Genetic and Phenotypic Analysis of the Genes of the Elbow-no-Ocelli Region of Chromosome 2L of Dvosophila Melanogaster

Hereditas 126: 67-75 (1997)

Genetic and phenotypic analysis of the genes of the elbow-no-ocelli region of chromosome 2L of Dvosophila melanogastev TERENCE DAVIS’, MICHAEL ASHBURNER2, GLYNNIS JOHNSON2, DAVID GUBB’ and JOHN ROOTE’ ’ Medical and Community Genetics, Kennedy Galton Centre Level SV, Northwick Park and St Mary’s NHS Trust, Harrow HA1 3UJ, UK

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK

Davis, T., Ashburner, M., Johnson, G., Gubb, D. and Roote, J. 1997. Genetic and phenotypic analysis of the genes of the elbow-no-ocelli region of chromosome 2L of Drosophila melanogaster. - Hereditas 126: 61-75. Lund, Sweden. ISSN 0018-0661. Received November 22, 1996. Accepted January 15, 1997

The elbow locus is found to be two genes elA and elB, each of which has a distinct phenotype when mutant. Mutations of the elA gene have a strong phenotype where the wing is markedly disrupted. Mutations of elB are weak, mainly affecting the alula and the wing bristles. The two genes are dominant enhancers of each other. Homozygous deletion of the complete elbow region results in lethality. Situated between the elbow genes is the pupal gene and a locus which when deleted causes a crippled leg phenotype. This locus may be a control region for elbow. Immediately adjacent on the proximal side of elA is the no-ocelli locus. The phenotypes of noc alleles vary from extreme, where the ocelli and associated bristles are absent, to weak where these structures are disrupted. The various noc phenotypes are associated with genetically distinct gene regions, mutations of which act as enhancers of each other. Alleles of el and noc show partial failure of complementation, heterozygotes having weak el or weak noc phenotypes. Alleles of both these genes interact with the antimorphic noc allele Sco.

Terence Davis, Medical and Community Genetics, Kennedy Galton Centre Level 8V, Northwick Park and St Mary’s N H S Trust, Harrow HA1 3UJ, UK

The chromosomal region surrounding the structural gene for alcohol dehydrogenase (Adh) has been described in several studies (WOODRUFF and ASH- BURNER 1979a; ASHBURNER et al. 1982a, b) and includes the genetic loci pupal bu) , elbow (el) and no -ocelli (noc), all separable by aberration breakpoints.

The el locus was originally discovered by E. M. WALLACE in 1935 (BRIDGES and BREHME 1944) as a spontaneous mutation, el ’. Phenotypically, elbow flies have small bent wings due to the absence or reduced size of the posterior compartment. The alulae are reduced in size with a reduced number of marginal bristles, and are often fused with the wing blade. When heterozygous with deletions of the el region, el alleles vary in their phenotype from very strong to almost wild-type, but the el alleles behave as if they fall into the same complementation group.

The noc locus was discovered by WOODRUFF and ASHBURNER (1979b) when it was noticed that certain trans-heterozygotes for deletions in the Adh region lacked the ocelli, the light sensitive organs on the back of the head of the adult fly. The noc phenotype of alleles associated with aberrations in the noc region varies considerably in strength; weak alleles have disrupted ocelli and associated bristles and in strong alleles these structures are absent. Present address: ’ CCRT, University of Honolulu at Manoa, 3050 Maile Way, Gilmore 408, Honolulu, HI 96822, USA

The pu gene was first described in 1925 (MORGAN et al. 1925). In pupal flies the wings do not expand after eclosion of the adult. The five known pu alleles all fall into a single simple complementation group (ASHBURNER, unpublished data).

These three genetic complementation groups can all be defined by chromosomal aberrations and point mutations. Some el and noc alleles, however, show partial failure of complementation. Flies heterozygous for some el and noc alleles frequently have phenotypes characteristic of either or both of these loci. Some noc alleles have el and noc phenotypes when heterozygous with an el- noc- deletion. The el and noc genes also interact with the antimorphic noc allele Scutoid (Sco), an X-ray induced mutation (KRIVSHENKO 1959) that is associated with a recipro- cal transposition between the noc and sna chromosomal regions (MCGILL et al. 1988). The Sco phenotype is a loss of the head and thoracic macrochetae from the adult fly, reducing their number from 40 to about 26 (ASHBURNER et al. 1982a). Many el and noc alleles enhance the Sco phenotype when in trans.

The el-noc region has been cloned in its entirety (DAVIS et al. 1990; CHIA et al. 1985). In this paper we present a detailed description of the el and noc phenotypes and the genetic and molecular organisation of this region and discuss the interaction between these genes and with Sco.

Hereditas 126 (1 997) 68 T. Davis et al.

Table 1. The recovery of new alleles of elbow

Mutagen vs. No. chromosomes No. new alleles

“points” abbs Dfs

y-rays e13 36,448 1“ 0 2b

EMS e13 144,249 0 1 (In)‘ lg EMS el ’ 50,920 6h 1 (T)’ 1’

y-rays el ’ 68,120 5“ 1 (In)d 6‘

b Adh‘f3 cn bw males were mutagenised and then crossed either to b el’rd” pr cn or b el’ homozygous females or to el3 pr/CyO females. New mutations were selected as phenotypically elbow flies in the F,. New aberrations are described in Table 2 a e129

e180i1, e181il el8, ell0, el”, el1’, ell3 In(2L)e19 e114, e115, e116, ell 7, ell& el28 In(2LR)e16

g e182f 1 el”, el”, el”, el26, elz7

MATERIALS AND METHODS

Stocks. - The chromosome aberrations used are described in DAVIS et al. (1990), GUBB et al. (1990), CHIA et al. (1985), and LINDSLEY and ZIMM (1992), with the exception of those recovered in this study (Table 2).

Crosses. - Routine crosses were done in vials on standard cornmeal/agar Drosophila media with incu- bation of cultures at 25°C.

Mutagenesis. -For EMS mutagenesis 3-day old males were starved for about 12 h and then fed a 25 mM solution of EMS in 1% sucrose overnight before mating en masse to appropriate females. The males were discarded after 5-6 days, so as to avoid the recovery of clusters of identical mutations from pre- meiotic germ-cell stages. For irradiation 3-day old males were irradiated, with 4-4.5 kR from either a 6oCo y-ray source or from an X-ray machine at about 150 R/min (160 kV, 14 mA, lmm Al+ Imm Cu filtration).

Cytology. - Polytene chromosomes were examined in temporary propionic acid-orcein-carmine stained preparations and interpreted with the revised maps of C. B. and P. N. BRIDGES (see LEFEVRE 1976).

Scoring of phenotypes. - The elbow phenotypes were examined after mounting wings in Canada balsam. The bristles used to score the phenotypes of Sco heterozygotes are listed in ASHBURNER et al. (1982a). Unless indicated otherwise, the mean (+ / - standard error) is given of counts of ten flies of each sex. A

JEOL 35 Scanning Electron Microscope (Department of Zoology, University of Cambridge) at 15kV was used to examine the ocellar phenotypes.

Nomenclature. -We have revised the names of the TE146 and TE36 derivatives to be in agreement with those used in LINDSLEY and ZIMM (1992) and Fly- Base.

RESULTS

The molecular breakpoints of the aberrations in the el-noc region discussed in this paper and the genes in this region are summarised in Fig. 2 and 4.

Screens for new elbow alleles

Four alleles of elbow existed before we made the screens for this study: the spontaneous el’ (DAVIS et al. 1990), the el2 and the temperature sensitive el3, EMS induced “point” alleles of G. MARONI (unpublished) and (WOODRUFF and ASHBURNER 1979b), respectively, and el4, an EMS induced T(Y;2) (ASH- BURNER et al. 1982b). New alleles were selected after both EMS and y-ray mutagenesis against the weak allele el3 and the strong allele el‘. New el alleles were relatively rare, the best recovery after EMS mutagenesis was less than 1 in 8000 chromosomes (Table 1). In addition to new mutations and the four listed above, two independent chromosome aberrations were found to have el mutations, T(Y;2)SD-a15 of T. LYTTLE (el5) and T(2;3)shv19 of W . M. GELBART (el?. The el mutations associated with aberrations are given in Table 2.

Hereditas 126 (1997) Genetics of the el-noc region 69

Table 2. Description of el aberrations

Allele Cytology Extenta Mutagen

T(Y;2)e14 Y;35B1 elB- EMS T(Y;2)a15, els Y;35A4-35B1 elB- y-rays T(2;3)shv19, el 22F1.2;35A1-4;97A elB- y-rays T(2;3)el 24 35B1.2;93C3-7 elA - EMS In (2L)eI' 34A2.3;35 A3,5 elB- y-rays In(2LR)eP 35B1-3;57C3-9 elB- EMS el n. v. (25kb deletion) elA - spont. Df(2L)ell4 n. v. (> 90kb deletion) elA - - nocc y-rays Df(2L)ell5 35B1.2;35C5 1(2)34F~- - 1(2)35Cb- y-rays Df(2L)ell6 n. v. (>90kb deletion) elA- - nocc y-rays Df(2L)ell7 34F1.2;35A4 wb- - Adh- y-rays Df(2L)el18 In(2L)35B;36C5 wb- - twec y-rays D f (2L)e120 34F3;35C5 wb- - 1(2)35Da- EMS Df(2L)e128 35B3;35D4 elA - - 1(2)35Eac y-rays

n. v., not cytologically visible. spont, spontaneous a Genetically determined limits

The elbow phenotype

The typical phenotype of el mutations is a reduction of the wing and halteres. In flies with a strong elbow phenotype, the posterior of the wing is considerably reduced, the alulae are missing, and the wing is bent backwards. Different alleles vary considerably in their expressivity when homozygous or hemizygous over an el- deletion (estimates of expressivity were made for mutations heterozygous with Df(2L)A47). Fig. 1A shows the phenotype of a very weak allele, el'. Very weak alleles are almost wild-type, their most obvious phenotypic manifestation is a reduction in the number of marginal bristles on the alulae and along the posterior wing margin and, sometimes, a shortening of wing vein L5. In some weak alleles the alulae are missing and the posterior crossvein is disrupted. Moderate alleles have a reduced wing and, especially, reduced alulae (e.g., e14 Fig. 1B). The strong el alleles (e.g. el24, Fig. 1C) show a marked reduction in wing size, especially in the posterior wing, shortened L5, and reduced halteres. The most extreme allele, el' (Fig. ID), has very disrupted wings. Both the posterior and anterior wings are severely reduced, wing vein L4 is shortened, and L5 and the alulae are absent. The submarginal cell (between L2 and L3), however, appears to be relatively normal. In some wings the costal cell is extended and the L1 (marginal) vein reaches the margin much more distally than normal. The triple row is reduced in the dorsal rows but not the ventral row (J. F. DE CELIS, personal commun.). Only three alleles can be classed as phenotypically strong, el', ell3 and el". Of these, el' is a small (circa 25-kb) deletion (DAVIS et al. 1990) and el" is a T(2;3). However, not all cytologically aberrant alleles are strong, el', eZ7 and eZ9 are

weak in phenotype (Fig. lA), and el4 and e16 are both moderate. (Fig. 1B). Heterozygotes between any two weak alleles are wild-type or nearly so, and heterozygotes between strong and weak alleles are usually weak in phenotype.

DAVIS et al. (1990) have shown that the chromosomally aberrant el alleles map to two loci, a proximal eIA and a distal eZB, by the molecular location of their breakpoints. These two groups are separated by sequences which when broken have no effect on the elbow phenotype. The strong el' and el24 alleles map to elA and the five weak and moderate el alleles map to elB.

The pu gene is located between the two el loci close to eIB as it is distal to the pu+ deletion Df'2L)ell4 and the elB- translocations (see Fig. 2). In pupal flies, the wings do not expand after eclosion of the adult. The five known pu alleles all fall into a single simple complementation group (ASHBURNER, unpublished data).

Homozygous deletion of the elbow-pupal region is very deleterious; the great majority of flies die as pharate adults (Table 3). Rare escapers show the pupal phenotype and have crippled legs and reduced halteres. For example, the heterozygote Df(2L)A400/ Df(ZL)b83d29a is homozygously deleted for - 60-kb of the elB-pu region (Fig. 2). The survival rate is only about 20% of expected (Table 3). This is not due to the pupal gene alone, since Df(2L)e114/ Df(2L)b83d29a is also relatively inviable. These flies not only have a weak elbow phenotype but also have crippled legs. Df(2L)ell6/Df(2L)b83d29a heterozygotes are similarly of low viability but are almost wild-type. These data might suggest that homozygous deletion of the - 190 to - 200-kb region results in the crippled leg phenotype. However, the

I0 T. Davis et a/. Hereditas 126 ( 1 997)

Fig. 1A-D. Illustrating the variation in expression of mutations at the elbow loci. All alleles were heterozygous with Df(2LA47. A very weak phenotype, el’; B moderate phenotype, el4; C and D strong phenotypes, elz4 and el’.

data with the more proximal deletion, Df(2L)b84a2, suggest that the situation is more complex, since deletion of the elA region enhances these phenotypes (Table 3). The heterozygotes with Df(2L)b8&2 give large numbers of pharate adults which, upon removal from the pupal cases, were seen to have crippled legs. It is interesting that complete deletion of elA heterozygous with complete deletion of elB is wild-type (e.g ., Df(2L)el ‘lDf(2L)b 83d29a and Df(2L)TE35BC- 8lDf(2L)b83d29a). The data suggest the possibility of a new locus responsible for the crippled leg phenotype between the breaks of Df(2L)el14 and Df(ZL)TE35BC-8 (Fig. 2). Alternatively, this region could be necessary for the correct expression of the el genes and/or the pu gene.

The noc locus

The noc locus was discovered by WOODRUFF and ASHBURNER (1979a) when it was noticed that certain trans-heterozygotes for deletions in the Adh region of chromosome 2L lacked the ocelli, the light sensitive organs on the back of the head of the adult fly. Further work showed that this phenotype could be assigned to the region between the genes outspread and elbow, and several alleles of noc have been ob- tained. The noc locus has two distinct phenotypes when mutant, an embryonic and larval lethal (CHEAH et al. 1994) and an adult ocellar phenotype. The

ocellar phenotype of the noc alleles varies from very strong where the ocelli are totally absent to weak alleles where the ocelli are merely disrupted. Fig. 3 shows scanning electron micrographs of the heads of flies heterozygous for noc alleles and the deletion Df(2L)b8lal, which deletes most of the DNA in the noc region. The wild-type fly is shown in Fig. 3A. Weak phenotypes are characterised by the disruption of the post-vertical (PVt) and the ocellar (Oc) macrochetae, the intra-ocellar microchetae (10), and a reduction in size of the lateral ocelli (LO) (the allele In(2L)noc2, Fig. 3B). The medial ocellus (MO) seems to be normal. In moderate phenotypes, the LO are absent with only the ocellar pits remaining and the MO is reduced in size (the allele Zn(2LR)noc7, Fig. 3C). The PVt and Oc macrochetae are reduced or absent, and the microchetae are misplaced. In strong phenotypes (e.g., homozygous In(2LR)noc4 and nocTE35B, Fig. 3D) the head of the fly is bald, the ocelli and the chetae being absent. The strong ocellar phenotype is unlike that of any other mutation affecting the ocellar region that we have examined by electron microscopy (sca, oc, emcD, fu , so ‘, so”, rdo, Ce2, and Oce). The phenotypes of trans-heterozygotes between weak and moderate, weak and strong, or strong and moderate noc alleles, are weak, similar to that of In(2L)nocz. Heterozygotes between lethal and visible noc “point” alleles usually have a

Hereditas 126 (1997) Genetics of the el-noc region 71

4 distal

-240 -220 -200 -180 -160 -140 -120 -100 kb

I elB elA noc

PU

1 - b84a2 - Ts( Y;2Lt)GT2+ Ts(YSt;2Rt)A8O - - Ts(Y;2Lt)els+Ts(YSt;2Rt)A80

I + TE35B-7 4 - b83d29a

* A400 - D Ts(Y;ZLt)el4+Ts(YLt;2Rt)RI5 D TE35B-12 + el14 - D el16 W f r t 2 D TE35BC-8 - el l

I b el28 - b TE35B-I - el4 m el5 - el7 - el9

el6

- A80

D TE35B-6 -fn3

=-* n7813 A446

-A178 -fn27 - el24

Fig. 2. A molecular map of the el region showing the genes and the aberrations mentioned in this study. Distal is towards the telomere. The co-ordinates are from DAVIS et al. (1990). The solid boxes are the maximum limits of the genes and the stippled box the area which when deleted gives the crippled leg phenotype. The solid lines show the areas of breakage of the aberrations and the thin lines show the extent and/or direction of the deletions.

weak noc phenotype as do the rare escapers of lethal noc trans-heterozygotes. The phenotype of noc is enhanced in heterozygous combinations with deletions.

Interactions of el alleles with noc

Some lethal noc alleles are weak and escape when heterozygous with a deletion of noc. The escapers have a weak noc phenotype. If the deletion also includes el the escapers have a weak el phenotype (ASHBURNER et al. 1982b). One allele of el, the E M S induced el2 of MARONI, is phenotypically weak but is semi-lethal when heterozygous with el+ noc- dele- tions (Table 4) and with all lethal alleles of noc

(ASHBURNER et al. 1982b). This lethality is enhanced by deletion of the el region. All other el alleles are viable when heterozygous with lethal alleles of noc (unpublished data).

Effects of el and noc alleles on the Sco phenotype

The curious behaviour of the Scuroid (Sco) mutation was discussed by ASHBURNER et al. (1982b, 1983). This unique antimorphic mutation of noc cannot be mapped to a single genetic interval by using noc deletions. The Sco phenotype is the loss of the head and thoracic macrochetae from the adult fly (ASH- BURNER et al. 1982b). In a wild-type fly, there are 40 of these. In Sco/+ flies this number is reduced to

72 T. Davis et al. Hereditas 126 ( I 997)

Table 3. Complementation of Df(2L)b83d29a (elB- p u - ) and Df(2L)b8&2 (elB- pu- eU -) with other deletions in the el region

Deletion Df(2Lb83d29a Df(2L)b84a2

Yo phenotype Yo phenotype

Ts( Y;2Lt)G T2 + Ts( YSt;2R t)A 80 2.9 el, crle, ph 8.9 crle, ph Ts(Y;2Lt)e15 4- Ts(YSt;2Rt)A80 5.4 crle, ph 6.6 crle, ph Df (2L)A 400 6.5 el, crle, pu 0 crle, ph Ts(Y;2Lt)e14 + Ts(YLt;2Rt)Rl5 0 crle, ph 0 crle, ph Df(2L)TE35B- 12 0.5 el, crle, ph 0 early lethal Df(2L)ell4 9.5 crle 0 crle, ph Df(2L)ell6 4.0 weak crle 0.3 el, crle, ph

29.6 weak crle 0.2 el, crle, ph early lethal

D f 2 L W Df(2L)TE35BC- 8 15.7 wild-type 0 el ' 45.6 wild-type 41.6 el Df(2L)el28 25.6 weak el 10.8 el Df(2L)TE35B- 1 36.6 wild-type 0 early lethal Df(2L)TE35B-6 31.4 weak el 15.1 el

The molecular breakpoints of these deletions are shown in Fig. 2. The percentage of non-balancer progeny over total progeny is given. All chromosomes balanced over CyO except el', which was homozygous. (Total numbers of flies scored varied between 169 and 1538.) Phenotypes: crle = crippled legs; pu = pupal; el = elbow; ph = pharate adults; se = semi- eclosed adults

25-28. Some alleles of el act as dominant enhancers of the Sco phenotype; when heterozygous with the Sco chromosome they reduce the number of chetae by between five and 15 per fly (Table 5). Of the el alleles which are associated with aberrations, the five elB alleles (el4, el', el6, el7 and el4 are enhancers of Sco, whereas neither el' or el", which map to elA, have this effect. The noc alleles that are associated

with aberrations are all strong enhancers of the Sco phenotype (Table 6) .

DISCUSSION

The el locus has been shown to consist of two genetically separable genes, elA and elB, both by the severity of the phenotype of the individual alleles, and by the molecular breakpoints of these alleles (DAVIS et al. 1990). The proximal gene, elA, has a strong phenotype when mutant, and the distal elB a weak phenotype. The two genes show failure of complementation with heterozygotes between weak and strong alleles, giving weak el phenotypes. The pu gene is found to be between the two el genes close to elB, as it is distal to the deletion Df(2L)e114 and proximal to the chromosomally aberrant elB alleles, with the exception of e f6 (Fig. 2). The two el genes can be physically separated by aberrations without causing any visible phenotype. Complete deletion of the el-pu region (e.g., Df(2L)A400/Df(2L)b8&2) causes lethality, though whether this is due to the el genes or a combined effect of deleting the el genes and the pu gene is unknown. As deletion of elA heterozygous with a deletion of the entire region (e.g., Df(2L)TE35BC-g/Df(2L)b842 or Df(2L)e114/ Df(2L)b8&2) is also lethal, this favours the latter hypothesis. Deletion of elB heterozygous with a deletion of the entire region (e.g., Df(2L)A400/ Fig. 3A-D. Electron micrographs of the heads of adult flies het-

erozygous for noc alleles and the noc- deletion Df(2L)bSlal. A wild-type; B In(2L)noc2; C In(2LR)noc7; D ln(2LR)noc4. MO, Df(2L)b83d29a) is, however, only semi-lethal. The medial ocellus; LO lateral ocelli; 10 intra-ocellar microchetae; PVt, region between the breaks of Df(2L)e114 and post-vertical macrochetae: Oc. ocellar macrochetae. Df(2L)TE35BC-8 could be part of the elB gene as the

Genetics of the el-noc region 73 Hereditas 126 (1997)

Table 4. Tlze lethality of e12

Molecular coordinates“ Deletion Extent“ %b

Df2Llfn27 noc+l- 24.0 -61.8- -62.2 Df(2L)Al78 noc- 35.2 -110.8- -113.0 D f(2L)A 446 noc- 17.2 -113.5- -117.5 Df(2L)n 7813 noc- 15.0 - 119.5- - 121.8 D f ( 2 L W noc- 18.9 - 124.9- - 126.4 Df(2L)TE35B- 6 elA- 16.9 - 146.8- - 150.8 Df(2L)TE35B-l elA- 0 - 146.8 - - 150.8 D f(2L)e128 elA- 6.8 -150.8- -151.3 Df(2L)TE35BC-8 elA - 0 - 166.1 - - 170.8 In(2L)TE35B- 13 etB- 0 - 196.0- -247.8 Df(2L)A400 elB- 1.5 -238.6- -243.5 Df(2L)TE35B- 7 1(2)35Aa ~ 0.5 -247.0- -247.8

a Distal limits of deletions. The proximal limits are beyond A&

(Total numbers of flies scored varied between 167 and 758) The percentage of non-balancer progeny over total number of flies from crosses between el’/Cy Roi and Df/CyO is given.

The molecular coordinates are from DAVIS et al. (1990) and CHIA et al. (1985)

elB aberration el6 is broken in this region. Alterna- tively, there may be a fourth gene situated between pu and elA. Deletion of this gene (e.g., Df(2L)e114/ Df(2L)b83d29a) results in the crippled leg phenotype and flies heterozygous for “crle-” elA - deletions and elB- “crle-” elA - deletions (e.g., Df(2L)e114/ Df(2L)b84a2) are lethal. Thus the “crle” locus acts as an enhancer of the el mutations. Whether this is a separate gene or a control region of the el genes will only be determined by a detailed molecular study.

Flies which are heterozygous for alleles of elA and elB are phenotypically elbow. This could be explained by a model based on structural interactions between the products of the two genes. For example, if the products combine to form a heterodimeric protein, then mutations at either loci could cause dysfunction

Table 5. The enhancement of the Sco phenotype by el mutations in trans with the Sco chromosome

Allele Bristle number Allele Bristle number

el ’ 27.45+/-0.48 elJ2 20.80+/-0.41 el2 14.80+/-0.45 ell3 25.1 5 +/-0.30 e13 17.45+/-0.43 el2’ 25.65+/-0.35 e14 20.15 +/-0.48 27.15 +/ -0.30 ei5 20.60+/-0.72 elZ3 22.60+/-0.37 el6 22.90+/-0.45 elz4 26.00+/-0.30 el7 20.28+/-0.57 el2’ 25.20+/-0.45 el8 23.85+/-0.35 22.20+/-0.47 e19 21.65 +/-0.47 el2’ 22.75 +/-0.40 ell0 25.25 +/-0.43 el29 24.45 +/ - 0.37 el ‘ I 23.45 + / - 0.45

The bristle count for Sco/+ is in the range 25-28 per fly. The mean bristle count +/ - standard error of ten flies of each sex is given, except for T(Y;2)a15, el’ (males only). The data for the alleles el’ to el4 are from ASHBURNER et al. (1982b).

of this protein. The severity of the phenotype would be dependent upon the number of mutant alleles. Thus, mutations of eIA heterozygous with mutations of elB would give stronger phenotypes than deletions of elA heterozygous with deletions of elB. This is seen to be the case as Df(2L&I1/Df(2L)b83d29a heterozygotes are wild-type (Table 3), whereas elA alleles heterozygous with elB alleles have weak el phenotypes. This model is similar to that proposed to explain the failure of complementation of mutations in the testis-specific tubulin genes (REGAN and FULLER 1988; HAYS et al. 1989). These authors found that point mutations of the testis-specific 82t tubulin gene failed to complement the mis-sence nc33 allele of the 84B a-tubulin gene resulting in male-ster- ile trans-heterozygotes, but that an 84B a-tubulin deficiency fully complemented P2t alleles. The two tubulins form a heterodimeric protein and the dimers are incorporated into microtubules. The presence of mutated copies of the a and 0 tubulins are thought to “poison” the dimeric complex and lead to microtubule dysfunction.

The noc region has been completely cloned, and the noc alleles associated with aberrations have been mapped to this DNA (DAVIS et al. 1990; GUBB et al. 1990; CHIA et al. 1985) (see Fig. 4). These aberrations map to a region of DNA of some 100 kb in length. A noc transcript has been identified and maps to the region -116 to -119 on this map (CHEAH et al. 1994). The only aberration which breaks in this transcript, is the lethal allele T(2;3)GT8. All of the alleles associated with a visible phenotype map to the region 3‘ to the noc transcript, the weaker the allele then the further away from the transcript it maps (Fig. 4). Trans-heterozygotes between lethal noc alleles and weak alleles or between strong and weak alleles in-

14 T. Davis et u1. Hereditas 126 (1 997)

Table 6. Enhancement of Sco by aberrations in the noc region in trans

Aberration noc mutation Bristle number

T(2;3)GT8 lethal nocA 12.85 +/-0.50 In(2LR)noc4 viable nocA 14.10 + /-0.49

T(2;3)Mpe wild-type 27.05 + / - 0.54 In(2LR)noc7 nocB 13.24-t / -0.45 fn(2L)noc' nocB 17.60+/-0.47 T( Y;2)GT1 wild-type 25.05 + /-0.52 T(2;3)G T7 nocC 21.80+/ -0.45

TE35B viable nocA 1 5.20 + / - 0.44

The bristle count for Sco/+ is in the range 25-28 per fly. The mean bristle count +/- standard error of ten flies of each sex is given

variably give weak noc phenotypes. One aberration, T(2;3)Mpe, maps between the strong noc alleles and the moderate allele Zn(2LR)noc' and is wild-type for noc. These data together with the phenotypic data from trans-heterozygotes suggest that the noc locus is divided into at least three distinct regions, lethal nocA, viable nocA and nocB, (MCGILL et al. 1988; see Fig. 4). The lethal is associated with the transcript and the visible alleles with a series of regions 3' to this transcript. The genetic organisation of the noc region has many similarities with that of the orthodenticle (otd) locus (FINKELSTEIN et al. 1990; ROYET and FINKELSTEIN 1995). Like lethal nocA, point mutations or aberrations of otd are homozygous lethal, and the otd transcript is expressed in the supraoesophageal ganglion during embryogenesis. otd is allelic with the gene ocelfiless (oc); mutants of which are homozygous viable and result in the lack of the ocelli and disruption of the associated bristles. Trans-heterozygotes between otd and oc have phenotypes similar to oc homozygotes. The oc region maps

nocA nocB nocC m ISQa -b

-120 -100 -80 -60 -40 -20 kb I I I I I I

GT8 - - noc7 - GTl

noc4 - - noc2 - GT7

sco - Mpe - . Distal

Fig. 4. A molecular map of the noc region. The co-ordinates are from CHlA et al. (1985), distal is towards the telomere. The boxes represent the maximum limits of the gene regions. The arrow beneath n o d is the noc transcript. The solid lines show the limits of uncertainty of the aberration breakpoints.

immediately 3' to the otd transcript. The similarities between the otd locus and nocA are striking.

The antimorphic noc mutation, Sco, maps molecu- larly to the nocA/nocB region (Fig. 4). Alleles of noc strongly enhance the SCO phenotype when in trans, resulting in 12-17 chetae per fly. Interestingly, the aberration T(2;3)GT7 has no noc phenotype but enhances Sco, whereas the noc+ aberration T(Y;2)GT1, which breaks between GT7 and the allele noc', does not enhance Sco (Table 6; Fig. 4). This suggests the presence of a fourth noc region (nocC) deletion of which enhances noc phenotypes.

It was shown by ASHBURNER et al. (1982b) that lethal noc alleles heterozygous with el- noc- deletions have a weak el phenotype when they escape. The weak el allele, elz, is semi-lethal when heterozygous with deletions of noc. This lethality is enhanced when the deletion includes elA (Table 4). el2 is not associated with a deletion of these genes as the phenotype of el2 heterozygous with deletions of elA would give a strong, rather than a weak el phenotype. The situation is more complex in that this el allele also strongly enhances the phenotype of Sco. This is typical of elB alleles but not of elA alleles. The strength of the enhancement of Sco by elz is much greater than for the aberrant alleles of elB (Table 5). These contradic- tory data could be explained by an interaction between the el genes and noc. If elz is actually an allele of noc and this gene interacts with elA, then heterozygotes between these two alleles would have weak phenotypes characteristic of either el or noc. This would also explain the strong enhancement of Sco by elZ, as most alleles of noc are strong enhancers of Sco (ASHBURNER et al. 1982b). The enhancement of Sco by elB alleles (Table 5) shows that this gene also interacts with the noc gene. Whether these gene interactions between el and noc are due to a physical interaction between the products of these genes, or reflect an overlap between the 5' regulatory region of noc and the el genes, remains to be determined.

In conclusion, we have shown that the el-noc region is complex. The el region is made up of at least two genes which show mutual enhancement. Both the elA and elB genes show interactions with the neigh- bouring locus noc, which is itself complex. These interactions differ for each gene. To understand the nature of these interactions a detailed molecular analysis of this region and its products will need to be undertaken.

ACKNOWLEDGEMENTS

This work has been supported by a Programme Grant from the Medical Research Council, London. We thank Gustavo Maroni for extensive co-operation in its early stages (we

Genetics of the el-noc region 15 Hereditas 126 (1997)

hope he remembers) and William Chia for a long-term collaboration in the molecular analysis of the noc region. The technical help of Terri Morley and Jill Regan has been invaluable. Our grateful thanks are also due to Jose F. de Celis for wing mounting and for helpful discussion.

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Genetic and Phenotypic Analysis of the Genes of the Elbow-no-Ocelli Region of Chromosome 2L of Dvosophila Melanogaster