reciprocal translocations and - genetics

RECIPROCAL TRANSLOCATIONS AND TRANSLOCATION DISOMICS OF ASPERGILLUS AND THEIR USE FOR GENETIC MAPPING1

ETTA KAFER Department of B,‘ology, McGill Uniuersity, Montreal, Canada

Manuscript received March 25, 1974 Reyised copy received August 27, 1974

ABSTRACT

Two new techniques are described for genetic mapping of reciprocal translocations in A. nidulans, which can be used to locate centromeres and meiotically unlinked markers. They both make use of unbalanced disomics from heterozygous translocation crosses. These are mainly hyperhaploids of two classes: either typical-looking n + 1 with a normal chromosome in addition to a haploid set containing the translocation, or translocation disomics. When large chromosome segments are involved, such disomics, as well as stable aneuploids and duplication types, show characteristic phenotypes and can be classified visually. The first method maps translocation breaks qualitatively, since translocated markers can be identified when translocation disomics are analyzed for heterozygous markers. The second method measures meiotic linkage of any marker to the translocation breaks when allele ratios in the balanced haploid sectors of either or both classes of disomics are determined: linked markers show reciprocal deviations from 1 : 1-In addition, it can be shown that frequencies of nondisjunction and recovery of specific translocation disomics both depend on the relative position of the break within a chromosome arm. Such information can provide a rough estimate of the positions of breaks for a new translocation.-Using these techniques, as well as mitotic mapping in homo- and heterozygous translocation diploids, four reciprocal translocations were mapped. From these results, information on the sequence and orientation of most of the “meiotic fragments” of the current maps (groups 111, VI, VI1 and VIII) was obtained, and the position of the centromeres of groups VI and VI1 were identified. Translocation disomics are also used to map meiotically unlinked single genes, e.g. oZiA of group VII, to specify chromosome segments.

ECIPROCAL translocations are found with high frequencies in UV-treated Rstrains of fungi. A large number of such aberrations has been successfully mapped by PERKINS (e.g. 1974) in Neurospora, since in this species “semisterility” shows up as distinct ascospore patterns. However, this essentially meiotic technique is not usable in Aspergillus, not least because markers located on the same chromosome, or even on the same arm, often show no meiotic linkages. Indeed, in spite of a large increase of genes mapped to the eight chromosomes in the past fifteen years, several centromeres and many fragments are still not sequenced with certainty in the recent linkage maps of Aspergillus (KAFER

Supported by operating grant No. 2564 from the National Research Council of Canada.

Genetics 79: 7-30 January, 1975.

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8 E. KAFER

1958; CLUTTERBUCK and COVE 1974). On the other hand, the mitotic mapping techniques for reciprocal translocations which have been worked out for Asper- gillus are independent of meiotic linkages, but depend for complete analysis on convenient selective markers in the chromosome arms containing the translocation breaks (KAFER 1962). Only recently, when the reciprocal translocation, T2(Z;VZZZ,), was found, which involved two well-marked arms, was it possible to carry out complete mitotic mapping in translocation diploids. In addition, the expected meiotic linkages between markers of two chromosomes were obtained for the first time, confirming and complementing the mitotic mapping (MA and =FER 1974). Non-reciprocal translocations are not considered here since, in Aspergillus as in Neurospora, they can be mapped in crosses by similar techniques, using duplication progeny (BAINBRIDGE 1970, PERKINS 1972).

An entirely different set of techniques, which will be demonstrated here, makes use of mitotic segregation in aneuploids which in Aspergillus can be visually identified and genetically analyzed, when these are produced by crosses heterozygous for reciprocal translocations and genetic markers. It is well known that in such crosses disjunction of the centromeres of the two structurally heterozygous chromosome pairs is disturbed, so that homologous centromeres may move to the same pole with relatively high frequencies (as reviewed, e.g., for higher plants by BURNHAM 1956). Often both homologous pairs segregate non- disjunctionally but to opposite poles (“adjacent-2” segregation), resulting in gametes that are nullisomic and disomic for certain chromosome segments in a complementary way. These types are not viable in haploid species but can be recovered in some diploid organisms, since complementary unbalanced gametes may produce balanced zygotes. In cases where a single chiasma per chromosome segment can be assured, these zygotes can be used for centromere mapping (e.g., in the mouse, by SEARLE, FORD and BECHEY 1971). On the other hand, segregation of one member of a translocation quartet to one pole and of the re- maining three to the opposite pole produces n - 1 and n + 1 meiotic products with varying frequencies. This type of segregation is often observed cytologically in higher plants, especially for small chromosomes. Also in Drosophila, n - 1 and n 4- 1 gametes for the small fourth chromosome may be recovered and detected genetically as haplo- or triplo-IV types (GRELL 1959; ANDERSON 1929). I n haploid organisms the n + 1 types are the only unbalanced products expected to survive, and such aneuploids can be detected visually in Aspergillus. All n + 1 grow slowly and regularly produce better-growing haploid sectors, but each type, disomic for one of the eight standard chromosomes, is identifiable and shows a specifically reduced growth rate and level of condiation (KAFER 1961 ; =FER and UPSHALL 1973). If ascospores from translocation crosses are plated at low density, typical-looking disomics, and translocation disomics with new modified phenotypes, are identifiable and are recovered with frequencies of 1-5% (POL- LARD, %FER and JOHNSTON 1968; UPSHALL and EFER 1974). As demonstrated here, such disomics can be used in two ways to map translocation breaks and indirectly to sequence centromeres and markers.

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TRANSLOCATIONS A N D GENETIC M A P P I N G 9

One method is qualitatively independent of meiotic recombination. It depends on the visual recognition of “somatic” loss of specific translocation chromosomes correlated with genetic marker segregation. Such a technique is now being de- veloped arrd exploited in human cell genetics to map markers to specific chromosome parts (e.g., RICCIUTI and RUDDLE 1973; GERMAN and CHAGANTI 1973).

The second method makes use of meiotic segregation of markers in the haploid sectors of a large number of disomics obtained from heterozygous translocation crosses. If “typical looking” disomics, which contain an extra standard chromosome and produce translocation haploids, can be distinguished from translocation disomics which produce standard haploids, these results identify even single markers linked to the breaks. Such meiotic linkages correspond to those obtained in higher plants when linkage between “semisterility” and genetic markers are determined (e.g., KASUA and BURNHAM 1965).

Several reciprocal translocations have now been mapped in A . nidulans combining results from these methods. Simultaneously, these translocations are used to sequence centromeres and meiotic fragments, and to map meiotically unlinked markers. In addition, the mapped examples demonstrate a quantitative relationship between the relative positions of breaks within a chromosome arm and the frequencies of aneuploids found in heterozygous translocation crosses. These can be used for rough estimates of the break positions for unmapped translocations.

MATERIALS A N D METHODS

Strains and mutants: All of the strains used here are descendants of the same wild-type strain (PONTECORVO et al. 1953), and most of the translocations are from stock strains in Glasgow (CLUTTERBUCK 1969) or at the Fungal Genetics Stock Centel. (Humboldt College, Arcata, Calif., BARRATT, JOHNSON and OGATA 1965; DORN 1967). Information on the genotypes of the crosses for which specific results are given are shown in Table 1. All strains used have been checked for translocations. Figure 4 gives the position of the breaks for the mapped translocations and of the markers used here. Gene symbols are explained in the legend of Figure 4 and the specifiq alleles used are indicated only there. These gene designations follow the recently adopted nomenclature for Aspergillus nidulans as given for all mapped genes by CLUTTERBUCK and COVE (1974). The mutant oliA(2) (=oligomycin resistance) has recently been isolated by ROWLANDS and TURNER (1973).

Translocations: Two of the four translocations mapped here have been reported on previously, namely Tl(1;VII) found in the original UV-treated biA; choA strain (KAFER 1958, 1962) and Tl(V1;VII) probably X-ray-induced in the original wbaA biA (%FER 1965). The other two were recently discovered (and preliminary results are included in UPSHALL and KAFER 1974). These are likely to be UV-induced since both of them are found to be associated with a UV-induced mutation; namely Tl(1V;VIII) with frA isolated hy ROPERTS (1 963), and Tl(1II;VII) with lysD(20) obtained by PEES (lysD is shown as Iys7 in the correct relationship to maZA and paZF, but with choA placed differently, in the map of DORN 1967). The reciprocal translocation TZ(1;Vlll) which is shown in Figure 4 and included in the DISCUSSION (and Table 4) has recently been mapped by MA and KAFER (1974).

Media: Standard media were used as devised by PONTECORVO et al (1953) and modified) later to obtain comparable phenotypes with easily available chemicals (described by BARRATT, JOHNSON and OGATA 1965). It was found, however, that aneuploids are extremely sensitive to suboptimal conditions. Therefore, complete media were regularly supplemented with up to four times normal amounts of lysine, adenine, phenylalanine or phosphate to obtain good allele ratios and normal phenotypes in aneuploids from crosses with the markers ZysD, adl, phenB,

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10 E. KAFER

palC or palF. In spite of this, some crosses heterozygous for 1ysD gave at certain times poor allele ratios, among haploid and n+l segregants (causing, e.g., a shortage of n + I I I types in cross 1743 compared to cross 1744, Tables 1 , 2, and 4 ) . Extensive analysis did not identify the cause for these results, but showed that they clearly were due to variation in the complete media which seemed to improve upon storage.

Methods: Standard techniques were used for the analysis of normal segregants from crosses (PONTECORVO et al. 1953) and for the analysis of diploids (PONTECORVO and KAFER 1958), most of them homozygous for translocations (MA and KAFER 1974). Haploids were selected from heterozygous diploids in various ways. Usually selective markers were available, especially suAadE. Occasionally haploids were obtained on complete medium supplemented with Na- arsenate (VAN ARKEL 1963), especially from diploids containing fpaB and phenB as important markers. For other diploids medium supplemented with p-fluorophenylalanine was used (LHOAS 1961, 1968). Analysis of “abnormal” meiotic segregants:

To permit recovery of abnormal, poorly growing segregants from crosses, fresh ascospores were plated at very low densities (15-30 per plate) on supplemented complete medium. Platings were incubated 2 days at 37”, then counted and inspected for abnormal colonies. “Abnormals” were replated after one or more additional days at room temperature, since lower temperature generally improves conidiation.

Presumed duplication types: Slightly abnormal, not sectoring, colonies were isolated onto fresh media by mass transfers, usually two per plate with a normal control added for comparison. Some of these turned out to be normal, while others showed restricted growth and produced rare normal sectors after four to six days of incubation, as is typical for duplication types (NGA and ROPER 1968). Reciprocal translocations only rarely produce such types and they were not analyzed further.

Sectoring aneuploids, mainly disomics: Unstable abnormals were purified for analysis by “needle platings” (KAFER 1961) as follows: Abnormal conidial heads of the n+l centers were touched with a fine platinum or glass needle, the adhering conidia were transferred in sequence into drops of saline on two petri dishes-usually one with thick and one with thin complete medium-and spread with a glass spreader. This produces high and low density platings, which demonstrate some of the phenotypic variability of each type.

Visual classification of aneuploids: All sectoring abnormals were classified visually. Any n f l , n+2 or 2724-1 types which contained extra standard chromosomes were identified by comparison with the well-known phenotypes of aneuploids from heterozygous diploids (as shown by KAFER 1973). However, from each heterozygous translocation cross two new types of aneuploids are expected, namely two types of translocation disomics, each containing one of the two translocation chromosomes in addition to a haploid set of standard ones (see Figures 1 and 2) . To identify the specific features of such new types, all replated disomics from any one cross are grouped by phenotype using as criteria size and shape of the aneuploid center, degree of conidiation o r formation of fruiting bodies, and mycelial color. If such objective classification produces more than four groups or a continuous range between two of them (e.g., translocation disomics from Tl(VZ;VZZ) may look like standard n+IV, see Figure 3 ) mixed platings are carried out under a variety of conditions, like high and low density, thin and thick media, high and low temperature. This establishes the phenotypic range of the new types and representa- tive cases are photographed for record and for comparison with disomics from later crosses. For most abnormal types classification becomes reliable in this way.

However, problems may arise later when translocations are crossed to markers which influence phenotypes on complete medium. For example, in crosses with Tl(VZ;VZZ) and palF new abnormal types were observed; these could be identified as typical-looking n+VI disomics when phosphate was increased in the medium to five times the normal amount. On the other hand, in the case of Tl(ZV;VZIZ) it turned out to be impossible to distinguish typical- looking n+IV types from “abnormal” ones on any media, especially when palC and nirA segregated in addition to fraA; these were therefore classified genetically (as explained below).

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Genetic analysis and identification of heterozygous markers in disomics:

To characterize specific translocation disomics genetically, heterozygous markers have to be identified. Since disomics from translocation crosses produce only one type of balanced haploid sectors-except in cases of rare mitotic crossing over (Figure 1)-the following two procedures were used which are partly complementary: (1) two haploid sectors from each aneuploid are tested for the markers segregating in the cross, to identify any requiring or sensitive alleles present in the haploid sectors; if the n+l type is heterozygous for any such marker it will grow, even if at the slow rate typical for disomics, on medium which selects against the haploid sectors. Therefore the n+l centers are transferred individually to each of the corresponding test media, usually in duplicate or triplicate (beside the nongrowing haploid sectors for comparison). This generally identifies n+l types as heterozygous for specific markers if they grow better than their haploid segregants. (2) When mitotic crossing over in disomics produces new types of haploid sectors, some additional heterozygous markers may be identified. This can easily be seen in the case of heterozygous color markers, when centers and sectors show wild-type green color. Mitotic crossing over may produce homozygous mutant patches or, after loss of one homolog, the occasional haploid sector of mutant color. To observe such crossover sectors disomics are plated at low density o n complete medium (5-20 plates, dependent on the position of the marker relative to the break and the centromere). Heterozygosis for y A and bwA was usually established by this method.

RESULTS

Unbalanced disomics from crosses heterozygous for reciprocal translocations can be used in two ways for genetic mapping for translocations, and indirectly of meiotically unlinked fragments and markers, and of centromeres. These disomics are the products of primary nondisjunction of either of the two chromosome pairs involved in the translocation. If all such disomics are viable and rec- ognizable, four types are expected from any one translocation cross (as shown in Figures 1 and 2). All four are structurally heterozygous; two of them contain the normal haploid set and one of the translocation chromosomes (called “translocation disomics”) , and two types containing the translocation in the haploid set and a standard chromosome extra. Since these latter look exactly like standard n 4- 1 they are called “typical-looking disomics.” However, these, as well as the translocation disomics, do not segregate 1 : 1 for heterozygous markers when haploid. sectors are formed, since only loss of a specific homolog produces a balanced sector (see Figure 1). Therefore, these disomics cannot be analyzed genetically by testing a few haploid sectors, as was possible for aneuploids from well-marked standard diploids (apart from the fact that potentially heterozygous markers may often be homozygous, because meiotic crossing over precedes nondisjunction). To analyze unbalanced disomics genetically, that is, to identify their het- erzygous markers, two methods are used (as described in detail above). (1 ) For biochemical markers it was found most convenient to check for differences in growth between potentially heterozygous n + 1 centers and their haploid sectors on appropriate test media, provided a marker showed the requiring or sensitive allele in the sectors. (2) For color markers, especially mutants which reduce conidiation just like disomy does (e.g., bwA), the only reliable method was to obtain mitotic crossover haploids. Mitotic crossing over in translocation disomics can produce balanced disomics if the exchange occurs between the break and the centromere. Presumably such disomics change phenotypes and at the same time

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12 E. KAFER

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FIGURE 1.-Four types of unbalanced disomics from a cross of T(Z;ZZ) x a b; c d standard, and crnssover haploids formed after mitotic crossing over in IIR.

become homozygous for many markers, so that it is difficult to prove such cases (e.g., one clear n + VI case segregated 1: 1 for bwA: bwt-). When haploid sectors from such crossover types are formed, it may become possible to recognize heterozygosis for some of the markers which show the dominant allele in the majority sectors (marker c in Figure 1 ) .

The four types of disomics from translocation crosses are two genetically complementary pairs, which provides useful cross checks on the genetic analysis of ally one type. However, some translocations do not regularly produce four identifiable types (UPSHALL and KAFER 1974) and in these mitotic analysis of hetero- and homozygous translocation diploids complements the analysis of disomics (KAFER 1962; MAandKXFER 1974).

A. Mapping of recriprocal translocations b y genetic analysis of the two comple-

Reciprocal translocations which regularly produce both types of translocation disomics in heterozygous crosses can be mapped by genetic analysis of these disomics alone. This is demonstrated here for the two cases of TI(ZIZ;VZZ) and TI (VZ;VZZ). To obtain well-marked n + 1 types, both translocations were crossed to all suitable markers available in standard strains which had been mapped on the two involved chromosomes. Several such crosses were carried out to avoid simultaneous segregation of interacting and epistatic markers and to

mentary types of translocation disomics.

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have markers in coupling as well as repulsion to the translocation (see Table 1 for genotypes).

1) To identify the intervals between mapped markers in which the breaks are located, translocation disomics are analyzed by phenotype and genotype: all aneuploids from heterozygous crosses were replated and classified visually. Both TI (ZZZ; VIZ) and TI (VZ;VZZ) produced two types of translocation disomics in addition to the two expected typical-looking n + 1 types. All disomics of the four groups were then checked for heterozygous markers. Information from the various crosses were consistent and results are shown for TI(ZZZ;VZZ) in Table 2 and Figure 2, and fo r TI(VZ;VZZ) in Table 3b and Figure 3. As expected, typical n + 1 are found to be heterozygous only for markers of the one linkage group for which disomy has been postulated on the basis of their phenotypes. The other n + 1 types, postulated to be translocation disomics, clearly show two complementary genotypes for each translocation. On the basis of these results the translocated markers and, therefore, the position of the breaks can be deduced (as shown in Figure 4).

For TI(III;VII) SA and probably galE, but not ActA, gulA and adl, of group 111 are translocated to VII, since the centromere is located between ActA and phenA (results from “diploid 2” by BAINBRIDGE 1970, were confirmed). Of the markers on VII, phenB, sF and palP are not on the translocated segment, while ma& choA and nicB are translocated to 111, distal to adz. (ZysD has not yet been separated from Tl(III;VII), but lysD+ is located on VII, since n + VI1 but not n + I11 are heterozygous for ZysD).

The same markers of group VI1 are also translocated in Tl(VI;VII), hut in addition paZF and sP are now separated from phenB and are on the translocated segment. Therefore, phenB must be the most proximal marker of VI1 used here. Of the markers on VI, sB and sbA are located on the distal segment, translocated to VI1 distal to phenB, while lacA and bwA are not.

These results are qualitative and very reliable provided a fair number of heterozygous cases can clearly be identified. This was possible for all except two markers: (a) bwA in Tl(VZ;VIZ) i s partly dominant and was tentatively identified in many n+l, but was proved to be heterozygous only in a very few cases which were checked for crossover sectors (Table 3b) ; (b) galE of TI(ZII;VII) was very difficult to classify in cross 1743 which segregated for many markers, and even though cross 1744 was analyzed mainly to identify gaZE/+ in one of the types of translocation disomics, very few cases were found (Table 2).

2) To obtain information on meiotic linkages between the translocation breaks and any segregating markers, the frequencies of marker segregation were determined in the balanced haploid sectors of all the analyzed disomics (and also in a sample of normal colonies from the same crosses). Specifically, results from typical disomics were compared with those from translocation disomics. When complementary patterns of allele ratios were obtained for these two classes, meiotic linkage of the markers in question to the translocation breaks is indicated (e.g., bwA was identified as linked to Tl(VZ;VZZ) in this way; Table 3a). Combining the complementary sets of data from all disomics, the approximate frequency of meiotic recombination between such markers and the breaks could be calculated. These values are shown for TI (VZ;VZZ) in Table 3a and indicated for all translocations in Figure 4. As expected, when markers on both chromosomes are identified as linked to the breaks, meiotic linkages between such mark-

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TRANSLOCATIONS A N D G E N E T I C M A P P I N G

TABLE 3b

Fraction of heterozygous disomics among testable cases from crosses heterozygous for TI (V1;VII) and markers of groups VZ and VZZ

19

Linkage group VI-Right arm

hcA 6wA sB s6A

Typical n + V I 34/64 3*/166 39/69 54/99

Translocation n + VI’ 17/26 lY/13 5/13 T/3 7 disomics n + VI1 0/28 0/76 0/31 0/48

disomics n + VII’ - 0/48 - 0/25 G/31 g / 4 1

Linkage group VII-Right arm

phenB SF palF malA choA nicB

0/78 0/11 0/41 0/69 O / l l l 0/69 Typical n + V I disomics n + VI1 ly/28 7 / 8 lF/21 8/24 28/47 15/24 Translocation n + VI’ 01/21 23/26 6/11 19/35 19/31 18/26

4%/62 0/4$ 01/40 - 01/57 0/51 0/38 disomics n + VII’

- - - - -

B. Mapping of translocation breaks b y genetic analysis of two or three disomic types and of translocation diploids.

Not all translocations produce both types of translocation disomics with high enough frequencies to analyze their heterozygous markers (see Table 4). Since the genotypes of the two translocation disomics from any one case are expected to be complementary, it is possible to obtain satisfactory results, even if only one such type is recovered, provided a large enough number is analyzed. This was the case for TZ (ZV;VZZZ) (Figure 4; details are given below). In other cases, as shown here for TZ(Z;VZZ), almost only typical disomics are obtained [see also T2(Z;VZZZ), Table 41.

In such cases, genetic analysis of disomics is either confirmed or supplemented with results from mitotic analysis, mainly of homozygous translocation diploids. Suitably marked strains for such diploids can be obtained among the haploid sectors from typical-looking disomics. As expected, these strains usually contain the translocation (a number were checked in heterozygous diploids or crosses). Pairs of strains can be combined to produce homozygous translocation diploids, heterozygous for as many of the relevant markers as possible. For each of the translocations analyzed here, over 100 haploids were selected from three to six such T/T diploids and analyzed for marker segregation. The unusual linkages found in these segregants corresponded to the translocation chromosomes postulated on the basis of the results with translocation disomics in all those cases where sufficient numbers had been obtained for genetic analysis [including

FIGURE 3.-Phenotypes of the four disomic types from crosses heterozygous for TI(VZ;VZZ) and comparison with standard n + IV. 3a, 3b and 3c: Typical disomics, (a) = n +VI (b) = n + VII, (c) = n f IV; 3d, 3c and 3E: Translocation disomics, (d) = n + VI’, (e) = n + VII’ on thick medium, (f) = n + VII’ on thin medium.

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20 E. KAFER

TI (ZIZ; VIZ) and cluded here; they KAFER 19741.

TI(VZ;VZZ) described above; details of these results are not in- . correspond exactly to those obtained for T2(I;VIIZ) by MA and

1) TI(1V;VIII): In this case the mapping of the breaks was helped by the fact that f r d , the marker induced by UV at the same time as TI(IV;VIII), appears inseparable from the translocation and also, that the markers of group IV can be sequenced meiotically (DORN 1967). Mainly n + IV-like disomics were obtained and almost 150 were analyzed from two crosses (1931 and 1933, Tables 1 and 4). These disomics could not be grouped by phenotype (see MATERIAL AND METHODS). But the marker frA could be used to distinguish typical n -b IV (with frA- sectors) from translocation-IV-dismics (with fra+ sectors; frA was also used in normal progeny to map the break on VI11 meiotically; see Figure 4). Results from the disomics were consistent with the assumption that the break is a t frA. They confirmed the meiotic mapping and identified the translocated markers.

a) The typical n + IV (with frA- sectors) were always heterozygous for f r A as well as other markers of IV but no markers of VIII. Of a total of 80 n + IV disomics the following fractions of testable cases were found to be heterozygous: for methG 44/53, for palC 35/39, for pabaB 27/36 and for pyroA 22/35.

b) In the other hand, the n + IV-like translocation disomics with frA+ sectors (total 63) were only heterozygous for methG of group IV (3/12 from the cross with methG and f r A in coupling, and 10/12 from the repulsion cross). In addition, the marker nir.4 of VI11 was found to be heterozygous with high frequency (30/37) but other markers of VI11 were not (e.g., 0/10 for riboB; 0/28 for SE). These results identify nirA of VI11 as the only translocated and therefore most distal marker of VI11 and the break position distal to riboB and SE (Figure 4). In group IV the break maps at frA, and the markers palC, pabaB and pyroA but not methG are located on the translocated segment.

2) TI(I;VII): This translocation produces mainly one type of disomics-namely n 4- VII- while typical-looking n + I are rare (and a very few translocated 1’-types have only been found in one cross, 1529, Tables 1 and 4 ) . However, in this case it was possible to use heterozygous diploids to identify the arms in which the breaks are located, namely the “left” arm of group I and the “right” arm of VI1 containing choA and nicB (KAFER 1962 and unpublished). In addition, over one hundred segregants from crosses of Tl(1;VII) to markers of I and VI1 were checked for presence or absence of TI(1;VII) in heterozygous test diploids. These results demonstrated linkage of the breaks to suAadE of IL (5-10% recombination) and absence of linkage to all then-available markers of group VI1 (choA, nicB, malA, palF and palD). In a final effort, TI(1;VIl) was recently crossed to two newly mapped markers of group VI1 which are both meiotically unlinked to the previously tested ones and to each other, namely phenB (SINHA 1967) and sF (GRAVEL et al. 1970). Results from heterozygous diploids indicated that phenB was the only marker not translocated of all VI1 markers tested here; also, analysis of a large number of typical n + VI1 disomics from the heterozygous crosses finally identified fairly close meiotic linkage of the break to phenB of group VI1 and showed that the break on I was distal to suAadE. On the basis of this information, the final mapping cross (1529, Table 1) was heterozygous also for fpaB and galD distal on IL. Using all 120 typical disomics from this cross the breaks were mapped close (2%) but distal to fpaB on IL, and fairly close (4%) and presumably distal to phenB on VI1 (as confirmed by the linkage values from 600 normal segregants, Figure 4).

In mitotic haploids from homozygous TI(1;VII) diploids, phenB was confirmed as the only marker of VI1 which is not translocated to IL; it segregated independently of all other group VI1 and group I markers which were completely linked to each other (51 recombinants out of 101 selected haploids; fpaB was not tested in T/T diploids because no fpaB-recombinant carrying TI(I;VII) has been obtained so far).

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TRANSLOCATIONS AND GENETIC MAPPING 21

C. Approximate mapping of breaks on the basis of aneuploid frequencies in het-

In Table 4 the following information is given for the four translocations mapped here and for the previously mapped T2(Z;VZZZ): frequencies of each disomic type, approximate position of a break in relation to centromeres and a viability rating of the aneuploid type. This rating is based mainly on growth rate

erozygous translocation crosses.

TABLE 4

Frequencies of different disomic types and position of breaks in mpped translocations

Disomics lor linkage groups a b

Typical n f l T,--disomics Typical n+l T,--disomics Total n f l Total Crosses No. % No. % No. % No. % No. % sample

TI(1II;VII) 1743 22 0.7 51 20 0.6 23 116 3332

2others 18 0.7 58 21 0.8 21 118 2550 4crosses* 31 1.1 47 9 0.3 26 113 2944

Total 116 1.0 176 1.5 66 0.6 79 0.7 437 3.7 11926

1744 46 1.5 20 16 0.5 9 90 31002

Viability ratingsf c41 [31 c51 [41 Break positions IIIL, median VIIR, med. distal

TI(V1;VII) 1921 111 2.5 32 51 1.1 64 258 4510 1922 78 1.6 30 50 1.0 23 181 4962 1941 26 2.5 18 7 0.7 18 69 IC60

2others 55 2.5 22 37 1.7 21 135 2208 4crosses* 35 1.0 13 30 0.8 22 100 3643

Total 305 1.9 115 0.7 175 1.1 148 0.9 743 4.5 16383 Viability ratingsf 151 [31 151 [41 Break positions VIR, prox. (-med?) VIIR, prox.-median

Tl(1;VII) 1529 12 0.3 3 100 2.3 0 115 44001: 3 others 7 0.1 1 146 2.7 0 154 5497

6crosses* 8 0.1 0 184 2.3 0 192 7858

Total 27 0.2 4 <O.l 430 2.0 0 ? 461 2.6 17755 Viability ratingsf r31 [I1 [51 [>5?1 Break positions IL, distal VIIR, pro'ximal

Tl(1V;VIII) 1931 64 1.2 44 3 0.1 0 111 5312 1933 16 0.4 19 4 0.1 4 43 3926

2 others 17 1.2 6 5 0.3 3 31 1450

Total 97 0.9 69 0.6 12 0.1 7 <O.l 185 1.7 10688 Viability ratingsf [51 [5 1 121 111 Break positions IVR, proximal VIIIR, distal

T2(I;VIII) 5 crosses* 14 0.4 0 <0.1 43 1.1 0 ? 57 1.6 3885 Viability ratings ~ 3 1 [<I?] [el [>5?1 Break positions IL, med. distal VIIIR, proximal

* Crosses of UPSHALL and KAFER 1974. 1. [5]-[l] for decreasing growth rates of n+1 centers. 2 Approximate totals.

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22 E. KAFER

and therefore size of the n + 1 centers, using a scale from [l] to [5]. Recovery and even detection of disomics depends on the size of colonies formed at 2 days when plates are inspected for abnormal colonies, and very small colonies are much more likely to be overgrown by normal ones.

Theoretically, equal numbers are expected of the two classes of disomics, typical n + 1 aEd translocation disomics, which result from nondisjunction of the same pair of centromeres. This has been found for some cases, but obviously not for others, especially not for those cases where no translocation disomics have been identified. In some of these cases the absence of such types might be caused by very poor viability and consequently poor recovery, if translocation chromosomes are extremely large [as, for example, the translocation-I’ type of T2- (Z;VZZZ)]. This was clearly demonstrated for two cases, namely the translocation-I’ type of TI (Z;VZZ) and the translocation VIII’ type of TI (ZV;VZZZ), both of which were recovered only occasionally and only at low densities in crosses free of viability-reducing markers. On the other hand, if translocation chromosomes are of very small size [like the translocation-VIIJ or -VIII’ chromosomes of TI (Z;VZZ) or T2(Z;VZZZ)] disomics might be indistinguishable from normal colonies (even standard n + IV are so close to normal that they are not easy to identify in some crosses and were probably underestimated in some of the large control platings of UPSHALL and KAFER 1974).

Therefore, only typical n + 1 can be considered suitable and can be used to judge the relationship of the relative positions of breaks to the frequencies of nondisjunction. For the mapped cases in Table 4 these typical disomics show a clear decrease in frequency with increased distance of the breaks from the centromeres. For example, for n + VI1 disomics 2.4%, 1.1% and 0.6%, respectively, are found in crosses with TI(Z;VZZ), TI(VI;VZZ) and TI(IZZ;VZZ); or for n 4- VI11 disomics 1.1 % and 0.1% in crosses with T2(Z;VZZZ) and TI(ZV;VZZZ). Prediction of approximate break positions might therefore be possible, based on information from heterozygous crosses, if a new translocation involves a chromosome arm, in which other breaks have been mapped. This was attempted here for the case of a V;VI translocation, specifically the break on VI. Crosses heterozygous for Til (V;VZ) gave about 1 % of n + V disomics and 0.5 % of n + VI when extensive results from two recent crosses were combined with previous ones (total plated almost 8000). From these results it is predicted that the break of Til(V;VZ) is closer to the centromere on V than on VI and probably further distal on VI than that of TI(VZ;VZI). These predictions are being tested and so far results are consistent with them (meiotic linkages of the breaks to pA in V and loosely to bwA in VI have been identified, but for final conclusions the precise mapping of the break in VI and of the centromere of V will be needed).

DISCUSSION

In organisms with genetically long linkage groups meiotic mapping of markers is very frustrating and success a matter of luck (like the linkages of pabaA and biA to y A found in one of the first crosses of A. nidulans; PONTECORVO et al. 1953). This is well known from mapphg attempts in man and equally holds for some fungal species, especially Aspergillus and to a lesser extent yeast.

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TRANSLOCATIONS AND GENETIC MAPPING 23

Recent advances in the use of systems with chromosomal segregation may solve the problem for yeast by identifying the “chromosome” to which each of the few known “fragments” belongs (e.g., CULBERTSON and HENRY 1973). On the other hand in man-and Aspergillus-such techniques only lead to classification of a large number of meiotically unlinked mutants into syntenic or mitotic linkage groups, each corresponding to one chromosome.

Qualitative mapping using translocations: Mapping of markers to specific chromosome parts can, however, be achieved by the use of reciprocal translocations. If loss of translocation chromosomes leads to segregation of genetic markers and can be followed in vegetative or somatic cells, such markers can be assigned to specific chromosome segments. If suitably overlapping translocations are available, meiotically unlinked markers cannot only be assigned to specific arms but also sequenced within a chromosome arm. Such work is now in progress in human cell genetics, e.g., sequencing of markers on the X chromosome (RICCIUTI and RUDDLE 1973; GRZESCHIK and GRZESCHIK 1973) and on chromosome A1 (MEERA KHAN et al. 1973; DOUGLAS, MCALPINE and HAMERTON 1973). In these hybrid cell systems the sets of mouse or Chinese hamster chromosomes are usually balanced and diploid and provide for cell growth, while the genetically marked human chromosomes may be retained or lost, as determined cytc~logically.

Similar information is also obtained in some higher plants in which gametes, disomic for a translocation chromosome, can be recovered and visually recognized in the progeny as “tertiary” or translocation trisomics; these have been used successfully for genetic mapping of translocation breaks, centromeres and markers (e.g., in tomato, by KUSH and RICK 1967).

In the corresponding Aspergillus system described here, translocation disomics are obtained from heterozygous translocation crosses and such “abnormal” disomics can be distinguished visually from standard n + 1. Two types of translocation disomics are expected and may be found, each containing one of the two translocation chromosomes in addition to the balanced haploid set of standard chromosomes (Figure 1) . Whenever the extra chromosome is lost, a standard haploid with improved growth rate is formed which shows up as a normally con- idiating sector. Any marker which segregates in this step must be located on the translocation chromosome present in the heterozygous disomic. In Aspergillus, such heterozygous markers can be recognized not only by comparing the growth responses of disomic centers with those of haploid sectors but, in addition, by identification of mitotic crossover segregants which may show up recessive alleles not recognized by the other method.

Thi? type of genetic analysis is expected and has been found to give complementary results for the two types of translocation disomics, which provides a check for the visual classification. In addition, if only one clearly identifiable type of translocation disomic is available in large enough numbers, the other expected one can be deduced, since it must be complementary. However, this type of indirect evidence should be checked in homozygous translocation diploids as was done for all cases in this investigation (e.g., for TI (ZV;VZZZ) the positions of paZC, pabaB and pyroA translocated to VI11 R were confirmed in T/T diploids).

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24 E. KAFER

This method of mapping based on translocation disomics is only usable when translocation disomics are found in fair numbers and are easily recognized even in crosses segregating for many markers, and when most of the crucial markers are clear-cut. The number of disomics to be analyzed has to be relatively large, because meiotic recombination preceeds nondisjunction and potentially heterozygous markers frequently become homozygous (unless they are closely linked to the breaks). Therefore, only an extensive analysis using many markers has made it possible to conclusively identify and make use of the disomic types postulated previously (POLLARD, KAFER and JOHNSTON 1968; UPSHALL and KAFER 1974). As a routine method, mitotic analysis of homozygous translocation diploids is much simpler, but has the disadvantage that strains containing markers in coupling with the translocation have to be obtained first (MA and KAFER 1974). Such strains are, however, easily isolated from typical-looking disomics produced by heterozygous translocation crosses, and identification of the eight typical-looking disomic types is not very difficult (KAFER and UPSHALL 1973).

Meiotic mapping of breaks linked to single markers in disomics from heterozygous translocation crosses: In addition to the qualitative mapping of translocation breaks which needs translocation disomics, breaks can be meiotically mapped to single segregating markers by analysis of any one specific class of disomics from heterozygous translocation crosses. This method depends on visual or genetic separatioE of “typical-looking” disomics, which carry a normal chromosome in addition to a haploid translocation set, from translocation disomics with an extra translocation chromosome in addition to a balanced standard chromosome set. For markers linked to the break the balanced haploids from these two classes give ccmplementary segregation patterns. Therefore, even just one clearly identifiable type obtainable in good numbers can be used, especially if viability differences are corrected for by using coupling and repulsion crosses.

The results obtained by this method are similar to those in higher plants when linkage of “semisterility” to genetic markers is determined (as discussed, e.g., by BURNHAM 1956) or the corresponding results obtained in Neurospora (PER- KINS 1967, 1974). But since no progeny testing is involved here, the Aspergillus technique is more efficient. In this respect it is comparable to the cytogenetic method used by SYBENGA (1970), who was able to map translocation breaks meiotically on the basis of chiasma frequencies in various segments of translocations in rye.

Mitotic crossing ouer in disomics: Linkage of a marker to the breaks may also be observed in mitotic crossovers from such disomics. Since mitotic crossing over is concentrated in the centromere area, such crossing over between the marker and the break is relatively frequent when markers are located on the opposite arm from the break. For example, fo r yA on the right arm of group I over 8% (55/663) crossover sectors were observed in various n+l disomics from TI (Z;VZZ) with a break distal to fpaB on IL (Figure 4) . On the other hand, for markers even loosely linked on the same arm as the break, such crossovers may become quite rare and none may be obtained for closely linked markers [e.g., very few (3/ca. 200) were obtained for bwA linked fairly closely to the break of Ti’(VZ;VZZ)] .

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TRANSLOCATIONS A N D GENETIC MAPPING 25

Mitotic crossing over may occasionally cause misclassification of balanced haploids from typical or translocation disomics. As a consequence of such an exchange, a disomic heterozygous for markers in a specific chromosome arm may become homozygous (this has been observed more than once in repeatedly replated standard n+l types). In disomics from translocation crosses, this may also eliminate the structural heteromorphism and change the phenotype of the disomic (Figure 1). A translocation disomic with a standard haploid set may then become homozygous for the standard chromosome as well as its markers. It will now look and be a standard n+l type. A few likely cases of this type were identified among the several hundred n f l from crosses with TI(ZZZ;VZZ), which is completely linked to ZysD. Such segregants were clearly of the typical n+III phenotype, but ZysD+; the noma1 VI1 chromosome is therefore expected to be present and the standard I11 chromosome is most likely homozygous in the n+III. Similarly, several unexpected apparent double crossover n+l from TI(VZ;VZZ) are probably mitotic crossovers. For two examples from each of these cases this was confirmed by checking a haploid sector in test diploids or crosses. When mitotic crossovers cannot be identified, the meiotic mapping technique, using disomics as described here, actually measures the sum of meiotic recombination between the marker and the breaks plus the mitotic crossing over between either the marker and the centromere, or the breaks and the centromere. This explains why generally these values are somewhat larger than expected from meiotic recombination in haploid progeny between markers which are linked to the breaks (e.g., for bwA-sF this latter value was only 16% while bwA showed 25% and sF 15% apparent recombination to the breaks). The recombination values given in Figure 4 are therefore of two types: for TI(Z1Z;VZZ) and TI(ZV;VZZZ) the markers at the breaks have been used and mitotic crossovers are excluded [similarly for T2(Z;VZZZ), MA and KAFER 19741, which is not the case for TI (Z;VZZ) and TI (VZ;VZZ). However, in either case such values do not correspond to meiotic linkages in standard crosses and only serve as relative measures of distances.

Approximate mapping of translocation breaks from aneuploid frequencies in heterozygous translocation crosses: The information obtained from absolute and relative frequencies of aneuploids in various translocation crosses can also be used to predict very approximate positions of the breaks of an unmapped translocation. If only typical-looking disomics are considered for which the relevant viability and the likely recovery rate are known, it seems clear from the mapped cases that nondisjunction decreases with distance of the breaks from the centromeres (Table 4). The differences shown for the different translocations are all highly significant when the combined values of all crosses are used, while there is con- siderable variability in some of the smaller samples of Table 4. However, since the more extreme cases can be traced to viability effects of linked markers which are magnified in poorly growing types like n f l , it is likely that fairly large samples (>1500, plated at low density on optimal media) from crosses with almost unmarked meiotic tester strains will provide quite reliable values for such estimates. While, admittedly, such results are very inaccurate, they may be

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26 E. KAFER

helpful in choosing markers of the right chromosome area to use for mapping of new translocations.

Map-construction: placing of fragments and centromeres

While mapping of translocations is of little interest in itself, the results may be very valuable for constructing genetic maps. When data from examples that involve a common chromosome are combined, the sequences of centromeres and meiotically unlinked “fragments” may be deduced. This is well illustrated for groups VI and VII, which each contain several fragments without indication of centromere positions, in the recent map of CLUTTERBUCK and COVE (1974). In Figure 4 all their markers have been sequenced with respect to each other and their centromeres. The complete sequence for group VI1 is especially well established, since it could be deduced from the analysis of TI(Z;VZZ) and TI(ZZ1;VZZ) alone, and subsequently confirmed by results from (TI (VZ;VZZ) (including the sequence of palF-lysD-malA-choA, which is different from that deduced by DORN 1967 on the basis of meiotic linkages alone, as also given by CLUTTERBUCK and COVE 1974; for group VI unpublished data from mitotic crossing over in diploids sB/+ selecting sB/sB on selenate media confirmed the centromere position and indicated the order of sbA distal to sB) .

The results from the mapping of TI (ZV;VZZZ) independently establish the orientation of the distal fragment of VIII, which so far had only been postulated on the basis of a small number of mitotic crossovers selected as chaA/chaA segregants (results of NIKLEWICZ, shown in MA and KAFER 1974).

One important result based on the mapping of TI(ZZZ;VZZ) is the placing of the fragment containing SA (which is closely linked to sC, and proximal to cnxH in the maps of BAINBRIDGE 1970, and CLUTTERBUCK and COVE 1974) very close and distal to adz on the left arm of group I11 (see Figure 4). The close linkage of SA to adz is more pronounced in, but not restricted to, translocation crosses and has also been obtained in several standard crosses between sC and adz (over 1000 segregants tested). These results resolve previous ambiguities in the map of group I11 as published by WEGLENSKI (1966) and by BAINBRIDGE (1970). They are in complete agreement with, and some confirm, the earliest mapping of suBpro (=Su4pro, KXFER 1958) to the “right” of phenA in the current maps. They also agree with the placing of suCpro on the other side of phenA (by WEGLENSKI) and therefore on the “left” arm, and the placing of the sC-cnxH fragment distal to suCpro (by BAINBRIDGE) and therefore distal on IIIL. The resulting map of I11 is shown in Figure 4. It has independently been confirmed by mitotic crossing over (selecting sC/sC segregants from se/+ diploids on selenate media) , which placed galE, sC, adz, methH, galA and ActA in this order on the same arm of 111 and phenA with suBpro on the other arm (details to be published elsewhere; this map disagrees only with two small sets of results of BAINBRIDGE, namely a cross with a sample of 156 tested which apparently produced two more double than single crossovers, and a diploid heterozygous for ActA and sC in coupling, which yielded only prototroph apparently resistant sectors on actidione media; however,

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TRANSLOCATIONS A N D GENETIC M A P P I N G

GROUPS

27

fpaB galD suAadE sulA riboA yAadE btA - - I 25

m t h H galA ACIA phenA SuBPro galE SA ad1 Ill 37 - .

39

methG f r A palC pabaB pyrnA IacA bwA SB sbA VI

m .

25 IV

V l l l

FIGURE 4.-Map positions of translocation breaks and meiotic linkages of markers in heterozygous translocation crosses (values not deviating significantly from 50% omitted). Recombina- tion frequencies from TI(VI;VII) are based on data of Table 3a, those for Tl(II1;VII) and TI(1V;VIII) on meiotic values from crosses of Table 1 [2001301) tested from each; for Tl(1;VII) see text, and for T2(I;VIll) see MA and =FER 19741.

Mutant genes (and alleles) used: Conidial color: bwA (1) = brown, chaA ( 1 ) = chartreuse, fwA(2) = fawn, y A ( 2 ) = yellow. Requiring mutants: adE(20), I (50) = adenine, biA(1) = biotin, choA ( 1 ) = choline, IysD (20) = lysine, methG ( 1 ) , H (2) = methionine, nicB (8) = nico- tinic acid, p a b d (1 ) B (22) = paminobenboic acid, phenA ( 2 ) , B ( 6 ) = phenylalanine, pyrwl (4) =pyridoxine, riboA(I), B ( 2 ) =riboflavine, s A ( 2 ) , B ( 3 ) , C(12), D(50) , E ( 1 5 ) and sP(211) = sulfite. Nonutilizers: fad?( 102) = acetate, frA (1) = fructose, gaZA ( I ) , D ( 5 ) , E(9) = galactose, lacA(1) =lactose, maZA(1) =maltose, nirA(14 = ni51) =nitrite, sbA(3) =sorbitol; paZB(7), C(4), F ( 1 5 ) = lacking alkaline phosphatase. suA(l)adE(20) = recessive suppressor of adE (20); SuB(4)pro = dominant suppressor of proline mutants. Resistance: ActA (1) = actidione, fpaB (37) = p-fluorophenylalanine, suZA ( 1 ) = sulfonilamide.

their genotypes were not analyzed, and our experience with this type of selection suggests that they probably were not ActAIAcrA) .

I n conclusion, it is evident that in Aspergillus nidulans reciprocal translocations, when mapped by a combination of the meiotic and mitotic techniques described here, can help to place centromeres and sequence meiotically unlinked markers very efficiently. Indeed, the results from only four cases investigated

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28 E. KAFER

here placed and connected practically all the fragments of the recent linkage map. However, the orientation of some of these (for example the nicB-palD fragment distal on VIIR) still needs to be identified and new fragments are also still being found.

Mapping of meiotically unlinked markers

As a further application, a set of Overlapping translocations can permit relatively easy mapping for new markers which are not meiotically linked to any of the markers placed in sequence on a very long chromosome arm. From crosses of the new marker to a set of mapped translocations, only a small sample of translocation disomics has to be tested to check for heterozygosis of the new marker. This will identify the interval between two breaks in which the marker is located. Such mapping can be carried out with completely unmarked translocation strains, testing for the new mutant only. However, since heterozygous translocation crosses show reduced meiotic recombination in the segments adjacent to the breaks, additional markers may be useful, and loose meiotic linkages to the new marker may become detectable.

Such mapping has been carried out for several markers of group VII, for example the meiotically unlinked marker oliA. Results from crosses of oliA to TI (VZ;VZZ) showed that oliA is heterozygous in the VIP-translocation disomics, and not in the VI’-disomics from this cross. In addition, linkage of oliA to sF (and pantoB which are closely linked meiotically) was detectable, but no significant linkage to phenB. These results map oliA proximal to the break of TI(VZ;VZZ) and therefore proximal but loosely linked to sF, and distal to phenB and the closely linked break of TI(Z;VZZ) (as shown in Figure 4).

The excellent technical assistance of MRS. P. MARSHALL is gratefully acknowledged as well as the expert photography of MR. R. LAMARCHE. DR. R. T. ROWLANDS kindly provided the oliA2 strain.

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Corresponding editor: F. H. RUDDLE

Influence of translocations on meiotic and mitotic Dow



reciprocal translocations and - genetics

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