EFFECTS OF ULTRAVIOLET IRRADIATION ON HETEROZYGOUS DIPLOIDS OF ASPERGZLLUS NZDULANS. 11. RECOVERY FROM UV-INDUCED MUTATION IN MITOTIC RECOMBINANT SECTORS ETTA KAFER Department of Genetics, McGill University, Montreal, Canada Received September 16, 1968 HE major effects of radiation on microbial cells or spores are killing, induced Tmutation and, at least in the case of diploid heterozygous fungal cells, increased mitotic recombination. In addition, poorly viable survivors have been observed in many organisms; these types are being investigated by pedigree analysis in unicellular organisms like yeast (JAMES and WERNER 1966; HAEFNER 1966) and bacteria (HAEFNER and STRIEBECK 1967). Most of them are found to produce lethal sectors in the first or in later divisions, but some recover com- pletely and revert to normal growth rate and viability (JAMES and SAUNDERS 1968). Similarly, when diploid conidia of Aspergillus nidulans are treated with increasing doses of y rays or ultraviolet (UV) light and plated on complete medium, an increasing frequency of slowly growing, poorly conidiating colonies is observed. These abnormal colonies frequently lorm completely normal sectors when they are incubated longer or transferred to fresh culture medium. If the treated diploid is heterozygous for markers on all chromosomes, most of the sectors show segregation for genetic markers. Since Aspergillus forms a multi- nucleated mycelium, it is not possible to separate the products of the first few divisions and, in this way, to determine directly the time and frequency of recovered and lethal sectoring colonies as is done in yeast or bacteria. It is, there- fore, difficult to ascertain whether the abnormally sectoring colonies of Asper- gillus correspond to the sectoring types in yeast. But it seems clear that in the radiation-induced abnormals of Aspergillus, sectors are formed much later and less frequently, and they may well originate by a different mechanism. While the early sectoring, observed in yeast and bacteria, may result from repair and segregation of chromosomal and cytoplasmic genetic units (JAMES and SAUNDERS 1968), the type of later sectoring in Aspergillus may occur when rare spon- taneous recombination of a suitable type eliminates radiation-induced, hetero- zygous mutations or chromosomal aberrations. Evidence for the latter mechanism has been obtained for some of the abnormal sectoring types induced by ionizing radiation (KAFER 1963). However, spon- taneous formation of rare, better growing, sectors has been found in other abnormal, poorly growing types in Aspergillus. In untreated diploid strains, the 1 Thx mvestigation was supported by operating grant No. 2564 from the hational Research Council of Canada Geneincs 63: 821-841 December 1969
Transcript
EFFECTS OF ULTRAVIOLET IRRADIATION ON HETEROZYGOUS DIPLOIDS OF ASPERGZLLUS NZDULANS. 11. RECOVERY
FROM UV-INDUCED MUTATION IN MITOTIC RECOMBINANT SECTORS
ETTA KAFER
Department of Genetics, McGill University, Montreal, Canada
Received September 16, 1968
HE major effects of radiation on microbial cells or spores are killing, induced Tmutation and, at least in the case of diploid heterozygous fungal cells, increased mitotic recombination. In addition, poorly viable survivors have been observed in many organisms; these types are being investigated by pedigree analysis in unicellular organisms like yeast (JAMES and WERNER 1966; HAEFNER 1966) and bacteria (HAEFNER and STRIEBECK 1967). Most of them are found to produce lethal sectors in the first or in later divisions, but some recover com- pletely and revert to normal growth rate and viability (JAMES and SAUNDERS 1968). Similarly, when diploid conidia of Aspergillus nidulans are treated with increasing doses of y rays or ultraviolet (UV) light and plated on complete medium, an increasing frequency of slowly growing, poorly conidiating colonies is observed. These abnormal colonies frequently lorm completely normal sectors when they are incubated longer or transferred to fresh culture medium. If the treated diploid is heterozygous for markers on all chromosomes, most of the sectors show segregation for genetic markers. Since Aspergillus forms a multi- nucleated mycelium, it is not possible to separate the products of the first few divisions and, in this way, to determine directly the time and frequency of recovered and lethal sectoring colonies as is done in yeast or bacteria. It is, there- fore, difficult to ascertain whether the abnormally sectoring colonies of Asper- gillus correspond to the sectoring types in yeast. But it seems clear that in the radiation-induced abnormals of Aspergillus, sectors are formed much later and less frequently, and they may well originate by a different mechanism. While the early sectoring, observed in yeast and bacteria, may result from repair and segregation of chromosomal and cytoplasmic genetic units (JAMES and SAUNDERS 1968), the type of later sectoring in Aspergillus may occur when rare spon- taneous recombination of a suitable type eliminates radiation-induced, hetero- zygous mutations or chromosomal aberrations.
Evidence for the latter mechanism has been obtained for some of the abnormal sectoring types induced by ionizing radiation (KAFER 1963). However, spon- taneous formation of rare, better growing, sectors has been found in other abnormal, poorly growing types in Aspergillus. In untreated diploid strains, the
1 Thx mvestigation was supported by operating grant No. 2564 from the hational Research Council of Canada
Geneincs 63: 821-841 December 1969
822 ETTA KAFER
most frequent of these are aneuploids of various types, especially diploids and haploids with one or two extra chromosomes. These grow slowly but form large, normal sectors usually by loss of single chromosomes in phenotypic patterns which are characteristic for the specific chromosome involved (KAFER 1961 ; UPSHALL and CROFT 1967; POLLARD, KAFER and JOHNSTON 1968). Spontaneous sectoring of another type, which does not appear to be related to mitotic crossing over between homologues, but may involve sister-strand exchanges, has been demonstrated in near-haploid duplication strains (NGA and ROPER 1968; BAIN- BRIDGE and ROPER 1966).
The investigation reported here was carried out to determine the mechanisms of sectoring found in abnormal colonies induced by ultraviolet irradiation of diploid strains. Quiescent as well as germinating conidia were treated since the latter are physiologically more comparable to the cells used in other organisms such as yeast. Also, UV treatment during germination might increase not only reciprocal mitotic crossing over (as discussed in WOOD and KAFER 1969) but also unequal crossing over and might produce a higher frequency of abnormal dupli- cation and deletion recombinants. In addition, it seemed of special interest whether UV could interfere with chromosomal segregation during mitosis and increase the frequency of aneuploids, possibly by causing nondisjunction of cross linked and belatedly replicated sister-strands.
MATERIALS A N D METHODS
Strains: Two diploid strains with markers on both homologues of all eight linkage groups were used (KAFER 1958). Their genotypes are given in Figure 1. Diploid A is identical to diploid R synthesized for the analysis of abnormal colonies induced by y rays (KAFER 1963; Table 2) except that it does not contain translocation T(VZ;VZZ). Diploid X is one of the diploids employed for the analysis of UV-induced mitotic crossing over that can produce visible twin-spots (WOOD and KAFER 1967). All strains are derived from the same original wild-type strains used by ~ONTECORVO, ROPER, BUETON, HF.MMONS and M4CDONALD (1953). Details of all the mutants used here have been published recently (DORN 1967a), except for ni21, an allele of ni50, which was isolated by Dr. M. BERLYN and kindly supplied by Dr. G. DORN.
Irradiation: A standard procedure was followed for all experiments (except where indicated otherwise in Table 1). A 15 watt G.E. germicidal lamp at a distance of about 50 cm with a measured output of 16 ergs/mm?/sec was used for treatment of conidia with ultraviolet light. 10 ml of a conidial suspension (with about 4 x IO4 spores/ml saline) were placed in an open Petri dish, irradiated for 234 min and plated onto complete medium at low density (aiming at 10 sur- viving conidiajplate) . To obtain germinating conidia of diploid X, which is homozygous for ad20, a conidial suspension (of 3 x 105 spores/ml) was incubated, with shaking, in liquid minimal medium supplemented with adenine at 37°C for 434 hr. Before irradiation this suspension was diluted 100-fold into saline. All manipulations were carried out as fast as possible under artificial light which is expected to have caused similar, negligible amounts of photo-reactivation in dif- ferent experiments.
Methods and media: Standard media and techniques were used for plating of conidia, replica testing of colonies and production of heterozygous diploids (ROPER 1952; PONTECORVO et al. 1953). The abnormal colonies were analyzed using the method of “needle plating” which has been de- veloped for the analysis of sectoring aneuploids (KAFER 1961). When sectoring colonies are re- plated, it is important to obtain conidia from the original abnormal type in the center as well as from all the different partially or fully recovered sectors. Suspensions were, therefore, prepared by touching single conidial heads in all the abnormal central areas with a fine platinum needle,
MITOTIC SECTORING IN ASPERGILLUS 823
a’ IU + + +-paba y ad20 + + rib02 + + - - b’ + f + -I-- + + ad20 b, + + + cha
DIPLOID A
LINKAGE GROUPS: I m - “(21 + paIB7+ + + + + + + ad20 bi - -
b su ribol an lu-paba y ad20 + + + + cha DIPLOID X
- a Acr +- + phm2 + +-wro4 + lyr5 13 + nlc8 +- - + w 2 n d -k 10 me; -I- n1c2 f f lac f cho A AND X
LINKAGE GROUPS: 1 m m P m m FIGURE 1.-Genotypes of diploids A and X with markers on both homologues (a and b) of
linkage groups I-VIII. Conidial color mutants: y = yellow; w2 (allele of w l ) = white; cha = chartreuse. Nutritional mutants: ad20 (allele of ads) = adenine; an = aneurin (= thiamine); bi = biotin; cho = choline; lac = inability to ferment laotose; Zu = leucine; Zys5 = lysine; meth = methionine; m.3 and ni21 (allele of ni50) = nitrite; nic2 = nicotinic acid; nid = nico- tinic acid or tryptophan; paba = para-amino-benzoic acid; paZB7 = lacking alkaline phosphatase; phen2 = phenylalanine; pyrol= pyridoxine; ribol and rib02 = riboflavine; SO(= s l2 ) and s3 = sulphite; thi4 = thiazole. Recessive suppressor of ad20: su (= sulad20). Semidominant re- sistance marker: Acr = acriflavine. (All unnumbered alleles have isolation number 1.)
rinsing them off in 0.2 or 0.6 ml of saline and plating directly, and after a 5-10-fold dilution, onto complete medium plates. Several such platings were made from complex abnormal colonies to recover all types. Any slowly growing types were then incubated as long as necessary (up to eight days) and kept several further days at room temperature to obtain maximum growth and conidiation.
To obtain information on the genotype of certain stable diploid sectors, transfers were made to complete medium supplemented with para-fluorophenylalanine (CM + fp). On this medium diploid types grow poorly while haploids form conidiating sectors (MORPURGO 1961 ; LHOAS 1961,1968).
To recover as many of the abnormal types as possible, quiescent conidia of diploid A and germ;nating conidia of diploid X (Figure 1) were treated with ultraviolet light and plated at very low density onto complete medium. A random sample of about 100 abnormals was chosen from each plating for analysis (percent survival and observed frequencies of abnormals are given in Table 2). Such a fairly large sample seemed needed since it was quite feasible that abnormal colonies might arise by different mechanisms, e.g., UV-induced mutation, nondisjunction or nonrxiprocal recombination, and certain types might be relatively rare.
To determine whether the observed phenotype of an abnormal colony was caused by a stable genetic change, all abnormals were replated using conidia from the most abnormal-looking cen- tral area, as described above. In a few cases only normal colonies were obtained and these were subsequently excluded from the analysis (Table 2, “almost normal”). In all other cases, abnormal colonies were obtained in at least some of the replatings and often it was possible to recover colonies which showed a phenotype similar or identical to the one found in the original colony. Original colonies and replatings were photographed to record phenotypic patterns, since these are found to be extremely characteristic for specific genetic types in Aspergillus (some are shown in Figures 3-11). To check for marker segregation in the abnormal colonies and their stable re- covered sectors, the original colony and a sample of its stable derivatives were replicated onto the various test media to identify homo- and hemizygous mutants (the results from the cases shown in Figures 3-1 1 are given in Table 5). Since they provide information about the genotypes of the original abnormals, spontaneous haploid sectors were preferentially isolated and tested. These rare mitotic segregants, if of a normal haploid type, have a selective advantage over any
824 ETTA KAFER
abnormal type and can often he found as sectors or as small hunchrz of colorrd conidia in poorly ronicliating arras.
In addition. a smaller snmplr of 26 ahnormals which showed zectors of mutant rolor were srlrctrtl for morr drtailrd analysis from platings of U\’-treated quirscent conidia of diploid X. In this tliploicl, linkage groups I, I1 and VI11 contain the most markers including the color mutants yellow, white and chartreuse (sre Figure 1) ; only in these linkage groups is it possible to distinguish crrtain rrosssvrr types from nondisjunctional mitotic recomhinants (see Tahlr 4). It was hoprtl. thrrefore. that thew srlectrd rases would he informatiw onrs. They werc replated and twelvr rases which showed continued srgregation for color markers in the replatings were analysed in detail. A large number of haploid and diploid sectors were tested. In addition. it was
MITOTIC SECTORING IN ASPERGILLUS 825 attempted to determine the genotypes of some of the diploid stable sectors. Over forty such sectors from seven abnormals were transferred to “CM + fp” and the resulting mitotic haploid segre- gants were tested. The results from three abnormal colonies are given in Table 6.
Only very few abnormals formed neither frequent sectors nor haploid segregants even when repeatedly transferred; they grew too slowly to be replicated and were not further analyzed (see Table 2, “abnormal without sectors” and Figure 4, transfers of colony X34 which show un- usually large areas of an abnormal type and only one conidiating sector after more than 8 days’ incubation).
RESULTS
a) Frequency of UV-induced abnormals among survivors in quiescent and germinating conidia treated with di#erent doses of UV: Table 1 gives the survival range and the frequency of abnormals at different doses of UV in quiescent conidia of diploid A (CHEN 1963) and similar data for germinating and quiescent conidia of diploid X. In both cases the frequency of abnormals increases with dose. However, the measurements are not accurate enough and depend too much on experimental variables, expecially plating density, to judge the exact relation- ships.
To compare the frequency of UV-induced abnormals at similar levels of sur- vival in quiescent and germinating conidia of diploid X, the dose was held con- stant ( 3 min) and the period of preincubation was varied. Generally the fre- quency of abnormals in germinating conidia is found to be significantly higher. The difference is largest at five to six hours’ preincubation (at 28°C). This period corresponds to the later part of DNA synthesis or the first mitotic division (Lu, personal communication). Since synchrony was incomplete, only general trends could be observed. Especially remarkable is the drop in the frequency of abnorm- als to about half its value, and an inverse increase in survival when preincuba- tion is increased from six to eight hours (between the two periods given in Table 1). Only part of this drop can be explained by the doubling of chromosomes or nuclei during this period (considering that survival is 10% and that 80% of the survivors are abnormals) , and some additional change, for example in sensitivity to UV action, appears to occur during this period.
rays: Table 2 represents an attempt to classify the large variety of abnormal types into
b) Relative frequency of major types of abnormals induced by UV and
FIGURE 2.-Plating of normal diploid conidia. FIGURE 3.-Plating of UV-treated, germinating conidia of diploid X showing two normal and
three abnormal colonies, and one cha-palB twin-colony (light green cha/cha colony with dark center palB/palB).
FIGURE 4.-Transfers of colony X34; stable abnormal type showing a single white haploid sector.
FIGURE 5.-Replating of abnormal X21 with many green diploid and small white haploid sectors.
FIGURES 6a and 6b.-Abnormals with only haploid sectors: a-replating of hyperhaploid X40 showing stepwise sectoring of the n -/- 3 type without marker segregation; b-transfers of X29 similar to breakdown types segregating for four linkage groups in small haploid patches.
FIGURES 7a and 7b.-Abnormals resembling preceding type but with rare diploid as well as the haploid segregants: a-transfers of X68; b-replating of X30.
826 ETTA KAFER
Fmt.tti.\ &I , ~ r i l l SI). ( , c ) l o r i i ~ ~ ~ of t l t r t\vin-spot I V J N , I X ~ I ) I , I I ~ ~ : ; I S 127 \ x i 1 1 1 ~ / I / I / ~ / I Q or palR/ palR colonic< of W I ~ I ~ * it1)norrititl typr; b-Xl20 with I ~ , , / I ~ I or , lcr/ / lrr co1onic.s of tlifferrnt ah- normal phenotype.
FIGURFS 9a and 9h.--Ahnorninls rescmhlinq hyper-tliploitls hut prwlucing colomcl sectors of the cmssowr type: a-Xi1 giving mainly w/w sectors nonr ni/ni; h-X87 with many y/ysu/+ sectors.
FIGURFJ 10a and lOh.-Ahnormal colonies with similar phenotypr. I ~ t h giving pale green bi/hi srctors: a-X31 not very different from 2n f l ( I I 1 ) type, original colony in insert: h-Xi14 similar to X31 hut crnters more aconidial; phrnotype resembling 2n 4- 1 ( I ) .
FIGURES 1 la and 1 1 h.-Abnormal colony XI 19: a-replittwl, producing frequent ahnormal sectors; b-stable abnormal sectors transfrrred, showing rare diploid, srcond-order sectors.
MITOTIC SECTORING IN ASPERGILLUS 827
TABLE 1
Frequency of abnormal colonies among survivors from quiescent and germinating conidia treated with different doses of ultraviolet light
Quiescent conidia DIPI+ID A* Survival
W dose in mmutes range Abnormals (distance 40 cm) (percent) (percent)
0 IOW 0.4 f 0.7
2 2- 5 13.3 f 7.4 1 8-14 2.2 f 0.5
3 0.5- 1 33.8 f 14.5 4 0.3-0.5 52.9 f 4.0 5 0.2-0.6 75.4 f 2.8
*Data of CHEN (1963). t Used as control value, set at 100%. $ Totals counted in each case: 280-750. s Range of four observations. '5 Range of several sets; in some cases incubation periods differing by '/2 or 1 hour.
1 1 Values from two sets with high or low plating densities; the latter usually give better recovery of abnormals, but sample size is smaller (50-200).
a few general categories. The major criteria used are frequency and ploidy of the better growing, normal, or almost normal, sectors and type of marker segregation. This type of classification by objective criteria was originally devised to classify the spontaneous abnormals. It was used previously for the abnormals induced by Y rays (&FER 1963). Since the results with UV treatment appear to be basically similar, the earlier r e d t s are given here for a comparison of relative frequencies. To make such comparisons meaningful, a corresponding survival rate (0.5 % ) was chosen for the analysis of UV-treated quiescent conidia which produced a similar high frequency of abnormals (about 80%-Table 2). On the other hand, when it became known that in germinating conidia induced crossing over was very frequent (WOOD and GFER 1969), a lower dose appeared preferable in order to reduce coincidence of too many types of segregation. As a result, the total survival in the two UV-treated series differs considerably, so that for com- parison of the quiescent and germinating series, only the relative frequencies shown in the lower part of Table 2 can be used.
Independent of any hypothesis about the processes involved, it can be stated that the cases of group A which produce normal diploid, often parental, sectors and which seem to be able to recover completely, are generally less abnormal than the cases of group B (without diploid sectors). Most abnormals of group A
828 ETTA KAFER
TABLE 2
Relative frequencies of different abnormal types induced by y rays and ultraviolet light in diploid quiescent and germinating conidia
Treatment uv Y rays Developmental state of conima Germinating Quiescent Quiescent
Survival 21 % 0.5% 0.5%
Total number replated 120 62 153
parental 8 3 13 mutant or mosaic 10 0 0
Diploid X A R
Total frequency of abnormals 4.0% 75 % 84%
Almost normal (misjudged?)
Abnormal without sectors Sectoring abnormals
4 1 11 98 58 129
Abnormal sectoring types: Number Percent Number Percent Number Percent
Total number analyzed in detail A. with diploid sectors
in diploid sectors a) parental b) mutant or mosaic
2) with segregation of markers
1 ) without segregation of markers
in diploid sectors a) some sectors parental b) no parental sectors
most sectors normal many sectors abnormal
B. without diploid sectors 1 ) with haploid sectors 2) with frequent abnormal sectors
(98) 100 (58) 100
(18) 18 (21) 36
(45) 47 (14) 26 (1 ) (1)
( IO) 10 ( 1 1 ) 26 (0) (4)
(129) 100
also form larger abnormal centers which conidiate better, so that the original abnormal type can easily be recovered in replatings, while in many of the very abnormal cases of group B it is doubtful whether the original abnormal was re- covered or one of its many possible derivatives.
The following two general findings are demonstrated by the figures in Table 2: 1) UV-induced mitotic recombination: It is evident that in the UV-treated
germinating set the diploid survivors (group A) are much more frequently all mutant (no parental type recovered) or partly mutant (i.e., of a mosaic type) than in the quiescent set. This segregation of markers must have occurred very early during the growth of the colony and is found equally in centers and sectors. Most likely it was induced by the UV treatment. In several germinating but in none of the quiescent cases mosaic colonies showed marker segregation of a re- ciprocal, twin-spot type (e.g., colonies X127, Figure 8 and Table 5 ) . This observed difference agrees with the findings in normal colonies which showed that UV- induced mitotic crossing over is at least ten times higher in germinating than in quiescent conidia (WOOD and KAFER 1969). Also, since the germinating conidia
MITOTIC SECTORING IN ASPERGILLUS 829
were irradiated at about the time of the first mitotic division, additional apparent mosaics are expected representing cases where two different abnormals were in- duced in the duplicated chromosomes of a single conidium.
2) Relative frequency of group B types and of cases with stable abnormal sectors: Comparing the frequency of cases in group B found after UV treatment in the quiescent series with the corresponding value in the germinating one, it can be seen that the latter is considerably smaller. This can be explained partly by the lower UV dose in the germinating series and partly by the fact that in cases which are mosaic for two different abnormal types, the more extreme group B types would often be selected against. These are especially frequent among the abnormals induced with Y rays. In addition, after treatment with Y rays, many cases gave better growing, but still abnormal sectors which appeared to be quite stable (over 20 cases each in group B and group A2, Table 2). Such stable ab- normal sectors are very rare in the UV-treated series (one case, colony X119, is shown in Figure 11 ) . These differences are assumed to result from the more drastic effect of ionizing radiation on chromosomes compared to ultraviolet light (and not from a difference in the process leading to sectoring and recovery after the treatment, as discussed below).
C ) Characteristic features of UV-induced sectoring abnormals which differ from those of spontaneous, sectoring aneuploids: Radiation-induced sectoring colonies can usually be replated and the original pattern of segregation can be obtained repeatedly. The sectoring must, therefore, occur spontaneously but it obviously depends on the genetic changes induced by radiation in the abnormal colony. The better growing sectors often show regular segregation for specific recessive markers in the well-marked diploids used here (as is evident for color mutants in Figures 7-9, and for all types of markers in Tables 5 and 6 ) . By analyzing in detail the patterns of genetic segregation in the sectors it was hoped to deduce the original changes caused by radiation in the abnormal centers as well as the processes eliminating them in the sectors.
There were, however, some cases found which showed no specific repeated segregation of markers in the normal sectors; their centers and sectors were ap- parently the same, either both of the parental type, like the original diploid, or both of the same mutant type.
For example colony X21, shown in Figure 5 and Table 5, was classified as such a type, since all diploid sectors were of a parental type (except two rare dark ones from one replated colony which were preferentially isolated, and found to be ni2i palB7) . The frequency of such cases is relatively small (24% in the germinating and 12% in the quiescent UV-treated series, Table 2) considering that only one of the eight linkage groups is marked well enough on both arms of both homologues to recognize most mitotic segregants while in all other groups many crossover types would go undetected. However, a few cases in this group may well be of an entirely different origin. They may, for example, produce sectors due to cytoplasmic segregation as found with low frequency among spontaneous abnormals (SURAK-SEIARPE 1958; ROPER 1958; KAFER 1961; DORN 1967b).
Of special interest was the question whether UV-treatment increases nondis- junction or any other type of abnormal segregation of whole chromosomes. This
830 ETTA KAFER
would produce aneuploids which, in diploids with markers on all chromosomes, always show segregation for some genetic markers and have sectoring phenotypes similar to some of the ones found here (e.g., Figures 9a, loa, and lob). The results from the UV-induced abnormals are therefore compared to those from spon- taneous aneuploid abnormals (Tables 3 and 4) and three basic differences are found:
1) Number of types with different segregation patterns: One important differ- ence which cannot be presented quantitatively becomes clear only when a large number of cases are analyzed and compared: Among spontaneous abnormals only eight basic aneuploid types with diploid sectors (group A) and eight types with haploid sectors (group B) are found. These correspond to the eight possible 2n 4- 1 and n + 1 types, trisomic or disomic for each of the eight linkage groups. On the other hand, every UV-induced abnormal appears to be a unique type and even those abnormals which segregate for markers on the same linkage group do not show identical phenotypes as aneuploids do (KXFER 1961; UPSHALL and CROFT 1967; POLLARD, K~FER and JOHNSTON 1968).
Among the UV-induced abnormals many cases are complex and segregate for more than one linkage group (as evident from Tables 5 and 6). But even the most similar simple cases, X31 and XI 14 (Figure 10 and Table 5 ) , are not identical. They show segregatlon for the same marker, bi, on homologue a of linkage group I which produces paler sectors on the suboptimally sup- plemented CM used here. But only one of them, X31, forms two types of segregant sectors, namely bi/bi sectors of the crossover type, still heterozygous su/+ on the other arm, as well as homozygous su+ bi sectors of an apparently nondisjunctional type (indicated as +a and a in Table 5). The other, X114, produced only the crossover ( f a ) sectors. The phenotypes of X31 and XI14 show centers and sectors of similar relative size but are not identical: while XI14 re- sembles a 2n + I (I) phenotype, X31, which actually gave nondisjunctional sectors like those found in 2n + 1 (I) trisomics inexplicably seems to resemble more a 2n + l(II1) phenotype but since only one homologue of I11 was recovered in the five tested haploids it is uncertain whether X31 was normal for 111.
2) Relative frequencies of diploid and haploid sectors in various types of ab- normals: Another striking difference between UV-induced and spontaneous cases which is difficult to express quantitatively is the difference in these frequencies. Spontaneous aneuploids either produce predominantly diploid sectors in hyper- diploids (with only the occasional haploid sector) or only haploid sectors in hyper-haploids. On the other hand, a continuous range is found among UV- induced abnormals, from cases with only or predominantly diploid sectors to cases with predominantly or only haploid sectors. The classification into groups A and B (Table 2) therefore separates two basic types among the spontaneous ab- normals, but not among the abnormals of the different irradiated series. In fact, in UV-induced abnormals, group B seems to contain two general types: one which is nearly diploid, and is assumed to be similar to cases with rare diploid sectors of group A, and a second nearly haploid type which may represent true hyper-haploids. The former types show up as the cases with 4-6 disomic linkage groups in Table 3 which have never been found among hyper-haploids, presum- ably because they are too inviable. Whether the few cases of the second, appar-
MITOTIC SECTORING IN ASPERGILLUS
TABLE 3
UV-induced abnormls with frequent haploid but no diploid sectors (Group B ) compared to spontaneous hyper-haploids
831
a. Numbers of cases with various numbers of segregating linkage groups Number of abnormals Number of “viable disomic” chromosomes
of group B 0 1 2 3 4 5 6
UV-induced Quiescent
Germinating
Total 24 4 4 2 1 6 5 2
(diploid A) 14 3 1 1 1 2 5 1
(diploid X) 10 I 3 1 0 4 0 1
Spontaneous Diploid A* 129 0 101 24 3 1
b. Relative frequency of identified disomy for each linkage group Linkage groups: I I1 111 IV v VI VI1 VI11
ently hyper-haploid type are really UV-induced aneuploids is difficult to decide. In several cases the original, extremely abnormal type seems to have been lost and the isolated aneuploids would then be an intermediate product of segregation (several complex cases produced such aneuploid intermediates). In addition, genetic segregation in UV-induced cases is rarely of the type found in spon- taneous hyper-haploids. The latter show the same number of visible steps as segregating linkage groups and give 1 : 1 ratios for the two alleles of all markers in disomic linkage groups. UV-induced cases, on the other hand, usually show more steps than segregating groups, possibly due to UV-induced recessive lethals.
A possible example is colony X40 (Figure 6a) which shows visual sectoring of 3 steps but produced only haploids of one type (Table 5). A case with rare diploid sectors and also multi- step sectoring is shown in Figure 7a for comparison. Two cases, with nearly diploid centers which are very different from aneuploids are shown in Figures 6b and 7b; one of these gave no diploid sectors (colony X24) while the other produced them with low frequency (colony X30).
It can, therefore, be concluded that very few or no true hyper-haploids were induced by ultraviolet light. This agrees with the data in Table 3 which show that in UV-induced abnormals, but not in hyper-haploids, all linkage groups are found to segregate for the two alleles of their markers with about equal frequency.
3 ) Types of marker segregation in diploid sectors from abnormal colonies: In spontaneous aneuploids with diploid sectors (hyper-diploids) all segregation is of a chromosomal type. When every homologue is marked, all 2n 4- 1 types give
832 ETTA KAFER
nondisjunctional segregant sectors homozygous for the markers of one homologue of the trisomic linkage group. Usually these are about half as frequent as diploid sectors of heterozygous parental type, indicating that loss of any one of the three homologues is random.
UV-induced abnormals also often show regular segregation of markers located on the same homologue of a single linkage group. However, in these UV-induced cases diploid segregants of a mitotic crossover type are on the whole more frequent than nondisjunctional ones (Table 4). Actually each individual case appears to produce its own specific ratio of crossover to nondisjunctional diploid segregants (in Table 4 cases are classified by their more frequent types).
Examples with more crossover than nondisjunctional sectors are colonies X127, X223, and X225. From replated centers of colony X127,29 su/+ bi/bi (+a) sectors of the crossover type and 21 homozygous su+ bi ( a ) were obtained (Table 5 ) ; and from colony X223, a few nondisjunc- tional sectors homozygous su ribol an Zu paba y were isolated besides many diploid sectors of all possible crossover types (homozygous for su, or su ribol, or su ribol an or su ribol an lu, Table 6 ) . In quite a few cases only diploid crossover sectors were found--e.g., colonies 51, 87, 114 and 120 from diploid X, while colony X31 produced both types with about equal frequency, and colony X201, which is discussed below, gave mainly sectors of a nondisjunctional type.
The patterns of marker segregation in UV-induced abnormals are, therefore, very different from the ones found in spontaneous abnormals of the sectoring trisomic type. And, not surprising, the pooled frequencies of the various segre- gating linkage groups and relative frequencies of segregation for markers of the different groups are also entirely different (see Table 4).
d) Evidence for lethal mutations and their elimination in normal sectors: When UV-induced abnormals are compared to those obtained after treatment with y rays it is found that most of the general features are the same. This holds for all the characteristics outlined in the previous part (c) . A likely hypothesis therefore, is that as in the case of y rays, UV treatment induces mutations which
TABLE 4
Relative frequency of segregation for markers of each linkage group in diploid sectors of frequently-sectoring abnormls induced by UV (Group A )
Number of Linkage groups I I1 111 IV V VI VI1 VI11 colonies
Type of segregation CO* N+ CO N CO or N CO involved
* CO = segregation of markers in one chromosome arm only, crossover type. t N = simultaneous segregation of markers in both arms, nondisjunctional type. $ KAFER 1961, summarized from Tables 3 and 4.
MITOTIC SECTORING IN ASPERGILLUS 833
are lethal when homo- or hemizygous but cause an abnormal phenotype in heterozygous condition. When spontaneous mitotic recombination happens to eliminate such a mutation, the resulting segregant grows normally and may become homo- or hemizygous for markers of the unaffected viable homologue. The abnormal phenotype and the pattern of sectoring and coincident genetic segregation would then depend on the size and location of these mutations. In many cases they might be chromosomal aberrations of various types and might possibly include some products of unequal crossing over. The following two pieces of evidence support this hypothesis:
1) Absence of segregation in haploid sectors for markers which segregate in diploid sectors: In all cases where many haploid and diploid sectors were found, the haploids fail to show segregation for the two alleles of some markers which
TABLE 5
Segregation of markers in centers and sectors of sectoring colonies shown in Figures 3-11 from diploid X induced by UV treatment of germinating conidia
(a or b = markers of homologues a or b of Figure 1; ab, +a, etc. = CO types; a = either homologue found in haploids; ? = likely, epistatic markers). b
Center ( = c ) Figure Colony or ploidy I I1 I11 IV V VI VI1 VI11 Number
appear to be heterozygous in the centers. Most significantly, only one type of haploid is usually isolated with respect to those linkage groups which segregate regularly in diploid sectors and are, therefore, most certain to be heterozygous in the central part of colony,
This can be found in almost all of the cases shown in Table 5, except in X51 where the two isolated haploids appear to be of the types reciprocal to the expected type. Similarly in Table 6, all three cases produced only one homologue of the following linkage groups which segregated in diploid sectors: colony X214 of groups I and VIII, X223 of groups I and I11 and X225 of groups VI and I (in the last case a few haploids of an exceptional type were selectively isolated, as discussed below).
In addition, the heterozygous original type oi UV-induced sectoring colonies is often found to be non-requiring for mutants which do not turn up in diploid sectors but are found in all the haploid segregants, presumably because the corresponding wild-type allele is present in the original colony but on an inviable homologue. Some of these cases are likely to be heterozygous for recessive lethal mutations. Such lethals do not seriously affect growth rate and are expected to be found equally in centers and sectors. (Colony X223, Table 6, may be such a case of a recessive lethal on the homologue a of linkage group VII; all haploid sectors showed the marker cho of homologue VI1 b even though no diploid types were homozygous for this marker.)
2) Recovery of markers on inviable homologues from diploid sectors: As indi- cated in Tables 5 and 6, it is sometimes possible to select rare types of segregants if they show a mutant color. Some of these are evidently resulting from more than one step of segregation. The cases which are relevant here are haploids which represent rare cases of a preceding crossover in the linkage group which is assumed to be heterozygous for a semidominant lethal. The markers of the inviable homologue which are recovered in such crossover haploids presumably indicate chromosome segments which are normal. X31 in Table 5 and X225 in Table 6 are given as examples, in which rare yellow haploids could be found, most of them of a crossover type, even though homologue Ib was apparently carrying the mutation which caused the original abnormal type.
Similarly, haploids selected from stable diploid crossover sectors are expected to segregate normally (1 : 1 ) for alleles from the originally inviable homologue (if any markers are still heterozygous in such sectors). Table 6 gives the results from three cases in which haploids selected from stable diploid sectors showed such marker segregation. This type of analysis is not often successful, partly because usually no heterozygous markers are left, as in the case of all analyzed chartreuse segregants, and partly because in some cases very few viable haploids are obtained.
Certain types of haploids show greatly reduced viability on the selective medium used (CM + fp, in Table 6). This is caused in some cases by specific markers, like phen2 on IIIn, or by a combination of many markers and especially by recessive lethals. If neither homologue of a linkage group is fully viable, haploids are difficult to isolate.
In the three cases of Table 6, new types of haploids were isolated from stable diploid sectors which could not be obtained from the original centers. In the sectors (but not in the centers), 1 : 1
MITOTIC SECTORING IN ASPERGILLUS 837
segregation for markers of the following linkage groups was observed: in colony X214 for linkage group I, in X225 for group VI and in X223 for group 111. As predicted, it is found in some of these cases that the newly-recovered homologue is of a crossover type.
Another case which gave this type of result was colony X201. This abnormal colony formed green centers and many white sectors. These were all diploid, homozygous w2 ni3, i.e., of an apparently nondisjunctional type for IIb. When yellow or chartreuse conidial heads from the original center were plated, these formed yellow or light green centers, again with frequent w2 ni3 sectors. The few haploids which were found were also white. However, a single green stable sector among over a hundred white ones was found which turned out to be Am/+ ni3/ni3. From this sector a few haploids were obtained which were of two types, some w2 ni3, others ACT ni3, the latter containing part of the previously inviable homologue IIa. It seems likely that in this case parts of both chromosome arms of IIa were abnormal so that a single exchange could not produce a normal diploid type. In addition, mutations in both homologues of another linkage group could explain why haploid segregants were exceedingly difficult to isolate.
DISCUSSION
The analysis reported here was restricted to one of the several effects of ultra- violet light observed after treatment of well marked diploid conidia of Aspergillus nidulans. Only abnormal-looking survivors were chosen for analysis. UV-induced segregation of genetic markers in normal-looking colonies has been reported in the previous paper (WOOD and KAFER 1969). Also, UV-induced reciprocal trans- locations and recessive lethals which do not change the normal phenotype of the diploid are not investigated here. Of these two types of mutations, the latter appears to be considerably more frequent and has been found with a frqeuency of about 25% at 2% survival in normal colonies formed by W-treated quiescent conidia ( KAFER and CHEN 1964). Both UV-induced mitotic crossing over as well as induced recessive mutations are, however, expected to occur in abnormal colonies as frequently as in normal ones and have to be considered in the interpre- tation of individual cases.
The main purpose of the present investigation was to determine whether the abnormally sectoring colonies, found after irradiation with ultraviolet light, are cases of induced mitotic segregation. The alternative would be that UV-induced mutations create differences in viability that lead to abnormal growth and a selective advantage of certain spontaneous segregants which have lost the muta- tions and, therefore, grow normally.
The two most likely types of induced mitotic segregation which would produce unbalanced and, therefore, abnormal types are nondisjunction and unequal crossing over. The latter should produce near-diploid types with corresponding duplications and deletions if the two strands involved segregated to different daughter nuclei. If both survived they would be expected to form twin colonies, each of the two types giving rise to normal diploid segregants of two reciprocal crossover types. No such case of reciprocal sectoring has been found, even in the case of germinating conidia where the survival was high (about 20%, see Table 2). However, since the almost normal duplication type might completely outgrow the less normal deletion type, such cases might easily go undetected. On the other hand, UV-induced nondisjunction (or breakdown of the mitotic spindle) is expected to produce aneuploids of the types found with low but regular frequency
838 ETTA KAFER
among spontaneous segregants ( KAFER 1961 ) . As secondary products, stable haploids ( PONTECORVO, TARR-GLOOR and FORBES 1954) and nondisjunctional diploids (PONTECORVO and KFER 1958) should also be increased. No such increase was observed among the normally growing colonies after UV treatment of quiescent or germinating conidia (CHEN 1963; WOOD 1967). Similarly, among the abnormal colonies, results differed radically from those obtained with spon- taneous aneuploids and not a single case with the type of marker segregation and sectoring pattern characteristic of aneuploids was lound.
It can, therefore, be concluded that the majority of abnormal and sectoring colonies obtained after UV irradiation of diploid conidia do not result from induced mitotic segregation. The only type of UV-induced mitotic segregation observed was mitotic crossing over, which occurred with similar frequencies in normal as well as abnormal colonies. In the latter it was found in centers and sectors, and preceded the formation of better growing sectors. I t was observed mainly after UV treatment of germinating conidia and often resulted in mosaic colonies, some of the twin-spot type.
The abnormal colonies found after UV treatment of quiescent or germinating conidia must, therefore, result from other effects of UV irradiation and the most likely one is induced mutation. The hypothesis is that the abnormal growth observed in many surviving colonies is caused by UV-induced semidominant lethal mutations. Heterozygous deletions, and possibly even point mutations in genes for essential functions, are expected to reduce viability and, therefore, growth rate and conidiation. Spontaneous mitotic recombination of both types, crossing over, and somewhat less frequently, nondisjunction, if occurring in the affected chromosome arm, could then eliminate such a mutation and produce a normal, much faster growing, segregant type. If heterozygous markers are present on the involved chromosome arm, haploid segregants should all show the alleles of the homologue not bearing the induced mutation and the normal diploid sectors are expected to become homozygous for these same alleles with varying frequen- cies, depending on the relative positions of centromere, markers and mutation. This is exactly what has been found in practically all the analyzed cases. If any diploid segregant sectors are still heterozygous for one of the markers of the involved linkage group, haploid second-order segregants from these should now show normal 1:i segregation for the two alleles (this could be demonstrated in a few cases; see Table 6).
Cases of abnormal colonies which show similar spontaneous sectoring have been isolated from translocation heterozygotes. If mitotic crossing over is selected for in one of the two chromosome arms involved in a reciprocal heterozygous translocation, an abnormally growing product is obtained. It presumably is tri- somic for one of the translocated segments and monosomic for the other (that is, heterozygous for a duplication and a deletion). On complete medium it forms spontaneous, normal diploid sectors, apparently by mitotic recombination in the other involved linkage group restoring a balanced diploid genotype (=FER 1961 and unpublished). Spontaneous sectoring in near-haploid duplication strains, which does not seem to involve exchanges between homologues, but possibly
MITOTIC SECTORING IN ASPERGILLUS 839
between sister-strands, has been analyzed in detail by NGA and ROPER (1968). Whether some of the cases which gave no marker segregation in the sectors were of this type, is impossible to decide.
The frequency of segregation in diploid sectors for markers of each linkage group and, therefore, according to this hypothesis, the frequency of semidominant lethal mutations appears to be very similar for the different linkage groups (Table 4 ) . In contrast to aneuploids, UV-induced abnormals segregating for the same markers may show different phenotypes, which is expected if the abnormal growth is caused by different mutations in the same chromosome. Indeed, each abnormal colony looked different from every other one and, in addition to the major features discussed here, some gave rare unexplicable segregants or isolates with deviant phenotypes.
These results with ultraviolet light are, therefore, directly parallel to those obtained earlier with Y rays. However, at the same level of survival, UV effects in quiescent conidia appear to be less drastic and easier to eliminate by mitotic crossing over: it is found that the frequency of stable abnormal types and sectors is much lower and that most sectoring abnormals produce normal diploid sectors. This is as expected and corresponds to similar observations in higher organisms- e.g., the different phenotypic changes in pollen of the F, grown from irradiated pollen in corn showed clear correlation with types and severity of induced chro- mosomal aberrations (STADLER 1941). On the other hand, the main difference between UV effects on germirmting and quiescent conidia appears to be the ad- ditional effect of induced mitotic crossing over in the former. The frequency of abnormals in germinating conidia at the same survival appears to be somewhat higher, especially at about the time of the first division. Some of this may be due to the fact that, for the same level of killing, a higher effective dose is needed in germinating conidia which have replicated their DNA and, possibly, their chro- mosomes.
It is well established that ultraviolet light can induce chromosome breakage and chromosomal aberrations in higher organisms ( STEINITZ-SEARS and SEARS 1957) as well as in fungal spores (e.g., PERKINS, GLASSEY and BLOOM 1962). The specific types of sectoring colonies in Aspergillus, however, appear to be unique and must depend on the relative viabilities of the various types. Some of the abnormals may correspond to the survivors among irradiated diploid yeast which, in addition to induced mitotic crossing over show lethal sectoring early during growth and later recovery in certain cases (JAMES and SAUNDERS 1968; HAEFNER 1966). Similarly, it has been postulated that the cause for the difference in the frequencies of mitotic haploids or aneuploids found in diploid strains of other organisms results from differences in the relative viabilities of aneuploids in the different species (e.g., in Ustilago, HOLLIDAY 1961 ; in Neurospora, PITTENGER 1954; or after treatment with p-fluoro-phenylalanine in Aspergillus niger, LHOAS 1961,1968; or in yeasts, GUTZ 1966; EMEIS 1966; STROMNAES 1968).
If we accept the hypothesis that most abnormal colonies are cases of hetero- zygous deletions, it becomes possible to use the frequency of abnormal colonies as a measure of the frequency of chromosomal aberrations. This is expected to be
840 ETTA KAFER
very useful, especially if combined with a system of markers which permits de- tection of mitotic crossing over as visible twin-spots. Simple inspection of colonies grown from treated conidia will be sufficient to obtain information about the effects of various agents or conditions on mutation and mitotic crossing over.
Sincere appreciation is expressed to Drs. F. C. FRASER and E. R. BOOTHROYD for helpful com- ments in the preparation of the manuscript and to PAT ZAMBRYSKI and PAT O'FARRELL for labora- tory assistance.
S U M M A R Y
UV treatment of heterozygous diploid, quiescent or germinating, conidia of Aspergillus nidulans was found to produce a high frequency of abnormal, fre- quently sectoring colonies. In replatings from the abnormal centers of sectoring colonies, the original type could usually be recovered, indicating that the for- mation of sectors occurred spontaneously. The frequency and type of sectoring was found to be specific for each case and to frequently coincide with a specific type of marker segregation. It is suggested that the abnormal growth of the orig- inal colonies is due to the presence of UV-induced mutations, possibly terminal deletions, which are eliminated by subsequent spontaneous mitotic recombina- tion. No evidence for induced segregation which could produce sectoring types (e.g., nondisjunction) was found. Cases with frequent diploid sectors of a single crossover type are interpreted to contain only one mutation in one homologue of the chromosome arm involved. This homologue is usually not recovered in hap- loids from the original center but parts of it can be isolated in haploids from the diploid segregant sectors. The most complex cases with only haploid sectors may contain more than one mutation in different chromosomes, while stable abnormal types could result from mutations in both homologues of the same chromosome. All eight linkage groups are affected with similar frequency which may indicate that the length of the different chromosomes is fairly similar.
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