Mol Gen Genet (1987) 208:226-229

© Springer-Verlag 1987

Isolation of autosomal mutations in Drosophila melanogaster without setting up lines

Satpal Singh*, Poonam Bhandari, Maninder Jit S. Chopra, and Devasis Guha Department of Biology, Guru Nanak Dev University, Amritsar, Punjab, India 143005

Summary. Mutants of Drosophila melanogaster are being used increasingly for studying different biological mecha- nisms. However, most attempts to identify new mutations have been restricted to the X-chromosome. It has been very difficult to identify new loci on the autosomes, as recessive mutations have to be made homozygous by setting up inde- pendent cultures for each mutagenized chromosome. We introduce a mutagenesis scheme which does not require set- ting up independent cultures. It uses meiotic recombination in compound autosomes to make recessive mutations ho- mozygous and allows the screening of tens of thousands of mutagenized chromosomes with relatively little effort. In a pilot experiment, we tested about 33,300 chromosomes for temperature-sensitive paralytic mutations. We obtained 62 independent paralytic mutations and a large number of other mutations. Eight out of 25 of the paralytic mutations are on the autosomes. This method makes autosomes, which constitute about 80% of the Drosophila genome, more accessible for mutational analysis of various biologi- cal mechanisms.

Key words: Drosophila - Autosomes - Mutagenesis - Com- pound chromosomes Temperature-sensitive

individual chromosomes. The method simply involves crossing mutagenized males to virgin females, transferring F1 progeny, as such, for a self-cross, and screening F2 flies for the presence of mutants. The method has been proposed and discussed briefly in a preliminary note (Singh 1983).

Materials and methods

Compound autosomes. The compound autosomes discussed here have two homologous copies of a chromosomal arm attached at the centromere (Fig. 1). They are also called attached autosomes or isochromosomes. Both the copies of the arm move together during meiosis and go to a single gamete. A heterozygous mutation on a compound arm can become homozygous during an event of meiotic recombina- tion (Fig. 2). Compound autosomes were first constructed by I.E. Rasmussen and E. Orias in E.B. Lewis' lab (Rasmus- sen 1960; Lewis 1967) for use in half-tetrad analysis in Drosophila (Chovnick et al. 1970). Their properties have been reviewed by Holm (1976).

Strains and cultures. Mutations used are gl (glass:3R-63.1), h 2 (hairy:3L-26.5), radius incompletus (ri:3L-47.0) and

Introduction

Drosophila mutants can be used as powerful tools in explor- ing biological mechanisms. They have been used effectively in studies on development (Lewis 1978), reproductive phe- nomena (Baker et al. 1976), neurogenesis (Campos-Ortega 1985), learning (Dudai 1985) and many types of behaviour (Hall 1982). However, the search for new mutations has been restricted mainly to the X-chromosome. Recessive X- linked mutations are expressed in males, which can be tested directly for the mutant phenotype. On the other hand, newly induced mutations on autosomes have to be made homozygous before testing the flies. For this, each single fly carrying a mutagen-treated chromosome is taken through the homozygosis procedure in an independent cul- ture (Gethmann 1974; Jackson 1983). This procedure is time consuming and limits the number of mutagenized chromosomes that can be tested. We describe a mutagenesis scheme in which it is not necessary to set up cultures for

* Pressent address: Department of Biology, University of lowa, lowa City, IA 52242, USA Offprint requests to: S. Singh

COMPOUND

L

AUTOSOMES

R

N o r m a l Autosomes

L • R

L • R

Fig. 1. Compound autosomes used in the experiment have homolo- gous copies of chromosomal arms attached at the centromere. Both the copies of an arm go to a single gamete during meiosis. L, left arm; R, right arm

,q7

> _._~

/77 /77

m

> Fig. 2. Homozygosis of a mutation in a compound chromosome. A mutation 'm' present on one arm of such a chromosome may become homozygous during meiotic recombination

Stubble (Sb: 3R-58.2). TM6 is a balanced third chromosome c a r r y i n g mult iple inversions. The nomenclature C(3L)RM; C(3R)RM refers to a compound (C) third (3) chromosome with the left arms (L) and the right arms (R) at tached in a reverse metacentr ic (RM) configurat ion (Fig. 1). See Lindsley and Grel l (1968) for descript ion of muta t ions and strains used. Cultures were mainta ined on cornmeal medium at 250-26 ° C.

Mutagenesis. C(3L)RM,ri; C(3R)RM males, aged 0-48 h, were fed 25 m M ethyl methanesulfonate (EMS) in 1% su- crose solution for 24 h (Lewis and Bacher 1968). Fur the r handling of the mutagenized chromosomes is described in the Results.

Detachment of compound autosomes. Females aged 3-10 d were X-irradiated, at a dose of approximate ly 2,000 R (Chovnick et al. 1970), and crossed to TM6/Sb males. The cross is sterile as it produces aneuploids. However, if the compound autosomes detach, and reat tach as normal chro- mosomes, it yields viable progeny (see Fig. 6A).

Temperature-induced paralysis. Flies, aged 0-2 d, were tested for paralysis at 37.0°_+0.5° C for 5 min. Flies that were paralysed were picked up as putat ive mutants. Pure cultures were set up after conf i rmat ion of the mutan t status (see Results).

Results

We have used meiotic recombinat ion in compound au- tosomes to make induced muta t ions homozygous (Fig. 2). EMS-t rea ted C(3L)RM, ri;C(3RJRM males were crossed to virgin females of the same genotype. An F1 fly from this cross would inherit one compound arm of a mutagen- ized chromosome from its father (Fig. 3). A newly induced muta t ion would be heterozygous at this stage. F1 flies were transferred to new bottles for a self-cross. Muta t ions which become homozygous during oogenesis (Fig. 2) can be iden- tified by testing F2 flies for the mutan t phenotype (Fig. 3). We tested the F2 for temperature- induced paralysis (Suzuki et al. 1971) and separated putat ive mutants. Putat ive mu- tant males or females were crossed to C(3L) RM, ri; C(3R) RM females or males (Fig. 4 A). Prog- eny, about half of which should be mutant , were tested for paralysis. Pure mutan t cultures were established by crossing mutan t males to virgin mutan t females (Fig. 4B).

Each F1 female inherits an independently mutagenized compound arm from its father. Each such arm contains two copies of half a chromosome. Hence the number of F I females used in the experiment reflects the number of mutagenized chromosomes screened. Al though the males also inherit a similar chromosome, they cannot be consid- ered for this purpose as they do not contr ibute to homozy- gosis of muta t ions due to an absence of meiotic recombina- tion.

F1 progeny were selfed in batches of about 100 females and 100 males. Each batch laid eggs successively in 3~4 bot- tles for 3 4 d each. Mutan ts obta ined from the progeny of different batches were considered to be of independent origin (Singh 1983). We tested the progeny of about 33,300 F1 females. This yielded 62 independent paralyt ic mutants and a large number of other mutants showing different behavioural and morphologica l defects. As the number of

227

EMS

P '-,>< X MALES

F2

m /'/" f'l

F, . . > < X .S L ,^ss CR°SS I

MEIOTIC RECOMBINATION

+ /7/ fg"

SCI~EEN FOR m .>< MUTANTS

Fig. 3. Procedure for obtaining autosomal mutations. EMS-treated males were crossed to females. F1 flies were self-crossed en masse. F2 flies were tested for the presence of mutants, m, a newly induced mutation; P, parents

07 rl" ,"1

->< X r,.>< 2 , ,

m fl" / ~ f/"

A

m r l 177 I ' I

m .)< X 1

D7 t ' l

B Fig. 4A, B. Setting up mutant cultures. A Putative mutants were propagated by crossing them to flies carrying compound chromo- somes. About half the progeny from this cross is expected to be mutant. B Mutant cultures were set up by crossing a mutant male and a mutant female

mutants was too large, we soon had to discard most of them except for those showing temperature- induced paraly- sis or those with striking morphologica l abnormali t ies. Out of 62 paralyt ic mutants , we have established pure cultures for 32 (Fig. 4). Some muta t ions could not be made into pure lines even though they could be readily mainta ined by se- lecting for the mutan t phenotype in each generation. The nature of these muta t ions is being studied after detaching the compound chromosomes (see below).

The meiotic propert ies of compound chromosomes al- low a muta t ion to be assigned to the left or the right arm of the chromosome (Fig. 5). Flies having muta t ions in the C(3L)RM, ri;C(3R)RM background were crossed to the marker C(3L) RM, h 2 ; C(3R) RM, gl flies. Mos t of the prog- eny from this cross are expected to be C(3L)RM, ri; C(3R)RM, gl and C(3L)RM, h 2; C(3R)RM (Fig. 5). A new muta t ion on the left arm of the third chro- mosome will go with C(3L)RM, ri; C(3R)RM,gl and a mu- ta t ion on the right arm with C(3L)RM, he; C(3R)RM. We tested 25 muta t ions in this way: three of these are on the

228

a m b c d

. , x > < , / k .

a m d ' / ~ c b

Fig. 5. Localization of mutations on chromosome arms. A cross between C(3L)RM, a; C(3R)RM, B and C(3L)RM, e; C(3R)RM, d should yield mainly two types of progeny. A mutation m on the left arm will go with markers a and d in the Ft . On the other hand, a mutation on the right arm will go with markers b and c. If the mutation is on the X-chromosome, FI males will show the maternal genotype and F1 females will be heterozygous. A mutation on an autosome, other than the third chromosome, will be in a heterozygous form in all FI flies. In our experiments, about 2%-3% of the Ft flies showed unexpected behaviour, perhaps due to a small level of abnormal disjunction in the strains carrying compound chromosomes

X RAYS / /

m Bg/

> K x _ : , k

v - - Oom m

A

Dorn

8al Bal

L

in ~ J ~ Bal

m • m B Fig. 6A, B. Linearization of compound chromosomes. A Females carrying compound chromosomes are X-irradiated and crossed to balancer males. The cross mainly yields aneuploids which are invi- able. If the compound chromosomes detach, and reattach like nor- mal chromosomes, viable progeny can be produced. B A cross between males and females obtained after irradiation yields homo- zygous mutant flies with linear chromosomes. Bal, balancer; Dora, a dominant marker; m, the newly identified mutation

left a rm of the third chromosome and three on the right arm. There are four dominan t mutat ions, two on autosomes other than the third chromosome and two on the X-chro- mosome. Fif teen recessive muta t ions are X-linked.

The chromosomal local izat ion of mutat ions, described above, was confirmed after detaching compound chromo- somes in five mutants (Fig. 6; see Mater ia ls and methods). These mutants bear the isolat ion numbers (with the chro- mosome or the arm in parentheses), D4(3R); P40(3R); S15(X); D36(X-dominant) and SS(2nd or 4th chromosome- dominant) . Homozygous mutan t cultures bearing linear chromosomes were established by crossing males and fe- males carrying detached compounds (Fig. 6 B). Muta t ions D4(3R) and P40(3R) were tested for recessiveness for tem- perature- induced paralysis and are both recessive. This rules out the possibil i ty that we might have obta ined only domi- nant muta t ions on the autosomes. D4 and P40 recombine with the marke r ri (3L-47.0) with frequencies of abou t 26% (160/617) and 28% (96/339) respectively. Since ri is located

0 7 1 , q 7 2

/% / T / I /7"/2

m, > < m . < Fig. 7. Construction of double mutants using compound au- tosomes. If two mutations ml and m2 lie on two different arms of the chromosome, a cross between them would yield double mu- tants in the progeny

near the centromere on the left arm of the third chromo- some and both D4 and P40 arc on the right arm, these two muta t ions are located on the right of the marker ri. D4 and P40 complement each other. The detai led character- ization of these mutants , o f the remainder of the 62 para lyt - ic mutants and of the morphologica l mutants is in progress.

Discussion

The existing methods for au tosomal mutagenesis (see Geth- mann 1974; Jackson 1983) are time consuming and require homozygosing of individual chromosomes in independent cultures. In addit ion, about 7 0 % - 8 0 % of these cultures yield nothing because o f recessive lethal muta t ions which show up only at the end of the procedure as an absence of homozygous flies in the cultures. Therefore this method can be used for testing only a few hundred chromosomes. The method in t roduced here makes newly induced muta- tions homozygous without going through independent cul- tures. As a result, tens of thousands of chromosomes can be screened with little effort.

The method can be used for identifying many types of muta t ions including those which affect behaviour, develop- ment, fertility, and sensitivity to chemical agents. F o r tests involving popula t ions o f flies, individual F2 flies can be crossed to other flies carrying compound chromosomes, to set up cultures (Singh 1983). This is equivalent to setting up cultures for X-chromosomes under similar constraints on testing procedures. Muta t ions that cannot be propa- gated in the homozygous form, can be obtained if their effect is condi t ional or if they affect the two sexes differ- ently. F o r example, male-sterile or male-lethal muta t ions can be obtained using F2 females. The method introduced here also has the advantage of the free recombinat ion scheme o f D.L. Lindsley (see Ge thmann 1974), i.e., some of the lethal muta t ions can be el iminated during recombina- tion.

C o m p o u n d chromosomes also make it easy to construct double mutants , which are useful for studying interactions between gene products affecting related functions. I t is gen- erally cumbersome to make double mutants for two muta- tions located on the same chromosome. I f muta t ions have been recovered using compound chromosomes, and if they are located on different arms of the chromosome, a cross between the two mutants would by itself yield double mu- tants in the progeny (Fig. 7).

Wi th the method described here, it is relatively more difficult to recover muta t ions that are close to the centro- mere, as the frequency of homozygosis is small for proximal loci. In our experiment (Fig. 3), we tested about 15 20 F2

flies for each F I female. This would permit the recovery of muta t ions that are a few recombinat ion units from the centromere. The possibil i ty of recovering more proximal muta t ions can be increased either by testing more F2 flies for each F1 female or by taking more F1 females. An inter- esting possibility, suggested D.L. Lindsley, is to use strains that carry large inversions spanning the centromere proxi- mal region. This would allow an easier recovery of muta- tions at loci that are centromere proximal , but which are located distally in the inversion strains.

There is a great dispari ty between the number of muta- tions available on the X-chromosome and those available on the autosomes. H o m y k et al. (1980) and others have urged that since muta t ions can be obtained with a good frequency in Drosophila, more autosomal muta t ions should be isolated even with the existing methods. Nevertheless, due to the difficulty o f obtaining autosomal muta t ions until now, this por t ion of the Drosophila genome is far from being fully explored. Our da ta show that autosomal muta- tions can be obtained easily by the mutagenesis scheme int roduced here. This method should help in extending the genetic utility of Drosophila for the analysis of different biological mechanisms, to the poor ly utilized 80% of its genome.

Acknowledgements. We thank A. Chovnick, B. Ganetzky, E.B. Lewis, D.L. Lindsley, J.D. Mohler, O. Siddiqi and C.-F. Wu for discussions. Work was supported by grant number 4/13/84 G from the Department of Atomic Energy, Government of India, to S.S. and research fellowships from the Council of Scientific and Industr- ial Research, India, to P.B., M.J.S.C. and D.G.

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Communica ted by O. Siddiqi

Received July 18, 1986 / February 17, 1987