a selective screen to recover chromosomal deletions and ...selective recovery of aberrations 379 i...

Copyright 0 1988 by the Genetics Society of America

A Selective Screen to Recover Chromosomal Deletions and Duplications in Drosophila melanogaster

D. Gubb, S. McGill and M. Ashburner Department of Genetics, University of Cambridge, Cambridge GB2 3EH, England

Manuscript received November 2, 1987 Accepted February 11, 1988

ABSTRACT A screen is described that will select for breakpoints within a restricted chromosomal region in

Drosophila. The aberrations recovered can be used to construct chromosomes carrying synthetic duplications and deletions. Such chromosomes have applications in the mapping of complementation groups at both the genetic and molecular level. In particular, breakpoints recovered after P element hybrid dysgenesis tend to be associated with P element insertion sites. Such aberration breakpoints can be genetically mapped, as synthetic deletions, and then used as transposon-tagged sites for the recovery of genomic clones.

T HIS study was undertaken to develop a selective screen to recover aberration breakpoints within

a small genetic region in Drosophila melanogaster. It is based on complementing the aneuploid lethality of the left and right-hand elements of pericentric inversions in their autosynaptic form (CRAYMER 1981). Chromosome aberrations are essential both for clas- sical genetic analysis (MULLER 1932; LINDSLEY et al. 1972) and for the precise identification of cloned DNA sequences with particular complementation groups. In addition, genomic clones can be recovered by walking across a breakpoint, from a known unique sequence (BENDER, SPIERER and HOGNESS 1983), or by transposon tagging from a transposon-associated aberration breakpoint (BINGHAM, LEVIS and RUBIN 1981).

CRAYMER (1981) demonstrated a novel type of rearrangement resulting from recombination between a normal sequence chromosome and a pericentric inversion. The recombinant products would normally segregate at meiosis and the progeny die due to aneuploidy (Figure 1). CRAYMER (1981) has shown a number of methods by which complementary aneuploid products can be recovered. CRAYMER termed the two complementary products of exchange the LS (levo-synaptic) and DS (dextro-synaptic) elements. He called an LS + DS stock “autosynaptic,” as each centromere carries homologous arms which will pair with themselves, at least along part of their length. This distinguishes them from the normal, heterosynaptic, form of a pericentric inversion in which pairing occurs between chromosome arms carried on different centromeres. Thus, autosynaptic stocks can be regarded as pericentric inversions in which the two breakpoints have been separated onto homologous centromeres (Figure 1).

Genetics 119: 377-390 Uune, 1988).

An alternative view of autosynaptics is to regard the complementary LS and DS products as reciprocal translocations between the left and right arms of an autosome (Figure 2). This suggests that autosynaptic elements could be directly formed as translocations following mutagenesis. Such products would normally be grossly aneuploid and would not, therefore, be recovered. In a cross to an autosynaptic stock, however, they become the only products that are viable. In order to complement aneuploidy, the breakpoints of novel autosynaptic elements would have to be close to those of the original stock. Given that novel breakpoints would not be identical to those of the original inversion, the recovered stocks will be aneuploid at both the left and right-hand breakpoints (Figure 2).

As hyperploidy is tolerated to a much greater extent than hypoploidy (LINDSLEY et al. 1972) duplication-bearing autosynaptics should be recovered at a higher frequency than deletion-bearing autosynaptics. However, given a set of autosynaptic stocks, the LS and DS elements can be exchanged (CRAYMER 1981). As with the proximal and distal fragments of T(Y;A) translocations (LINDSLEY et al. 1972), a given element can be used to synthesize either deletions or duplications, depending on the relative positions of the breakpoints in the LS and DS elements. The heterosynaptic form of such aneuploid inversions can be recovered by mating autosynaptic females to wild- type or balancer-carrying males. The only surviving progeny will result from recombination within the pericentric inversion (CRAYMER 198 1).

As pointed out by CRAYMER (1981), the lack of recombination in male Drosophila is crucial for the generation and maintenance of autosynaptic stocks. Thus, autosynaptic males can be used to select recom-

378 D. Gubb, S. McGill and M. Ashburner

hiring configuration Non-pairing configuration

\ 4! """" ~

FIGURE 1.-The generation of autosynaptic elements by recombination between the breakpoints of a chromosome carrying a pericentric inversion and a normal sequence chromosome. Note that the same products will be generated whichever side of the centromere the recombination takes place. These elements will segregate at meiosis and will be lethal unless fertilized by sperm carrying complementary elements. On the left side of the Figure, the heterosynaptic inversion and wild-type chromosomes have been drawn as synapsed. The LS and DS elements, however, have been separated, one above the other, for clarity.

"""

binants from a heterosynaptic female carrying a pericentric inversion to give a stable stock.

In this study, autosynaptic stocks are used to select for novel chromosomal breakpoints. In most cases, aberrations are recovered in hyperploid flies. As a result, any recessive mutation that might be associated with the breakpoint will not affect viability. Once established in stocks, autosynaptic elements can be interchanged so that a given breakpoint can give either a synthetic deletion or duplication.

MATERIALS AND METHODS

Stocks: The chromosome aberrations used are listed in Table 1. Mutations used as markers are described in LINDSLEY and GRELL (1968) with the exception of the recessive lethl(2)pawn (1(2)pwn, 2-55.4) carried on the Zn(2LR)TE146(Z)GV6 chromosome (GUBB et al. 1985) and no-ocelli, noc, (2-50.0) (ASHBURNER, TSUBOTA and WOODRUFF 1982). The P element carryin stock 7r-2 was used to induce hybrid dysgenesis. In(2LR)Sc$+' and Zn(2LR)Sco R + 9 were recovered by ASHBURNER et al. (1983). Zn(2LR)TE146(Z) GR15 was isolated by GUBB et a1 (1986).

For some experiments (series 3, 5 , 6, 7, 8 and 9, Table 2) "virginator" stocks were used to produce large numbers of virgin females carrying a compound X chromosome. These virginator stocks carry a temperature-sensitive lethal mutation, shibire-ts (shi'), on the male X chromosome (HALL 1973). When grown at 30" during the pupal stage, the shi' males die, leaving bottles from which only females hatch. The compound X chromosomes used were either C ( I ) R M , y (series 7, Table 2) or C( 1)DX, y w f. The C( I)DX, y w f stock (series 3, 5, 6, 8, 9 and 10, Table 2) carries an unidentified lethal mutation mapping in the y region. In the stock this lethality is covered by Dp( l ;Y)y+ , but an out-

TABLE 1

Description of chromosomes

Chromosome Cytological breakpoints

Aberrations: Zn(2LR)Sc8+' Zn(2LR)ScoR +

Zn(2LR)noc4, b noc4cn bw In(2LR)TE146(Z)GV6, a1 dp b

Zn(ZLR)TE146(Z)GR15, a1 dp b

Zn(2LR)DTD43, h 2 GlalCyO i.e.:

nocTE146 pr l(2)flwn cn

nocTE146 pr 1 (2)pwn cn

Zn(2LR)Gla, Gla 1 (2)34D1?/ Zn(2LR)O, Cy dpxUr pr cn2

C(2L)SH3, + : C(2R)VK2, bw Df(2L)75c

Df(2L)Al78, b r 8 cn Df(2L)A260, b cn bw

Df(2L)b75 Df(2L)b84a7 Df(2L)bBOcl Df(2LYn3, pr cn D f ( 2 L Y d 7 , pr cn Df(2L)ospl8, p cn Df(2L)TE146(Z)GW4, a1 dp b p

Df(2L)TE146(Z)GW6, a1 dp b pr

Df(2L)TE146(Z)GW7, a1 dp b p

Df(2L)TE146(Z)GWll, a1 dp b p r

Df(2L)b-L

l(2)pwn m

l(2)pwn cn

l(2)pwn cn

l(2)pwn cn

Visible and lethal mutations: Adnn7 lac?G34

Adhn7 smHc3' b Adn& r 8 pr m

b e12 AdhF b noc6 cn bw

b 1(2)35Aa4 A&* pr cn b 1(2)35Bb' pr b 1(2)34Fc6 Adhn4 b 1(2)35Dg) pr cn bw b wbSFZ5 Adh* pr cn Zn(2L)dpd6 Dp(2;2)DTD48, h g

Zn(2L)C163.41, 1(2)35Eb' Zn(2L)NS, 1(2)35De' jsF7 Adhup rd' pr cn 1(2)34Fa' Adh"' pr cn 1(2)35Bd Adh"" cn vg rk4 T(2;3)DTDl4, d#' 1 (2)35Ec' T(2;3)H16, h 2 1(2)35Dd' GI H

1(2)35Ea'

35D1.2;44C4.5 35D1.2;41het 35B1.2;41het 35B;41het

35B;44DE

34D;41het

Multiple Multiple 40het;4lhet Df 35A4;35D1-4 + In

Df 35B 1.2 Df 35B 1.2

27E1.2;35A1.2

Df 34D3;34E3-5 Df 34D4-6;34E5.6 Df 34C1;35B1.2

Df 35B1;35B3.4 Df 35B1;35D1.2 Df 35B1.2;35C4.5 Df 34F1.2;35A2

Df 35B1.2;35Cl

Df 35A3.4;35B2

Not visible

Df 34D3;34DS-E1.2

Cytological locations: lac: 35D3.4 sna: 35D1.2 osp: 35B1.2 rd: 35C3 el: 35A4-Bl b: 34D4.5 noc: 35B1.2 35Aa: 35A3.4 35Bb: 35B4 3 4 F ~ 34F4-35A1 35Dg: 35D5-7 wb: 34F3 35Ea: 35E1.2

35Eb: 35E3-6 35De: 35D5-7 j:34E3-5 34Fa: 34F1.2 35Ba: 35B 1.2 rk: 34E3-5 35Ec: 35E3-6 35Dd: 35D5-7 SU(H): 35B9-10

The H mutation, which maps to the third chromosome, was used to test for duplications that carried the Su(H)+ gene on the second, see text.

Selective Recovery of Aberrations 379

I

FIGURE 2.-The induction of novel autosynaptic elements as translocations in irradiated females. Breaks in the left arm of one chromosome and the right arm of its homologue produce four chromosome fragments. This is followed by rejoining of the distal left and proximal right fragments, to give an LS, or the distal right and proximal left, to give a DS. (Although drawn as a symmetrical event, only one of the products will be included in the oocyte nucleus, so that even were reciprocal elements formed, only one would be recovered.) In the example diagrammed, breaks are induced in 36 and 43. After mating to an autosynaptic male with 35D1.2;44C4.5 breakpoints, LS(2)Sc8+It net/ / D S ( 2 ) S c 8 + l , bw s$, a novel LS element would be recovered in a fly carrying a 35-36 duplication and a 43-44 deletion. A novel DS would be recovered in a fly with a 35-36 deletion and a 43-44 duplication.

cross to males with a wild-type Y chromosome gives only sons. In one series (9) the paternal autosynaptic males carried Dp(l;Y)y+ allowing survival of daughters.

Crosses: Mass crosses were made in 10-cm vials with standard yeast-glucose-agar food or Philip Harris instant medium. In the experiments to recover irradiation-induced elements, 40 females were mated to 20 autosynaptic males. These crowded conditions prevent yeast and bacterial in- fections from becoming a serious problem when there are few, or no, larvae. In the dysgenic experiments, 20 autosynaptic females were mated to 20 dysgenic males. In both series of experiments, adult flies were transferred to fresh food every 3 days and vials were examined for any surviving progeny 6 days later. Most vials contained only dead eggs and were discarded. In occasional vials a surviving larva or pupa was seen. These were transferred to fresh vials to prevent single eclosing adults from sticking to the surface of stale food.

Estimates of the number of fertile eggs laid under these conditions were made by mating irradiated females of the same genotype as those used in series two, seven and nine to wild-type males. Nonirradiated control crosses were also made but replicas were transferred daily to prevent larval competition.

A similar protocol was used for the dysgenic experiments. Twenty autosynaptic females were mated to twenty dysgenic males in vials at 25". Flies were transferred to fresh culture medium every three days for four replicas. The cross used to generate dysgenic males was cultured either at 18" or 25". The different temperatures did not appear to affect the recovery of autosynaptic elements significantly, although higher temperatures are known to cause gonadal sterility (KIDWELL and Novv 1979).

Complementation crosses were scored from the 10th to the 18th day after setting up.

Cytology and in situ hybridization: Temporary pro- pionic-orcein-carmine-stained squashes of larval salivary gland chromosomes were interpreted using the revised polytene chromosome maps of BRIDGES (see LEFEVRE 1976).

In situ hybridization to polytene chromosomes followed the procedure given in GUBB et ul. (1984). A tritium-labeled probe, p6.5, with homology to a P element, was used (p6.5 was kindly supplied by G. M. RUBIN). In cytological preparations, the autosynaptic and heterosynaptic forms of the same inversion cannot be distinguished.

"Resolution" of autosynaptic inversions: To convert autosynaptic inversions to their corresponding heterosynaptic forms, virgin females of the autosynaptic stock were crossed to GZulCyO males (Table 1). Surviving heterosynaptic Glazed or Curly progeny were backcrossed to GhlCyO flies to recover a stock. In practice, several vials containing 40 females and 20 males needed to be set up to recover two or three heterosynaptic progeny using inversions with breakpoints in regions 35 and 40. (For inversions with more widely separated breakpoints, fewer autosynaptic females would be required.) The frequency of recovery can be increased about tenfold by using the "interchro- mosomal effect" to increase recombination within the pericentric inversion (LUCCHESI and SUZUKI 1968). The C(1 )M4 chromosome used by CRAYMER (1981) is particularly useful in this respect as it is retained in females of an autosynaptic stock. The TMl balancer chromosome also gives a tenfold increase in the frequency of recombination in Zn(2LR)noc4/ + ; TMl/+ females mated to LS(2)noc4/ /DS(2)noc4 males (our unpublished observations).

As seen in Figure 1, resolution of an autosynaptic inversion can give two types of heterosynaptic product, either an inversion or a chromosome with a wild-type sequence. In general, these alternative products cannot be distinguished phenotypically as they will carry the same marker mutations. Resolved heterosynaptic inversions were therefore identified cytologically. Synthetic deletions associated with the inversion breakpoints were mapped by crossing to standard chromosomes carrying simple deletions or point mutations in sections 34-35 of the 2L chromosome arm (Table 1).

Nomenclature: This follows CRAYMER (1981) with minor modifications. The levo-synaptic element derived from


In(2LR)noc4, b n0c4 cn bw is designated LS(2)noc4, b indicating that it is homozygous for b, which maps distal to the inversion break oint. Similarly, the dextro-synaptic element DS(2)nocf cn bw is homozygous for cn and bw. Balanced pairs of autosynaptic elements are designated with a double slash thus, LS(2)noc4, bllDS(2)noc4, cn bw. This notation emphasizes the differences between heterosynaptic and autosynaptic stocks and can be abbreviated as in n0c4, bl h o c 4 , cn bw.

Mutations mapping proximal to the breakpoint, within heterosynapsed regions, are indicated after a second comma. Thus, LS(Z)TEI46(Z)GV6, dp b, pr is homozygous for dp b and hemizygous for pr. Recombination within the inversion of heterozygous I ~ ( Z L R ) S C O ~ + ~ , +/net bw sp females will yield the autosynaptic products S C O ~ + ~ , [net]/ I S C O ~ + ~ , [bw sp]. The brackets indicate heterozygous recessive markers. These will tend to "float" in the stock and to be lost by recombination, unless selected to give a homozygous stock i.e.: S C O ~ + ~ , net1 I S C O ~ + ~ , bw sp.

Markers proximal to the inversion breakpoint will not be lost, as single crossovers are lethal. In this case brackets indicate uncertainty about the position within the inversion loop of the recombination event. Thus, In(ZLR)TE146(Z)GV6, a1 dp b pr l(2)pwn cnl + + LS(2)TE146(Z)GV6, [a1 dp b], [prll lDS(Z)TE146(Z)GV6, [1(2)pm cn], [pr]. (The LS element will carry pr if the recombination event were distal, and pr+ if the recombination were proximal, to pr locus. Conversely, the DS will carry pr+ if generated by recombination distal to pr and pr following recombination proximal to pr. In neither element can the pr mutation be gained, or lost, by recombination.)

A particular case arises when an inversion breakpoint is associated with a recessive mutant phenotype. For example, the left-hand breakpoint of In(2LR)noc4 is associated with the noc4 mutation. As a result the LS(2)noc4 element will carry the 35Bl41 noc4 breakpoint, while DS(2)noc4 carries the reciprocal 41135B breakpoint. In such cases, the following notation is adopted: In(2LR)noc4, b n0c4 cn bwlb cn bw + LS(2)noc4, 61 lDS(2)noc4, cn bw. This autosynaptic pair is hemizygous for n0c4 with one wild-type noc allele carried on the LS element. Because the n0c4 mutation is associated with the inversion breakpoint, noc4 is not regarded as mapping to either the LS or DS element. The product of recombination between In(2LR)noc4 and b noc6 cn bw is designated as follows: Zn(2LR)noc4, b n0c4 cn bwlb noc6 cn bw + LS(2)noc4, b, noc6/ lDS(2)noc4, cn bw. LS elements of this type have applications in mapping DS breakpoints (see RESULTS).

Mutagenesis: Aberrations were induced either with gamma-rays in females, or by hybrid-dysgenesis in males. Females were irradiated rather than males because the fertility of males is severly reduced by irradiation during premeiotic stages of spermatogenesis (SANKARANARAYANAN and SOBELS 1976). Premeiotic events can readily be induced in either the male or female germ-line using hybrid dysgenesis (ENGELS 1983). P element dysgenesis has the additional advantage that transposon-associated breakpoints might be recovered (ENGELS and PRESTON 1981, 1984).

For y-irradiation, virgin females carrying cytologically wild-type chromosomes were irradiated with 4.5 kR from a 6oCo source with a dose rate of approximately 1 kR min". Irradiated females were mated to males carrying an autosynaptic inversion on the second chromosome. Nine series of crosses were made using the chromosome combinations listed in Table 2. A control (series 10) used nonirradiated females.

In series 1, 2 and 3 (Table 2), parental flies were transferred to fresh vials every 3 or 4 days for 3 weeks.

Vials set up after the first week, however, gave very few progeny so only three replicas were made in subsequent series. Vials were labeled so that clusters of progeny laid in successive replicas could be identified.

Putative autosynaptic elements were backcrossed to flies from the paternal stock and selected in the F2 generation. Newly induced elements were distinguished from those of the paternal autosynaptic stock by the differences in the recessive markers carried (see Stock design)

Hybrid dysgenic flies were recovered as the progeny of a cross between females of an M strain and males of a P strain, IT-2 (ENGELS 1981). This cross was made at either 25", for the first series or 18", for the second and third series. The two different temperatures at which dysgenic males were reared did not significantly affect recovery, although gonadal sterility is more severe at higher temperatures (KIDWELL and Novv 1979). The M strain used was homozygous for net bw sp in the first series of crosses and homozygous for net pr p k cn sp in the second and third series. The dysgenic males from these crosses, either net bw spl+ or net pr p k cn spl+ , were crossed to a female autosynaptic stock carrying appropriate homozygous recessive markers (see Stock design).

Stock construction: Autosynaptic stocks heterozygous for recessive markers were constructed by crossing females heterozygous for a heterosynaptic inversion over a marked wild-type sequence chromosome to males of the L S ( ~ ) S C O ~ + ~ I I D S ( Z ) S O ~ + ~ stock (obtained from L. CRAY- MER). Exchange between homologous arms in subsequent generations allowed elements homozygous for the desired markers to be selected. Thus, L S ( Z ) S C O ~ + ~ , + I / D S ( Z ) S C O ~ + ~ , + males crossed to In(ZLR)Sc8+', +/net bw sp females gave L S ( ~ ) S C ~ + ~ , + I lDS(Z)Sc8+', [bw sp] progeny which were crossed interse. This stock was subsequently selected for homozygous bw sp flies and used to recover the LS element of Sc8+' as follows: male L S ( Z ) S C O ~ + ~ , + I lDS(2)ScoR+', bw sp were crossed to female I~(ZL.R)SOC?+', +/net bw sp to give LS(2)ScoR+', (net)/ IDS(2)ScoR+', bw sp progeny which were then crossed inter se and selected for net bw sp.

This sequence of crosses can be shortened if the heterosynaptic chromosome carries suitable markers. Thus the construction of the LS(2)noc4, bl lDS(2)noc4, cn bw stock was much simpler: male L S ( Z ) S C ~ + ~ , +I I D S ( ~ ) S C O ~ + ~ , + crossed to female In(2LR)noc4, b cn bwlb cn bw gave LS(Z)Sc8+', +llDS(2)noc4, cn bw and LS(2)noc4, b l l D S ( Z ) S C O ~ + ~ progeny, which were crossed inter se to give the LS(2)noc4, bl lDS(2)noc4, cn bw stock in the next generation. (In this case, the breakpoints of the In(2LR)noc4 and I ~ ( Z L R ) S C O ~ + ~ inversions are close enough for both the hypoploid, LS(2)noc4, bl I D S ( Z ) S C ~ + ~ , + , and hyperploid, LS(Z)SCO~+~, +l lDS(2)noc4, cn bw autosynaptic stocks to survive.)

Stock design, y-irradiation experiments: Stocks were made carrying homozygous recessive marker mutations on both chromosome arms. Markers were chosen to allow flies carrying newly induced autosynaptic elements to be unambiguously distinguished from either parental stock. For example, in series one (Table 2) the maternal genotype is b cn bw while the paternal autosynaptic stock carries dp b, pr on the LS element. Newly induced LS elements were recovered as b flies while newly induced DS elements were recovered as dp b cn bw flies (Figure 3). This feature of the experimental design is critical, because occasional C~US-

ters of matroclinous (b cn bw) or patroclinous (dp b) flies can occur following nodisjunction of second chromosome centromeres. The extent to which nonsegregational gametes are produced in autosynaptic males is unknown.

Selective Recovery of Aberrations 38 1

9

Series 1

a' DS

Series 8

FIGURE 3.-The phenotype of flies in which novel y-ray-induced elements were recovered. The centromeres of the maternal chromosome are drawn as solid cicles while the paternal centrometes are open. Series I : females carried to the homozygous markers b cn bw. The paternal LS carried a 35B1.2;40 breakpoint marked with dp b, pr. The DS carried a 41;35D1.2 breakpoint without marker mutations. Novel LS elements were recovered in b, and novel DS elements in dp b cn bw, flies. Series 2: females were homozygous for dp b cn bw. The paternal LS element, however, carried a 2R breakpoint in 44C4.5, distal to the cn locus. Thus novel DS, elements were recovered in flies with a bw, rather than a cn bw phenotype. Novel LS elements were recovered in dp b flies. Series 8: females were heterozygous net bw spldp b cn b w , while the paternal autosynaptic stock was marked with b on the LS and carried a genetically wild-type DS. As the maternal chromosomes have the bw mutation in common, novel DS elements are recovered in flies homozygous for b and bw. Novel elements can, however, be induced as translocations between sister-strands (carrying the same markers), or non-sister-strands (carrying different markers). As a result, novel DS elements will be recovered in flies of three phenotypic classes: b cn bw, b bw and b bw sp. Novel LS elements will be recovered in net, wild-type or dp b flies.

Irradiation of females will, however, result in the recovery of nullo-2 and diplo-2 gametes (GAVIN and HOLM 1972). Their recovery, as matroclinous and patroclinous progeny, respectively, in these screens demonstrates that a proportion of the gametes from autosynaptic males are nonsegregational. In series two, new LS elements were recovered as dp b flies and new DS elements as bw flies.

The same combination of recessive markers on the maternal second chromosome (dp b cn bw) was used in series six. However, C ( I ) D W , y w f ; dp b c n Incr females laid so few eggs that the cross had to be modified in series 7 and 8. In these screens the maternal second chromaomes were homozygous for bw and heterozygous for net dp b cn and

sp while the paternal LS element was homozygous for 6. New LS elements were recovered as bw+ flies and new DS elements as b bw flies (Figure 3).

Autosynaptic elements can be recovered as translocations between the 2L and 2R arms involving either sister chromatids or non-sister chromatids. Sister-chromatid translocations would give LS elements homozygous for net or dp b and DS elements homozygous for cn bw or bw sp while non-sister-chromatid translocations would give phenotypically wild-type LS elements (ie.: netldp b) and bw DS elements ( i e . : cn bwlbw sp). In either case, however, an independently occurring recombination between non-sister-chromatids could exchange recessive marker mutations.

382 D. Gubb, S . McGill and M. Ashburner

TABLE 2

Genetic crosses and recovery of y-ray-induced autosynaptic elements

Novel elements Clusters Paternal genotype

Series Maternal genotype LS I/ DS LS DS Paternal Maternal vials frequency No. of Estimated recovery

1 2 3 4 5 6 7 8 9

10

b cn bw dp b cn bw C ( I ) D X ; + dp b cn bw C ( I )DX; + C ( I )DX; dp b cn bw C ( I )RM; bw" C ( I ) R M ; bw" C( I )DX; + C ( 1 ) D X ; +

TEI46(Z)GV6. dp b, prllScoRf9, i

noc4, bllnoc4, cn bw n0c4, b l l S ~ o ~ + ~ , + TE146(Z)GV6, dp bllnoc4, cn bw

D l , b l / S ~ o ~ + ~ , +- n0c4, b I l S c ~ ~ + ~ , + ScoR+', netllScoR+', bw sp TEI46(Z)GV6, dp 6, prllnoc4, cn bw

&OR+', +-IISCOR+9, +

scoR +9, i lISCOR + 9 , +

1(0) 0 2(1) 5 0 2 0 0 1(1) 4 0 0 3(0) 14 0 0 l(1) 10 0 1

0 15 1 in 2.2 X io4 2 21 1 in 1.4 X lo4 0 59 1 in 2.6 X lo4 0 12 0 in 7.2 X lo4 0 124 1 in 3.7 X lo4 0 12 0 in 1.8 X lo4 6 63 1 in 1.2 X lo4 0 9 1 in 2.7 X lo4

17 139 1 in 8.3 X lo5 2 75 1 in 1.1 X io5

The number of novel elements recovered is indicated by numbers of survivors of the expected phenotype followed by the number of stocks established, in parentheses. Clusters of flies with the paternal or maternal phenotype presumably resulted from nonsegregational gametes.

a In series 7 and 8, females were heterozygous for two recessively marked second chromosomes that carried the bw mutation in common i.e.: dp b cn bwl net bw sp.

FIGURE 4.-The phenotype of flies in which novel hybrid dysgenesis-induced elements were recovered (series 2). The diagram shows the phenotype of viable combinations of gametes, lethal combinations are left blank. The M chromosome carried the markers net pr pk cn sp while the P chromosome was wild-type, so the majority of the sperm will carry heterosynaptic net p pk cn sp or + second chromosomes. These will be lethal with the majority of gametes from the maternal autosynaptic stock carrying either LS(2)TEI46(Z)GV6 marked with dp b, p r or DS(2)noc' marked with cn bw. Occasionally, however, heterosynaptic sperm will fertilise oocytes carrying one of the alternative products of recombination within the autosynaptic inversion; either In(2LR)TE146(Z)GV6L n 0 P or a cytologically wild-type chromosome. Both of these types of maternally derived heterosynaptic chromosome will carry either dp b cn bw or dp b p cn bw, depending on which side of pr the recombination took place. Surviving heterosynaptic flies will thus be either cn, or pr cn, if they inherit the paternal P chromosome, and wild-type if they inherit the paternal M chromosome. Novel LS elements will carry either + , [net] , pr , [net], or net, p r depending on whether they arose as sister-strand or non-sister-strand translocations. They will be recovered in either cn bw, or net cn bw flies. Novel DS elements will be recovered in d p b, d p b pr, or d p b pr pk cn sp flies.

Stock design, hybrid dysgenesis experiments: The ap- ically wild-type heterosynaptic chromosomes, Figure 1. proach is similar to that used in the y-irradiation experi- These heterosynaptic chromosomes will be viable with wild- ments. Two additional factors, however, have been allowed type chromosomes from the dysgenic male. It must be for. First, since the autosynaptic elements are introduced possible to recognize and discard such survivors. Second, through the female, recombination can occur. This will it seemed likely that dysgenic events would occur predom- resolve the autosynaptic to give either Zn(2LR) or cytolog- inantly between non-sister-chromatids ( i e . : one P and one

Selective Recovery L - Aberrations 383

M chromatid). To see whether this was the case, M chromosomes carrying the recessive markers net bw sp or net pr pk cn sp were used. In the first and second series, the maternal autosynaptic stock was marked with dp b, p r on the LS and cn bw on the DS i.e.: LS(2)TE146(Z)GV6, dp b, pr l lDS(2)noc4, cn bw. In the second series, with net pr pk cn sp on the M chromosome, resolution of LS(Z)TE146(Z)GV6, dp 6, pr l lDS(2)noc4, cn bw will give + , cn, or pr cn survivors i.e.: I T Z ( ~ L R ) T E ~ ~ ~ ( Z ) G V ~ ~ no^^^, dp b [pr] cn bwl+ , dp b [pr] cn bwl+ , In(2L.R)TE146(Z)GV6L no^^^, dp b [pr] cn bwlnet p r pk cn sp or, dp b [pr] cn bwlnet p r p k cn sp (Figure 4). New LS elements will be recovered in flies carrying DS elements marked with cn bw. The flies will be phenotypically either cn bw or net cn bw depending on whether the novel LS derived from two M chromatids (net prlnet pr ) , an M and a P chromatid (net p r l + ), a P and an M chromosome ( + / net) or two P chromatids ( + I + ) . Although these classes differ for the eye-color mutation p r they cannot be distinguished as homozygous cn and bw flies have white eyes. New DS elements will be recovered as dp b pr pk cn sp, dp b pk cn sp, or dp b flies, with the dp b, p r LS element, and carry the markers pk cn sp, pr, pk cn sp, pr' or [pk cn sp], pr' . The classes obtained with net bw sp on the M chromosome are analogous, but differ in some cases for eye-color markers.

In the third series of experiments the M chromosome markers were net pr p k cn sp while the maternal auto synaptic stock was homozygous for dp b bw sp i.e.: LS(2)TE146(Z)GR15, dp b, pr cnl lDS(2)ScoR+', bw sp.

Comparative recovery of LS and DS elements from the female germline: The ratio of LS to DS elements recovered in the progeny of females carrying a heterosynapticgeri- centric inversion was estimated by crossing LS(2)noc , b l l DS(2)noc4, cn sp, males to In(2LR)noc4, b n0c4 cn bwl+ females. In the progeny, maternally derived LS elements were recovered as cn sp flies (i.e. : LS(2)noc4, [b]l lDS(2)noc4, cn sp and DS elements as b or b bw flies (Le.: LS(2)noc4, bl lDS(2)noc4, [cn bw] or, LS(2)noc4, bl lDS(2)noc4, [cn] bw. DS elements homozygous for bw result from double recombination, one crossover occurring within the pericentric inversion and the other between cn and bw. Maternally derived LS elements homozygous for b would not be recovered, since b maps close to the noc4 breakpoint, within the region where recombination would be suppressed.

RESULTS

Recovery of y-ray-induced elements: Seventy-six flies were recovered with the phenotypes expected for novel autosynaptic elements (see Stock design). In series 1-8 and the non-irradiated series, 10, the paternal autosynaptic chromosomes carried right- hand breakpoints in the centric heterochromatin. This heterochromatin forms a large target which was expected to increase the frequency of recovery of new elements. In series 1-8, one putative novel element was recovered in about every 30 vials. In series 9, the paternal autosynaptic inversion had both breakpoints in euchromatic regions (35B;44C). As expected, the recovery of novel elements was significantly reduced, to about one in every 140 vials.

These experiments do not allow a direct measure of the frequency of recovery per irradiated chromosome. An indirect estimate was made by crossing

26 neterosynaptic Dp(l;Y)y+ ; net bw sp males to 40 C( I )DX, y w f l o p ( 1;Y)y + females in vials and trans- ferring the parents to fresh food every day to prevent larval overcrowding. Under these conditions, about 3000 progeny were recovered from the replicas of a single vial over a 14-day laying period. Based on this measurement, the frequency of recovery for series 1-8 was between 1 and 4 novel autosynaptic elements per 10,000 eggs (Table 2). In series 9 the frequency was 1 in 830,000 eggs. The spontaneous frequency of recovery of autosynaptic elements (series 10) was an order of magnitude lower than the y-ray induced frequency (series 1-8). This is despite the lack of irradiation-induced sterility, which would increase the number of potentially viable gametes by about threefold. (In a test cross of 40 C( I ) D W , y w f / Dp(l;Y)y + females with 20 Dp(1;Y)y + ; net bw sp males, under the same conditions, 2 183 progeny were recovered from nonirradiated females and 7 15 progeny were recovered from females irradiated with 4.5 kR y-rays.)

To recover stocks, putative autosynaptic survivors were crossed to flies of the particular paternal autosynaptic stock used in each screen. Stocks were established from 32 of the 76 putative autosynaptic flies. In the first four series, virgin wild-type females were added to any cross in which a putative autosynaptic male was apparently sterile after 1 week of mating to autosynaptic females. Flies were then transferred to fresh medium and examined after a further week. No cases were found, however, in which putative autosynaptic males were fertile with wild-type females. Similar test crosses confirmed that survivors carrying the maternal recessive second chromosome markers were fertile only with heterosynaptic flies and those carrying paternal markers were fertile only with autosynaptic flies. A striking feature of the recovered elements (Table 2) is the preponderance of LS elements. The ratio of putative LS to DS elements is almost 9 to 1 (68: 8). This bias is even more extreme among elements that gave fertile stocks (Table 3), about 15 to 1 (29:2).

Distribution of y-ray-induced aberration breakpoints: The cytological analysis of new autosynaptic elements is summarized on Table 3. Surprisingly, a large proportion of the novel LS elements appeared to have identical breakpoints to the paternal autosynaptic. On close examination these stocks, LS(2)D4, 08 , DIO, D I I , 013, 0 1 4 , D 1 6 , D I 7 , 018, D l 9 and D2I-D31, carried a large duplication from 35B to 40. In these LS elements both breakpoints are within the centric heterochromatin and invisible in salivary gland preparations. This results in a configuration very similar to that of a pair of autosynaptic elements with precisely reciprocal breakpoints. The 35B to 40 region on the DS element pairs with both copies of the proximal 2L carried by the novel LS. This gives


TABLE 3

Cytological breakpoints of y-ray-induced autosynaptic elements

New elements Induced breakpoint Series Paternal breakpoint 2L Aneuploidy 2R Aneuploidy

LS(2)DI , b DS(2)D2, cn bw LS(2)D3, d p b LS(2)D4, b LS(2)D5, + DS(Z)D6, + LS(2)D7, + LS(2)DR, dp B LS(2)D9, dp b LS(2)DI0, + LS(2)DI I , + LS(2)D12, + LS(2)DI3, [net d p b] LS(2)DI4, [net dp b ] LS(2)D15, d p b LS(Z)DI6, [net dp b ] LS(2)D17, [net dp b ] LS(P)DIB, net LS(2)DI9, [net dp b] LS(2)D20, [net d p b] LS(2)D2I , [net dp b ] LS(2)D22, [net dp b ] LS(2)D23, [net dp b ] LS(2)D24, net LS(2)D25, [net dp b ] LS(2)D26, [net d p b ] LS(2)D27, [net d p b] LS(2)D28, net LS(2)D29, net LS(2)D30, dp LS(2)D31, [net dp b] DS(2)032, +

36C;4 1 het

36D1.2;4 1 het 40het;4 1 het

41het;35D 35F1.2;4 1 het 40het;4 1 het 35BI-3;41het 40het;4 1 het 40;4 I het 35D1.2;4 1 het 40het;41het 40het;4 I het 35C4;4 1 het 40het;4 I het 40het;4 1 het 40het;4 1 het 40het;4 1 het 34E4-F2;41 het 40het;4lhet 40het;41 het 40het;4 1 het 40het;4 1 het 40het;4 1 het 40het;4 1 het 40het;4 1 het 40het;4lhet 40het;4 I het 40het;4 1 het 40het;4 1 het 34F;4 1 het

41B3-9i34D4.5

36C1.2;42A16-19

1 2 2 1 3 5 5 2 2 5

10 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 9

3

41het;35D1.2 44C4.5;35D1.2 4 1 het;35D 1.2 4 1 het;35D 1.2 41 het;35B 1.2 35B3-8;41 het 4 1 het;35B 1.2 41het;35D1.2 41het;35D1.2 41het;35B1.2 4 1 het;35B 1.2 41 het;35B 1.2 41het;35D1.2 41het;35D1.2 4 1 het;35D 1.2 41het;35D1.2 41het;35D1.2 41het;35D1.2 4 1 het;35D 1.2 41het;35D1.2 4lhet;35Dl.2 4 1 het;35D 1.2 41het;35D1.2 4 1 het;35D 1.2 4 1 het;35D 1.2 41he~35D1.2 41het;35D1.2 4 1 het;35D 1.2 4 1 hec35D1.2 41 het;35D1.2 4 1 het;35D1.2 44C4.5;35D1.2

Dp 35D1.2;36C Dp 35D1.2;34D4.5 Dp 35D1.2;36D1.2 Dp 35D1.2;40het Dp 35B1.2;36C1.2

Dp 35B 1.2;35B 1.2 Dp 35D1.2;40het

Dp 35s 1.2;40het Dp 35B1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het

Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D 1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D1.2;40het Dp 35D 1.2;40het Dp 35D1.2;40het Df 34F;35D1.2

Df 358 1.2;35B3-8

Df 3.iB 1-3;35D 1.2

Df 34E4-F2;35D1.2

het

het het

het het het het het het het het het het het het het het het het het het het het het het het het het het

Dp 41A;44C4.5

Dp 41B3-9i44C4.5

Dp 41Ai42A16-19

a salivary gland configuration showing a thickened region between 35B 1.2 and the chromocenter (Figure 5). Unless the chromosomes are asynapsed, this configuration looks very similar to that of a euploid pericentric inversion with both left- and right-hand euchromatic breakpoints in 35B 1.2.

Autosynaptic elements with both breakpoints in centric heterochromatin can be regarded as a limiting case. They have been known for many years as compound chromosome arms (RASMUSSEN 1960; HOLM 1976).. The identity of the C ( 2 L ) arms recovered in the y-ray-induced autosynaptic screen was confirmed by crossing to a standard compound arm stock. In this test cross, the survival of two classes of progeny is diagnostic of a novel compound arm (e.g.: LS(2 )D8 , dp b/ / D S ( ~ ) S C O ~ + ~ , + X C(2L)SH3, +; C ( 2 R ) V K 2 , bw + LS(2)D8, dp b; C(2R)VK2, bw and C(2L)SH3, +I / D S ( ~ ) S C O ~ + ~ , +). Survival of only a single class indicates that the LS element carries a euchromatic breakpoint in the 2 L and is lethal when recovered over C(2R)VK2, bw e .g . : LS(2)DI , bl / D S ( ~ ) S C O ~ + ~ , + x C(2L)SH3, +; C(2R)VK2, bw +

C(2L)SH3, + / /DS(2)ScoR+’, + . Flies of the missing

a i

2L ie C

FIGURE 5.-Polytene chromosomes of autosynaptic stocks. (a) LS(2)D25/ / D S ( ~ ) S C ~ ~ + ~ . LS(2)D25 is equivalent to a C(2L)RM chromosome, the stock thus carries a duplication from the 35D breakpoint of DS(Z)ScoR” to the centromere ( i e . : 21----40het/ 4 1 het----21 + 60----4 lhet/35D--O----60). T h e DS(2)ScoR” breakpoint is marked with an arrow and the partially synapsed proximal 2L region is bracketed. (b) LS(2)PI7/ IDS(2)noc4. LS(2)PI7 is a complex element that can be regarded as a C(2L)RM with a deletion from 36A to 38B. T h e 2R breakpoint is in 42A giving LS(2)PI 7 an order of 2 1----0--42/3RC--40het/35F--2 1. T h e stock thus carries a duplication from 38D to 40het and from 4lhet to 42A. (c) LS(2)noc‘l /DS(2)P9. DS(2)P9 has breakpoints in 34B7- 12 and 40het. With LS(2)noc4, the stock carries a duplication from 34B7-32 to 35B1.2, which is indicated with brackets.


TABLE 4

Cytological breakpoints of hybrid-dysgenesis-induced elements

New elements Induced breakpoint 2L Aneuploidy 2R Aneuploidy

L S ( 2 ) S I , [net] 36C;41het Dp 35B1.2;36C het LS(Z)S2, [net] 35D;41het Dp 35B 1.2;35D het LS(2)S?, [net] 26C;35C;42A Dp 35B1.2;35C Dp 41A;42A LS(2)S4, [net] 35B;41het Dp 35B1.2;36A het LS(2)S6, [net] 36C;42E Dp 35B1.2;36C Dp 41A;42E LS(2)S8, [net] 35DE;42B Dp 35B1.2;35DE Dp 41A;42B LS(P)PI , [net] , Iprl 35E1.2;41het Dp 35B1.2;35E1.2 het LS(2IP2, [net], [prl 35D5-7;41het Dp 35B1.2;35D5-7 het L S ( W 3 , [net] , [prl 37B1.2;41het Dp 35B1.2;37B1.2 het LS(W'4, [net], pprl 25F;35E;41het Dp 35B;35E het DS(21P5, [ p k cn spl, pr 41het;34A7-11 Dp 35B1.2;34A7-11 het LS(W'6, [net] , [prl 25EF;28B;35B1-3;41het DpiDf 35B het LS(2)P7, [net] , [pr pk ] cn 34F5;47A1.2 Df 34F5;35B1.2 Dp 41A;47A1.2 L S ( W 8 , [net] , [ P I 37B;41het Dp 35B1.2;37B het DS(21P9, [ p k cn spl , pr 41het;34B7-12 Dp 34B7-12335B1.2 het LS(2)PIO, [net] , [prl 35BC;41het Dp/Df 35BC het LS(P)PII , [net], [prl 25EF;35D;41het Dp 35B1.2;35D het LSWP12, [net] , [prl 35E1.2;41het Dp 35Bl.2;35E het L S W ' I ? , [net] , [prl 35D;41 het Dp 35B1.2 het LS(2)P14, [net], [pr pk ] cn+ 35B;44D1.2 Dp/Df 35B Dp 41A;44D1.2 LS(2)Pl5 , [net], [pr P A ] cn+ 35Ci47Al-4 Dp 35B1.2 Dp 41A;47A1-4 L S ( W 1 6 , [net] , [prl 37DE;41het Dp 35B1.2;37DE het L S ( W 1 7 , [net] , [prl 35F;38C;40het;42B Dp 38CD;40het Dp 4 1 het;42A LS(21P18, [net], [prl 36B1.2;41het Dp 35B1.2;36B1.2 het

Elements S1 to S8 were induced in series 1 and P1 to P18 in series 2. The maternal autosynaptic stocks used to select for new elements carried 35B1.2;41het breakpoints.

class, LS(2)Dl , b/ lC(2R)VK2, bw, would carry a synthetic deletion from 36C to 40.

Disregarding the compound-21 stocks, 7 LS elements and 2 DS elements were recovered with a single euchromatic breakpoint, LS(2)Dl , 3 , 7, 9, 12, 1 5 , 2 0 and DS(2)D6 and 32 (Table 3). In addition, a single LS and a single DS were recovered with both breakpoints in euchromatin, LS(2)D5 and DS(2)D2.

Recovery of elements induced by hybrid-dysgenesis: Three series of dysgenic crosses were made. In the first two, dysgenic males were crossed to females carrying autosynaptic elements with 35B;41 breakpoints. In the third, the maternal autosynaptic elements carried 35D1.2;44C4.5 breakpoints.

In the first series, from 46 vials, ten putative LS elements were recovered and six stocks were established (Table 4).

The second series gave 44 putative LS elements and 2 putative DS elements from 91 vials. From these putative elements, 18 LS and 2 DS stocks were established. Given that only 20 females per vial were used, the frequency of recovery of stocks per female in the first two series is about three times that in the irradiation screens (series 1-8, Table 2). Autosynaptic elements were recovered in flies of four phenotypic classes: cn bw, bw, dp pr and net cn bw. The first class contained 28 putative LS elements as cn bw flies. Of these flies, nine were sterile but the remainder gave fertile autosynaptic stocks. The second class

consisted of thirteen putative LS elements as bw flies, of which all but two were sterile. These elements, LS(2)P14 and LS(2)P15, had right-hand breakpoints distal to section 44 (Table 4). As a consequence the LS element carried a wild-type copy of cn, which suppressed the phenotype of the homozygous cn mutation carried on the DS element. Similarly, the phenotype of the flies in which the two DS elements, DS(2)P5 and DS(2)P9, were recovered, was affected by the DS breakpoints being distal to b, a recessive marker carried on the LS. These flies were dp pr, indicating that the heterosynapsed region of the DS carried b+ and pr, derived from the paternal M chromosome. The fourth phenotypic class consisted of three putative LS elements recovered as net cn bw flies, all three of which were sterile. The net phenotype indicates that they carried both 2L arms derived from the paternal M chromosome (Figure

Thus, the majority of putative LS elements (43146) were either homozygous or heterozygous for wild- type alleles of marker mutations within their auto- synapsed regions. They therefore derived either from sister-strand events between P chromatids or non-sister-strand events between one P and one M chromatid. T o distinguish between sister-strand and non-sister-strand events, 14 of the novel LS stocks were scored for net progeny in subsequent generations. In eight stocks occasional net progeny were

4).


found while the remaining six stocks gave no net progeny in over 500 flies.

These data indicate that novel elements frequently derived from P + P or P + M events, but only rarely as M + M events (3146 cases). In addition, there were no cases in which the reciprocal products were recovered as matched LS and DS elements in different flies.

In the third series, only two putative LS elements and one putative DS element were recovered from 158 sets of vials. All three flies were sterile.

In addition to autosynaptic elements, heterosynaptic chromosomes were recovered in all three screens. These result from either recombination within the inversion in heterosynaptic females or nondisjunc- tion. In the second series, 36 putative heterosynaptic chromosomes were recovered as pr cn or cn flies and 96 as wild-type flies. The corresponding classes in the third series were 29 pr cn sp flies and 58 wild- type flies. P element distribution: Salivary gland chromo-

somes of stocks from the first series of dysgenic screens were examined using in situ hybridization. Four of the six chromosomes showed P element homology at the inversion breakpoint, LS(2)S2, LS(2)S4, LS(2)S6 and LS(2)S8 (Figure 6). LS(2)SI had no P element at the breakpoint, although, like the other novel autosynaptic elements, it carried P elements at other sites. LS(2)S3 carried a P element at its paracentric breakpoint (26C;35C), but not at its pericentric breakpoint (35C;42A).

Genetic mapping of y-ray-induced aberration breakpoints: The cytological breakpoints (Table 3) were confirmed by resolving autosynaptic stocks as synthetic deletions and mapping the 2L breakpoints of the corresponding heterosynaptic inversion. In most cases this required exchanging LS and DS elements between stocks to recover the novel elements in autosynaptic stocks with synthetic deletions. Thus, LS(2)Scdi”, +I lDS(2)D2, cn bw X LS(2)DTD43, ho2 I lDS(2)DTD42, bw sp + LS(2)DTD43, ho2/ lDS(2)D2, cn bw. This hypoploid autosynaptic was resolved to give In(2LR)DTD43L D2R, ho2 cn bw which carries a deletion distal to the left-hand breakpoint of Df(2L)b84a7 in 34C1 ( i . e . : In(2LR)DTD43L D2Rl Df(2L)b84a7 survives). I n ( 2 L R ) n o ~ ~ ~ D6R carries a deletion from noc to l(2)35Dg (Of 35B1.2;35D5-7). In(2LR)D12L D6R is deleted from 1(2)35De to 1(2)35Dg (Of 35D5-7). In(2LR)DF is deleted from osp to l(2)35Da (Of 35B1;35D1-2). In(2LR)D2@ D32R is deleted from rk to l(2)34Fa (Of 34E3.4;34F1.2). It was not possible to map the 36C breakpoints due to the lack of an available inversion with 36D-37A;41 breakpoints from which to recover an appropriate DS element. In the case of L S ( 2 ) D l , b, however, it was confirmed that the 2L breakpoint was proximal to the Su(H), 1(2)35Bh, locus by resolving it with

FIGURE &-In sifu hybridization of polytene chromosomes of LS(2)S6/ lDS(2)noc4 with a P element probe. LS(2)S6 has the order 2 1----36/42E--O----2 1 . Several sites of P-element hybridization can be seen including one of the 36C142E breakpoint (arrow).

DS(2)noc4, cn bw. The corresponding heterosynaptic inversion, In(2LR)DlL no^^^, b cn bw, carries a duplication from noc, in 35B1.2, to 36C. This duplication, which includes l(2)35Bh+, enhances the phenotype. of the Hairless ( H ) mutation (ASHBURNER, TSUBOTA and WOODRUFF 1982).

The 2 R euchromatic breakpoint of LS(2)D5 was confirmed by resolution with DS(2)TEI46(Z; SR36)SZ4. In(2LR)D5L TE146(z;SR315)SZ4~ carries a synthetic 35B-36C duplication and a 42A16.19 to 43A1 deletion which fails to complement prickle ( p k ) .

A particular problem was encountered in mapping D S ( 2 ) D 2 . I n ( 2 L R ) n o ~ ~ ~ D2R is homozygous viable im- plying that the DS breakpoint is distal to the noc locus. To map this breakpoint with respect to rickets (rk), in 34E3.5, an LS stock hemizygous for rk4, was constructed from In(2LR)D2@ ~ o c ~ ~ , net cn bw. Fe- male In(2LR)D2@ 7 2 0 ~ ~ ~ ) net cn bwlrk were mated to male LS(2)noc4, bl lDS(2)noc4, cn sp to give LS(2)D20, [net], rkl lDS(2)noc4, cn sp and LS(2)noc4, 61 lDS(2)noc4, [cn bw]. The phenotype of LS(2)D20, [net], rkl I DS(2)noc4, cn sp is rk cn sp while that of LS(2)D20, [net], rkl lDS(2)D2, cn bw is rk’ cn bw. This indicates that the 0 2 breakpoint is distal to rk, so that DS(2)D2 carries rk’. Having demonstrated this, DS(2)D2 was resolved with an LS having a 34D;41 breakpoint, LS(2)DTD42, ho2.

Ratio of LS to DS elements recovered after recombination in the female germline: After mating female In(2LR)noc4, b not4 cn bwl+ females to LS(2)noc4, bl I DS(2)noc4, cn sp males, 82 LS elements carrying wild- type markers were recovered compared to 36 wild- type and 7 bw DS elements. This gives a ratio of 1.9 : 1 LS to DS elements. About one-fifth of the DS elements were homozygous for bw. These bw elements result


from double recombinants, one crossover being within the inversion and the other between cn and bw .

DISCUSSION

As shown in this study, it is possible to recover novel aberration breakpoints by complementing the aneuploid lethality of autosynaptic elements. Large numbers of chromosomes can be handled with little effort, particularly if the females used are derived from a “virginator” stock. The method is limited to regions of the major autosomes where appropriate pericentric inversions are available, but should become generally applicable as autosynaptic stocks are recovered in other regions.

Breakpoint distribution, irradiation experiments: As expected, the majority of novel elements (29132) were recovered in flies carrying synthetic duplications. Only three flies carried synthetic deletions in the 2L. Given this result, the distribution of left-hand breakpoints was surprisingly restricted: three of the left-hand breakpoints fall within division 34, five within 35 and three within 36C (Table 3). It is clear, however, that extensive hyperploidy is tolerated in Drosophila. LINDSLEY et al. (1972) showed that only when flies are hyperploid for about one-quarter of a chromosome arm is their viability reduced to 10% that of euploids. CRAYMER (1981) recovered several autosynaptic stocks hyperploid for four to six numbered divisions. Thus, DS elements with breakpoints distal to salivary gland division 34 and LS elements with breakpoints in the 36C-40F interval would have been expected and the failure to recover them is surprising.

It is clear that large duplications can survive the autosynaptic screen; 21 of the novel elements corre- sponded to compound-2L arms carrying both their left and right breakpoints within centric heterochromatin. These stocks are duplicated from 35B 1.2 to 40F. Flies with newly induced duplications of this size are slow moving and semisterile, although stocks that survive a few generations in culture become reasonably healthy. Thus, it seems likely that only a small proportion of C(2)s survive the screen, but they are induced at a high enough frequency to form a significant fraction of the recovered elements. In euploid flies, one compound arm is recovered per 20 to 35 irradiated females (HOLM 1976). This frequency is about 15 times greater than that of (2L) arms recovered in hyperploid autosynaptics carrying a 35B1.2 to 40F duplication (see RESULTS).

The high frequency of induced heterochromatic lesions is also reflected in the right-hand breakpoints recovered. Only 3 of the 32 novel elements had breakpoints in 2R euchromatic regions, resulting in duplications of 2 to 4 numbered salivary gland chromosome divisions.

Breakpoint distribution, hybrid dysgenesis experiments: After hybrid dysgenesis in the male germline, a similar set of breakpoints to those induced by y-irradiation was recovered. The major difference is a much reduced tendency to recover heterochromatic breakpoints. Thus, although 15 of the 24 elements had right-hand breakpoints in centric heterochromatin, no double heterochromatic breakpoint elements were recovered as C(2L) arms. The remaining nine elements had right-hand breakpoints in euchromatin and were thus aneuploid at both breakpoints (Table 4). On the 2L, 3 of the breakpoints fell within division 34, 14 within 35, 4 within 36, 2 within 37 and 1 within 38. Thus, although the spread of breakpoints is greater than after irradiation, the majority of 2L euchromatic breakpoints recovered still fall within division 35. On the right arm, 15 of the breakpoints fall within centric heterochromatin while the remainder were between 41EF and 47A1.2.

Biased recovery of LS compared to DS elements: An unexpected result of the autosynaptic screens was the extreme bias in recovery of LS compared to DS elements. The source of this bias was investigated by comparing the ratio of LS and DS elements recovered from the heterosynaptic noc4 stock. Recombination within the pericentric inversion in Zn(2LR)noc4/ + females will generate asymmetric dyads each having an autosynaptic chromatid and a noncrossover chromatid. In one dyad, the LS chromatid will be shorter than the noncrossover chromatid; while in the other dyad, the DS chromatid will be longer than the noncrossover chromatid. As shown by NOVITSKI (1951), the smaller element of an asymmetric dyad is preferentially included in the egg nucleus, while the larger element is included in the polar body. Thus, preferential recovery of the LS element would be expected following recombination in Zn(2LR)noc4/ + females. The observed bias of 1.9: 1 LS : DS elements was much lower than the ratio of 8.5 : 1 putative LS : DS elements recovered in the irradiation screen. If, however, the recovered C(2L) arms are dis- counted, on the basis that the corresponding C(2R) arms would not survive, the ratio of LS:DS elements among the established stocks becomes 3 : 1. This ratio remains greater than the estimate of 1.9: 1 following recombination. A differential bias in favor of smaller elements between asymmetric dyads resulting from meiotic recombination, and those resulting from induced chromosome breakage, would not be surprising in the light of MARK and ZIMMERING’S (1976) evidence that the degree of bias toward recovery of the smaller element from an asymmetric dyad is affected by the physical location of the crossover that generated the dyad.

It should be noted that the size difference between elements during chromosome segregation might be greater than that observed in polytene chromosomes,


where the centric heterochromatin is under-repli- cated. LS elements having a 2L euchromatic breakpoint and a 2R heterochromatic breakpoint would be deleted for 2R heterochromatin while complementary DS elements would be duplicated for 2R heterochromatin (J. ROOTE, personal communica- tion).

Unexpectedly, LS and DS elements were also recovered with very different frequencies from dysgenic males. The ratio of LS : DS elements was 27.5 : 1. A weak preference toward recovery of the smaller element of an asymmetric dyad following X-ray induced exchange in males was demonstrated by ZIM- MERING and BENDBOW (1973). The basis of this bias is unknown, but has been attributed to sperm dys- function (ZIMMERING 1976). NOVITSKI, GRACE and STROMMEN (198 1) found a very strong bias in favor of nullo-2 sperm compared to diplo-2 sperm derived from C(2)EN males. The degree of this nonrandom recovery is similar to that with dysgenesis-induced elements in the current experiments. In autosynaptic females, an equal number of LS and DS eggs should be produced, so the ratio of diffferent elements recovered through the male should not be altered. Recombination within the inversion loop may, however, give asymmetric dyads. This would result in biased recovery of the alternative (Zn(2LR) and +) chromosomes when autosynaptic females are crossed to wild-type males.

Availability of inversions and construction of autosynaptic stocks: CRAYMER (1981) described a number of approaches to converting heterosynaptic inversions to the autosynaptic form. The simplest of these is to use a preexisting autosynaptic stock to recover novel recombinants in an aneuploid stock. This stock, carrying the LS element from one inversion and the DS from the other can then be used to recover the euploid autosynaptic stock of the new inversion (CRAYMER 1981). This approach CRAYMER termed the “inchworm” method. It is simple to use providing an autosynaptic stock having breakpoints similar to those of the heterosynaptic stock is available. In order to distinguish the original autosynaptic element from the novel element, appropriate recessive marker mutations distal to the inversion breakpoints can be introduced (see Stock construction).

The inchworm method could be used with the set of second chromosome inversions isolated by GEL- BART (1982) and SMOLIK-UTLAUT and GELBART (1987). This set includes sufficient inversions with euchromatic 2L and heterochromatic 2R breakpoints to construct autosynaptic stocks in most regions of the 2L by progressive iteration of the inchworm technique.

For most chromosome arms, however, too few inversions are available for the inchworm method to be applicable in all regions. In such cases a modifi-

cation of CRAYMER’S (1981) ideas should simplify recovery of autosynaptic inversions. In principle, synthetic autosynaptic elements could be constructed to mimic any second or third chromosome inversion using T(Y;A) translocations from the collection of LINDSLEY et al. (1972). (This is a particular case of the product-mimic method suggested by CRAYMER 198 1 .) Briefly, females carrying an attached-X chromosome and a heterosynaptic inversion heterozygous with a T(Y;A) translocation mimicking the left-hand breakpoint of the inversion are crossed to males with a T(Y;A) translocation which mimicks the right-hand breakpoint of the inversion heterozygous with a balancer chromosome. DS-bearing sons are recovered following recombination in the female. The synthetic LS element is provided by the maternally-derived Y p 2O element and the paternally derived YD 2p. These males can be crossed to females carrying an attached- X chromosome and the heterosynaptic inversion (heterozygous with a chromosome carrying appropriate recessive markers) to recover the standard LS element of the autosynaptic stock.

General considerations in design of autosynaptic screens: Many of the particular tricks in designing screens are described under Stock construction and Stock design and are concerned with unambiguously distinguishing novel elements by the use of appropriate combinations of markers. There are a number of other features to be considered:

It is an advantage to use autosynaptics with one breakpoint in euchromatin and the other in centric heterochromatin. Not only will such elements be recovered at a higher frequency than those with two euchromatic breakpoints (see RESULTS), but aneuploidy at the heterochromatic breakpoints can be ignored when constructing synthetic duplications and deletions. This is analogous to constructing segmental aneuploids using the T(Y;A) translocations of LIND- SLEY et ad. (1972) in which the second site breakpoints are in the Y chromosome heterochromatin.

Elements having one euchromatic and one heterochromatic breakpoint can perhaps most simply be visualized as compound arms that include either a deletion or a duplication. Thus, inversions such as Zn(2LR)noc4, with a euchromatic left-hand and a heterochromatic right-hand breakpoint, give “hypo- compound” LS elements and “hyper-compound” DS elements. As we have shown, a potential problem with using such hypo-/ lhyper-compound arms in screens is that if the euchromatic breakpoint is suf- ficiently proximal, occasional true compound arms will survive as flies duplicated for proximal euchromatic regions. The problem is not serious, as compound arms can be identified and discarded by crossing to a standard compound arm stock before further analysis (see RESULTS). Compound arm chromosomes should cease to be recovered when using


hypo-/ /hyper-compound stocks with euchromatic breakpoints more distal than about one quarter of a chromosome arm from the centromere (as duplications larger than this do not survive in segmental aneuploids, LINDSLEY et al. 1972). With stocks carrying a euchromatic breakpoint near the tip of a chromosome arm, however, an analogous problem will be encountered as hyper-compound elements are recovered with elements that correspond to free chromosome arms, e.g.: F(2L) or F(2R) (HOLM, FITZ- EARLE and SHARP 1980). Such free arms could be identified and discarded by using a standard free arm stock in an analogous manner to which compound arms were identified in this study.

An alternative approach is to design screens so that survivors carrying putative compound, or free, arms are phenotypically distinguishable from those carrying the desired hypo-compound elements. This prop- erty can be built into a screen by constructing autosynaptic elements homozygous for proximal recessive markers. For example, we constructed a marked compound arm stock by irradiating females homozygous for b pr pk cn sp and mating to a standard compound arm stock, C(2L)SH3, +; C(2R)VK2, bw to recover C(2L)C6, b pr; C(2R)C2, pk cn sp (Unpub- lished results). Using this stock, hyper-compound LS elements with breakpoints between pk and cn can be recovered as cn sp flies (i.e.: LS(2), pk+llC(2R)C2, pk cn sp) while C(2L) arms will be recovered in pk cn sp flies. Similarly, hyper-compound DS elements with breakpoints between b and pr will be recovered in b flies while C(2R) arms will be recovered in b pr flies.

This approach can clearly be extended to identify novel breakpoints falling between any pair of recessive mutations carried on a complementary element. Thus, DS elements recovered in flies carrying LS(2)noc4, b, noc6 will be phenotypically b noc+ if the DS breakpoints fall between b and noc. In principle it does not matter whether homozygous markers, distal to the breakpoint, or hemizygous markers, proximal to the breakpoint are used. In practice, however, recombination is strongly suppressed close to a breakpoint, so that elements carrying homozygous markers close to the inversion breakpoint can only be recovered by inducing inversions on chromosomes that already carry the required markers. Markers proximal to the breakpoint, on the other hand, are simple to introduce into an unmarked element, as they map to the other side of the centromere to the inversion breakpoint (c$ : construction of the LS(2)020, [net], rk element above, Genetic mapping of y-ray-induced aberration breakpoints).

In regions for which suitable hypo-/ lhyper-compound stocks are not available, but an autosynaptic stock with both breakpoints in euchromatin exists, it should be possible to increase the frequency of recovery of novel elements by introducing a block of

heterochromatin at one of the target breakpoints. For this purpose the Y chromosome would be ideal and Y insertions into most autosomal regions are available in the form of T(Y;A) translocations (LIND- SLEY et al. 1972). As an example, breakpoints in segment 47 could be recovered using the LS(2)TE146(Z)GR226, b/ lDS(2)TEl46(Z)GR226, sp stock which has 35B1-2;47B10-14 breakpoints (M. ASHBURNER, unpublished observations) and T(Y;2)R15, Dp(l;Y)Bs Dp(l;Y)y+ with a 2L breakpoint at 35B9-C 1. Thus, irradiated female C( 1 )RM, y ; T(Y;2)R15, Dp(l;Y)BS Dp(l;Y)y+/net bw sp crossed with male LS(2)TE146(Z)GR226, b/ I DS(2)TE146(Z)GR226, sp would give novel LS elements in [net] b+ sp flies and novel DS elements in b [bw sp] flies. (Neither of the novel elements would carry Dp( 1;Y)y + or Dp( l;Y)Bs, which would have been deleted together with the bulk of the Y chromosome.)

To recover dysgenesis-induced aberrations in males, as in this study, requires mating dysgenic males to autosynaptic females. As a consequence of using the autosynaptic stock in females, a proportion of the progeny will carry the original inversion in the heterosynaptic form. With small inversions, such as In(2LR)noc4, the frequency of recombination within the inverted region is too low for this to be a problem. The occasional heterosynaptic recombinants can be identified by use of appropriate marker mutations (see Stock design). With larger inversions, however, it would be an advantage to cross dysgenic females to autosynaptic males to avoid recovering large numbers of heterosynaptic chromosomes.

Autosynaptic screening techniques might be de- signed for use with species other than D. melanogaster. Autosynaptic inversions have been described in the domestic chicken (BITGOOD et al. 1982), onion-fly, Hylemya antiqua (VAN HEEMERT 1977), and the lily Gasteria (BRANDHAM 1970). A basic problem, however, is that in species in which recombination occurs in both sexes, stocks carrying pericentric inversions will be unstable. Whatever the initial state of the stock, both autosynaptic and heterosynaptic individuals will be formed. This alternation between autosynaptic and heterosynaptic forms of a pericentric inversion has been demonstrated in the chicken (BITGOOD et al. 1982). There are a number of species of Diptera, however, such as the onion fly and the Australian sheep blowfly, Lucilia cuprina (see FOSTER 1982) to which the technique might be applied.

We would like to thank LORING CRAYMER for preprints of unpublished work, encouragement and a whole series of enigmatic letters over the years. The DTD chromosomes were a gift from S. M. SMOLIK-UTLAUT and W. M. GELBART. Particular thanks are due also to PAM THOMSON, who helped with the dysgenic screening and DARIN COULSON who completed the genetic mapping of some of the abberration breakpoints. The diagrams were drawn by


BELINDA DURRANT. JOHN ROOTE spent a lot of time discussing autosynaptics and how to make them with us. Financial support for this study came from grants to M.A. from the Medical Research Council.

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Communicating editor: A. CHOVNICK

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