QUANTITATIVE INTRACHROMOSOMAL CHANGES ARISING AT … · 2003-07-22 · QUANTITATIVE INTRACHROMOSOMAL CHANGES ARISING AT MITOSIS IN ASPERGILLUS NIDULANS B. H. NGA AND J. A. ROPER Department

QUANTITATIVE INTRACHROMOSOMAL CHANGES ARISING AT MITOSIS IN ASPERGILLUS NIDULANS

B. H. NGA AND J. A. ROPER

Department of Genetics, The Uniuersity, Sheffield, England

Received August 11, 1967

novel and spontaneous process, giving quantitative change of genotype at A mitosis in Aspergillus nidulans, was described by BAINBRIDGE and ROPER (1966). Segregants carrying a segment of linkage group 111 in duplicate were derived from crosses of strains free of translocations to strains with an apparently non-reciprocal III-VI11 translocation. These duplication strains had reduced linear growth rate and a distinctive morphology; they were designated “crinkled.” Crinkled colonies showed vegetative instability by producing sectors which, in varying degree, approached wild type in growth rate and morphology. Genetic analysis showed that the sectors had lost a variable amount of the chromosome segment carried in duplicate; loss occurred from either the untranslocated segment of linkage group I11 or from that segment of linkage group 111 translocated to VIII.

The investigations reported here extend these observations by seeking answers to the following questions. First, is the phenomenon of chromosome loss confined to the particular linkage group I11 duplication or, as might reasonably be sup- posed, is it of general occurrence? Second, are the losses terminal and are there indications of preferential regions of loss? This required, ideally, duplications with several markers in heterozygous condition. Third, does loss involve exchange of markers between the homologous segments? The answer would cast light on the mechanism involved. Finally, and this is to some extent related to the third question, is the change always one of loss and never of gain? Losses might arise by, for example. unequal mitotic crossing over or by a mitotic version of the intrachromosomal meiotic process proposed by LAUGHNAN ( 1955, 1961 ) , PETER- SON and LAUGHNAN (1961, 1963) and BOWMAN (1965). Either mechanism would generate, in addition to losses, tandem duplications. Such duplications might he detected by a further deterioration in phenotype.

MATERIALS A N D METHODS

Media, Minimal medium (MM) was Czapek-Dox medium with 1% w/v glucose. Complete medium (CM) was a complex medium containing yeast extract, hydrolysed casein, hydrolysed nucleic acid, vitamins, etc. Solid media contained 2% agar.

Organisms: Strains of A . n i d u l a q all derived from Glasgow stocks, were kept at 5” on CM

slopes. They were purified every six months by single colony isolation and auxanographic char- acterisation. Except when otherwise specified, all strains were free from translocations. “Master” strains (MSE and MSF). carrying markers on all eight linkage groups, were those of MC€ULLY

(;ericti< F 5 8 : 19?-200 Felebrtml-y 19(iR

VI 53 VII n!ca Vlll ribo?

FIGURE 1.-Mutant alleles used in this study determined the phenotypes: y, yellow conidia; w3 (epistatic to y / y f ) white conidia; ad20, bil , meth3, nicl, pabab, prol, pyro4, rib02 and s3, requirement, respectively, for adenine, biotin, methionine, nicotinic acid, p-aminobenzoic acid, proline, pyridoxin, riboflavine and thiosulphate; Acrl, resistance to acriflavine; jacA and gall , inability to use, respectively, acetate and galactose; SUI-ad20, suppressor of ad20. MSE had the genotype sul-ad20 y ad20; w3; gall; pyro4; jacA; s3; nicl; ribo2. MSF differed from MSE only in having Acrl instead of w3 as linkage group I 1 marker. Meiotic linkage values and centromeres are given for linkage groups I and 11. w3 showed free recombination with the point of attachment of the duplicate segment.

and FORBES (1965). Mutant alleles of particular importance in this work are shown in Figure 1 . Diploid strains were prepared from heterokaryons by the method of ROPER (1952). Symbols used for mutant alleles, tandem duplications, translocated duplicate segments and deletions are illustrated by an example. I dl ( y ad20) bil/I-I1 y ad20 bil Dp designates the genotype: linkage group I , with deletion of the y and ad20 loci and carrying the mutant allele bil; duplicate segment of I translocated to 11, carrying the mutant alleles y, ad20, bil , and a tandem duplication of unspecified length. The haploid components of diploid strains are separated by the symbol//.

Incubatiom At 37" except where otherwise specified. Methods of analysis: General techniques of genetic analysis were those of PONTECORVO,

ROPER, HEMMONS, MACDONALD and BUFTON (1953). Mitotic haploidisation was used for the assigning of genes to their linkage group or groups (FORBES 1959), for the detection and prelimi- nary analysis of translocations (KAFER 1962) and for chromosome substituiton. The detection of haploid segregants arising mitotically from diploids was facilitated by the use of CM with 1/10,000 w/v DL p-fluorophenylalanine FA) (MORPURGO 1961). Since PFA inhibits haploids which carry a duplication, CM alone was used when such segregants were expected.

RESULTS

The linkage group I duplication: This duplication strain (PRITCHARD 1956) was isolated from a selective plating of apparently normal haploid ad20 bij conidia on MM -I- biotin. Its mode of origin was unknown but it was shown by PRITCHARD to have the genotype I ad20 bil/I-I1 ad20 b i l ; two doses of the leaky ad20 allele permitted its selection from the parental normal haploids. The strain had a distinct crinkled morphology and reduced linear growth rate.

The following analyses confirmed the location, arrangement and extent of the duplication and also established correlation between the duplication itself and crinkled morphology. In addition, they provided a segregant duplication strain suitable for the study of vegetative instability.

From the cross, I ad20 bil/I-I1 ad20 bil x pro1 paba6 y; w3, was selected a

GENOTYPIC CHANGE AT MITOSIS 195

TABLE 1

Haploids from the diploid I prol paba6 y ad20+ bil +/I-I1 y+ ad20 bil//MSF

Linkage group

I1 111 IT \’ V I VI1 VI11 _ _ _ r _ _ _ _ _ _ _ _ _ - - ~

I

Colour/Morphology pro+ paha+ pro paba Acr+ Acr gal+ gal pyro+ pyro fact fac s+ s nic+ nic rho+ ribo

Green crinkled 6 7 1 3 0 6 7 7 6 6 7 7 6 8 5 5 8 Yellow normal 5 8 0 1 3 7 6 6 7 6 7 1 0 3 4 9 5 8

All segregants were ad+ hi+.

green crinkled recombinant with probable genotype I pro1 palm6 y &20+ bil+/ 1-11 y+ ad20 bil. This segregant was combined with MSF to form a diploid. The results of haploidisation on CM are shown in Table 1. Segregation of morphological types was correlated with segregation of linkage group 11; the factor responsible for crinkled was carried on linkage group I1 of the parent with putative duplication. Some green crinkled haploids combined linkage groups I of MSF with I1 of the duplication strain, showing that the latter linkage group did, in fact, carry a segment of linkage group I with the y+ allele. No effective part of linkage group I was translocated in the duplication strain; this was shown by recovery of haploids combining linkage group I of the duplication strain with linkage groups I1 to VI11 of MSF. These particular haploids also confirmed the linkage group I genotype, in the duplication strain, as pro1 paba6 y ad20+ bil+.

Table 2 summarizes results of the cross I pro1 paba6 y ad20+ bil+/I-I1 y+ ad20 biz x y; pyro4; meth3. Although recovery of crinkled and normal differed significantly from 1 : 1 (P < 0.001 ) , the result was presumed to reflect a 1 : 1 segregation with reduced viability of the morphologically abnormal type. Most of the crinkled segregants were green and in the parents the y+ allele was carried on a translocated, duplicate segment of linkage group I. This suggested either that the duplication itself was responsible for the crinkled phenotype o r that the 11-1 linkage group complex carried a mutant allele determining this abnormal mor-

TABLE 2

Segregants from ihe cross I prol paba6 y ad20+ bil +/I-I1 y+ ad20 bil x y; pyro4; meth3

Crinkled 200 green 22 yellow

Normal 28 green

272 yellow

b) pro+ pabaf pro paba pro paba+ pro+ paba Yellow normal 47 45 5 7

c ) ad+ bi+ ad bi ad+ bi ad bi+ Yellow normal 103 1 6 0 Green nxmal 0 27 0 0

a ) Total coloriies fwm a plating of ascospores; b) and c) randomly picked normal yellow and green segregants.

196 B. H. NGA A N D J. A. ROPER

phology. The latter possibility was excluded by analysis of vegetative segregants; normal morphology was restored by loss, from the duplication parent, of either of the duplicate chromosome segments. The frequencies of green normal and yellow crinkled segregants permitted tentative calculation of the recombination frequency between the y locus and the point of break as 9 to 10% ; the calculation takes no account of possible preferential pairing between particular homologous segments. The 1 :I segregation of the alleles of pro and paba showed that the duplication did not extend to their loci; this agreed with the tentative estimate of the proximal point of break. Normal green segregants arose by crossing over between one or other homologue of linkage group I and the duplicate segment, followed by appropriate segregation of chromatids. Normal yellow bi segregants arose simi- larly. If the duplicate segment were inverted or located interstitially or if it did not include the right end of linkage group I, then each class could have arisen only by double crossing over. In fact, the frequency of normal green and normal yellow bi types indicated a noninverted duplicate segment of linkage group I, including its right end, terminally located on linkage group 11. The translocated duplicate segment carried the alleles y+ ad20 biz.

The strain I pro1 paba6 y ad20+ bil+/I-I1 yf ad20 biz was used as original parent for the main part of these studies. It showed a high frequency of vegetative segregation as detected by the following methods; i) As sectors of modified morphology or non-parent conidial colour growing out from the parent colony. These sectors were usually of improved morphology and faster growth rate but rare exceptions were detected. ii) As small patches, within the parent colony, bearing conidia of non-parental colour. iii) As colonies with non-parental morphology or conidial colour amongst those growing from conidia plated at low density. I t was appreciated that yellow segregants could arise by standard mitotic crossing over between the homologous linkage group I segments. The yellow sectors and patches arose with far higher frequency than those arising from a diploid by mitotic crossing over; furthermore, subsequent analysis showed that no yellow segregants taken for analysis did, in fact, arise by mitotic crossing over. Some conidial platings yielded a very high proportion of non-parental variants of extremely diverse types and no systematic analysis of these has yet been undertaken. In- stead, variants obtained by methods i) and ii) were used on the assumption that they were more likely to be only one change removed from the parent duplication type.

Vegetative instability gave both phenotypic improvement and deterioration. Improved sectors were sometimes indistinguishable from wild type but more commonly they were intermediate between parent and wild type in growth rate and their degree of crinkled morphology. In the latter cases further stepwise improvements occurred towards wild type. On one occasion an improved sector produced a deteriorated variant. Deterioration was detected rarely; it was seen as brown pigment in the mycelium, coupled with crinkled morphology and a slightly less than parental growth rate. Under appropriate conditions the “browns”, as they were designated, showed stepwise improvement towards normal, though they only rarely reached normal. The overall range and lineage

GENOTYPIC C H A N G E AT MITOSIS 197

I m"* Y f + PWnMQmDiype: + * NI

1 - 1 1

Phenotype Crgmen + +

. . . Y

cr b green + + 06) cryellow+ +(I)

cr br yellow

I yellow + + (2)

I

crbgreen++(u)

cr br yelbw + +(U) I

yellow + +@) t br green - - no)

FIGURE 2.-Lineage of some of the phenotypic classes ahsing by vegetative instability. Pheno- types refer to: c r (crinkled, with different degrees of expression in different classes); br (brown pigment); yellow or green conidial colour; -, requirement for or +, independence of adenine and biotin: + and --A+ represent phenotypic improvement and deterioration respectively.

of types arising by vegetative instability are shown in Figures 2 and 3. Vegetative instability of crinkled strains was apparently greater at 34" and 42" than at 37".

Analysis of variants wising by vegetatiue instability. Improved uariants: Strains of class 8 were stable, green ad bi and morphologically normal; it seemed likely that they had lost most or all of the segment of linkage group I which carried the markers y, a&0+ and bil+ and that they had the genotype I dl ( y ad20+ biZ+)/I-II y+ mi20 biz. This was shown for strain 8A. White haploids derived from 8A//MSE (Table 3) on CM never carried linkage group I of 8A, suggesting a lethal on that linkage group. This was confirmed by the cross 8A x MSE (Table 4) . The 2: 1 segregation of morphological types was consistent with the suggestion that 8A carried a translocation but little or no duplication. The approximately 2:l segregation of green : yellow showed close linkage of a recessive lethal to the y allele of 8A. This lethal, representing the proximal point of break, was located by nutritional test of a particular class of progeny (Table 4). Normal white hi+ progeny carried the bil + allele of MSE; most were presumed to carry the y allele of MSE but this could not be checked visually because of the epistasis of w?. Normal white bi+ pro (or paba) types arose by recombination in the region between the pro2 (or pabab) locus and the proximal point of break. The break was located about 8 units to the right of paba6 on linkage group I of 8A. In 147 segregants of the cross, 8A x bil; Acrl w3; ribo2, there were no yellow or bi+ types. This showed that y and biz+ were lost from 8A; if they had been sup- pressed, rather than lost, they should have been recovered free from their suppressor on outcrossing.

198 B. H. N G A A N D J. A. ROPER

I;IGUI<I; 3.-Yegt~tativt* instaI)ility i r i i l ~ p ~ ~ p i l l i ~ . ~ nirlulrrns. A. sho\ving iiiiprovctl yt,llo\v ;md grrcw srctws and 13. with a siriglr tlrtrrioratctl srctor: tmth from rrntral inorulum of conidia of genotype I pro1 pnha6 y ad20+ bil+/I-II y+ ad20 bil. C, sectored colony from a central inoc- ulum of conidia of deteriorated brown variant. 11A. D. colonies from a conidial plating of strain 16B, genotype I pro1 pabad y ad20+ bil+ Dp/I-I1 y+ ad20 bil . All at 37".

Strains of class 3 were stable, morphologically normal, yellow ad+ bi+. They seemed likely to have the genotype I y nd20+ bil+/I-I1 dl (y+ ad20 biz), with, perhaps, a small and variable part of the translocated segment remaining on linkage group 11. The cross 3A x MSE gave 70 yellow and 77 white normal, 4 yellow and 4 white crinkled segregants. No green, ad or bi segregants were obtained


E

3 2

i 2 2

8 % M w

4

2 % 2 - 4 s


TABLE 4

Segregants from the cross 8A x MSE

Crinkled Normal

a) 38 green 34 green 23 white 63 white

2 yellow 28 yellow

b) pro + Pro White normal bi+ 45 8

White normal bi+ 49 4 c ) paba+ paba

a) Total colonies from one plating of ascospores; b) and c) randomly picked normal white bi+ from a number of platings.

showing that these alleles were lost from 3A. In contrast to the above similar cross of 8A, few crinkled segregants were obtained; 3A behaved like an almost normal haploid. However, the recovery of a small proportion of crinkled segregants indicated that linkage group I1 retained part of its duplication. The remaining fragment included part of the proximal end of the duplication since it permitted, by crossing over, regeneration of the full crinkled phenotype. Five more class 3 strains gave similar results and each, on outcross to normal, yielded about 5% crinkled segregants (Table 5) . This implied that these six class 3 strains had a roughly similar proximal point of break in the translocated segment. Two further class 3 strains differed from the other six in only one respect: from crosses to normal they gave no crinkled segregants. They appeared to have lost either the whole of the translocated duplicate segment, or at least that proximal part which could, by crossing over, have permitted regeneration of crinkled.

Individual strains of class 4 gave sectors of three different, further improved, types (Figure 2). If morphological improvement involved loss of chromosomal material, then the lineage indicated that loss must have been, at least sometimes, interstitial. For example, if 4A arose by terminal loss from either of the duplicate segments, then further terminal loss to give 5A, 6A and 7A would not have been consistent with viability. Analysis of one class 4 strain, fully representative of

TABLE 5

Segregations from crosses of class 3 uariants x y; pyro4; meth3

Class 3 parent Normal

3A 147 3B 295 3c 4.00 3D 183 3E 21 7 3F 154 3G > 200 3H > 200

Crinkled Per cent crinkled

8 15 21 9

11 8 0 0

5.2 4.8 4.9 4.7 4.8 4.9 0 0

GENOTYPIC CHANGE AT MITOSIS

TABLE 6

Haploids f rom the diploid 4A / /MSE

20 1

Linkage mouu

I 11 -

Coloui /.\Iorphology pro+ paba+ pro paba A<l+ .4cr

Green crinkled 3 0 Yellow normal 14 0

3 0 a 14

the two analyses made, is given here. The diploid 4A//MSF gave, on CM, no yellow haploids carrying linkage group I of 4A (Table 6). Presence of a recessive lethal on that linkage group was confirmed by the cross 4A x MSF (Table 7). Strain 4A carried a lethal about 32 units to the right of pabab. In view of the looseness of this linkage value, further crosses were made; all confirmed a lethal, located about 30 units to the right of paba6. This, and the variants arising by vegetative instability, showed that 4A had the genotype I y ad20+ bil+ dZ/I-II y+ ad20 big.

Strain 5A, derived vegetatively from 4A, was crossed to MSF (Table 8). Normal green segregants, all ad bi, suggested that there was no lethal distal to y+ on the translocated segment of 5A. Deficiency of paba types, among the normal yellow segregants, showed a recessive lethal about 33 units to the right of pabad.

TABLE 7

Segregants from the cross 4 A x MSF ~~~ ~

Crinkled 165 green

6 yellow

~

Normal 22 green 95 yellow

b) pm+ paha+ pro paba pro paha+ pro+ paba Yellow normal 60 30 3 0

a1 Total colonies from a plating of ascospores: h i randomly picked normal yellow segregants

TABLE 8

Segregation f rom the cross 5 A x MSF

Crinkled 110 green

7 yellsw

Normal 8 green

59 yellow

b) Yellow normal

paba+ 37

paba 18

C) ad+ bi+ ad bi ad+ hi ad bi+ Green normal 0 8 0 0

a ) Total coliiniei from a plating of ascospores; b) and c i randomly picked nonrial yellow and green segregants

202 B. H. NGA AND J. A. ROPER

TABLE 9

Segregation from the cross I A x y; pyro4; meth3

a) Crinkled Normal 201 yellow 209 yellow

b) ad+ bi+ ad bi ad+ bi ad bi+ 293 0 3 0

~~~~~~

a) Total colonies from a plating of ascospores, b) randomly picked segregants

Any further loss of chromosomal material in the event 4A -+ 5A probably took place either proximally to y+ on the translocated segment, or distally to the lethal detected on the untranslocated segment.

Strain 6A, obtained vegetatively from 4A, had normal morphology and was yellow ad+ bi+. From its cross to y; pyro4; meth3, no crinkled, green or bi segregants were obtained in 400 progeny. This established loss, from 6A, of all or most of the translocated segment and certainly of the markers y+ and bil. Strain 6A was probably a balanced lethal of genotype I y ad20+ bil+ dl/I-II dZ(y+ ad20 bil); the fragment on linkage group I1 must have included the region homologous to the deleted part of linkage group I.

A further instance of phenotypic improvement following interstitial loss was provided by a class 1 variant. The cross 1A x y; p y r d ; meth3 (Table 9) gave normal to crinkled segregants in 1 : 1 proportion. In this respect 1A behaved like the duplication parent. However, all 410 segregants were yellow and, of 296 tested, all were ad+. This showed that y+ and ad20 had been lost from 1A. Test for biotin requirement showed that 1A still carried the bil allele; the low frequency of bi segregants indicated that bil was still located on the translocated segment. The genotype of 1A was I y ad2O+ bil+/I-I1 d2 ( y+ ad20) bil.

Phenotypic improvement arose, then, by loss of chromosomal material from either the translocated or untranslocated duplicate segment. There was sometimes apparently total o r near-total loss of a whole duplicate segment; more frequently there was partial loss leaving scope for further losses. The loss was sometimes, perhaps always, interstitial and there was no evidence that the process of loss involved exchanges between homologous segments. One series of improved variants, class 3, gave tentative evidence of a preferential point of loss.

Deteriorated variants: Five instances of phenotypic deterioration, giving rise to brown variants, were observed. Four were independent, apparently one-step events from the duplication parent; the fifth arose from an improved variant. Two brown strains, 11A and 16A, were extremely unstable at 34", 37" and 42". Genetic analysis was not attempted since there would have been undetected changes during the heterokaryotic stage which precedes meiotic o r mitotic analysis.

Strain 16B was suitable for analysis; sectors arose freely at 34" and 42" but only rarely at 37". This apparent difference in stability may have reflected only a possible difference in the selective value of new variants at the various temper- atures. Brown haploids, from diploid 16B//MSF (Table lo) , had linkage groups

I

2 ,

a 4 ’

w , d

b


F: 2 n . \I , ? - ..

- 2 3 4 $ i b i 4

5

i - L


I and VI11 of 16B. However, brown haploids from the diploid 1 GB//MSE (Table 11) showed random segregation for the members of linkage group VIII; the determinant of brown was located on linkage group I of 16B. Haploids with linkage group I of 16B, linkage group I1 of MSF or MSE and any combination of linkage groups I11 to VI11 were examined. They had yellow (I1 of MSF) or white (I1 of MSE) conidia and were brown, crinkled, ad+, bif. They were inhibited by pFA which was known (BAINBRIDGE 1964) to inhibit duplication strains. Furthermore, they segregated vegetatively to give improved, though still brown, variants. This evidence strongly suggested that brown was determined by a duplication; since it was carried on linkage group I it was probably a tandem or, less likely, an insertional duplication. Variants of classes 17, 18, 19 and 20, all of which arose from 16B, have not yet been analysed. Their phenotypes suggested that brown remained as long as the markers ad20+ biz+ were present (classes 17, 18, 20), but loss of these markers permitted return to fully normal morphology (class 19) by elimination of brown along with most or all of the untranslocated duplicate segment of linkage group I. The implied genotype of 16B was I y ad2O+ big+ Dp/I-II y+ ad20 bil, though the exact location and extent of the tandem duplication remained uncertain.

Strain 11B was like 16B in its apparent stability at 37" and instability at 34" and 42". Haploids from 1 lB//MSF (Table 12) showed i) that linkage group I of 11B was unchanged compared with its parent and ii) that the determinant of brown was carried on linkage group I1 or on the attached linkage group I segment. Haploids combining linkage group I of MSF, the linkage group 11-1 complex of IIB, and any combination of linkage groups I11 to VIII, were unstable, brown and crinkled and were inhibited by pFA. Variants of classes 12, 13, 14 and 15 all arose from 11B but the only normal types (class 15) were yellow ad+ bi+. These probably arose by elimination of brown along with most or all of the translocated linkage group I segment. The implied genotype of 11B was I y ad20+ bil +/I-I1 y+ ad20 bil Dp, though, again, the precise extent and location of the tandem duplication were uncertain.

Strain 13B was an improved sector from 11B. Haploids from 13B//MSE (Table 13) showed: i) that linkage group I of 13B was unchanged compared with 11B and ii) that the determinant of brown was still carried on the 11-1 linkage group complex. The cross 13B x MSE gave no green segregants in 270 ascospores tested. Of these segregants, 267 were bi+ and 3 bi. This showed that 13B had lost y+ but retained the bil allele; the low frequency of bi segregants indicated that bil was still carried on the translocated linkage group I segment. Strain 13B, a further instance of interstitial loss, had the probable genotype I y ad20f bil+/I-I1 dl (r+ ad20) bil Dp.

Overall, it seemed likely that phenotypic deterioration resulted from further duplications, tandem or insertional, and of undetermined length and orientation. All the new duplications so far analysed probably occurred on one or other of the duplicate linkage group I segments, but there was no indication that their origin involved inter-segment exchange. Strains yvith such duplications were usually very unstable. For example, conidia of strains 16B and 11B gave colonies of which

4 2

iq c

\

? h 2 3

3 .c I u a -

2 - c s


g

3:

i %

w @

b

-3 n i s d

2 3 3 2

--,

Y


more than 50 % were conspicuously non-parental in morphology. Some colonies showed further deterioration in gross morphology and growth rate, others showed improvement. Completely normal types arose only following probable total or near-total loss of the chromosome segment carrying brown.

DISCUSSION

Answers can be given now to some of the questions posed earlier. In strains of A. nidulans carrying a chromosome segment in duplicate, loss at mitosis of all or part of a segment is frequent. This has been shown definitively for two duplication strains and tentatively for a third strain with a different linkage group I duplication (NGA and ROPER unpublished). Possibly similar effects have been demonstrated by NEWMEYER and TAYLOR (1967) in a duplication strain of Neurospora crassa, though there are points of probable difference as well as of similarity between their results and ours. It seems likely that the process of chromosome loss occurs in all duplication strains.

In the present study loss of chromosomal material was sometimes, perhaps always, interstitial and there was no evidence that it involved exchange between homologous segments. There was tentative indication of preferential points of break. Rare phenotypic deterioration has also been detected; this can be explained plausibly, but not definitively, by new tandem duplications whose origin did not appear to involve inter-homologue exchange. The results point to an intrachromosomal process or processes for both the loss and duplication of chromosome segments. Unequal sister strand exchange was proposed by SLIZYNSKA (1963) to explain formaldehyde-induced repeats in Drosophila and was also suggested by NGA and ROPER (1966) for the present case. In its consequences, unequal sister chromatid exchange is in part like exchange in an intrachromosomal loop. This latter idea has some attraction since various meiotic and mitotic versions of it have been demonstrated in, or proposed for, very diverse situations (LAUGHNAN 1955, 1961; CAMPBELL 1962; BOWMAN 1965; S. BEERMANN 1966; WHITEHOUSE 1967a); furthermore, it may need only one basic intrachromosomal process to generate interstitial deletions, tandem duplications and even circular fragments. There is insufficient information to justify further speculation on the precise processes producing this type of instability in A . nidulans and we have no evidence about its possible occurrence in normal haploid and diploid strains. More details may be provided by a search for possible reciprocal products of one mitotic error, by a search for tandem duplications in haploids and interstitial loss in diploids, and by study of environmental and genetic factors which modify the frequency of vegetative instability.

Consideration of any purposeful end that could be served by mitotic losses and duplications requires more information than is available now. For the present we are concerned mainly with the association between quantitative intrachromo- soma1 change and genetic stability. At least at the level of observation, and perhaps even at the level of mechanism, there are points of similarity between our system and many other instances of instability. Some features of the complex

GENOTYPIC CHANGE A T MITOSIS 20 7

instability patterns ascribed to controlling elements (MCCLINTOCK 1951, 1956) and controlling episomes ( SMITH-KEARY 1958; DAWSON and SMITH-KEARY 1963) lend themselves to explanation by tandem duplication and interstitial losses and gains. To account for a high frequency of reversible, premeiotic mutation in some white mutants of Drosophila, JUDD (1 967) has already proposed intrachromo- soma1 recombination within a duplicate locus. And for the instability of a gaZ mutant of Escherichia coli, MORSE (1967) has also suggested a duplicate locus and recombination events. HARRISON and FINCHAM (1964) have pointed out the similarities between unstable genes, systems involving controlling elements, and position effect variegation. Our strains were unsuited to the ready detection of position effect variegation; but BALL (1967), with a situation in some respects closely analogous to ours, found vegetative instability resulting in both morphological change and variable degrees of suppression of a mutant allele determining methionine requirement. It is tempting to think that the correspondence between vegetative instability in Aspergillus and other cases of instability is more than merely observational.

A further important phenomenon, antibody diversity, appears to call for somatic instability. Two recent hypotheses to account for antibody diversity require, as one of their features, something like interstitial loss. EDELMAN and GALLY ( 1967) suggested unequal somatic crossing over between sister chromatids or homologous chromosomes in a series of non-identical tandem duplications. WHITEHOUSE’S ( 1967b) hypothesis included intrachromosomal crossing over between members of a master-slave series of non-identical, duplicate genes (cf. CALLAN 1967). Among many unknown details of our system are the upper and lower size limits of the intrachromosomal events observed in and proposed for Aspergillus; and with the possible exception provided by BALL (1967), we do not know whether the present instability can give variable expression of a single gene. Clarification of these points could yield experimental evidence indirectly relevant to the problem of antibody diversity.

The authors are indebted to DR. B. W. BAINBRIDGE, DR. C. BALL and DR. R. A. WOODS for valuable discussion and to Mx. E. FORBES and PROFESSOR R. H. PRITCHARD for strains. Support from the SINO-BRITISH TRUST for one of us (B.H.N.) is gratefully acknowledged.

SUMMARY

A particular type of vegetative instability has been shown in further strains of Aspergillus nidulans. These strains, which had distinctive morphology and reduced growth rate, carried a duplicate chromosome segment in translocated position. Instability produced a wide range of variant types; most had improved morphology and growth rate but a few showed deterioration. Improved variants arose by loss, at mitosis, of part or all of one of the duplicate segments. Loss, which was at least sometimes interstitial, was extremely frequent compared with normal mutation rates. Deterioration was probably due to new duplications arising at mitosis; the proposed duplications were probably tandem and were of unknown length and orientation. Strains with these putative duplications, and without the


original duplicate segment, were unstable. Interstitial loss and new duplications arose without evidence of crossing over between the duplicate segments. The mitotic origin of both the losses and proposed gains may be explained by unequal sister chromatid exchange or by sister strand crossing over in an intrachromo- soma1 loop. It is argued that quantitative intrachromosomal change occurring at mitosis might explain some other cases of instability.

LITERATURE CITED

BAINBRIDGE, B. W., 1964 Genetic factors affecting the morphology of Aspergillus nidulans. Ph.D. thesis, The University, Sheffield.

tion in Aspergillus nidulans. J. Gen. Microbiol. 42 : 41 7424.

Genet. Res. (In press).

Cyclops furcifer. Genetics 54: 567-576.

BAINBRIDGE, B. W., and J. A. ROPER, 1966

BALL, C., 1967

Observations on the effects of a chromosome duplica-

Chromosomal instability related to gene suppression in Aspergillus nidulans.

BEERMANN, S., 1966 A quantitative study of chromatin diminution in embryonic mitoses of

BOWMAN, J. T., 1965 Spontaneous reversion of the white-ivory mutant of Drosophila melanogaster. Genetics 52: 1069-1079.

CALLAN, H. G., 1967

CAMPBELL, A., 1962 Episomes. Advan. Genet. 11: 101-145.

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QUANTITATIVE INTRACHROMOSOMAL CHANGES ARISING AT … · 2003-07-22 · QUANTITATIVE INTRACHROMOSOMAL CHANGES ARISING AT MITOSIS IN ASPERGILLUS NIDULANS B. H. NGA AND J. A. ROPER Department