Interactions and Developmental Effects of Mutations in the Broad … · 2002. 7. 8. · Allele...

Copyright 0 1988 by the Genetics Society for America

Interactions and Developmental Effects of Mutations in the Broad-Complex of Drosophila melanogaster

Istvan Kiss,’ Amy H. Beaton, Jil Tardiff,2 Dianne Fristrom and James W. Fristrom3 Department of Genetics, University of California, Berkeley, California 94720

Manuscript received March 17, 1987 Revised copy accepted October 24, 1987

ABSTRACT The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at

the end of the third larval instar. The region contains a complex genetic locus, the Broad-Complex (BR-C) composed of four groups of fully complementing (br, rbp, l ( l )2Bc , and 1( l ) 2 B d ) alleles, and classes of noncomplementing ( n p r l ) and partially noncomplementing I( l )2Bab alleles. BR-C mutants prevent metamorphosis, including the morphogenesis of imaginal discs. Results are presented that indicate that the BR-C contains two major functional domains. One, the br domain is primarily, if not exclusively, involved in the elongation and eversion of appendages by imaginal discs. The second, the 1(l)2Bc domain, is primarily involved in the fusion of discs to form a continuous adult epidermis. Nonetheless, the two domains may encode products with related functions because in some situations mutants in both domains appear to affect similar developmental processes.

T HE metamorphosis of Drosophila melanogaster is induced by the steroid hormone 20-hydroxyec- dysone (20-HE) at the end of the third larval instar. The first major event of metamorphosis is puparium formation which begins the prepupal period. The prepupal period ends 12 hr later with pupation and is followed by the pupal stage. Extensive morphogenesis of imaginal epidermal structures occurs during prepupal development. This morphogenesis in- cludes three distinct processes (FRISTROM et al. 1987): the elongation of appendages, the eversion of the appendages to the outside of the animal, and the fusion of the discs to form the continuous imaginal epidermis of the head and thorax (FRISTROM and FRISTROM 1975; POODRY 1980; MILNER, BLEASBY and KELLY 1984).

The rise in 20-HE titer at the end of the third larval instar induces a series of puffs, sites of active transcription, in the polytene chromosomes of salivary glands. One of the earliest induced puffs, an “early ecdysone puff’ (ASHBURNER 1972), develops from the 2B5 band of the X chromosome. Physiolog- ical (ASHBURNER 1972), cytogenetic (BELYAEVA et al. 1981), and molecular (CROWLEY, MATHERS and MEY- EROWITZ 1984) results suggest that the locus encodes a trans-acting regulator of expression of other genes. The 2B5 region contains a complex genetic locus, now called the Broad-Complex (BR-C; LINDSLEY and ZIMM 1986). BELYAEVA et al. (1980) proposed the existence of six complementation classes within the

’ Current address: Institute of Genetics, Biological Research Center, * Current address: Department of Genetics, Albert Einstein College of ’ To whom correspondence should be addressed.

Hungarian Academy of Sciences, Szeged, pf521 Hungary.

Medicine, Bronx, New York 10461.

Genetics 118: 247-259 (February, 1988)

BR-C. Using the nomenclature of LINDSLEY and ZIMM (Table l) , these include a totally noncomplementing nonpupariating (nprl ) class, a partially noncomplementing, pupariating class (1( 1 )ZBab), and four complementation groups: broad (br) , reduced bristles on palpus (rbp), 1(1)2Bc, and 1( 1)ZBd that exhibit complementation for lethality with each other. According to BELYAEVA et al. (1980), mutants in the partially noncomplementing class do not complement mutants of the br and rbp groups but do complement those of the I ( 1 )2Bc and I( 1)ZBd complementation groups.

BR-C mutants have different stage- and tissue- specific phenotypes. All, however, have nearly normal larval development. They are deficient in prepupal or pupal development or both, especially in the morphogenesis of imaginal discs. In some viable combinations of alleles, the adults have severely malformed legs and wings (the mlf phenotype). Even with lethal combinations of alleles, the differentiation of the imaginal discs, i.e., the formation of cuticular structures, still occurs (FRISTROM, FEKETE and FRIS- TROM 1981). Because differentiation occurs, the absence of disc morphogenesis is not caused by the death of the tissue. In the most severe BR-C mutants, e.g., npr13 homozygotes, pupariation does not occur and metamorphosis is blocked in all tissues. Devel- opmental arrest is not caused by the absence of a hormonal stimulus but rather is an autonomous defect in the responding tissue (KISS et al. 1976).

As described in a subsequent paper (BEATON et al. 1988), BR-C mutants can be used to identify genes that may actually mediate disc morphogenesis. Here, we describe our efforts to determine the developmental effects of BR-C alleles to learn if different

248 I. Kiss et al.

TABLE 1

Mutant symbols and abbreviations

Mutant class designation* Previous designation Origin Comments Reference Current

broad, 1(1)2Ba*

reduced bristles on palpus, 1(1)2Bb*

Partially noncomplementing, 1(1)2Bab*

lethal(1 )prepupal-I, 1(1)2Bc*

lethal(I)prepupal-2, 1(1)2Bd*

nonpupariating 1(1)2Bad*

Other stocks

br' b? b 8 b$ b?5 b?6 b?7

rbp'

1(1)2Bab' 1(1)2Bad

1(1)2Bc' 1(1)2B2

l(1)Bd'

nprl ' npr13 nprI ' nprI

Df(I)S39 Df(1)sta l ( I ) ~ t a ' ~ ~ Df(I)RA19 YY67gI 9. I Dp(1 ;Y)Sz280 l(1 )dol"sl 1(I)swPo0

br lt35t It103 It366

None None

It99

Df(I)pn7b

It4 It143

It10 It76

It252

l(1)d. nonn.-l" nfrI npr2 None Df(I)S39 Df(1)sta lt3 6 Df ( l )RA19

DpYSz280 dorlt81 swi"O0

D P ( W ' 6 7 g

Spontaneous EMS EMS EMS X-ray DEB DEB

EMS

EMS EMS

EMS EMS

EMS

EMS EMS EMS DEB

Viable Lethal In(1)2B3-4;3Cl Lethal Df(l)E1-2;2B4-5 Df(l)D-E;2B5 Viable

Lethal

Lethal Lethal

Lethal Lethal

Lethal

Lethal Lethal Lethal T(1;3)2B5;61F3-4 Df(l)lE1-2;2B5-6 Df(l)lE1-2;2B3-5

Df(l)lE3-4;2B11-12 Dp(l;Y)lA;2B17-18 Dp(l;Y)lA;2C1-2**

* Based on LINDSLEY and ZIMM (1986). ** Contains an internal deficiency, 2B3-4;2B7-8. References: (1) MORGAN, BRIDGES and STURTEVANT (1925); (2) this study; (3) BELYAEVA et al. (1982); (4) KISS, MAJOR and SZABAD (1978);

(5) STEWART, MURPHY and FRISTROM (1972); (6) BELYAEVA et al. (1980).

alleles regulate different aspects of imaginal disc morphogenesis. We also describe experiments to determine which 2B5 alleles are amorphic and to determine the nature of any interactions between members of the different complementation groups in order to infer the number of independent functional domains present in BR-C product(s).

The results indicate that the broad and 1(1)2Bc complementation groups contain amorphic alleles that identify two major independent functional domains. The broad domain is involved in the elongation and eversion of appendages. The 1(1)2Bc domain appears primarily to mediate the fusion of discs. Products encoded by the two domains may, however, have similar functions. Because of the absence of amorphic rbp alleles and because the rbp phenotype arises in broad mutants, we doubt that the rbp phenotype results from defects in an independent functional domain in BR-C product(s), but rather is a phenotype that often arises from partial loss of activity in or near the broad domain. Finally, we

suggest that 1(1)2Bd' is a viable allele of the rbp complementation group, and does not constitute a complementation group of its own.

MATERIALS AND METHODS

Mutant stocks and crosses: The mutants and stocks used are described in Table 1, LINDSLEY and GRELL (1968), and LINDSLEY and ZIMM (1986). In an effort to conform gen- erally with the nomenclature set forth in LINDSLEY and ZIMM, we use mutant designations that are modified from those used previously (Table 1). Various alleles are designated by their complementation group and a superscript notation. For example, lt366 (BELYAEVA et al. 1980), a lethal allele of the br complementation group, is designated as br7.

Standard yeast-cornmeal-agar medium that contains pro- pionic and phosphoric acids as mold inhibitors was used. Crosses were made at 25". Eggs were collected for 12 to 24 hr. After removing the parents, the developing offspring were maintained at 18" or 29" (k 1") until all F1 adults emerged.

Mutagenesis and isolation of new BR-C mutants: Males (y u) were fed 0.005 M diepoxybutane (DEB) in 1% sucrose solution for 24 hr (MOHLER and PARDUE 1984). The DEB-

Developmental Roles of the BR-C 249

TABLE 2

Viability of some homoallelic and heteroallelic combinations of BR-C mutants

Percent survival of mutant combinations (no. of balancer heterozygotes) with female parent: Temp.

Male parent ("C) b?6 P7 np17 2Bab' np13 b? rbp' 2Bc' sta 2Bd'

b?6 29 O(83) lOO(52) O(78) O(45) O(956) l(215) lOO(87) lOO(51) O(64) - 18 O(92) lOO(72) O(64) O(51) O(1112) O(103) 100*(59) lOO(62) O(45)

b g 7 29 - 59(251) - O(70) O(121) O(175) 97(78) lOO(57) lOO(106) - -

18 - 99(124) - 41(81) O(115) 3*(90) 73(63) lOO(37) lOO(46) - nprI ' 29 - O(79) O(103) l(184) O(27) O(115) O(63) O(113) 96(27)

- O(105) O(75) O(74) O(39) O(94) O(93) O(76) lOO(25) - 2Bab' 29 - - - O(79) O(53) O(70) 3*(307) 75(329) -

18 - - O(86) O(118) O(136) 21*(256) 67(162) - br' 29 - - - - 48*(153) - -

18 - 10*(50) - - - - b? 29

18

2Bc' 29 -

- 18

- -

"

- - - -

- - - - - - - - - O(64) 73(227) lOO(163) - -

- - lOO(156) O(107) - - - - lOO(73) O(132) - -

- - - - - o(7) 81(89) 91(91) - - - - -

18

2Bd' 29 - 18

- - - - - - lOO(78) - lOO(155) lOO(90) lOO(110) - 91(139) - - lOO(123) - lOO(97) lOO(81) lOO(86) - 88(80) -

Crosses: male parent = BR-C mutandfY67gl9.1, female parent = BR-C mutandBinsn. Allele abbreviations: 2Bab' = 1(I)2Bab1; 2Bc' = I(1)ZBc'; sta = Z(l)sta"6; 2Bd' = Z(I)2Bd1.

% survival = number of mutants (homozygotes or heterozygotes)

number of balancer heterozygotes x 100.

The % survival is the first number given and is separated from the number of balancer-carrying heterozygotes by parentheses. Values equal to or greater than 100% are not distinguished. Results that are underlined indicate malformation of legs; those marked by * indicate the rbp phenotype is expressed (100% except for one of three b 2 / b g 7 survivors. See text for details). -, not reported or not tested.

treated males were crossed to C ( I ) R M , y shiL."lfY67g19.1 females at 25"; the parents were transferred daily to fresh food for a total of 3 replicates. After removing the parents, the bottles were kept at 29" for 24 hr, then returned to 25". This temperature regimen killed the shP' temperature- sensitive female offspring. fY67gZ9.Z covers the distal tip of the X chromosome, 1 A1 to 2B 17- 18, so the only survivors were y w*ly%7g19.1 males with no new lethals proximal to 2B18. These males were individually crossed to y I ( 1)nfn-1' w shi"'lFM6b ( $ I d B 1(1)69j) females. Parents were transferred daily to fresh vials. The same temperature regimen used above was followed to eliminate shi*' male offspring. Only females survive. The female progeny were checked for y w*ly 1( 1 )nprl' w shzP females; their absence indicated that they w* chromosomes carried a new BR-C lethal allele. Three new mutants, b?6, b?', and nprZ7 (Table I), were isolated from 2647 mutagenized chromosomes. Stocks of the new mutants, balanced over Binsn, were established from y w*lFM6b survivors.

Cytological preparations: Chromosome squashes were made according to ATHERTON and GALL (1972) and examined by phase microscopy.

Determination of lethal phases: Eggs were collected for 12-24 hr at 25" in bottles which were then transferred to 18" or 29". Additional yeast was added as necessary to assure optimum feeding. Wandering larvae were removed from the sides of the bottles 4 (29") or 9 (18") days after egg collection. Mutant hemizygous male larvae or homozygous female larvae (with yellow mouth hooks) were selected and transferred to vials with fresh food. (Homo- zygous lethal female larvae resulted from crosses involving males carrying a lethal BR-C allele covered by a duplication

on the Y chromosome with females heterozygous for a lethal BR-C allele.) A few normal sibling larvae carrying the balancer chromosome were included for comparison. During these procedures care was taken to keep the larvae at the specified temperature. The vials were incubated further at the indicated temperatures. All larvae pupariated within 2-3 days. Freshly formed puparia were removed daily, transferred onto moist bibulous paper, sealed in Petri dishes, and kept at the specified temperature until normal sibling adults eclosed (8-9 days after egg laying at 29" and 20-2 1 days at 18").

Before dissection, mutant lethal pupae were grouped according to their gross morphology after examination under water (to improve visibility) with a dissecting micro- scope. Prepupae, pupae, and pharate adults were grouped separately. If possible, subgroups were formed according to more detailed morphological features visible through the pupal case. At least 5 representative specimens were dissected for each group in each experiment. Gross morphology was observed and the body was opened to evaluate internal tissues for histolysis and adult differentiation. Notes were made on each dissected specimen for subsequent comparisons.

RESULTS AND DISCUSSION

This section contains two major parts. First, we describe the viability and the phenotypes of adults carrying various combinations of BR-C alleles. Based mainly on these data (Table 2) and the hypomorphic nature of rbp' (Table 5 ) , we suggest an alternative

250 I . Kiss et al.

npr 1

1(1)2Bab

br rbP 1(1)2Bc 1(1)2Bd

FIGURE l."Complementation map of the BR-C based on BEL. YAEVA el al. (1980).

nprl {nprl and I(1)ZBab alleles}

br ibr, rbp , and 1(1)2Bd alleles} 1 ( 1 ) 2 ~ ~

FIGURE 2.-An alternative complementation map of the BR-C based on the results presented in this paper.

(Figure 2) to the BR-C complementation map proposed by BELYAEVA et al. (1980). Second, we describe the stages of developmental arrest caused by various combinations, particularly those involving one or two doses, of BR-C alleles. We deduce from these results (Tables 4 and 5) whether particular BR-C alleles are amorphic or hypomorphic, and propose major developmental functions for the wild-type genes.

Complementation and abnormal adult phenotypes of heteroallelic combinations

BELYAEVA et al. (1980, 1982) reported complementation and temperature-sensitive interactions between BR-C alleles from which they derived a complementation map (Figure 1). We undertook further inves- tigation of the interactions by determining the viability and phenotypes of adults carrying combinations of alleles representing different BR-C complementation classes. We report those data that augment the results of BELYAEVA et al. (1980), or that establish the complementation classes of new DEB-induced mutations, All crosses were made in parallel at 18" and at 29". The crosses were made reciprocally to test for any maternal influence. Frequencies of survival, compared to balancer-carrying sibs (Table 2), and the presence of structural malformations of legs and wings (br and mlf phenotypes) or of a reduction in the number of bristles on the palpus (rbp phenotype), were determined for the heteroallelic combinations. Because no maternal influences were observed, data are presented for only one of each reciprocal cross. Using germ-line somatic crossing-over, PERRIMON, ENGSTROM and MAHOWALD (1984) previously dem- onstrated the absence of a maternal effect for nprpr13 and npr14.

Properties of new DEB-induced mutants: TWO new DEB-induced mutations, b?6 and b?7, behave as alleles of the br complementation group. They exhibit nearly complete lethality with b? and high viability with both rbp' and 1(1)2Bc' (Table 2). Cyto-

logically, b?6 is associated with a deficiency with breakpoints in 1D-E and 2B4,5 (Figure 3a). Further- more, bP6 is lethal with l ( 1 ) ~ t a " ~ the nearest distal flanking mutant. b?7 is cytologically normal. A third DEB-induced allele, nprl 7, behaves as an nprl allele. It is lethal with b?, rbp', and 1(1)2Bc'. Cytologically, npr17 is associated with a translocation between the X and third chromosomes with breakpoints in 61F3- 4 and near or in 2B5 (Figure 3b). Thus, two out of three of the newly induced DEB mutants are associated with visible rearrangements.

Interactions between br and rbp alleles: BELYAEVA et al. (1980) reported interactions between br and rbp alleles. For example, flies heteroallelic for b? with any allele of the rbp complementation group exhibited the rbp phenotype, and br6 with either r b f (t144) or rbp4 (t358) produced both the br and rbp phenotypes. These results indicate that br mutants are deficient in rbp function and rbp mutants in br function or, alternatively, that altered products caused by mutants of the two complementation groups interact to produce both phenotypes. We sought to distinguish between these alternatives by determining the viability of brlrbp heterozygotes, and whether br mutants expressed an rbp phenotype in the absence of an rbp allele. Indicating only partial complementation between lethal br and rbp alleles, we found a slight reduction in viability, particularly at 18", of b?/rbp' and bP7/rbp' heterozygotes (Table 2). The rbp phenotype was expressed in bp'lb?, b?61rbp', brJlnpr13, bd lDf(1 )S?9, and b?6/Dp( 1;Y)SZ28O adults. Dp( I;Y)SZ28O contains an interstitial deficiency that removes all BR-C function. The expression of the rbp phenotype among bP7/b?, b?"/Dp( 1;Y)Sd8O, and br'lDf(1 )S39 survivors demonstrates that br alleles are themselves deficient in rbp function. Fur- thermore, examples of all three types of br alleles (lethal, amorphic; lethal, hypomorphic; viable, hypomorphic; see below) exhibit an rbp phenotype. In each instance where an rbp phenotype was observed, the palpus was smaller than normal. In addition to an rbp phenotype, rbp'll ( I )2Bab' escapers exhibit a weak br phenotype (as in Figure 5d) and other malformations of the wings, indicating that rbp' lacks br function. Finally, +lDf( l )S39 females exhibit a slight br phenotype and a smaller than normal palpus (data not shown) indicating that a reduction in normal BR-C products affects both wing an palpus development. No malformations of the legs and wings, however, are observed.

Properties of 1(1)2Bd': In our hands the single member of the 1(1)2Bd complementation group, 1( 1 )2Bd' has 90% viability as a homozygote (Table 2). When heterozygous for a deficiency, this allele has 20-40% viability. According to BELYAEVA et al. (1980), I ( 1 )2Bd' exhibits at 18" the rbp phenotype over a deficiency ( D f ( l ) R A - 1 9 ; Df(l)E3-4;2Bll-12). Using

Developmental Roles of the BR-C 23 1

""I

FIGURE 3."CytoIogv of two new DEB-induced BR-C mutants. (a) b?6; Df( 1)D-E;BBS; (b) nprl'; T( 1 ;3)2BS;61 F3-4.

FIGURE 4.-Third legs from (a) wild type, (b) I (1)2Babf /1(Z)2Bcf , and (c ) br'lnprl' at 18".

FIGURE S.-\Vings from (a) +/+, (b) br'lnprl', ( c ) b?'/b?', and (d) br1/6r1.

252 I. Kiss et al.

a smaller deficiency with a more distal proximal breakpoint, D f ( I ) S 3 9 (Df(l)E1-2;2B5-6), we observed only a slight rbp phenotype at 18" or 29" with I( I)2Bd'. We found that I(I)2Bd1 was fully complementing for viability with b?, rbp', 1(I)2Bc1, and 1(I)2Bab1 (Table 2).

In summary, the results of BELYAEVA et al. (1980) and our current ones indicate that all members of the br, rbp, and Z(I)2Bd complementation groups are deficient in rbp function. Furthermore, rbp' appears to be deficient in br function. Hence, under some circumstances, br alleles are rbp alleles; rbp alleles are br alleles. Consequently, we suggest that the rbp, br, and 1(1)2Bd complementation groups are functionally dependent.

Interactions between 2(1)2Bab and 1(1)2Bc alleles: We have identified previously undescribed interactions between I( I )2Bab and l( I )2Bc alleles. I ( I )2Bab'l l( I )2Bc' heterozygotes have reduced viability (Table 2) at both 18" and 29". Furthermore, all of the survivors at both temperatures exhibit a specific malformation in the third pair of legs (Figure 4b). In addition to shorter and thicker femurs and tibias, the legs have a genotype-specific malformation of the basitarsus. These results indicate that in addition to defects in br and rbp function that 1(1)2Bab', and other partially noncomplementing alleles, partially lack 1(1)2Bc function. More important, the effect on leg structure demonstrates that l ( I )2Bc , at least in this genotype, is involved in appendage elongation (see below). No interaction occurs between b? and I( I )2Bc' for either viability or adult phenotype. Like- wise, l(I)2Bc' and rbp' are fully complementing for viability (Table 2).

The homozygous viable allele br' in combination with npr13, or with any complete deficiency of the 2B5 band, or with other amorphic members of the nprl complementation class, is 90-99% lethal at 18" and 40-50% lethal at 29" (Tables 2 and 4). Survivors have extremely malformed (mlf) wings and legs (4 1 % at 29"; 100% at 18") compared with wild type (Figures 4 and 5). Wings are short and severely broad, often with interrupted veins (Figure 5b) and legs, particularly the third pair, exhibit the mlf phenotype with short, bulbous and often twisted segments (Figure 4c). The proboscis and palpus are reduced in size, the latter has an rbp phenotype. The malformations are more extreme in survivors raised at 18" than at 29". Similar, though less extreme malformations are seen in b?'/b?, b?'/b$, and b?6/Dp(l;Y)Sz280 combinations.

BR-C may encode two major functional domains We turn here to discuss the complementation data.

As first described by BELYAEVA et al. (1980), br, rbp, 1(1)2Bc, and 1(1)2Bd fully or partially complement for viability. From these results they concluded that

the BR-C contains four complementation groups in addition to the partially and completely noncomple- mentating classes (Figure 1). We believe, however, that the results from their and our complementation studies are formally compatible with another interpretation (Figure 2).

We suggest that the BR-C mutants exhibit two forms of allelic complementation. One form is exemplified by the rudimentary locus in D . melanogmter (RAWLS and FRISTROM 1975; JARRY 1979). Here, a multifunctional polypeptide encodes several functions. Defects in one function are fully complemented by alleles that are defective in another function. Individual mutants can be amorphic for particular functions and allelic sites of a complementation group are clustered. A second form of allelic complementation is exemplified by multimeric proteins such as alkaline phosphatase in Escherichia coli (GAREN and GAREN 1963; SCHLESINCER and LEVINTHAL 1963). Interaction of differentially defective subunits in heteroallelic cells substantially restores function. Such allelic complementation typically involves hypomorphic alleles with partially overlapping distribu- tions of mutant sites within a gene that encodes a unifunctional polypeptide. BR-C mutants have attributes characteristic of both types of allelic complementation.

broad and 1(1)2Bc alleles are fully complementing and independent: The br and l ( I ) 2 B c complementation groups are typical of the first form of complementation. The two complementation groups identify independent functional domains. Both groups have amorphic (no activity) alleles (e.g., b? and 1(1)2Bc', see below and Tables 4 and 5) . There is no interaction between amorphic alIeles for viability or adult phenotype (Table 2) (BELYAEVA et al. 1980). b? and l(I)2Bc' have been mapped to well separated sites (0.015 m.u.) within the BR-C (AIZENZON and BE- LYAEVA 1982).

broad and rbp alleles interact: br and rbp alleles may exemplify the second form of allelic complementation. They do not fall into clearly separate functional domains. br alleles interact with rbp alleles to produce both br and rbp phenotypes. Moreover, br alleles in the absence of an rbp allele can produce the rbp phenotype. Thus, br alleles can also be rbp alleles. Conversely, rbp' appears to be a h allele. b? maps distally to rbp', but b$ may map either distally or proximally to rbp' (AIZENZON and BELYAEVA 1982). These mapping data suggest that the mutational sites of br and rbp alleles overlap. The developmental effects of the br and rbp alleles appear to be related. The br alleles prevent the appendages from reaching their full extension. The rbp phenotype is associated with a reduction in size of the palpus. Hence, both br and rbp mutants prevent disc-derived structures from reaching their normal size. Furthermore, re-


TABLE 3

Stages of developmental arrest and phenotypes of BR-C mutants

developmental Stage of

arrest Key genotypes Phenotype

Late larval npr~'InprI' No pupariation. Larval tissues do not degenerate. Discs form bloated vesicles Index = 0 nprl'/Df(l)S39

Dp(l;Y)Sz28U/Df((r)S39

Early prepupal b?lW Untanned puparia form. No elongation or eversion of appendages. Discs do not Index = 1 b?/Df(l)S39 fuse and remain as vesicles

1(1)2Bab'IDf(l)S39

Late prepupal 1(1)2Bc1/1(1)2Bc'* Abnormal puparia form. Appendages elongate and evert. Partial fusion of discs; Index = 2 1(1)2Bab'lld head and thorax incomplete

Early pupal 1(1)2Bc1/1(1)2Bc'** Abnormal puparia form. Appendages elongate and evert. Fusion of discs not Index = 3 1(1)2Bc'lDf(I )S39 complete. Partial head emergence (pupation)

bFlDf(l)S39 P I n p r I '

Late pupal 1(1)2Bab1Il(l)2Bab' Normal puparia. Head emergence complete, disc fusion usually complete. Little Index = 4 b@/bP or no pigment in eyes and bristles

b?7/Df(l)S39 bP/w

Pharate adult rbp'lrbp' Normal puparia. Adults fail to eclose but are essentially normal with pigmented Index = 5 rbp'/l(l)2Bab' bristles and eyes. The rbp phenotype is expressed

p 7 / b ? 1(1)2Bd'/Df(l)S39

Early adult br'lbr' Adults are viable or die after eclosion***. Wings may be broad; mouth parts rbp Index = 6 1(1)2Bab'/rbp'

1(1)2Bd'll(l)2Bd'

* at 29'; ** at 18"; *** 1(1)2Bab'lrbp'.

gardless of genotype, most individuals examined whose development stopped in the pharate adult stage exhibited the rbp phenotype (see below). (The exception is e1676 which, however, is associated with an inversion.) Thus, the rbp phenotype appears to be dependent on the stage of developmental arrest, and is not a specific manifestation of rbp alleles. Finally, no amorphic (no activity) rbp alleles have been isolated (see below, Table 5). Because hypomorphic (reduced activity) rbp mutants develop into pharate adults (Table 3), if there was an rbp function independent of br function, we would expect amorphic rbp alleles, causing pupal developmental arrest, to have been isolated. We propose that br and rbp phenotypes are different stage-specific manifes- tations of mutations that affect a single product and that complementation between br and rbp alleles may depend on compensatory interaction between defective multimeric subunits.

Our proposal that allelic complementation involving multimers occurs would be strengthened if complementation occurred between hypomorphic alleles of the same complementation group, e.g., br alleles. We have found no unequivocal instance of such complementation. bg6 and bg7, however, may complement. Although bg7 is a credible candidate for a haplo-specific allele (Tables 2 and 4), in females at

29" it is only 58% viable as a homozygote. As noted above, bP6 is associated with a deficiency, and is fully lethal as a homozygote female. In a male, protected by a duplication (Dp(I;Y)Sz280) which covers all of the deficiency except that in the 2B5 region, b 8 6 is only 11% viable at 29" (Table 4). Nonetheless, suggesting allelic complementation, bPIb72' is 100% viable at 29" (Table 2).

Z(Z)2Bd' may be an rbp allele: We hypothesize that the E( I)2Bd' allele, assigned to a separate complementation group by BELYAEVA et al. (1980), is a weak allele of the rbp complementation group. We have confirmed the results of BELYAEVA et al. (1980), showing that 1(1)2Bd' complements for viability with b?, rbp', 1(I)2Bc', and 1( I)2Bab', but is partially lethal with a 2B5 deficiency. Indicating that l(I)2Bd' is deficient in rbp function, BELYAEVA et al. (1980) found that I( I )2Bd' deficiency hemizygotes expressed the rbp phenotype. The rbp phenotype and the absence of lethality in 1(1 )2Bd'lrbp' heterozygotes does not require that these two alleles be in separate complementation groups. Both alleles are hypomorphic; I( I )2Bd' is a haplo-specific lethal as defined by NASH and JANCA (1983). As pointed out by these researchers, heterozygosity for two haplo-specific alleles can be fully viable. In addition, the combined defects of a hypomorph and a haplo-specific allele may also not

254 I . Kiss et al.

be sufficient to cause either lethality or, in this case, an rbp phenotype. A comparable situation is found with the b?6 and b e 7 alleles. For these reasons, and because this allele is the only member of the 1(1)2Bd complementation group, we suggest that 1(1)2Bd’ is really an rbp allele. I( 1 )2Bd’ has not been mapped so its position in the BR-C is unknown.

In conclusion, we propose an alternative view of the functional organization of the BR-C than that of BELYAEVA et al. (1980). We suggest that the BR-C contains two major functional domains, the br domain and the 1(1)2Bc domain (Figure 2). The rbp alleles, including I( 1 )2Bd1, are viewed as belonging to the br domain. We further suggest that the br, rbp, and 1( 1 )2Bd alleles exhibit allelic complementation among themselves. As one possible simplistic molecular model, we can imagine that the BR-C produces by differential processing two polypeptides, one affected by br, rbp, and 1(1)2Bd alleles, the other by I ( 1 )2Bc alleles. Both polypeptides would be affected by 1( 1 )2Bab and nprl alleles. The data, however, do not allow one to determine conclusively the validity of either complementation model. We hope juxtaposing two contrasting views will have heuristic value.

Dosage compensation The phenotypes of representative alleles were com-

pared in hemizygous males to those in homozygous females (Tables 4 and 5 ) and were found to be indistinguishable from each other. We conclude that all of the tested alleles dosage compensate.

Dosage effects on lethal-effective phases Complementation classes of the BR-C have char-

acteristic and distinct lethal phenotypes (KISS et al. 1980; AIZENZON, BELYAEVA and ZHIMULEV 1982). The stages of developmental arrest and the major phen- otypic characteristics of key BR-C genotypes are summarized in Table 3. Depending on genotype, developmental arrest can occur as early as the end of larval development (e.g., npr13/npr13) to as late as shortly after emergence of the adult (rbp111(l)2Bab1).

We sought to determine which of the developmental syndromes result from amorphic (no activity) and which from hypomorphic (reduced activity) alleles. T o this end, we characterized and compared the lethal phenotypes, including the times of developmental arrest, of representative alleles from each of the complementation classes as homozygotes and as deficiency hemizygotes with Of( 1 )S39. Because of the absence of dosage compensation in females (for review, see STEWART and MERRIAM 1980), we assume that the functional output of a mutant gene as a hemizygote (one effective dose) in a female is half that of a homozygote (two effective doses). Conse- quently, the phenotype of a hypomorphic allele is expected to become more severe in a hemizygous

female, while that of an amorphic allele should remain unchanged (MULLER 1932). To facilitate pre- sentation of the data, we have assigned a developmental index (Table 3) to each stage of developmental arrest. An index of 0 denotes larval arrest, 1 = early prepupal arrest, and so forth up to an index of 6 which indicates eclosion of the adult. The values of the developmental indices are arbitrary and do not imply any linear relation. For example, in terms of time, the difference between late prepupal (index = 2) and early pupal arrest (index = 3) is a matter of 6 to 12 hr, between late pupal (index = 4) and pharate adult arrest (index = 5), 24 to 48 hr.

nprl: The phenotypes of npr131npr13, npr131 Of( I )S39, and Of( 1 )S39lDp( l;Y)SZ28U (0 effective doses, data not shown) individuals were practically indistinguishable (Table 4). They all fail to pupariate (index = 0) and have imaginal discs that form swollen vesicles (Figure 6b). These observations agree with previous descriptions of nprl’ and other fully noncomplementing, nonpupariating alleles (KISS et al. 1980; FRISTROM, FEKETE and FRISTROM 1981). Df(l)S?9IDp(l;Y)Sz28U lacks the 2B5 band and per- haps parts of the flanking interbands (BELYAEVA et al. 1981). Hence, the late larval developmental arrest of npr13/npr13 is equivalent to the amorphic phenotype of the entire BR-C. Two other nonpupariating and noncomplementing late larval lethal alleles, npr14 and nprl‘ (KISS, MAJOR and SZABAD 1978; STEWART, MURPHY and FRISTROM 1972) were tested with iden- tical results (data not shown).

broad: The br complementation group is composed of both amorphic and hypomorphic alleles (Table 4). Amorphic alleles cause early prepupal developmental arrest (index = 1). Hypomorphic alleles cause late pupal or pharate adult developmental arrest, or are viable.

b? is an amorphic allele (Table 4) that causes early prepupal developmental arrest and an untanned puparium. Its discs (Figure 6c) remain as vesicles because little elongation and no eversion of appendages occurs. Another allele, b$, behaves like b J (data not shown).

Other lethal alleles of the br complementation group ( b f l , b?6, and b?7) develop beyond the prepupal stage into pupae or pharate adults (index 4- 5). In contrast to b?, their phenotypes are more severe in females as hemizygotes than as homozygotes (Table 4). Therefore, we regard these alleles as hypomorphs.

bP6 is associated with a deficiency which removes several essential genes. Its br phenotype cannot be ascertained in males with a normal Y chromosome, or & homozygous females. Assuming dosage compensation, its developmental potential can be determined in b?6/Dp(l;Y)Sz28U males (two effective doses) and in be6/npr13 females (1 effect dose, assum-


TABLE 4

Effect of dose on stage of developmental arrest of nprl and br alleles

Developmental index (% survival) No. of specimens Effective Temp.

dose ("C) np13 b? wi bP6** b?' w 29 0 29

18

2 6 6 29 18

1 9 0 29 18

1 P P 29 broad 18 n p r ~ -

0(0%)221 0(0%)198

0(0%)200 0(0%)138

0(0%)240 0(0%)172

NA

NA

1.0(0%)64 1.0(0%) 17

1.0(0%) 1 17 1.0(0%)81

1.0(0%)206 1.0(0%)65 -

-

- -

4.1(0%)160 4.1(0%)76

3.0(0%)48 3.0(0%)144

- -

NA

S A

4.5( 1 1 W)50 4.1(0%)34

NA

NA

3.0(0%)96 3.0(0%)71

5.2(59%)251* 6.0(99%)124*

- -

4.3(0%)278 4.1(0%)143

4.2(0%) 12 1' 4.1(0o/c)l15*

6.0(99%)227*

6.0(100%)193* - -

5.0(43%)204 4.1(10/0)260

- -

Crosses: mutandfY67gl9.1 males X mutantlBznsn, Df(l)S39/Binsn, or npr13/Bznsn females. Effective dose: number of copies, 1 or 2, of the mutant allele present assuming dosage compensation in males and its absence in females. * Viability calculated as in Table 2. ** bP6 is associated with a deficiency. See text for the genotypes used to emulate 1 and 2 copies. broad refers to any br allele. Data entries: the first number, in bold, is the average developmental index. The average developmental index is the sum of the

developmental indices for all animals, lethal and nonlethal, divided by the total number of animals. The stage of developmental arrest corresponding to each developmental index is given in Table 3. The second number, in parentheses, is the percent viability. Unless otherwise indicated, percent viability was determined directly (the number of mutant survivors divided by the total number of mutants) and not from the number of balancer heterozygotes. The third number is the number of animals, both lethal and viable, scored. Thus, for npr13/npr13 at 29", the developmental index is 0, the viability is (OW), and 221 larvae were counted. -, not determined; NA, not applicable.

ing nprl' is amorphic). Some bP6/Dp( l;Y)SZ28O males escape at 29" (index = 4.5 at 29"; 4.1 at IS"), but b86/npr13 females are fully lethal (index = 3.0 at 29" and 18"). Consequently, b P is hypomorphic. When heterozygous with the amorphic b? allele, 1 % of b P / b? individuals survive at 29" (Table 2). The developmental index of b?/b? at 29" is 4.1 (based on 104 specimens), that of b?26/npr13 is 3.0. Thus, heterozygosity of b?26 with an amorphic nprl allele is pheno- typically more severe than heterozygosity with an amorphic br allele. Another br mutant associated with a deficiency, b P (BELYAEVA et al. 1980), behaves like b? (data not shown).

b?7 is partially viable as a homozygote in females and as a hemizygote in males, but is fully lethal in a single dose in females or with nprl' (Table 4) and consequently is hypomorphic. The lethal specimens are blocked as pupae (Table 4) and, to a lesser extent, as pharate adults. The frequency of late pupal developmental arrest is increased at 18". Like b+ b127/ br5 (developmental index = 5.0 at 18" based on 98 individuals) is developmentally less severe than b727/ nprl' (developmental index = 4.1 at 18", Table 4). The lethal b?/b?7 pharate adults exhibit the rbp phenotype.

The original BR-C allele, br', is viable when homozygous in females or hemizygous in males and has wings that are slightly shorter and wider than wild type (Figure 5d). In combination with Of( 1 )S39, br' is semilethal at 29" and almost fully lethal at 18"

(Table 4; also BELYAEVA et al. 1982). At 29", about 75% of the lethal specimens die as pupae, 25% as pharate adults. At 18", late pupal developmental arrest predominates (Table 4). Pupal developmental arrest is often associated with the presence of gaps in the thoracic integument (Figure 7). The lethals have the same, but more severe, morphological abnormalities as the survivors. Pupal wings and legs are very short. At 18" the legs are extremely malformed with twisted and swollen segments.

rb$~ and 1(1)2Bd: One member of the rbp complementation group, rbp', was examined (Table 5). This allele was chosen because, as a homozygous lethal, it is among the most extreme in the rbp group. In two doses, rbp' has part late pupal (42% at 18"; 65% at 29"), part pharate adult developmental arrest (Table 4). In one dose at 29", rbp'lDf(1 )S39 is almost entirely a late pupal lethal (88% at 18"; 97% at 29"). Thus, rbp' behaves as a hypomorph. The rbp pupae have no obvious gross anatomical abnormalities, except that the thorax and appendages are somewhat smaller or shorter than normal. The pharate adults exhibit the rbp phenotype.

I ( 1 )2Bd' homozygotes are viable. I( 1 )2Bd'/Df( 1 )S39 hemizygotes are partially lethal, particularly at 29", but have substantially greater viability than reported by BELYAEVA et al. (1980). These researchers reported 0% viability at 29" and 26% viability at 18". At both 18" and 29" the lethals die primarily as pharate adults. Thus, the stage of developmental arrest is similar to

FIGURE 6.-(a) A leg disc from a 0 hr Oregon R prepupa. The disc epithelium (d) and the peripodial epithelium (p) form a vesicle that persists until eversion is complete 4-5 hr after puparium formation. (b) Leg disc from an nprl' late larva. (c) Leg disc from a 4-hr bJ prepupa. Discs from these two mutants never progress beyond the vesicle stage.

FIGURE 7.-br'lnpr13 at 18". This pupa was dissected and fixed with osmium. Osmium stains tissue only where it can penetrate through gaps in the body wall. (Wild-type pupae do not stain with osmium.) Gaps, when present, typically occur mid-ventrally between head and thorax and ventral to the wing blade (arrows).

Developmental Roles of the BR-C

TABLE 5

Effect of dose and genotype on the stage of developmental arrest of 1(1)2Bc, 1(1)2Bb, r&, and &1)2Bd alleles

257

Developmental index (% survival) No. of specimens Effective Temp.

dose (“C) 1(1)2Bc’ 1(1)2Bab’ rbp’ 1(1)2Bd’

2 P P 29 2.7(0%)107 3.9(0%)79 4.3(0%)63 5.9(91%)139* 18 3.0(0%)132 4.0(0%)86 4.6(0%)204 5.9(88%)80*

2 d d 29 2.9(0%)75 4.1(0%)63 4.6(0%) 172 18 3.0(0%)43 4.0(0%)40 4.5(0%)150

29 2.9(0%)58 1.0(0%)88 4.0(0%)170 4.9( 18%)304 18 3.0(0%)46 1.0(0%)29 4.2(0%)68 5.3(42%)486

Genotype 1(1)2Bab1 29

npr13 18

1(I)2Bab1 29 br5 18

rbp’ 18 1(I)2Bab1 29

NA

NA

NA

NA

NA

NA

1.7(0%)53 1.0(0%)118

2.8(0%)70 2.5(0%)106

4.8(3%)57 5.0(0%)61

N A

N A

NA

NA

NA

NA

NA

NA

N A

NA

NA

N A

See Table 4 for an explanation of the entries and crosses. Crosses induded the use of b?/Binm and rbp’iBinsn females. * Viability calculated as in Table 2.

that of rbp’, supporting the view that Z(1)2Bd’ is an rbp allele (see above).

Z(1)2Bc: 1(1)2Bc’ is an amorphic allele that causes either late prepupal or early pupal developmental arrest (Table 4). Extreme expression of this mutant is seen at 29” where, in 12-26% of the specimens, no head or dorsal thorax form because fusion of the epidermis in the thorax and head regions is defective. The appendages elongate and evert. The leg discs fuse ventrally and to the anterior edge of the abdom- inal epidermis. Failure to fuse dorsally appears to result from a failure of the proximal disc regions to spread after eversion has occurred. In most cases early prepupal morphogenesis occurs more or less normally, the discs reaching a stage equivalent to a 7-8-hr prepupa (Figure 8b).

In less extreme cases, typically at 18”, all the discs fuse, with a few gaps, and partial to full head eversion occurs. In these cases we classify 1(2)2Bc’ as an early pupal lethal. No further morphogenesis, however, takes place and the appendages remain as short structures characteristic of the late prepupa (like those in Figure 9). This suggests a role for Z(I)2Bc’ in appendage elongation. Because gene dose does not change the lethal phenotype, 1(2)2Bc’ behaves as an amorphic allele. Another allele, Z(2)2B2, gave similar results (data not shown). These results and conclusions agree with those of BELYAEVA et al. (1980).

Z(I)2Bab: As noted before (Table 2), 1(2)2Bab’ only partially complements Z(2)2Bc’ for viability and fails to complement the br and rbp complementation groups (Table 5 ) . l(I)2Bab’ behaves as a hypomorph

for the whole locus. In males, Z(2)2Bab’ exhibits late pupal developmental arrest with only a few specimens at 29” becoming pharate adults (Table 5) . Indicating that br function is defective in 1(1)2Bab’, the legs remain short and are malformed (Figure 9). In a single dose in females, the lethal phenotype is substantially more severe, resembling that of b? homozygotes (Table 5). At both 18” and 29” the imaginal discs form large swollen vesicles without forming a head and thorax. Another Z( I )2Bab allele, Z( 2 )2Bab?, has similar properties (data not shown).

The above results demonstrate that the Z(2)2Bab alleles in their total effects are hypomorphic. The activity states of the individual br, rbp, and Z(I)2Bc functions in 1(1)2Bab alleles were also investigated. Because the heteroallelic combination Z( I )2Bab’l I( I)2Bc’ has about 70% viability (Table 2), we conclude that the 1(2)2Bc function in 1(2)2Bab alleles is slightly reduced. I( 2 )2Bab’/b? heterozygotes have substantially earlier developmental arrest than that found in I( I )2Bab’ homozygotes, demonstrating that br function is hypomorphic. The 1(2)2Bab’/bJ individuals reach late prepupal and early pupal stages like those of Z(1)2Bc’ homozygotes (Table 5 ) . Hence, they reach a developmentally more advanced stage than Z( 2 )2Bub’/Df( I )S39 individuals. Almost all I( 2)2Bab’/rbp’ heterozygotes become pharate adults, whereas approximately 50% of rbp’ homozygotes stop developing as pupae, demonstrating that rbp function is hypomorphic in Z( 2 )2Bab’ (Table 5) . Similar results have been obtained with I( I )2Bab?. Consequently, the br, rbp, and I( 1 )2Bc functions are all hypomorphic in .

FIGURE 8.-(a) Leg discs from an 8-hr wild-type prepupa. The discs have elongated and everted and are beginning to fuse. (b) Four fused leg discs from a I(I)ZBc’ male with late prepupal developmental arrest.

FIGURE 9.--1(1)2Bab1 males at 18”. The short, distorted legs are visible through the pupal case (22 days after egg laying).

258 I . Kiss et al.

l(1)BBab alleles. Assuming that 1(1)2Bd’ is an rbp allele, then l(1)2Bab alleles are hypomorphic for all functions of the 2B5 locus. Consequently, 1(1)2Bab alleles, better named 1(1)2Babc alleles, are hypomorphic forms of nprl alleles.

Functional roles of BR-C alleles As suggested above, the br and 1(1)2Bc alleles may

affect the structure of functionally independent polypeptides (hereafter called the br and 1(1)2Bc polypeptides). Indeed, from three perspectives, the br and 1( 1 )2Bc domains appear to encode independent functions. One, as already discussed, amorphic br alleles fully complement amorphic 1( 1 )2Bc alleles for both adult phenotype and viability. Two, amorphic br alleles prevent elongation and eversion of appendages; 1(1)2Bc alleles primarily prevent disc fusion. Thus, each class of mutant appears to affect different aspects of disc morphogenesis. Three, Stubble mutants interfere with appendage elongation but not with disc fusion. Correspondingly, Stubble alleles interact with br alleles, but not with 1(1)2Bc alleles (BEATON et al. 1988).

Other evidence, however, suggests that the two hypothetical products may be functionally related. First, completely noncomplementing nprl alleles prevent pupariation, but none of the individual complementing alleles do so. Two types of possible explanations, simplistically framed in a molecular context, are evident. Both br and l(1)2Bc alleles affect pupariation. The amorphic br alleles produce untanned puparia and the amorphic 1(1)2Bc alleles produce misshapen puparia. Possibly, either the br+ product or the I( 1 )2Bc+ product is sufficient for pupariation. Consequently, the loss of function of both domains would be required to block pupariation. If so, a br 1(1)2Bc double mutant would be nonpupariating. nprl mutants are viewed as disrupting transcription of the entire BR-C. The second explanation, which we currently favor, is that nprl alleles affect a polypeptide domain essential for the function of any BR- C product. Conceivably, the hypothetical br and 1( 1)2Bc polypeptides each contain an nprl domain essential for their function. As a consequence, nprl alleles would not complement, and 1(1)2Bab alleles only partially complement br and 1(1)2Bc alleles. In as much as an nprl polypeptide, not involving the br or 1( 1 )2Bc genetic regions might be synthesized, there are no obligatory expectations for the phenotype of a br 1(1)2Bc double mutant.

A second result suggests that br and 1(1)2Bc can encode functionally related products. Our analyses suggest that 1(1)2Bc function is hypomorphic in I( 1 )2Bab alleles. l( 1 )2Bab’/l( 1 )2Bc’ heterozygotes have short, swollen legs somewhat similar to those found in b#/Df( 1)S39 individuals. Indeed, the legs are also short in I ( 1 )2Bc’/l( 1 )2Bc’ early pupae. These results indicate that loss of 1(1)2Bc+ function, like

loss of br function, can affect leg elongation. If the br and 1(1)2Bc polypeptides share a common (nprl ) domain, sharing a common function is not unreasonable.

A third type of result that involves the amorphic allele b? can be interpreted as suggesting that the proposed br and I( 1 )2Bc polypeptides are functionally related, but can be readily interpreted in other ways. We have described three instances where heterozygosity of a BR-C allele partially deficient in br function (b?6, bP7, and 1(1)2Bab’) with the amorphic b? allele, is less severe than heterozygosity with the amorphic npr13 allele (or with D f ( l ) S 3 9 ) . If we assume the br and l( 1 )2Bc products are functionally independent, it is seemingly surprising that, for example, b?6/b? develops farther (index = 4.1) than b?6/npr1’ (index = 3.0). Two, somewhat trivial explanations to these results are worth immediate mention. First is the possibility that the earlier developmental arrest of b?6/npr13 individuals is a nonspecific result of loss of half of all BR-C functions as opposed to loss of only br+ function. Second, is the possibility that the bP6/ b? genotype exhibits partial allelic complementation, but the b?61npr13 genotype does not. A third simplistic molecular explanation, suggesting functional redundancy, is that in the combination of a hypomorphic br allele with b?, the normal 1(1)2Bc polypeptide with a wild-type nprl domain would be produced at normal levels and could compensate for loss of functional br polypeptide. In the combination with an nprl allele, the level of functional l(1)2Bc polypeptide would be reduced so that less compensation could occur.

Despite the three examples cited immediately above, it is evident that br+ function is particularly important for early disc morphogenesis (prepupal appendage elongation and eversion); E( 1 )2Bc+ function for late disc morphogenesis (disc fusion and pupal appendage elongation). We cannot, however, discount the possibility that the products encoded by the two genetic domains share related functions. Recalling that the BR-C appears to encode a transcriptional regulatory protein (e .g . , CROWLEY, MATH- ERS and MEYEROWITZ 1984), it is possible, as specu- lation, that the br and I ( 1 )2Bc polypeptides are DNA binding proteins and that the nprl encoded region is essential for function. The BR-C has been cloned (CHAO and GUILD 1986; GALCERAN et al. 1986) and its genetic structure partially analyzed (BELYAEVA et al. 1987). Our analyses of BR-C cDNA clones (D. WITHERS and J. W. FRISTROM, unpublished data) indicate substantial transcriptional complexity which is compatible with our genetical deducations. None- theless, only a detailed molecular analysis will produce any real understanding of the locus.

2B5 mutants isolated by BELYAEVA et al. (1982) were generously provided by E. BELYAEVA and I. ZHIMULEV. H. GYURKOVICS kindly


aided in the determination of cytogenetic breakpoints. Supported in part by U.S. Public Health Service grant GM-19937.

LITERATURE CITED

AIZENZON, M. G., and E. S. BELYAEVA, 1982 Genetic loci in the X-chromosome region 2A-B. Drosophila Inform. Serv. 58: 3- 7.

AIZENZON, M. G., E. S. BELYAEVA and I. F. ZHIMULEV, 1982 Cytogenetic analysis of 2B1-2-2B9-10 region of Dro- sophila melanogaster X-chromosome. IV. Phenocritical and effective lethal phases of mutants. Genetika 18: 73-83.

ASHBURKER, M., 1972 Puffing patterns in Drosophila melanogaster and related species. pp. 101-152. In: Results and Problems in Cell Differentiation. Developmental Studies on Giant Chromosomes, Vol. 4, Edited by W. BEERMANX. Springer Verlag, New York.

ATHERTON, D., and J. GALL, 1972 Salivary gland squashes for in situ nucleic acid hybridization studies. Drosophila Inform. Serv. 49: 131-133.

BEATON, A,, 1. KISS, D. FRISTROM and J. W. FRISTROM, 1988 Interaction between the Stubble-stubbloid region and the Broad-Complex in Drosophila melanogaster. Genetics. Submitted.

BELYAEVA, E. S., M. G. AIZENZON, V. F. SEMESHIS, I . KISS, K. KOCZYA, M. BARITCHEVA, T. D. GORELOVA and I. F. ZHIMULEV, 1980 Cytogenetic analysis of the 2B3-4-2B 11 region of the X-chromosome of Drosophila melanogaster. I. Cytology of the region and mutant complementation groups. Chromosoma 81: 281-306.

BELYAEVA, E. S., I. E. VLASSCVA, Z. M. BIYASHEVA, V. T. KAKPAKOV, G. RICHARDS and I. F. ZHIMULEV, 1981 Cytogenetic analysis of the 2B3-4-2B 1 1 region of the X-chromosome of Drosophila melanogaster. 11. Changes in 20-hydroxy-ecdysone puffing caused by genetic defects of puff 2B5. Chromosoma 8 4 207- 219.

BELYAEVA, E. S., M. G. AIZEKZON, I. KISS, T. D. GORELOVA, S. PAK, G. H. UMBETOVA, P. G. N. KRAMERS and I. F. ZHIMULEV, 1982 New mutants. Drosophila Inform. Serv. 184: 184-190.

BELYAEVA, E. S., M. 0. PROTOPOPOV, E. M. BARICHEVA, V. F. SEMESHIN, M. L. IZQUIERDO, and I. F. ZHIMLLEV, 1987 Cytogenetic analysis of region 2B3-4-2B11 of the X- chromosome of Drosophila rnelanogaster. VI. Molecular and cytological mapping of the ecs locus and the 2B puff. Chro- mosoma 95: 295-310.

CHAO, A. T., and G. M. GUILD, 1986 Molecular analysis of the ecdysterone-inducible 2B5 ‘early’ puff in Drosophila melanogarter. EMBO J. 5: 143-150.

CROWLEY, T. E., P. H. MATHERS and E. M. MEYEROWITZ, 1984 A trans-acting regulatory product necessary for expression of the Drosophila melanogaster 68C glue gene cluster. Cell 39: 149- 156.

FRISTROM, D., and J. W. FRISTROM, 1975 The mechanism of evagination of imaginal discs of Drosophila mehogaster. 1. General considerations. Dev. Biol. 43: 1-23.

FRISTROM, D. K., E. FEKETE and J. W. FRISTROM, 1981 Imaginal disc development in a non-pupariating lethal mutant in Dro- sophila melanogaster. Wilhelm Roux’ Arch. Dev. Bioi. 190: 11- 21.

FRISTROM, D., A. BEATOX, I. KISS and J. W. FRISTROM, 1987 Cellular and genetic mechanisms of imaginal disc morphogenesis in Drosophila. pp. 235-246. In: U C o l Symposia on Cellular and Molecular Biology. The Molecular Biology of Inverte- brate Development, Edited by J. D. OCONNOR. Alan R. Liss, New York.

GALCERAN, J., C. GIMINEZ, J. E. EDSTROM and M. IZQUIERDO, 1986 Microcloning and characterization of the early ecdysone puff region 2B of theX chromosome of Drosophila melanogaster. pp. 249-254. In: Ecdysone: From Metabolism to Regulation of Gene Expression, Edited by M. BOWNES. Pergamon Press, Oxford.

GAREN, A., and S. GAREN, 1963 Complementation in vivo between structural mutants of alkaline phosphatase from E . coli. J. Mol. Biol. 7: 13-22.

JARRY, B. P., 1979 Genetical and cytological location of the structural parts coding for the first three steps of pyrimidine biosynthesis in Drosophila melanogaster. Mol. Gen. Genet. 172:

KISS, I., J. MAJOR and J. SZABAD, 1978 Genetic and developmental analysis of puparium formation in Drosophila melanoguster. Mol. Gen. Genet. 164: 77-83.

KISS, I., G. BENCZE, A. FODOR, J. SZABAD and J. W. FRISTROM, 1976 Prepupal-larval mosaics in Drosophila melanoguster. Na- ture 262: 136-138.

KISS, I., j. SZABAD, E. S. BELYAEVA, I. F. ZHIMULEV and J. MAJOR, 1980 Genetic and developmental analysis of mutants in an early ecdysone-inducible puffing region in Drosophila melanogaster. pp. 163- 18 1 . In: Development and Neurobiology of Droso- phila, Edited by 0. SIDDIQI, P. BABU, L. M. HALL and J. C. HALL. Plenum Press, New York.

LIXDSLEY, D. L., and E. H. GRELL, 1968 Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627.

LISDSLEY, D. L., and G. ZIMM, 1986 The genome of Drosophila melanogarter. Drosophila Inform. Serv. 64: 7-8.

MILKER, M. J., A. J. BLEASBY and S. L. KELLY, 1984 The role of the peripodial membrane of leg and wing imaginal discs of Drosophila melanogaster during evagination and differentiation in vitro. Wilhelm Roux’s Arch. Dev. Biol. 193: 180-186.

MOHLER, J., and M. L. PARDCE, 1984 Mutational analysis of the region surrounding the 93D heat shock locus of Drosophila melanogaster. Genetics 106: 249-265.

MORGAK, T. H., C. BRIDGES and A. H. STURTEVANT, 1925 The genetics of Drosophila. Bibliogr. Genet. 2: 145.

MULLER, H. J., 1932 Further studies on the nature and causes of gene mutations. Proc. Sixth Int. Congr. Genet. 1: 213-255.

NASH, D., and F. C. JANCA, 1983 Hypomorphic lethal mutations and their implications for the interpretation of lethal complementation studies in Drosophila. Genetics 105: 957-968.

PERRIMOK, N., L. ENCSTROM and A. P. MAHOWALD, 1984 The effects of zygotic lethal mutations on female germ-line functions in Drosophila. Dev. Biol. 105: 404-414.

POODRY, C. A., 1980 Imaginal discs: morphology and development. pp. 407-441. In: The Genetics and Biology of Drosophila, Vol. 2d, Edited by M. ASHBURNER and T. R. F. WRIGHT. Academic Press, New York.

RAWLS, J. M., and J. W. FRISTROM, 1975 A complex genetic locus that controls the first three steps of pyrimidine biosynthesis in Drosophila. Nature 255: 738-740.

SCHLESIKGER, M. J., and L. C. LEVINTHAL, 1963 Hybrid protein formation of E . coli alkaline phosphatase leading to in vitro complementation. J. Mol. Biol. 7: 1-12.

STEWART, B., and J. MERRIAM, 1980 Dosage compensation. pp. 107-140. In: The Genetics and Biology of Drosophila, Vol. 2d, Edited by M. ASHBURNER and T. R. F. WRIGHT. Academic Press, New York.

STEWART, M., C. MURPHY and J. W. FRISTROM, 1972 The recovery and preliminary characterization of X chromosome mutants affecting imaginal discs of Drosoplrila mehogaster. Dev. Biol.

199-202.

27: 71-83.

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Interactions and Developmental Effects of Mutations in the Broad … · 2002. 7. 8. · Allele...

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