Drosophila melanogaster

The region of the second chromosome of Drosophila melanogaster defined by DJ(2R)vgB was screened for recessive lethal and visible mutations. Fifty-eight new recessive alleles fall into 17 complementation groups. Many new vg alleles were also isolated in a screen for new ng deficiencies. The breakpoints of the new ug deficiencies were nonrandomly distributed. The distal breakpoints of twelve of 20 deficiencies overlapping Df(2R)vgB are genetically identical to that of DJ(PR)ngD, coinciding with the position of a complex, pleiotropic locus, 1(2)49Ea-Psc-Su(r)2. T HE merger of molecular and genetic analyses in recent years has permitted, in a few chromosomal regions, a very detailed analysis of the organization of genes on chromosomes and the relationships between genes and their nearest neighbors. Although maintenance of the wild-type linkage order may not, in general, be essential (HILLIKER AND TRUSIS-COULTER 1987), such studies reveal in some cases a more complicated genetic organization than a simple linear arrangement of unrelated genes. The most striking example of functionally related genes clustered together may be the region constituting the Antennapediu complex, in which genes specifying morphological identities along the anterior-posterior axis are arranged in a proximal-to-distal pattern (KAUFMAN, 1986). The Ubx, abd-A, and Abd-B loci are arranged similarly within the Bithorux complex (KARCH et al. 1985). However, examples of linkage of functionally related genes are not limited to homeotic genes. For instance, genes specifying tropomyosin and a flightmuscle specific actin are closely associated at 88F (KARLIK et al. 1984). In other cases, apparently unrelated genes overlap. HENIKOFF et al . (1986) described a pupal cuticle protein gene which is located completely within an intron of the Gart gene (which encodes three purine pathway enzymatic activities). The two nested genes use opposite DNA strands as coding strands. CHIA et ul. (1 985) report the location of osp(outspread) inversion breakpoints to both sides of the Adh gene, suggesting perhaps a similar organization. We are engaged in the study of a single-copy gene encoding a 20-kD muscle protein, named mp-20 (LASKO AND PARDUE 1985, LASKO 1986). Cytological LEWIS AND WAKIMOTO 1980; GEHRING AND HIROMI I Present address: Department of Genetics, University of Cambridge, Downing Street, Cambridge, England CB2 3EH. Genetics 1 2 0 495-502 (October. 1988) localization of the cloned gene indicates that it lies within the region deleted by Df(2R)vgB (polytene bands 49D3.4-50A1.2) (MORGAN, BRIDGES AND SCHULTZ 1938). As an initial step in analyzing the chromosomal context of this muscle-specific gene we have isolated recessive lethal and visible mutations within the chromosomal region defined by this deficiency. We have also isolated a series of new vg deficiencies to subdivide the region. Aside from the muscle gene, the chromosomat region defined by Df(2R)vgB contains numerous interesting loci. Included among these is the vestigial gene, a locus whose mutation results in the reduction of wings and halteres and whose many alleles differ widely in degree of expressivity (LINDSLEY AND GRELL 1968). Mutations at Aristapedioid (Arp; ADLER 1984) and Posterior sex combs (Psc; NUSSLEIN-VOLHARD, WIESCHAUS AND KLUDINC 1984), also located in this region, cause homeotic transformations of adult organs: from aristae to legs in the case of Arp, and from posterior legs to anterior legs in the case of Psc. A dominant suppressor locus, Suppressor of zeste-2 (Su(z)2), is linked to, and interacts with, Psc (C.-T. Wu, R. S. JONES, P. F. LASKO AND W . M. GELBART, unpublished data). The maternal-effect polarity gene, bicaudal, is also believed to lie within Dfi2R)vgB (BULL 1966; NUSSLEIN-VOLHARD 1977). This paper describes new recessive lethal and visible mutations which we isolated in this region, as well as their relationships with each other and with previously known genes. MATERIALS AND METHODS Culture of Drosophila: Flies were grown on standard cornmeal-molasses-yeast medium supplemented with live yeast. Growth was at 21 O , unless otherwise noted. Screen for recessive lethal mutations: All balancers and markers not described herein are included in LINDSLEY AND 496 P. F. Lasko and M . L. Pardue GRELL (1968) or LINDSLEY AND ZIMM 1986). a1 b c sp males were mutagenized with ethyl methanesulfonate (0.0 125 M) by the method of LEWIS AND BACHER (1968), or with y-rays (4000 R) from a 6oCo source. These mutagenized males were then mated en masse to SMS/Sco virgin females. F, SMS/al b c sp males were then individually crossed to 2-3 SMSIDf(2R)ugB virgin females. The presence of a recessive lethal mutation on the mutagenized a1 b c sp chromosome was indicated by the absence of the Df(ZR)ugB/al b c sp (straight-winged) class among the FP flies. Hybrid dysgenic screen for recessive lethal mutations: Pi-2 males were mated en masse to a1 b c sp ( M ) females. F1 Pi-P/al b c sp females (P/M) were then crossed to SMS/Sco (P) males. This SMS/Sco (P) stock was constructed by five generations of backcrossing of appropriate males to Pi-2 females. Individual FP males, of either the SM5/Pi-2 or SM5/ a1 b c sp genotypes, were then crossed to SMS/Df(ZR)ugB (P) females (constructed as for SMS/Sco (P)) , in order to isolate individual mutagenized chromosomes. As before, recessive lethals were scored by the absence of straight-winged flies among the progeny. Screen for new vg deficiencies: Canton-S or Oregon-R wild-type males were mutagenized with 4000 R of gammarays from a 60Co source, and mated en masse with a1 ug virgin females. F1 progeny flies of d u g phenotype carry a new ug mutation. To determine if these new ug alleles were in fact recessive lethals, as would be expected for deficiencies, the al+ug flies were crossed individually to SMS/al Df(2R)ugB. If the original mutation were isolated in a male, the absence of the al+ug class indicated that the newly induced ug allele included a recessive lethality within the bounds of the ugB deficiency. A stock would then be made from the SMS/al+ progeny flies. If the original mutation were isolated in a female, a1 could not be used to sort the parental chromosomes, as recombination would often separate it from ug. Individual SM5/al male progeny were then crossed to SMS/DJZR)ugB females to be scored for recessive lethality. A stock was made from the SMS/al flies from one of these final crosses, if lethality were observed. Complementation testing: One to three males and two or three females of the balanced lethal or deficiency stocks were mated, and the cross was incubated at 25". The parentaI flies were subtracted from the resulting F1 progeny data. Failure to complement was concluded if no Cy' flies were found in at least 50 progeny. Determination of embryonic lethality: Five males, heterozygous for a recessive lethal allele and for the Canton-S wild-type chromosome, were crossed to ten Df(ZR)ugB/+ females in a small shell vial containing standard cornmealmolasses-yeast food. After 2 days at 25", the females were transferred to an empty half-pint milk bottle. The bottles were each covered with a 15 X 60 mm Petri dish containing grape agar (ELCIN AND MILLER 1978) sprinkled with live yeast. The bottles were inverted, and embryos were collected for 24 hr on the grape plate. The plates were incubated for another 30 hr, at which time the egg cases, embryos, and larvae were washed onto a 116 mesh Nitex screen. The egg cases and unhatched embryos were then counted. RESULTS AND DISCUSSION Data from recessive lethal screens: Over 12,000 mutagenized chromosomes were examined for new recessive lethal alleles within the bounds of Dj(2R)vgB. Dj(2R)vgB is large, lacking approximately 28 polytene chromosome bands, and normal development ceases in homozygous embryos about 10 hr after fertilization at 25" (BULL 1952, 1956). 8788 chromosomes were screened after EMS mutagenesis, 2473 after 7-ray mutagenesis, and 1527 after mutagenesis by hybrid dysgenesis. Many of the mutagenized males were rendered sterile; 38.8% of the EMS-treated males, 28.7% of the y-ray-treated males, and 22.8% of the males from dysgenic crosses produced no F1 progeny. A total of 71 recessive alleles were isolated: 58 from the EMS screen, nine from the y-ray screen, and four from the hybrid dysgenic screen. Thus the frequencies of isolation of mutations were 0.66% for EMS, 0.36% for y-rays, and 0.27% for hybrid dysgenesis. Data from deficiency screens: Approximately 425,000 wild-type chromosomes were screened for the induction of new vg mutations; 399,000 from Canton-S and 26,000 from Oregon-R flies. From these, 126 new vg alleles were isolated (a frequency of 0.030%). Of these 69 were sterile, 29 were recessive viable and vestigial, three were dominant vestigial, nine were recessive lethal but complemented both of the lethal complementation groups adjacent to vg, and 16 were recessive lethal and failed to complement one or both of the adjacent lethal complementation groups. These deletions were then used to map the other complementation groups. Mapping: The results from the inter se complementation crosses and deficiency mapping experiments are illustrated in Figure 1. The 58 lethal alleles fall within 18 complementation groups, the vg alleles comprise a nineteenth. The complementation groups are described in Table 1, while the deficiencies used for the mapping are listed in Table 2. The extensive crosses performed generate the map shown in Figure 1. Most of the complementation results were simple, as has been the general rule in such screens (HOCHMAN 197 1, JUDD, SHEN AND KAUFMAN 1972; GVOZDEV et aZ. 1975; WOODRUFF AND ASHBURNER 1979; HILLIKER et al. 1980; GAUSZ et al. 1981; WRIGHT et al. MACINTYRE 1983; NICKLAS AND CLINE 1983; MOHLER AND PARDUE 1984; ROBERTS et al. 1985; CROSBY AND MEYEROWITZ 1986; ASHBURNER et al. 1988). In most crosses, no or a very few non-Curly flies were recovered when flies of a given complementation group were crossed with all other alleles of the same complementation group. The few exceptions are discussed below. 49Ea1, 49Ea4, 49Ea4: C-T. W U , R. S. JONES, P. F. LASKO AND W . M. GELBART (unpublished data) have shown that 49Ea makes up part of a complex pleiotropic locus, as 49Ea" shows allelic interactions with Psc and with three alleles of Su(z)2. The other 491% alleles are assigned to the same complementation group on the basis of their reduced viability when heterozygous with each other and/or 49Ea'. They all 198 1 ; ZHIMULEV et d . 198 1 ; KOTARSKI, PICKERT AND Mutations of vg Region 497