Copyright 0 1985 by the Genetics Society of America

A GENETIC AND CYTOGENETIC ANALYSIS OF THE

DROSOPHILA MELANOGASTER REGION SURROUNDING T H E LSP-1 @-GENE IN

DAVID B. ROBERTS, HUGH W. BROCK,' NANCY C. RUDDEN AND SUSAN EVANS-ROBERTS

Genetics Laboratory, Biochemistry Department, University of Oxford, South Parks Road, Oxford, England

Manuscript received May 8, 1984 Revised copy accepted September 15, 1984

ABSTRACT

The region surrounding the gene coding for the &polypeptide (2 1 D-22C) of the major Drosophila melanogaster larval serum protein, LSP-I , has been studied in detail. Seven new 7-ray-induced deficiencies of the region have been used, together with the two extant deficiencies, to map the position of the @-gene and of the 55 newly induced ethyl methanesulfonate mutants uncovered by one of the largest deficiencies. No lethal mutation of the &gene was found.

ARVAL serum protein one (LSP-1) is the major protein of the serum of L Drosophila third instar larvae (ROBERTS, WOLFE and AKAM 1977). It is synthesized only by the fat body (SATO and ROBERTS 1983). Synthesis begins immediately after the second larval ecdysis and continues until the wandering larva stage (POWELL et al. 1984). LSP-1 is a family of heterohexamers which is made by the apparently random association of three polypeptides, LSP-1 a, LSP- 1 0 and LSP-1 y (WOLFE, ROBERTS and AKAM 1977). These polypeptides are coded by genes that probably have evolved by the duplication of an ancestral gene and the subsequent dispersal of the duplicates throughout the Drosophila genome (ROBERTS 1983). The three coding sequences have been mapped genet- ically, cytogenetically (ROBERTS and EVANS-ROBERTS 1979) and by in situ hybrid- ization (SMITH et al. 198 1).

One of the major goals of geneticists is to understand the mechanism of gene control in higher eukaryotes. An approach to this goal is the analysis of coding sequences and adjacent sequences which are likely to be involved in control. This approach is being carried out in the genetic analysis of genes coding for major proteins and the analysis of the environs of these genes (see inter alia CHOVNICK, GELBART and MCCARRON 1977; WOODRUFF and ASHBURNER 1979a,b; WRIGHT et al. 1981; KOTARSKI, PICKERT and MACINTYRE 1983).

A prerequisite for any detailed genetic analysis of a gene is the establishment of its position relative to the array of neighboring genes. This paper sets out to analyze the gene coding for the LSP-1 0-polypeptide which is one of three coordinately controlled genes (POWELL et al. 1984) scattered throughout the

' Present address: Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T PA9 Canada.

Genetics 109 145-156 January, 1985.

146 D. B. ROBERTS ET AL.

Drosophila genome and which may have features of control not found for the singleton genes cited before.

In addition to investigating the control of LSP-1 synthesis, the analysis of the LSP-1 P-region also has a bearing on the evolution of the LSP-1 genes. The evidence that the LSP-1 genes are homologous and have evolved by gene duplication is overwhelming and is shown not only by the similarity in their peptides (BROCK and ROBERTS 1980) but also by the cloned DNA of any one gene hybridizing with DNA from all three genes (SMITH et al. 1981). What is of present interest is how these genes were dispersed. Were they dispersed discretely or as part of an array? We suggested (ROBERTS and EVANS-ROBERTS 1979; ROBERTS and BROCK 1981) that duplication and dispersal may have been one and the same event mediated by some transposable element, but the discovery that in some Drosophila species LSP-1 duplicates are still adjacent to each other in the genome (BROCK and ROBERTS 1983) argues against this.

One way of studying how large a portion of the genome moved when the LSP- 1 genes were dispersed is to compare the genetic environment of the three LSP- 1 -coding sequences.

MATERIALS AND METHODS

Culture conditions: Stocks were maintained on standard yeast-cornmeal-agar medium in half-pint milk bottles at 25". Nipagin was added as an inhibitor of moulds. For cytological studies flies were reared on yeast-glucose-agar medium (10% yeast, 10% glucose and 3% agar autoclaved after mixing).

Stocks: The strain Oregon-R was used as the standard wild-type strain in this work, and it has been maintained in this laboratory for more than 10 years. The Bacup stock carrying the &chain electrophoretic variant has been described previously (ROBERTS and EVANS-ROBERTS 1979). The stocks ast ho; net dp ; ds dp ; Df(2L)al, al/Zn(2L)Cy, E(S)Cy; and shr bw abb sp/SM5 were obtained from the Stock Center at Bowling Green. The deficiencies Df(2L)S2/SMZ and Df(2L)S3/SMI were obtained from the Stock Center at the California lnstitute of Technology. The stocky; TMZ mwh ve;Dp(l:3)sc J 4 , y;Dp(l:3)scJ4 jv tra red was obtained from the Medical Research Council Laboratories in Cambridge. The stock 8(TE6Z)w; ds TE61 dp/SM5, Cy was a gift from GUNNAR ISING, University of Lund. The chromosome carrying the &null mutation was found in the y; TMI mwh ue;Dp(l:3)scJ4, y;Dp(1:3)scJ4 j u tra red stock (D. B. ROBERTS, T. JOWETT and S. A. OWEN, unpublished result). Unless stated otherwise in this paper all chromosomes used in this work carry the wild-type electrophoretic allele of LSP-10. All other stocks used in this work have been kept in this laboratory for more than 5 years. Genetic markers and chromosomes used are those described by LINDSLEY and GRELL (1 968) unless stated otherwise.

Mutagenesis: Four-day-old Oregon-R or a1 d p male flies were irradiated with a cobalt source receiving 5000 and 3000 rads, respectively, at a rate of about 1000 rads/min. The irradiated males were crossed to ast ho females (two males and three females per vial). After 5 days the flies were transferred to fresh vials, and the progeny were scored for the asteroid phenotype. These chromo- somes carrying the asteroid mutations were recovered as shown in Figure 1. Each deficiency was recovered from a separate mating.

Four-day-old net d p male flies were treated with ethyl methanesulfonate according to the protocol of Lewis and Bacher (1968). Lethal mutations in the asteroid region were recovered as shown in Figure 2. Lethals were selected as being uncovered by Df(2L)S2 or Df(2L)ast 4. Cytology: The putative deficiencies selected after irradiation with the cobalt source were examined

cytologically. The DflBalancer stock was crossed to Oregon-R and temporary propionic-orcein- carmine squash preparations of salivary gland chromosomes were prepared by standard techniques. The analyses of the deficiencies or inversions were made on the heterozygotes with the wild-type Oregon-R homologue and the breakpoints are described with reference to the revised map of BRIDGES (1 942).

UP-I @-REGION 147

/ Oregon-R $8 ostho 9 9 i

i

select D f / o s t tlies (very small eyes)

DfiZLISZ/SMl, Cy 4

Df ast- DfU LI 52 Df lZ LI S2 SM1,Cy SM1,Cy Df ast

star eyes wild type eyes dies S/ast eyes(very small) curly * curly

)c Sib mating to establish stock

FIGURE 1.-The scheme used to select for deficiencies of the ast+ gene.

dEMS 4 1

net dp ry Df/SMl,Cy

netdpX/SM1,Cy x DflZ LIS2 or DflZLIasWSM1,Cy

net dp" netdp* SMlCy Df(2LIS2 DfiZ LIS2 SM1,Cy SM1,Cy SM1,Cy

dies if lethal curly dies curly,star mutation un- covered by Df I2 L I 52 otherwise non-curly, star

*Mutagenised chromosome I f no non-curly flies the lethal mutant stock is established by crossing curly non-star flies inter se

- -

FIGURE 2.-The scheme used to select for lethal/visible mutants uncovered by Star or ast deficiencies.

Complementation: Complementation crosses were done between mutations balanced over In(2LR)SMI,Cy, the absence of non-Cy flies in more than 100 progeny being taken as the criterion for failure to complement.

Where two lethals occurred between the same two deficiency breakpoints they were crossed inter se. The absence of non-Cy flies among the progeny was again taken as the failure of these two chromosomes to complement one another.

Known recessive visible mutations in the region were crossed to all deficiencies and were mapped between deficiency breakpoints according to which deficiencies uncovered the mutations. They were then crossed to all lethal mutations in the region. Failure to complement was revealed by either the expression of the visible mutant phenotype in lethal/visible mutant heterozygotes or by the absence of non-Cy progeny.

The &coding sequence was mapped between deficiency breakpoints in the following way. All of the deficiencies were induced in chromosomes carrying the wild-type electrophoretic allele of /3. The

148 D. B. ROBERTS E T AL.

S M I , Cy stock also carries this allele. All Df/SMl stocks were crossed to the Bacup stock homozygous for the fast allele of 8. If the Dfchromosome lacks the @-coding sequence, then 50% of the progeny of this cross should have only the fast polypeptide chain; the other 50% have both. If the Dfcarries the @-coding sequence, then all progeny will have both polypeptides. Hemolymph from ten wandering larvae from each cross was analyzed (ROBERTS and EVANS-ROBERTS 1979). The failure to find a single larva with only the fast polypeptide chain was taken as a demonstration that the Dfchromosome carried the @-coding sequence.

Recombination: We used two strategies to measure recombination in this region. Recombination between a lethal mutant distal to the S/ast locus and one proximal to the locus was calculated by crossing l(a)/l(b) females X Df(2L)S2/SMl, Cy. The recombination frequency was the twice the number of non+ flies expressed as a percentage of the total number of flies eclosing. Only 50% of the nonrecombinant 1/Sml, Cy flies survive and only 25% of the recombinants (++/Of) are recog- nized.

For recombination between mutants distal to the S/ast locus dp was crossed out of the lethal chromosome and the cross net l (a) dp/net l(b) dp+ females X Df(2L)SZICy U DTS 513 was set up in small population cages. The Cy U DTS 513 chromosome carries a dominant temperature-sensitive lethal mutation which allows the stock to survive at 25" but kills the stock at 29". The eggs were harvested from the yeast-glucose-agar medium twice daily, washed well, blotted dry and weighed. Knowing the weight of a known number of eggs treated in the same way we were able to estimate the number of eggs from the weight. The eggs were placed in bottles of medium at 29", but two bottles were kept at 25" which allowed us to calculate fertility. Apart from a few escapers recognized by Cy wings, only wild-type recombinants (++/Df(2L)S2) survived at the higher temperature, which represents 25% of the recombinant progeny. In extreme cases in which there were no escapers, only one wild-type recombinant appeared in a bottle that at the lower temperature would have given more than 1000 flies. It was relatively easy to analyze large numbers of progeny from many crosses simultaneously using these techniques. The wild-type recombinants were crossed to net dp, and depending on how the original cross was set up, net d p or net dP+ progeny from this testcross establish the proximodistal order for these closely linked lethal mutants.

To map the &coding sequence we used a semilethal mutant, an allele of ds, which had been induced on a /+null chromosome. Recombinants between this mutant and 1(2)neh-5 and 1(2)neh-7 were crossed to a homozygous @-null stock, and the progeny were analyzed for the presence of 8. As shown in Figure 5 the presence of a 8' recombinant allowed us to position the 8-gene with respect to these loci.

The established map positions for the mutations used in these recombination experiments are as follows: net 2:O.O; ds 2:0.3; S 2:1.3; ast 2: 1.3 and ho 2:4.0.

RESULTS

Mutation: The LSP-1 @-coding sequence had been mapped to 2:1.9 close to the Starlast locus (ROBERTS and EVANS-ROBERTS 1979). T o make deficiencies of this region we sought mutagenized chromosomes that uncovered ast. We estab- lished seven ast deficiencies from the 9 188 irradiated chromosomes examined which together with the two extant Star deficiencies, Df(2L)S2 and Df(2L)S3, gave us a total of nine deficiencies in this region. We also established an inversion Zn(2L)ast-I and four putative S alleles (D-80; 7, 8, 11).

In four experiments involving ethyl methanesulfonate we recovered a total of 58 chromosomes (n = 4400) that carried lethals uncovered either by Df((2L)S2 or Df(2L)ast-4 (a frequency of 0.001 lethal mutants/band with the former deficiency or 0.003 lethal mutants/band with the latter). Three of these mutants were either lost or reverted to wild type, and a total of 55 newly induced ethyl methanesulfonate mutants together with point mutations or small deficiencies picked up after the irradiation are considered here.

Cytology: The putative deficiencies were all examined cytologically. The break-

up-1 @-REGION

TABLE 1

A description of the second chromosome aberrations used in this work

149

Aberrations Breakpoints Origin

Df2L)ast-1 Df2L)ast-2 Dj72L)ast-3 Dj72L)ast-4 Dj72L)ast-5 Df2L)a~t -6 Dj7ZL)ast- lo" Dj72L)S 2' Df2L)S3'

In(2L)ast-1

2 1C7-8-23A 1-2 21 D1-2-22B2-3 2 1 D1-2-2 I E1-2 2 1 D1-2-2 1 El-2 21 E1-2-21F3-22Al 21E1-2-2lE2-3 2 1 D2-3-22A1-2 2 1 C6-Dl-22A6-BI 2 1 D2-2 1 F3-22A1

2 1 El-3-centromeric heterochromatin rays

a Deduced from complementation data. LEWIS (1945).

points of the deficiencies and the inversion studied by us and of the deficiencies studied by LEWIS (1945) are given in Table 1. Photographs of most of these aberrations are given in Figure 3.

Complementation: No two deficiencies complemented each other. All 55 lethal mutations and the three Star mutants picked up after y-irradiation, the T E stocks, the Znv(2L)ast-1 stock and the @-fast stock, were crossed to all nine deficiencies. This established nine intervals between proximal and distal break- points, and all mutants were placed in one or the other of these intervals. Mutants occurring within the same interval were crossed inter se. This established 18 new lethal complementation groups designated Z(2)neh-1 - 1(2)neh-18, additional alleles of the mutants ds and S/ast and the @-complementation group. The sobriquet neh stems from the loci net and held-out(ho) which limit the region studied. The mutants and their complementation groups are given in Table 2. A diagram illustrating the deficiencies and the complementation groups is shown in Figure 4.

Mutants were recovered in all breakpoint intervals defined by cytological analysis except between the distal breakpoints of Df(2L)ast-1 and Df(2L)ast-2. Moreover, we were able to resolve breakpoints which by cytological analysis were apparently identical by lethal complementation groups that were uncovered by one deficiency and not by the other.

The chromosome TE61 is lethal due to the insertion of a transposable element which has been mapped to 2L:0.8;2 1 EF (G. ISING, personal communication), and it failed to complement some of the ast deficiencies. The lethal phenotype associated with TE61 occurs between the proximal breakpoints of Df(2L)ast-5 and Df(2L)ast-6. This TE also failed to complement 1(2)neh-10 and I(2)neh-1 1.

Recombination analysis: The interval between the distal breakpoints of Df(2L)ast-4 and Df(2L)astd is small, at most three bands, and contains the p- coding sequence in addition to four other complementation groups. This interval was obviously of the greatest interest for our study of the @-coding sequence and

150 D. B. ROBERTS ET AL.

FIGURE 3.--ast deficiencies. A, LSP-I @-gene identified by in situ hybridkition using the cloned B DNA. B. Df(2L)asf-l/Oregon-R. C, Df(2L)ast-2/0regon-R. D, Df(2L)asf-4/0regon-R. E. Df(2L)Ost- 3/0regon-R. F, Df(2L)~st-5/0regon-R. G. Df(2L)ast-6/0regon-R.

its environs. We set up crosses between lethals of the various complementation groups in this region which allowed us to order unambiguously their proximo- distal relationship. It also gave an estimate of the map distance between these mutants. In addition we set up crosses between mutants from some of the complementation groups in this interval and mutants in adjacent intervals. The relative positions of these complementation groups is given in Figure 4. The recombination data are given in Table 3.

In the first cross to map the B-gene with respect to 1(2)neh-5 (Figure 5), two of the seven recombinants survived, were fertile and both carried the &null allele. These data are two few to allow any conclusion to be drawn as to whether the &null allele is proximal or distal to 1(2)neh-5. In the second cross two of five recombinants survived and were fertile. Both carried the /3+ allele, which slows that the &coding sequence is distal to 1(2)neh-7.

LSP-I &REGION 151

TABLE 2

Complementation groups in the S/ast region and their alleles

Locus Allele Locus Allele Locus Allele Locus Allele

ds ds I (2)neh-6 J4 - I 0-80 5-K H3- 1 l(2)neh-7 24-B 7 37K L1-1 N I - 1 5.2- I 7 L

l(2)neh-1 37-L 1(2)neh-2 J4-4 l(2)neh-3 S3-I 1(2)neh-4 27-A

14-4 1(2)neh-5 11-3

s3-2 14-C 35-A 12-L

14-5 LSP- 1 8-fast

8-null l(2)neh-8 Y2-1

Y5-2 4K F

H I - 3 20-M s2-2 E- 2

ast

(1(2)neh-9 J2-6

Slast S

8 I 1

1(2)neh-l0 24-1 I(2)neh-I1 28-K 1(2)neh-12 Y5-I

27-K MI-4

24-K J3-3

1(2)neh-13 X I - 1 1(2)neh-14 25-K

J4-3 H5-2 26-K

1(2)neh-15 V L2-I L I - 2 F2 Y X3-2

l(2)neh-16 214 38-K 38L G H

1(2)neh-l7 J2-8 5%.

1(2)neh-18 X I - 3

All mutations were induced by ethyl methanesulfonate, except that the ds allele, the two LSP- 1 alleles and the first two S/ast alleles were of spontaneous origin and the last four Slast alleles were induced by y rays.

DISCUSSION

These results are similar to those reported from analyses of other small regions of the Drosophila genome (CHOVNICK, GELBART and MCCARRON 1977; WOOD- RUFF and ASHBURNER 1979a,b; WRIGHT et al. 1981; KOTARSKI, PICKETT and MACINTYRE 1983).

From a total of 4400 ethyl methanesulfonate-mutagenized chromosomes we recovered 50 lethal and five semilethal (ds) mutations that occurred within the region defined by the breakpoints of Df(2L)S2. In addition we found four new alleles of the Star locus, all of which were induced by y rays. We found no such mutations after treatment with ethyl methanesulfonate. It may be that these new mutations are all small deficiencies which are not detected cytologically nor are they detected as deficiencies by their inability to complement more than one complementation group.

These lethal and semilethal mutations have been put into 18 complementation groups, as have the visible mutants ds and S/ast and the coding sequence for LSP-lp, giving a total of 2 1 complementation groups in this region. The number of mutations in the complementation groups in this region is distributed as a Poisson distribution by the tests of BARRETT (1 980). We were, therefore, able to estimate the number of mutationally silent loci in the region as one giving a total of 22 complementation groups or loci in the region 21C6-Dl-22A6-22B1, an interval of no more than 19 bands. This is in good overall agreement with the approximate ratio of one locus to one band found in most other studies, inter alia JUDD, SHEN and KAUFMAN (1 972), HILLIKER, CLARK and CHOVNICK (1 980), WOODRUFF and ASHBURNER (1 979a), HOCHMAN (1 97 l) , WRIGHT et al. (1 98 1)

152 D. B. ROBERTS ET AL.

FIGURE 4.-Diagrammatic representation of the distal part of the left arm of chromosome 2 showing the deficiency breakpoints; the positions of the Complementation groups vis d vis the breakpoints; and the recombination frequency in percentage X lo-’ between the complementation groups. Where two or more complementation groups occur between the same two breakpoints their order is arbitrary unless (1) recombinants have been recovered between mutants in the two groups, in which case the proximodistal relationship is known, or (2) both have been crossed to other complementation groups, in which case their order is the best order for the recombination frequencies available.

and KOTARSKI, PICKERT and MACINTYRE (1983). However, in the interval between the distal breakpoints of Df(2L)ast-4 and Df(2L)ast-6 we find at least four complementation groups in a two-band region, and between the distal breakpoints of Df(2L)ast-3 and Df(2L)ast-4 which cytologically are indistinguish- able we find two complementation groups.

The lethal TE61 that had been mapped to this region was analyzed. TE61 caused a mutation that failed to complement both l(2)neh-IO and 1(2)neh-ll, two lethal mutations which complement each other. This result can be explained either by assuming that both of these lethal mutants are mutations of the same gene with intracistronic complementation or that the TE61 is inserted between the two genes such that it affects both. This result places the TE cytologically between 21E2-3 and 21F3, in good agreement with the observed position of 2 1 EF reported by G. ISING (personal communication).

The fine structure mapping gives some indication of the organization of this

UP- 1 &REGION

TABLE 3

Recombination data for crosses between mutants in the S/ast regzon

153

Cross

ds x ast ds X 1(2)neh-7 ds x I(2)neh-5 1(2)neh-2 X I(2)neh-7 l(2)neh-3 X l(2)neh-7 l(2)neh-4 X 1(2)neh-7 1(2)neh-4 X 1(2)neh-6 1(2)neh-5 X 1(2)neh-7 1(2)neh-5 X 1(2)neh-6 1(2)neh-6 X l(2)neh-7 1(2)neh-7 X I(2)neh-lO 1(2)neh-7 X 1(2)neh-9 1(2)neh-7 X ast l(2)neh-7 X I(2)neh-8 l(2)neh-8 X l(2)neh-lO l(2)neh-8 X I(2)neh-9

Recombi- nants"

140 40 28 8 16 64 8

124 24 24 72 20 98 40 20 24

Total

12,200 14,000 13,500 7,600 18,500 73,000 39,000 193,000 173,000 86,000 1 1,000 5,700 42,000 13,000 4,000 5,000

Map distance (%)

1148 x 10-3 285 x 10-3 207 x 10-3 io5 x 10-3

21 x 10-3 62 x 10-3 14 x 10-3 28 x 10-3

655 x 10-3 350 x 10-3

307 x 10-3

480 x 10-3

86 X lo-' 88 X lo-'

230 X lo-'

476 X lo-'

"These represent the total number of recombinants, that is, the observed number of recombinants adjusted by the factor that takes into account the double mutant/Df and the ++/DTS recom- binants that do not survive.

ds Bo + Df A DTS

+ I + H m l cross over in I survivor :- t b o + D f ie. po cross over in 11 survivor:- t t t I Df ie ,B'

t m + I 11

cross over in I survivor :- + + B o / D f ie Bo no survivor i f cross over in I1

FIGURE 5.-The scheme used to map the @-gene. m is either 1(2)neh-5 or 1(2)neh-7.

region. LEFEVRE (1 976) has estimated the crossover frequency per band for the whole genome to be 0.057%. JUDD, SHEN and KAUFMAN (1972) gave a similar value of 0.054% for the z-w region, and WOODRUFF and ASHBURNER (1979a) give an estimate of 0.07% for the Adh region. The most detailed fine structure genetic analysis in Drosophila is of the rosy region (CHOVNICK, GELBART and MCCARRON 1977). r j has been placed cytologically at 8 7 D 8 - 1 2 (LEFEVRE 1971) and karmoisin at 87C3-C7 (ISH-HOROWICZ, HOLDEN and GEHRINC 1977), which

154 D. B. ROBERTS ET AL.

gives a minimum of 14 bands and a maximum of 19 between Kar and ry. The frequency of crossing over between these two loci is 0.35%, giving a minimum of 0.018%/band and a maximum of 0.025%.

The mutants ds and S have been mapped cytologically to 2 1 D1-2 and 21E1-2, respectively (LEWIS 1945), and to 21D2 and 21E1-2 (this paper), making ds and S a maximum of five and a minimum of four bands apart. The crossover frequency between ds and ast is 1.1 % (this paper) and 1 .O% (LEWIS 1945), which gives a frequency of between 0.2 and 0.25/band. This is some five times greater than for the genome as a whole or for the Adh region or particularly the z-w region, which is about the same distance from the tip of chromosome 1 as this region is from the tip of the left arm of chromosome 2. It is some ten times greater, however, than the frequency per band for the kar-ry region. There is no obvious reason for this considerable difference but, if the elevated level of crossing over extends evenly throughout this region, then the genetic distance between physical points is five to ten times greater here than elsewhere.

The cytogenetic placing of the ,&coding sequence in bands 2 1 D3-4 is in good agreement with its position from in situ hybridization with the &probe (SMITH et

One of the prime reasons for a detailed study of this region was to investigate the LSP-1 P-coding sequence and the genes surrounding it: first, as a prelude to an investigation of the homologous LSP- 1 -coding sequences on other chromo- somes and their immediate surroundings in order to learn something of the events that took place during the evolution of these genes in D. melanogaster and, second, as a detailed study of a small part of the Drosophila genome.

We have, by rudimentary genetic analysis, placed the @-coding sequence distal to 1(2)neh-7 and we have shown that the &polypeptide is not essential for normal development under laboratory conditions as witnessed by the fact that @-null homozygotes and P-nullldeficiency heterozygotes survive. It may be that, al- though LSP-I is essential, the a and polypeptides can substitute for p. We have a y-null chromosome and ,&null y-null homozygous flies survive, so the amount of LSP-I required for survival under laboratory conditions is small. We have recently found an a-null chromosome, and although a-null, &null, y-null flies survive, they show a complex phenotype which is still being studied.

al. 1981).

We wish to thank JEAN MATTHEWS for feeding our flies and WINIFRED EDMONDS for looking after their general welfare. MICHAEL AKAM, MICHAEL ASHBURNER and TREVOR JOWETT were kind enough to comment on various drafts of this paper and so to them we express our thanks. The work was supported by a grant from the Science and Engineering Research Council to D. B. R. H. W. B. was a recipient of a Commonwealth Scholarship.

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Communicating editor: A. CHOVNICK