred (Section 111, RESULTS). Therefore, our simplified models require modification to account for the absence of a 1 : 1 : 1 : 1 ratio among the four subclasses. Inequalities in the ratio would be expected if, in a hybrid DNA segment containing mismatched base pairs, the initiative in base correction were more often taken by the intact single DNA strands in Model I or, in the event of an exchange as shown in Model 11, by the strand in each chromatid which remains intact at the proximal end. Thus, the correction would more often be to the allele initially carried by each chromatid, and this would account for the greater proportions of restoration over substitution corrections. This idea agrees with the data presented in Table 8 and discussed in Section IX, part 3, of RESULTS. On the other hand, the deviations of the subclasses from the 1: 1: 1: 1 ratio might also be explained on the basis of hybrid DNA formation at a mutant site in one chromatid but not in the homolog. But in this case it would be difficult to explain the absence of a polaron-like effect in the data.

( 5 ) Maximum numbers of conversion ascus types. In mutant x wild type crosses, five visually distinguishable aberrant ascus types have been studied in Sordaria fimicola (KITANI et al. 1962; KITANI and OLIVE 1967), S . breuicollis (W. G. FIELDS, unpublished data) and Neurospora crassa (CASE and GILES 1964), and four in Ascobolus immersus (EMERSON and YU-SUN 1967). The presence of the normal appearing 4 f : 4m conversion type was predicted by WHITEHOUSE (1963), EMERSON (1966), and KITANI and OLIVE (1967) and confirmed in the present study (Section IX, part 2, RESULTS). The existence of only six types of conversion asci stresses the fact that only two chromatids are involved in a single conversion event. The rare occurrence of 7: 1 (KITANI et al. 1962; KITANI and OLIVE 1967) and 8:O (EMERSON 1969) asci indicates that the other pair of chromatids may on rare occasions participate simultaneously and probably independently in the conversion process at the same locus.


From the various combinations among the three base correction modes in the two participating chromatids, no more than 45 different conversion ascus genotypes are expected in heteroallelic crosses (Figure 4b). However, if HOLLIDAY’S unmodified model, which entails a re-annealing of already corrected DNA single strands to the original half-chromatids, were applicable an even greater variety of ascus genotypes would be expected, since wild type-double mutant spore pairs would also be present, and these were not found in our studies. (This is avoided in HOLLIDAY’S modification of the model to exclude one of the mutant sites from hybrid DNA.) In the present investigation all but four of the 45 predicted genotypes were found (and 20 out of the 21 visually distinguishable phenotypes) , but not the 54 types predicted by EMERSON (1969), who included asci with the hypothetical but undemonstrated wild type-hyaline spore pair. Other data op- posing a re-annealing model have been presented in Section IX, part (2) of RE-

(6) Correlation between frequency of wild-type recombinants and distance between heteroalleles. The great majority of reports on fine structure mapping in fungi have generally indicated that increased distances between heteroalleles usually lead to increased frequencies of wild-type recombinants, though apparent exceptions are often found in these studies. The present data show some degree of correlation between relative positions of sites as indicated by complementation tests and frequencies of wild-type spores in interallelic crosses.

The origin of recombinant wild-type progeny in interallelic crosses has been variously interpreted by different investigators. ( 1 ) RIZET’S group has concluded from studies on Ascobolus immersus that they commonly result from gene conversion that gives a 6+:2m segregation at one of the mutant sites, and a similar conclusion has been reached by STADLER and TOWE (1968) for Neurospora. (2) In studies of Ascobolus (LISSOUBA et al. 1962), Neurospora (MURRAY 1963) , Aspergillus ( SIDDIQI and PUTRAMENT 1963) , and Saccharomyces ( FOGEL and HURST 1967), it has been suggested that recombinant wild types have two origins-one via a reciprocal event and the other via a non-reciprocal event. (3) Most fine structure mapping appears to have been based on the assumption that the wild-type progeny have resulted from reciprocal recombination (e.g., WOOD- W A R D ~ PARTRIDGE, and GILES 1958; CATCHESIDE, JESSOP and SMITH 1964; SU- YAMA, LACY and B ~ N N E R 1964; FINCHAM 1966).

The present data at first appeared to conform to the second group. Most of the aberrant ascus types (Figure 3) are visually identifiable as not having resulted from reciprocal chromatid exchanges. In fact, only M asci appear phenotypically to be of the latter type. From a total of 46 sufficiently analyzable M asci from crosses between group 2 h mutants and g , (Table 5), 9 contained a double mutant spore pair, 17 only one double mutant spore, and 20 no double mutant spore. Of the first group, 7 showed recombination of outside markers and two had the parental alignment. Though it can not be disproved that the former 7 resulted from conventional reciprocal exchange, the proportion of this group would not be unexpected from gene conversion. In this connection, it should be noted that I

SULTS.


type asci, which are phenotypically like M except for the substitution of a gray for a dark spore in the wild-type pair, are clearly the result of conversion, and this group is second in abundance to M in gl x h, and gl x h, crosses.

The foregoing observations and the previously discussed evidence for the equal involvement of all sites of the cistron in hybrid DNA formation suggest that recombinant wild-type frequencies in interallelic crosses can not be directly correlated with exchanges between sites, which increase proportionately with distances between the sites. Rather, it appears more likely that the sites have mu- tually modifying effects upon each other in conversion and that these effects may be related in part to their distances apart.

This discussion of items listed in Table 9 suggests the need of some modifications in the models that we have proposed. These will be listed after an analysis of characteristics of the normal 43: 4m conversion asci.

In addition to the above stipulations, consideration must also be given to differences between original and derived mutants in conversion behavior in both mutant x wild type and interallelic crosses and to their significance in the map- pings of sites (Section VIII, RESULTS). The actual mode of origin of the modified mutants is unknown, but it is probable that each occupies essentially the same position as the original mutant from which it was derived, since no wild-type spores are recovered from crosses between them (see footnote 6). This would indicate that the relationship of one site to another in gene conversion can not be based entirely upon their relative positions but at least in part on the nature of the mutants themselves.

Those conversion asci in which one heteroallele showed normal 4+: 4m segregation exhibited a number of characteristics different from those of other types. Also, the frequencies of these asci were in large part responsible for the apparent correlation between distances separating sites and frequencies of wild-type spores. In Table 10 three different postulated methods by which this type may have originated are tested against the observed characteristics of the group. None of the methods (items A-C) satisfied all of the listed characteristics. Least satisfac- tory is item A, which proposes the origin of these asci by reciprocal interallelic recombination. Only characteristics l-a and l-b agreed with the expectations.

Item B, which proposes that part of the cistron may sometimes be excluded from the hybrid DNA region, at first appeared to agree best with the listed characteristics. The 1: 1 ratio of Ra:Rp classes, instead of the 1.4: 1 ratio obtained among pooled asci (characteristic 4), was fully satisfied only by item B, and the extremely asymmetrical proportions observed among the subclasses agreed most satisfactorily with this item (characteristics 5-a, 5-b, 6-a and 6-b) . However, the postulate definitely disagreed with an important observed character, namely, the persistent correlation between increased frequency of conversion asci with normal 4+:4m segregation at one site with the decreased frequency of aberrant 4+:4m asci failed to affect a number of other ascus types (see characteristic 3) . This item would also not explain the observation that derivative mutants (e.g., h4,,) may give proportions of aberrant ascus types in crosses with wild type and gl that differ from those of the original mutants (e.g., h4) in corresponding crosses (not


TABLE 10

Comparisons between observed characteristics of normal 4$ :4m conversion asci in interallelic crosses and the predictions from three hypothetical modes of origin

Hypotheses C

B Distally intact

Observed Refer Interallelic chromatids not always has initiative A Part of cistron in both DNA single strand

characteristics of normal to text reciprocal involved in hybrid 44- :h conversion asci sections: recombination DNA formation+

-

1 -a. Very high proportion IV I-b. Correlation with distance VI

between alleles 2. No alteration in total I1

frequency of conversion asci in interallelic cross

3. Increased frequency primarily correlated with and IX-(3) decreased frequency of aberrant 4+:4m asci without affecting several other types

4. Ra:Rp ratio in pooled asci IV and V 1:l rather than 1.4:l

restoration group

substitution group

IV, V

5-a. Ra-IpRp-2 in I11

5-b. Ra-2<(Rp-l in I11

6-a. Ra-1 )>Ra-2 in pooled asci 6-b. Rp-2<(Rp-l in pooled asci*

I11 I11

7. M:I ratio near 1:l. IX-1 2g ,h< l g , h S n o g l h

Possible Possible Obligate Obligate

Would increase Presumably the apparent unexpected frequency of (see text) conversion asci

Unexpected Unexpected

0:l expected 1: l expected

Expected

. . . _ . . Expected

. . . . . . Expect e d Only Rp-2 Expected for m, site

Only Rp-1 for m2 site

Unexpected Possible Unexpected Possible

in base correction for its own allele

Possible Expected

Expected

Possible

Either 1 :1 or

Expected 1.4:l possible

Expected

Expected Expected

Expected Expected

* The reverse of observations in all other ascus types. -f Expected fit poor if confined to one chromatid.

listed in Table 10; see section VI11 of RESULTS). Furthermore, if item B were applicable, a significant increase in proportions of conversion asci with normal 4f : 4m segregation at one site in the interallelic crosses should be accompanied by correspondingly increased total frequencies of conversion asci (see characteristic 2 ) . Among all conversion asci, those showing normal 4 f : 4gl segregation would not be conversion asci for the gl site, while all other types, including those showing normal 4 f : 4h segregation, would represent conversion asci for that site. This would apply similarly to any h site. Hence, the observed frequency of all conversion asci minus the frequency of those showing normal 4+: 4gl segregation should be equal to the constant conversion frequency observed for all g heteroalleles. In other words, the total frequencies of all conversion asci in interallelic crosses would be higher than the constant frequency observed among

64 Y. K I T A N I A N D L. S. OLIVE

heteroalleles in crosses with wild type, and the increase would be correlated with increased frequencies of conversion asci with normal 4+: 4m segregation at either site.

Earlier it was suggested that the initiative in base correction in a hybrid DNA segment may belong to the strand which has remained unbroken proximally. If, as in item C, it is postulated that in interallelic crosses the initiative may to some degree be taken by the strand which remains distally intact, then all the observed characteristics in Table 10 could be reconciled with the expectations. In Model I (Figure 13) the DNA single strands which are intact at the proximal ends are also intact at the distal ends, but in Model I1 the strands that are proximally intact have broken at the distal ends. If the proximal initiative goes into effect first and is responsible for the general preference for restoration base correction in aberrant asci, and the distal initiative occurs secondarily and tends to correct any remaining mismatched pairs, the correlation between increased frequency of conversion asci with normal 4+:4m segregation at one site and the decreased frequency of 4+:4m asci with aberrant segregation for the same site would be expected.

Also expected would be the observed reversal in the Rp-2 (restoration) : Rp-1 (substitution) ratio in normal 4+:4m conversion types from that found in other types, with the concurrent maintenance of the superiority of Ra-1 (restoration) over Ra-2 (substitution). The effect of the distal initiative on ascus proportions in interallelic crosses would presumably be greater with respect to heteroalleles (e.g., h, and h4) in the distal part of the cistron. No such effect appears to be present in the cross gl X h,, where both heteroalleles are believed to be close together in the proximal portion of the cistron. Thus it would seem that conversion asci with 4 + : 4m segregation at one site can be better explained by item C rather than B in Table 10.

Several possible modifications of our two models, previously discussed in an analysis of Table 9, may be considered in conclusion: (1) A moderate difference in frequencies of Models I and I1 exists in favor of the former; (2) the choice of base correction mode is influenced by a proposed proximal initiative in base correction; (3) a distal initiative in base correction operates secondarily in interallelic crosses, increasing base correction and leading to an increase in normal 4+: 4m segregation (with corresponding decrease in aberrant 4+: 4m segregation) at a site; (4) the mode of base correction at one mutant site is modified by the heterozygous presence of a second mutant allele.

The writers gratefully acknowledge the valuable technical assistance of MRS. M4RJORIE CHRISTIANSEN POLLICE and the critical reading of the manuscript by DR. STERLING EMERSON.

SUMMARY

A total of 862 conversion asci, representing virtually all types and numbers present in observed material, were recovered from interallelic crosses involving heteroalleles (gl, h,, he, and h4) at the gray spore (g ) locus in Sordaria fimicola. The results are compared with those from mutant x wild type crosses. The following observations on interallelic crosses may be made in summary: (1) 20 of


the 21 possible visually distinguishable conversion ascus phenotypes and 41 of 45 predicted genotypes were obtained from six different interallelic crosses; (2) total frequencies of conversion asci were not different from those obtained in mutant x wild type crosses; ( 3 ) a large amount of recombination of outside markers was observed, and asymmetries in ratios of recombination classes Ra: Rp (recombination of outside markers absent or present, respectively) and among their subclasses were found; (4) the expression of asymmetry in conversion asci with normal 4+:4m segregation at one allele was different from that in all other ascus types; ( 5 ) proportions of the different aberrant ascus types varied among interallelic crosses (gl x h alleles), and these proportions varied from those of the corresponding mutant x wild type crosses; (6) conversion characteristics of heteroalleles in repulsion and coupling crosses were similar; (7) certain derivative mutants (e.g., h45) showed conversion behavior different from that of the original mutants (e.g., h4) from which they were derived. Previously described hybrid DNA gene conversion models are discussed and adjustments in our recently described models (KITANI and OLIVE 1967) are proposed.

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Documents

GENETICS OF SORDARZA FZ-MICOLA. VII. GENE CONVERSION