studies of normal and position-affected ...storrs, connecticut 06268 manuscript received march 17,...

Copyright 0 1986 by the Genetics Society of America

STUDIES OF NORMAL AND POSITION-AFFECTED EXPRESSION OF ROSY REGION GENES IN DROSOPHILA

MELANOGASTER

STEPHEN H. CLARK AND ARTHUR CHOVNICK

Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06268

Manuscript received March 17, 1986 Revised copy accepted July 16, 1986

ABSTRACT

Transformant complementation, intragenic deletions and Northern blot anal- yses provide unambiguous localization of the 1(3)S12 gene immediately proximal to the 5’ end of the rosy locus. We have characterized an array of transformants with respect to 1(3)S12 and rosy expression. The 1(3)s12 gene is exceedingly sensitive to euchromatic site-specific position effects. Unlike the rosy locus, 1(3)S12 is insensitive to heterochromatic position effect in rearrangements, as well as in a transformant located in heterochromatin. Cotransformants for both 1(3)S12 and rosy elicit no apparent pattern of concordance with respect to euchromatic site-specific position effects. Heterochromatic-euchromatic rearrangements are examined with respect to position effects on expression of the rosy region genes 1(3)12, rosy, snake and piccolo, as well as suppressor effects. Clear distinction is seen between euchromatic and heterochromatic effects.

N a prior report (DANIELS et al. 1986), we examined the underlying bases I for expression differences in stable, rosy locus transformants. We concluded that these line-specific expression differences were due largely to chromosomal landing-site-specific position effects that must result from influences of neighboring genomic sequences upon transposon expression. Two classes of position effects were seen. One class, represented by the only rosy locus transposon inserted into heterochromatin, exhibits classical heterochromatic position effect variegation, as seen in earlier work with heterochromatic rearrangements (RUSHLOW, BENDER and CHOVNICK 1984; RUSHLOW and CHOVNICK 1984). Such expression is characterized by (1) underexpression measured in whole organisms, (2) all-or-none variegated expression visualized in histochemical preparations of Malpighian tubules and (3) modification of expression by the Y chromosome. The second class, represented by all of the other transposons examined (14 lines), differed from the former as follows: (1) The transposons were located at various euchromatic chromosomal sites. (2) Variation in rosy locus expression included a full range of expression differences including overproducers as well as underproducer lines. (3) Variation is not subject to Y chromosome modification. (4) Malpighian tubules of underexpression trans- Genetics 114: 819-840 November. 1986

820 S. H. CLARK AND A. CHOVNICK

ry snk Hsc 70-2 -200 -190 -180 - 170 - 160 ~ 150 - 140

' 1 1 1 1 1 1 1 1 l I ' I I I 111 I I I I 1111 II ni I 1111 1 1 I I I M I 1 1 1 1 1 1 1 II

8 8 RRBRRHRR HRR R H R S SHBHSH BRBBBHRSR RB H R H BSBHRRH HB BR SB B

4 .,. ...... -b f--- .......... O f f 3 R I r ~ ~ ~ Df (3 R l t X d

......& Df (3UI kor eG27

t B -Born H I H -Hind Ill SSaI I R-€CO R I

rY ps 1N36

CbxUbx

(pic 1

FIGURE 1.-Genetic and molecular map of the immediate region flanking the rosy locus. More detailed descriptions on the rearrangements are to be found in HILLIKER et al. (1980) and KUSH- LOW and CHOVNICK (1984). The DNA map is that of BENDER, SPIERER and HOGNESS (1983), and the various deficiency endpoints are presented in GAUSZ et al. (1986). The dashed lines represent the uncertainty in localization of breakpoints.

posons exhibit uniform underexpression like that seen with underexpression control variants and leaky coding element mutants.

The present report extends our study of transposon expression variants to include the 1(3)S22 gene, which is located adjacent to the rosy locus and is present with rosy in the same transposon. Consequently, we are able to compare the expression of both genes in each transposon examined. In addition, the study of heterochromatic position effect is extended to include several heterochromatic rearrangements, and their spreading effects upon several adjacent rosy region genes. Finally, we extend observations made in earlier studies (HEN- IKOFF 1979; RUSHLOW and CHOVNICK 1984) indicating that the heterozygous deletion of polytene segment 87E2-F2 is a general suppressor of heterochromatic position effect.

THE GENETIC SYSTEM

Figure 1 presents a restriction map of the rosy region located on the right arm of chromosome 3. We define the genetic interval pertinent to the present discussion as that extending from the centromere distal break point of Df(3R)ka~"~' to the distal break point of D f ( 3 R ) r ~ ' ~ . In a prior report (HILLI- KER et al. 1980), three complementation groups were localized to this interval. These complementation groups include, beginning with the most proximal genetic unit, l(3)S12, rosy and piccolo(pic). The l(3)S12 and rosy genes are localized to the DNA segment to the right of the distal break of D f ( 3 R ) k a ~ " ~ ~ and to the left of the distal break of D f ( 3 R ) ~ y ~ ~ .

The l(3)S12 complementation group (HILLIKER et al. 1980) consists of three mutant alleles. The X-ray-induced mutation, l(3)S12, and the EMS mutation, l(3)B21-4, are completely lethal in hemizygotes, whereas Z(3)GZ (EMS-induced

ROSY REGION GENES IN DROSOPHILA 82 1

i

1 i

: I

I

I

I

I 1 i

FIGURE 2.-Scanning electron micrographs illustrating the bristle phenotypes associated with I(3)S12 and piccolo mutants. A, T h e extreme bristle phenotype observed among infrequent survivors of the genotype 1(3)G1/1(3)821-4. T h e thinner and shorter bristle morphology illustrated in this scanning electronmicrograph is a characteristic phenotype associated with partial 1(3)S12 complementation (see discussion in HILLIKER et tal. (1980). B, Full complementation of the 1(3)S12 morphological phenotype in adults (l(3)SI2/ry"). C , T h e pic visible phenotype associated with In(3R)ry" (In(3R)ry"/hr2 ry'wpic'c2'; X/X female). Note the missing and short posterior scutellar bristles. D, T h e suppression of the pic visible phenotype associated with In(3R)ry" by the addition of extra heterochromatin (In(3R)ry"/hrz ryrospic'c2J; X . Y / X female). T h e individual in this micro- graph has normal posterior scutellar bristles.

mutation) is associated with low hemizygous viability. Surviving hemizygotes of l(3)GZ express a phenotype of very thin and short thoracic bristles (Figure 2). A pattern of allele complementation is seen in that 1(3)SZ2 fully complements with 1(3)G1 and neither fully complements with l(3)B2I-4. Further definition of the l(3)SZ2 locus is provided by the rosy position-effect mutation, ryf"'"36, which does not complement l(3)S12. This mutation is associated with a heterochromatic-euchromatic rearrangement involving a euchromatic break between the Hind111 site a t -174 kb and the Sal1 site a t -174.4 kb (Figure l ) (RUSHLOW, BENDER and CHOVNICK 1984). In contrast to the Y-suppressed rosy mutant phenotype associated with ryf""'36, the 1(3)S12- phenotype is not Y suppressed. This is consistent with the notion that the rearrangement break is located within the l(3)SZ2 gene.


Variation at the rosy locus has been the subject of prior review (CHOVNICK et al . 1977; CHOVNICK et al. 1980). The locus codes for a polypeptide of 150,000 daltons, which functions as a homodimer as the enzyme xanthine dehydrogenase (XDH). Total inactivation of the enzyme is associated with a visibly, brownish mutant eye color. However, individuals with low levels of enzyme activity (e.g., “leaky” coding mutations, “underproducer” control variants and position-effect variants) exhibit a wild-type eye color.

The pic gene has been localized to the right of the distal break of Df(3R)ryj6 and located in the DNA segment missing in both D f ( 3 R ) ~ y ~ ~ and Df(3R)Z26d. Further definition of pic is provided by the location of three rearrangement breakpoints associated with pic- mutations (Figure 1). A rearrangement associated with a reversion of Deformed, Tp(3;3)Dfd+Rx’6, is pic- and is associated with a break within the 0.8-kb EcoRI fragment at -152 (SCOTT et al. 1983). Another pic allele, isolated as a revertant of Cbx, Cbx Ubx2’988B, has an inversion breakpoint in the 4.2-kb EcoRI fragment at -148 to -152 (BENDER, SPIERER and HOGNESS 1983). Additionally, the rosy position-effect mutation, T(3;4)ry@”49, does not complement pic and is associated with a heterochromatic rearrangement involving a euchromatic break (Figure 1) at -152 within the 0.8-kb EcoRI fragment (RUSHLOW, BENDER and CHOVNICK 1984). The pic- phenotype is not Y suppressed (as is the rosy phenotype), consistent with the breakpoint being located within the pic locus DNA. The pic locus (SCHALET, KERNAGHAN and CHOVNICK 1964; HILLIKER et al. 1980) consists of a group of 31 recessive lethal and semilethal alleles, and the locus is characterized by allele complementation. Surviving mutants (homozygotes or mutant heteroal- lele combinations) are associated with reduced viability and exhibit the pic phenotype; namely, shortened and missing thoracic bristles, abnormal tergite morphology, and occasional wing defects (Figure 2).

The positions of two additional genes located within this region are indicated in Figure 1. Snake(snk), a maternal effect gene, was placed in this region by virtue of its inclusion in the region of Df(3R)ryj6 and exclusion from D f ( 3 R ) k ~ r “ ~ ’ (ANDERSON and NUSSLEIN-VOLHARD 1984). Additionally, these workers reported snake to be nonallelic to 1(3)S12 and rosy. Snake has been further localized to the DNA segment immediately distal to rosy (R. DELOTTO and P. SPIERER, personal communication). A heat-shock cognate gene (Hsc 70- 2), defined by CRAIG, INGOLIA and MANSEAU (1983) and located in the 87D region by in situ hybridization, was placed at position -159 on the DNA map by BENDER, SPIERER and HOGNESS (1983).

MATERIALS AND METHODS

Special chromosomes, rearrangments, balancers and mutants: Th,-oughout the manuscript several abbreviated chromosomes designations have been employed, including MKRS = Tp(3)MKRS, M(3)S34 kar rf Sb; YFX.YL = YsX.YL, In(l)EN, B f U y.y+. A complete description of mutants and rearrangements used in the present study may be found in SCHALET, KERNACHAN and CHOVNICK (1964); LINDSLEY and GRELL (1968); HILLIKER et al. (1980); and RUSHLOW and CHOVNICK (1984).

Strains employed: Exceptional lines containing P-element transposons were obtained from three sources. ALLAN SPRADLINC provided several ry+ transformed lines used in

ROSY REGION GENES IN DROSOPHILA 823

this study. A complete description of these strains is found in SPRADLING and RUBIN (1983), and their nomenclature is continued here. Ddc transformants were provided by JAY HIRSH (SCHOLNICK, MORGAN and HIRSH 1983). Finally, several lines were produced in this laboratory. A detailed description of these may be found in DANIELS et al. (1986). Flies were reared at 25 O on cornmeal-sucrose-yeast extract Drosophila medium.

Y s X . Y L / O ; h r 2 ryio6 ~ ~ c " ~ ~ / M K R S males were employed to generate XXY genotypes. Snake alleles were provided by KATHRYN ANDERSON (ANDERSON and NUSSLEIN-VOL-

HARD 1984). Northern analysis: RNA was extracted, chromatographed on oligo-dT, electropho-

resed, transferred to nitrocellulose and hybridized with radiolabeled probes, using procedure similar to those described by RUSHLOW, BENDER and CHOVNICK (1984).

Southern blot analysis: Genomic DNA was extracted from adult flies by the method described in CLARK et al. (1984). The procedures for restriction enzyme digestion, agarose gel electrophoresis, gel blotting and filter hybridizations are described in RUSH- LOW, BENDER and CHOVNICK (1 984) and CLARK et al. (1 984).

Plasmids: pry8.1. This plasmid contains an 8.1-kb Sal1 ry locus DNA fragment isolated from a Canton-S strain cloned into the plasmid vector pBR322 (provided by WELCOME BENDER).

Plasmid DNA was prepared by standard ethidium bromide, cesium chloride equilib- rium centrifugation following sodium dodecyl sulfate (SDS) lysis of chloramphenicol- amplified, plasmid-bearing bacteria (MANIATIS, FRITXH and SAMBROOK 1982).

Isolation of DNA fragments for nick translation: SaZI-SstI and XhoI-Sal1 fragments were liberated from the plasmid by appropriate endonuclease digestion. The liberated fragments were separated by agarose gel electrophoresis and were recovered from the gel by electrophoresis onto ion exchange paper (NA 45 Schleicher and Schuell, Keene, New Hampshire). The bound DNA was recovered by elution from the ion exchange paper and ethanol precipitation (DRETZEN et al. 1981).

Rocket immunoelectrophoresis: This procedure is described in MCCARRON et al. (1979).

Scanning electron microscopy: Flies were fixed in glutaraldehyde/Aerosol (a wetting agent) solution. Fixed animals were subject to critical point drying and were subsequently coated with gold. Coated specimens were examined with a Coates and Welter HPS-50 scanning electron microscope.

RESULTS

Physical separation of the Z(3)S12 gene and rosy: Z(3)SZZ and rosy are believed to constitute separate genetic units on the basis of classical complementation analysis. Recombination mapping placed the l(3)S12 mutation to the left of the leftmost sites within the rosy locus (CHOVNICK et al. 1976; MCCARRON et aE. 1979). Unequivocal separation of these genes was lacking since there were no classical rearrangements that separated these complementation groups. However, distinction is accomplished by the combination of transposon complementation and deletion complementation tests summarized in Figure 3. The introduction of functional ry+ DNA into the Drosophila genome was first achieved by P-M dysgenesis-mediated transformation involving an 8.1-kb Sal1 DNA fragment of the rosy region (Figure 3A) inserted into a P-element transposon (RUBIN and SPRADLING 1982). In subsequent experiments, a transposon carrying a smaller fragment of the rosy region (7.2-kb Hind111 fragment) (Fig- ure 3B) also was found to provide ry+ function (SPRADLING and RUBIN 1983). Still another ry+ transposon is a modified 8.1-kb Sal1 fragment with the dopa decarboxylase (Ddc)-bearing sequence inserted into a PstI site at 0.3 kb from


Complementotion

t ( 3 ) S l Z ry

A. I ; : I 8.1 SuPI t t

ROSY REGION DNA

S H P H S

I 8.1 sui I , a - + H S

Ddc insert into Psi I site

Ddc insertion

S - 6 t I H - Hmd III P - PstI

FIGURE 3.-Rows A, B and C present relevant restriction maps of several transposons. 1(3)S12 and rosy gene expression associated with each transposon i s indicated. Row D presents a restriction map of Df(?R)ry506 and indicates l(?)Sl2 and rosy expression.

the left end Hind111 site (Figure 3C) (SCHOLNICK, MORGAN and HIRSH 1983). In contrast, deletions, insertions and other genetic rearrangements with breaks in the rightmost or distal 4.0 kb of the 8.1-kb Sal1 DNA segment are known to eliminate ry' function in vivo (CLARK et al. 1986; COTi et al. 1986). Figure 3D illustrates one such deletion mutation, ry506, which is missing more than 3 kb of the distal, 3', end of the rosy DNA. In summation, these observations serve to limit the ry+-specific DNA to the distal 6.9 kb of the 7.2-kb Hind111 fragment.

With respect to l(3)SlZ' function, we note that ry5'06 homozygotes as well as ry5u6/Df(3R)ry36 individuals are fully viable and are wild type with respect to the E(3)S12 morphological phenotype (Figure 2). Tests of the ability of the several transposons of Figure 3 to introduce l(3)S12+ function as transformants was accomplished by the crosses outlined in Figures 4A and B. The complementation results, summarized in Figure 3, indicate that 1(3)S12+ function is limited to the left end of the 8.1-kb Sal1 fragment, clearly separable from the rosy locus expression of these transposons. The Z(l)S22 and rosy transcripts: The combined genetic and molecular data

discussed above indicate that the 1(3)SZ2 gene includes only a relatively small segment of the 8.1-kb Sal1 DNA. This notion is supported further by the results of large-scale mutagenesis studies carried out over many years; only three mutant alleles of l(3)S12 were recovered, in contrast to the extensive recovery of mutations of rosy and other loci in the region (HILLIKER et al.

In an effort to identify a E(3)S12 transcript, poly-A+ RNA was extracted from whole third-instar larvae. This developmental stage was selected since previous studies had shown that the rosy transcript is readily detected at this

1980).


A

PI 2nd' 2' X

CyO, MKRS dd Tf23JopXo

X IY dd Znd* - CY0 ; MKRS

&orP t(3JSfZ ry4Oz MKRS 99 FI

X 2nd'. karP C(3JSf2 ry4OZ &orZDf /3RJ U 3 J s 1 2 - r ~ - 2nd ' MKRS *' F2 MKRS 99

score r y + Sb+

X &or ' Df GQ/ (3JSf2-ry- X ', &orz t(3JSf2 ry402dd X ' MKRS Y MKRS PP - -

score r y + Sb' FIGURE 4.-A, The mating scheme employed to assess the 1(3)SI2 expression associated with

several y+ transposons inserted into chromosome 2. The second chromosome carrying the y+ transposon is denoted by 2nd*. The kar' D f ( 3 R ) 1 ( 3 ) S I 2 - y - l R S females used in the F2 mating represent two deficiency bearing stocks employed in this study (kar' Df(3R)ry7'/A4KRS and kar' Df(3R)ry"68/MKRS). Expression associated with the fourth chromosome transposon [R401. I] was tested using a similar protocol. B, The mating scheme utilized to examine X-linked y+ transposons for 1(3)SJ2 function. In this case X* denotes the X chromosome bearing the transposon. The deficiency females used in this mating were kar' Df(3R)ry JJs8/MKRS.

stage and would provide a convenient internal control in the analysis (CLARK et al. 1984). Poly-A+ RNA was electrophoresed and transferred to nitrocellulose paper. Figure 5 illustrates RNA blots probed with the indicated "P-labeled DNA probes. Figure 5, panel A illustrates the result obtained from a filter probed with a plasmid bearing the entire 8.1-kb Sal1 fragment. Two strong signals are detected: a large signal (approximately 4.6 kb) and a much smaller signal estimated to be 0.6-0.8 kb. Since the large signal had previously been identified as the rosy transcript, we infer that the much smaller signal represents the 1(3)S12 transcript. This is confirmed by the results illustrated in Figure 5, panel B. The SalI-SstI fragment probe (panel B, lane 2) produces a dramatic increase in the signal intensity associated with the very small fragment, whereas only a modest signal is observed for the 4.6-kb rosy transcript. The predicted reverse signal intensities are observed with the XhoI-Sal1 probe

826

A

I kb U

S. H. CLARK AND A. CHOVNICK

4.6 4

.6-.8 + +

B 3 . '

FIGURE 5.-Northern blot autoradiogram of poly-A+ RNA extracted from late third-instar larvae of the transformed line [R310.1]; 'y42/ry42. which contains multiple copies of the 8.1-kb SulI fragment (SPRADLING and RUBIN 1983; RUBIN and SPRADLING 1983). A, Three lanes representing serial dilutions of poly-A+ RNA hybridized with a '*P-labeled 8.1-kb Sal1 probe. B, Lanes 1 and 2 illustrate poly-A' RNA probed with a "P-labeled XholSulI and SalISstI fragments, respectively. DNA fragments used as probes are illustrated below the panels, and the approximate location of l(3).S12 and roq is indicated.

(panel B, lane 1). Together these data point to the small 0.6-0.8-kb RNA as being transcribed from the segment of DNA shown to be associated with the l(3)SZ2 gene. The faint signal observed for the 0.6-0.8 transcript (panel B, lane 1) is believed to result from probe contamination resulting from the isolation procedure (see MATERIALS AND METHODS). The faint 4.6-kb signal (noted in panel B, lane 2) is due to probe contamination, as well as to a small region of homology between the rosy transcript and the SalISstI fragment.

Variation in 2(3)S22 expression associated with transformants involving the 8.1-kb Sal1 region: As noted above (Figure 3), the transposon that carries the 8.1-kb Sal1 fragment produces transformants that complement both l(3)SZ2 and rosy mutants. Following the breeding protocols of Figure 4A and B, we have examined l(3)SZ2 expression associated with 18 different second and X-chromosome insertions and the single documented case of a transposon, [R40Z.Z], located in or near the fourth chromosome heterochromatin (SPRADLING and RUBIN 1983; DANIELS et al. 1986). In all instances, we examined individuals whose third chromosomes are hemizygous, 1(3)SZ2/-. The autosomal insertions were examined as a single doses of the transposon in both males and females. For X-chromosome insertions, we examined only hemizygous, singledose females. Table 1 provides a summary of our observations on the expression of the 19 transposons. As in several other studies (reviewed in DANIELS et al. 1986), the transposons exhibit insertion-specific differences in

ROSY REGION GENES IN DROSOPHILA

TABLE 1

1(3)S12 phenotypes for 19 transformants carrying the 8.1-kb Sal I fragment bearing transposon on the indicated chromosomes

827

Chromosomal location

Phenotype" 4 heterc-

1 2 chromatin Totals

I( 3)s 1 2+ 3 1 1 5 l(3)S 1 2* 1 1 Mild bristle phenotype 2 8 10 Extreme mutant bristles 2 2 K3)S 12- 1 1

a See Figure 2 for bristle phenotype. 1(3)S12* = largely wild-type progeny, occasional individual with slight bristle phenotype. 1(3)S12- = no ry+ Sb' survivors among more than 75 progeny (see Figure 4). Extreme mutant bristles = one of the two is a semilethal, in that only a few ry+ Sb+ progeny survived among more than 75 progeny.

TABLE 2

Comparison of the XDH CRM levels of the indicated genotypes (Figure 6) with their 1(3)S12 expression (Table 1)

Transposon" XDH" CRM' l(3)S12 Genotype location activity level phenotype Transposon

[R303.1 ];ryr2/rySo6 42AB 0.38 >L,


FIGURE 6.-Rocket electropherogram. Matched extracts from adult males were run against anti- XDH serum. Samples were applied to wells as follows: ( 1 ) [R303.I]/-; ryi2/ry’06; (2) [R301.1]/-; ry*’/ry”; (3) [R;IOS.I]/-; ry4’/ly506; (4) [R304.1]/-; ry4’/ry”; (5) 25% ry+‘/rym; (6) 50% ry+4/

XDH CRM data summarized in Table 2. Thus, [R303. Z] and [R304. I ] illustrate phenotype concordance ([R303. I ] exhibits low XDH expression and a l(3)SZ2- phenotype, whereas [R304.Z] appears to be normal with respect to these char- acteristics). In contrast, [R30I. I ] and [R305. Z ] exhibit differing degrees of phenotypic discordance.

Transposon expression and DNA sequence organization: Prior reports (HAZELRIGG, LEVIS and RUBIN 1984; DANIEU et al. 1985, 1986) document the mutagenic effect of P-M dysgenesis on a P-element transposon. Consequently, we examined the DNA sequence integrity of the several transposons of Table 2 by Southern blot analysis. The logic for the restriction analysis is presented in Figure 7, and the Southern blots are presented in Figure 8. Figure 8 (panels A and C) presents blots of PvuII and BglII digestions of DNA from each of the transformant lines. Three points are documented by these blots: (1) The orientation of the 8.1-kb Sal1 fragment of each transposon. (2) That each line contains only one transposon is determined by the presence of only one variable fragment in each lane in the PvuII blot and two variable fragments in each lane in the BglII blot (see Figure 8 legend and CLARK a i d Chovnick 1985). (3) Gross perturbations of the transposon DNAs are not found. In an effort to examine this issue further an additional experiment w a s conducted with the restriction enzyme HindIII. The HindIII digestion (Figure 8, panel B) examines the integrity of the small 1.1- and 0.6-kb fragments (associated with the l(3)SZ2 segment of pry1 and pry3, respectively), as well as the other fragments in the transposon. In aggregate, these experiments failed to find any indication of perturbation of the DNAs of the several transposons of Table 2. However, very small alterations, such as single base changes or rearrangements involving only a few bases would have escaped detection by this analysis.

Transposon expression and euchromatic position effect: In a prior report (DANIELS et al. 1986), we demonstrated that the low level of rosy locus expression associated with the transposon [ ~ y + ~ ~ - ’ ’ ] , which is not subject to Y-chromosome modification, is due to a euchromatic position effect. This was accom-

ly”; (7) 100% ry+*/ry”.


I(3)SIZ row

3.75 1.0 2.45 - -)mI

1.1 - 7.2 ) Hind IlI

829

A

Pry I

B

Pry 3

C

ry 42

V 1.4 3.4 -r* Y“) Bgt H

rosy &3k3Z H P W P P E P E E B H H

1.3 2.45 -- 1.0 V -

- 1.2 22 ) Hind lU V 1.4 0.6

4) 8g1 I 1.4 - V - - 3.4 -

&3lSl2 rosy E H P H B E E P B P PHB P B H U/ : : : : : : :: : : : :: : : I &

6.5.- HindII[ 1 IB 7.2 ~ , -10 1.4 3.4

B d J I M - k

I kb - FIGURE 7.-A and B, Restriction maps of transposons pry1 and pry3 that contain the 8.1-kb

SalI fragment from the rosy region (the solid line denotes the 8.1-kb Sal1 fragment, and P-element sequences are indicated by hatched boxes; vector DNA not shown). Below each transposon is illustrated the restriction fragments expected in whole genome Southern analysis of a transformed line (containing the respective transposon) hybridized with a radiolabeled 8.1-kb SalI probe. Frag- ment sizes are indicated in kilobases, and V denotes variable fragment diagnostic of each inde- pendent insertion site. C, A restriction map of the 9‘’ allele. The thin line represents the limits of the 8.1-kb SalI fragment. Below the map are the restriction fragments observed in a whole genome Southern analysis utilizing radiolabeled 8. I-kb SaEI DNA as probe.

plished by relocation of the transposon, intact, to a different, euchromatic site in the genome by P-M dysgenesis, and by demonstrating that the relocated derivative, [ ~ y ‘ ~ - ’ ~ - ~ ] , has normal rosy locus expression. Table 3 summarizes these observations and extends the phenotypic characterization to include the l(3)S 12 phenotype. In this instance, we note a concordance of position-affected phenotypes, in that [r~+’~-’~], at polytene segment 57F, exhibits underexpression for both 1(3)S12 and rosy. Upon relocation to polytene segment 68A, the derivative transposon [7y+i4-’a-4] exhibits wild-type expression of 1(3)S12 and a normal rosy locus XDH level.

Effect of heterochromatin on I(3)S12 expression: Among the transposons examined for 1(3)S12 expression (Table l), we included [R401.1], which is the


A B C

1 2 3 4 1 2 3 1 2 3 4

"- 34-

2.0- '

- 7T-7

CSYI

72--1

I

4.2 -

2.45- 1.4-

I .8

1.3- .(I)- , 1.2. I .I

6-

.7 .6

FIGURE 8.-Autoradiogram of whole-genome Southern blot of several transformed lines. DNA samples digested with the indicated restriction enzymes were loaded into all lanes as follows: lane (1) [R301.1]; ry4'/tyYl2; lane (2) [R303.4 ry'2/ry42; lane (3) [R304.I]; ry"/ry"; lane (4) [R305.1]; ry"/ty". The pry8.1 DNA was used as probe, and sizes of restriction fragments are indicated. The arrows in panel A denote the single fragment associated with each insertion (see text and CLARK and CHOVNICK 1985). The single diagnostic fragment in lane (2) is slightly smaller than the 2.18-kb ry" genomic fragment.

TABLE 3

Comparison of the XDH CRM levels of the indicated genotypes with their 1(3)S12 expression ~~~ ~~~~

Transposon' XDH' CRM I(S)S12 Genotype location level phenotype Transposon

[lytic" l;tym/rym 57F >L,


TABLE 4

Euchromatic-heterochromatic position effects in the rosy region: effects on expression of the indicated genes”, and effects of suppressors and enhancers

83 1

Gene expression

Rearrangement (transposon) 1(3)S12 ry snk pic

[401.1]

Position-effect suppression ( X X Y ) Position-effect enhancement ( X O ) Position-effect suppression

(Df 87E2-F2)

In(?R)ryJJJ36

Position-effect suppression ( X X Y ) Position-effect enhancement ( X O ) Position-effect suppression

(Df 87E2-F2)

In(3R)ry54 Position-effect suppression ( X X Y ) Positioneffect enhancement (XO) Position-effect suppression”

(Df 87E2-F2) T(?$)ryPJJJ49

Positioneffect suppression ( X X Y ) Position-effect enhancement ( X O ) Position-effect suppression

(Df 87E2-F2)

+ NA

NA -

Lethal

+ NA

NA

+ NA

NA

Underex- pression + + +

Underex- pression + + +

v- - NA -

Underex- pression + + NP

NA

NA NA NA

Female sterile

NP

+ NA NP NA

Female sterile

NP NP

NA

NA NA NA

Underex- pression

Lethal

+(lethal) +(Pi4

+(pic)

Lethal

All phenotypes were examined among progeny of reciprocal crosses. No parental effects were observed (see review, SPOFFORD 1976).

[401.1]: SPRADLING and RUBIN (1983); DANIEKS et al. (1986). In(?R)ry”‘36: RUSHLOW and CHOVNICK (1984). In(3R)ryS4: SCHALET, KERNAGHAN and CHOVNICK (1 964); LEFEVRE (197 1); COT^ et al. (1 986). T(3;4)rypsJJ49: RUSHLOW and CHOVNICK (1984). NA, Not applicable; NP, test not possible.

chromosome modification (DANIELS et al. 1986), a feature of heterochromatic position effects. Of particular interest is the fact that this transposon is associated with wild-type expression of the 1(3)S12 gene (Table 4).

The table summarizes observations on heterochromatic position-affected expression of the rosy region genes in three rearrangements, as well as in [R401. I ] . T(3;4)ryPs1149 has a break in the pic locus (Figure l), and the insertion of heterochromatin results in severe position-affected expression of the snake and rosy genes more than 10 kb away. In the case of rosy, the heterochromatic insertion is more than 15 kb from the 5’ end of the locus. This distant heterochromatic insertion, 3’ to the rosy locus, exerts a severe, variegated position effect on rosy expression, which is subject to suppression by the addition of extra Y-chromosomal heterochromatin to the genotype. Yet, less than 2 kb


further upstream lies the 1(3)S12 gene, apparently unaffected by this heterochromatic perturbation.

Similarly, Z r ~ ( 3 R ) r y ~ ~ has a break in the rosy locus, and the insertion of chro- mocentric heterochromatin exerts a severe Y-suppressed position effect on p ic locus expression some 15 kb away. This rearrangement has no effect on expression of the adjacent 1(3)S12 locus. However, it should be noted that the breakpoint in rosy separates 1(3)S12 and pic , placing them adjacent to different heterochromatic segments. Thus, the failure of 1(3)S12 to respond to the heterochromatic position effect, in this instance, may reflect differences in the respective heterochromatin adjacent to l(3)S12 and to pic , rather than to an intrinsic feature of the E(3)S12 locus.

Znn(3R)~y”’~~ has a break in the 1(3)S12 locus (Figure l ) , and the insertion of heterochromatin at this point exerts a position effect on expression of genes distal to the rearrangement (Table 4). However, the 1(3)S12 mutant phenotype associated with this rearrangement is not a position effect, but represents inactivation of the locus by the rearrangement. It is not subject to Y chromosome suppression (Table 4). Thus, for three heterochromatic-euchromatic associa- tions that are associated with position-affected expression of rosy region genes, and for which we might have expected to see a position effect on 1(3)S12 gene expression, we have failed to see one. Although it is possible to explain away this failure in one instance, the remaining failures are significant.

The spreading effect: A classic feature of heterochromatic position effect is the observation that the disturbed gene expression brought about by the euchromatic-heterochromatic rearrangement may extend for some distance and involve several genes (see reviews: LEWIS 1950; BAKER 1968; SPOFFORD 1976).

We have examined this phenomenon with respect to three euchromatic- heterochromatic rearrangements: Zn(3R)ryJ’”1136 , T(3;4)ryps1149 and I n ( 3 R ) r ~ ~ ~ . As noted above (Figure 1 and Table 4), In(3R)ryP’”’36 has a break in the l(3)S12 locus and is associated with a Z(3)S12- phenotype that is not amenable to Y chromosome position-effect suppression. However, the rosy locus exhibits variegated underexpression and responds to classical heterochromatic position- effect modification (Table 4). Although untested for modifier effects, this rearrangement break produces mutant effects on snk and p ic more than 15 kb distal to the rearrangement break. In tests of two snk alleles, F1 females, snk/ Zn(3R)ryP”1‘36 are infertile, producing large numbers of inviable embryos. We have examined and failed to find mutant effects on the genes proximal to 1(3)S12 (“A, mes-B and 1(3)G9) as well as on l (3 )SS , located distal to pic [see HILLIKER et al. (1980) for complete genetic map of this region].

Similar experiments were carried out with T(3;4)ryPs’’49, which has a break in the p ic locus DNA (Figure 1) and is associated with a pic- phenotype that is not modified by heterochromatic position-effect modifiers (Table 4). This rearrangement affects both snk and ry expression. The rosy locus, some 15 kb proximal to this breakpoint, exhibits classical heterochromatic position-effect variegation and responds to position-effect modifiers. The snake gene, located immediately distal to rosy (between rosy and the breakpoint), is severely affected, as with Zn(3R)ry22236. In this experiment, since D f ( 3 R ) ~ y ~ ~ is snk- (Figure


I), we examined females of the genotype T(?;4)ryPS”49/Df(3R)ry36 as well as T(3;4)ryP””49/snk. Both classes of females are infertile, producing large numbers of inviable embryos. In contrast, the survival of the D f ( 3 R ) ~ y ~ ~ heterozygous individuals with wild-type bristles (similar to those of the Z(3)S12/ry506 fly shown in Figure 2B), as well as individuals of the genotype T(3;4)r~P””~~/Z(3)S12 (Ta- ble 4) indicates failure of the heterochromatic position effect to spread to the 1(3)S12 locus (Table 4). Moreover, the wild-type phenotype associated with these individuals indicates failure of the position effect to spread proximally to include mes-A, mes-B or Z(3)G9 loci; nor does it have an effect on expression of the distally located 1(.3)S8 locus in a separate test against 1(3)S8.

and T ( 3 ; 4 ) ~ y ” ~ ~ , we fail to observe a spreading effect with Z n ( 3 R ) r ~ ~ ~ , which has a euchromatic- heterochromatic break within the rosy locus DNA and is associated with a ry- phenotype that does not respond to position-effect suppressors (Table 4). In this case, the pic locus, 15 kb distal to rosy, exhibits a classical heterochromatic position effect (Table 4). Yet, we have failed to observe a position effect on snake, which is located only 2.5 kb distal to the ryj4 breakpoint. Moreover, we have examined and failed to see evidence of a position effect on the locus of l(3)S8, which is distal to pic. As noted earlier (Table 4), the ~y~~ rearrangement has no apparent effect on 1(3)S12 expression. In addition, we have examined, and have failed to find, a position effect of this heterochromatic rearrangement upon the mes-A, mes-B and 1(3)G9 genes as well.

The observation that Zn(3R)ry54 has no apparent effect on the adjacent snk locus, yet exerts a severe position effect on the more distally located pic gene, is particularly surprising. We note, in comparison, the effect associated with the more distant break of Zn(3R)ry1”36, which has a most severe effect on snk and a less severe effect on pic.

Position-effect modification by deletion of 87E2-F2: Another well-established modifier of position effect is associated with polytene region 87E2-F2. HENIKOFF (1979) demonstrated that heterozygous deletion of this region results in suppression of a lethal allele of a vital gene located in 87C. This lethal mutation arose in association with a nearby euchromatic-heterochromatic rearrangement. Additionally, he showed that this deletion heterozygosity suppressed the wm4 position effect. Subsequently, RUSHLOW and CHOVNICK (1984) extended this suppressor effect to include suppression of the position effect on rosy locus expression associated with the euchromatic-heterochromatic rearrangement, Zn(3R)ryfi””’36. In these earlier studies, four different deletion-bearing chromosomes were shown to have suppressor activity, and all are missing the polytene region 87E2-F2. Several other chromosomes with deletions in this region were tested, and all failed to exhibit suppressor activity. The latter do not have deletions that extend into the segment 87E2-F2.

We have extended these suppressor-effect observations utilizing two rosy region deletion chromosomes, Df(3R)r~’~ (Df(3R) 8 7 0 1 - 2 ; 8 7 0 1 4 - E I ) and D f ( 3 R ) ~ y ” ~ ~ (Df(3R) 87B15-Cl; 87B9-12) (HILLIKER et al. 1980). Only D f ( 3 R ) r ~ ” ~ ~ , which is missing polytene segment 87E2-F2, suppresses the heterochromatic position effects on rosy and pic expression (Table 4 and Figure

In contrast to the spreading effect seen with Zn(3R)ry

1 2 3 4 5 6 7

FIGURE 9.-Rocket electropherogram. Matched extracts from adult males were run against anti- XDH serum. Samples were applied to wells as follows: ( 1 ) 100% ry+'/Df(3R)ry", (2) 50% r y " / Df(3R)ry', ( 3 ) 25% 7y+'/Df(3R)ryy)6. (4) [ry+'""]/hrZ Df(3R)ry7', (5) [ry*'-'']/hr2 Df(3R)ry"", (6) [R401. I ] / h r z Df(3R)ty". (7) IR401. I ] / h r 2 Df(3R)ry"68.

9). Thus, with the addition of the pic locus, heterochromatic position effects on four different genes have been suppressed by the 87E2-F2 deletion. Like the Y chromosome, this deletion appears to behave as a general suppressor of heterochromatic position effects.

In an effort to assess the effect of the deletion of 87E2-F2 on rosy locus expression associated with transposons [ry+i4-fo] and [R402.2], crosses were conducted involving Df(3R)ry7'/MKRS and D~(~R)~Y"~~/MKRS mated to transformant lines [ t ~ + ~ ~ - " ] and [R402.2]. In an earler report (DANIELS et al. 1986), the transposon [y+i4-fo1 located at polytene segment 57F, was not subject to Y- chromosome modification, whereas the transposon [R402.2], located by in situ hybridization to the chromocenter at the base of chromosome 4, did show Y- chromosome modification. Figure 9 presents XDH CRM data illustrating the effect of Df(3R)ry7' and Df(3R)r~"~~ on XDH expression associated with transposons [ ~ y + ~ ~ - " ] and [R402.2]. Clearly, the deletion of the 87E2-F2 region does not significantly affect the XDH expression of [ry+i4-fu] (Figure 9, wells 4 and 5), whereas a marked increase in XDH CRM is recorded for [R402.2] (Figure 9, wells 6 and 7).

Crosses were carried out (Figure 4) to examine the possibility of position- effect suppression by Df(3R)ry7' and Df(3R)ryJf6' on the modified 1(3)S22 locus expression associated with the transposons of Table 1. Genotypes were produced to permit examination of all autosomal transposons in the presence of each rearrangement. There were no phenotypic effects that could be attributed to the positioneffect modifier, since the effect of transposon complementation of the 1(3)S12 phenotype was identical with both deletion chromosomes. A p parently, heterozygous deletion of polytene segment 87E2-F2, although effective in modification of heterochromatic position effect in a transposon, is ineffective in modification of euchromatic position effect. Confirmation of this point comes from the fact that the euchromatic fa''"* position effect also is not


subject to modification by deletion of 87E2-F2 (W. J. WELSHONS, personal communication).

DISCUSSION

Transformation as a tool in genetic mapping: Demonstration of two sep arate genetic units by classical cytogenetic analysis requires functional distinction (i.e., identification of separate, nonoverlapping complementation groups) as well as physical separation of those groups by rearrangements or recombination. When dealing with instances of very closely linked complementation groups in higher organisms, the latter requirement is very difficult to obtain and often lacking, as was the case for 1(3 )S12 and rosy. The present study illustrates the ease of accomplishment, and increased resolution brought about by the utilization of molecular genetics in the resolution of this issue.

Transformant site-specific variation in gene expression: In a prior report (DANIELS et al. 1986), we described the results of studies on transformant site- specific variation in rosy locus expression and presented evidence that two features of the experimental system are responsible for such variation. The first relates to transformant insertion site-specific position effects. The second, and far less likely basis for such variation, relates to the fact that transformation involves dysgenic perturbation of a P-element transposon, a process associated with a well-established mutagenesis (HAZELRIGG, LEVIS and RUBIN 1984; LEVIS, HAZELRIGG and RUBIN 1985; DANIELS et aE. 1985, 1986).

In this report, we extend our observations on transformant site-specific variation in rosy locus expression to that of the 1(3 )S12 locus. Several features of the 1(3 )S12 gene expression in transformants are of interest:

1, Like the element responsible for chorion gene amplification in Drosophila (DECICCO and SPRADLING 1984), the 1(3 )S12 l o c ~ s appears to be exceedingly sensitive to euchromatic site-specific position effects. Thus, only one of the 12 examined second-chromosome transposons fully complements the mutant, 1(3 )S12 allele, yielding wild-type progeny. In view of the small “target” size of 1(3)S12 relative to rosy, we are drawn to site-specific position effects as the underlying basis for most, if not all, of this variation. Support for this view is seen in the phenotypic change brought about by relocation of one such transposon (Table 3). For the most part, these transformants are associated with varying degrees of “ underexpression,” as inferred from their reduced viability and visibly mutant phenotype. Indeed, one transformant is clearly unable to complement the lethal phenotype, and another produces only a few very de- fective survivors. Had we restricted our study to a small sample of transformants and found only the latter, we would have failed to recognize that the 1 ( 3 ) S I 2 gene lies within the 8.1-kb Sal1 fragment. This point is of particular relevance to the use of transformant complementation of specific mutants as a means of confirmation or proof of a cloned DNA segment’s genetic identity. One should examine a sample of several transformants at different locations to obviate the position-effect problem. 2. Comparison of 1(3 )S12 and rosy expression among the euchromatic trans-

formants reveals no pattern of concordance or discordance. Perhaps the only


conclusion to be drawn about gene expression in cotransformants is that no inference should be drawn about one gene’s expression from observations made on the expression of neighboring gene(s) in the same transformant.

3. Although the expression of 1(3)SZ2 appears to be quite sensitive to euchromatic position effect, it is notably insensitive to heterochromatin, as evi- denced by both transposon and rearrangement data (Table 4).

The extreme lability of the l ( 3 ) S 1 2 locus to euchromatic position effect may relate to the fact that the 5’ end of the l (3 )SZ2 locus lies at the end of the 8.1-kb Sal1 fragment adjacent to the P-element sequence in the transposon (F. L. DUTTON and A. CHOVNICK, unpublished results). Study of the molecular organization of the locus is still incomplete; it is quite possible that some of the 5’ noncoding region is not included within the transposon-hence the sensitivity of l ( 3 ) S 1 2 expression of the transposon to genomic sequences.

Euchromatic us. heterochromatic position effect: In a prior report (DANIELS et al. 1986), we called attention to two clear experimental distinctions between euchromatic and heterochromatic position effects based on their effects on rosy locus expression: (1) In histochemical preparations of XDH activity in Mal- pighian tubules, XDH activity is variegated in hererochromatic position-affected larvae. XDH activity levels per individual are typical of “underexpression” variants, but the variegation observed is consistent with “all-or-none” activity at the cell level. In contrast, histochemical preparations from euchromatic position-affected larvae exhibit uniform underexpression of XDH, just as do leaky “underexpressing” rosy locus structural mutants and the i1005L “underproducer” control variant. (2) Euchromatic position-affected rosy locus expression is not subject to Y-chromosome modification, as is heterochromatic position-affected expression. The present report provides additional observations extending these distinctions as follows: (a) Further evidence is presented to demonstrate that the position-effect modifier of polytene region 87E2-F2 (HENIKOFF 1979; RUSHLOW and CHOVNICK 1984) is, in fact, a general modifier of heterochromatic position effects; like the Y chromosome, it also is ineffective in influencing euchromatic position-affected gene expression. (b) Expression of the l ( 3 ) S 1 2 gene is exceedingly sensitive to neighboring euchromatin, yet appears to be insensitive to adjacent heterochromatin.

In the case of the faxwb euchromatic position effect (WELSHONS and KEPPY 1975; KEPPY and WELSHONS 1977; WELSHONS and WELSHONS 1985, 1986), the observed phenotypic effects are mostly readily understood as gene expression alterations in response to an alteration in a DNA sequence coupling. This is brought about by a euchromatic rearrangement in a restricted chromosomal interval in the white to Notch region. In the case of the wDzL mutation (ZACHAR and BINGHAM 1982; LEVIS and RUBIN 1982), associated with a transposable element insertion adjacent to, but outside of, the white locus, molecular studies (BINGHAM and ZACHAR 1985) provide a specific example of such an alteration in DNA sequence coupling resulting in a position effect.

Clearly, euchromatic position effects result from DNA sequence rearrangements within a very restricted interval. Modifiers of such euchromatic position effects must have direct effect on these sequence arrangements. Hence, such


modifiers should be highly specific in their effects, and, for the most part, be located in the immediate region of the position effect. This expectation stands in contrast to heterochromatic position-effect modifiers that function from more remote gecomic locations with little specificity. Indeed, WELSHONS and WELSHONS (1 986) demonstrate the very localized nature of rearrangements that modify the faswb position effect; the present report and DANIELS et al. (1 986) provide a demonstration that heterochromatic position-effect modifiers (Y chromosome and 87E2-FZ deletion) are unable to modify the euchromatic position effects on l(3)S12 and rosy expression.

Heterochromatinization and position effects: In contrast to specific DNA sequence interactions, heterochromatic position effects have been interpreted generally in terms of changes in the state of the chromatin of a euchromatic gene placed adjacent to heterochromatin. Such changes are associated with apparent all-or-none effects on gene expression in somatic cells. When dealing with a cell autonomous gene product, the result seen is that of variegated expression (noted above). A possible basis for our failure to see a heterochromatic position effect on 1(3)S12 expression is simply that the cell product of this gene is nonautonomous and that variegated expression would yield a wild- type phenotype. SPOFFORD (1976) discusses several reasons why a gene might not not be subject to heterochromatic position effect. For example, since the time of heterochromatinization is not earlier than blastoderm formation, a gene that functioned only during the early cleavage stages would not be subject to heterochromatic position effect.

The spreading effect of heterochromatic position effect represents a dramatic demonstration of the notion of gene inactivation by an all-encompassing heterochromatinization (see reviews: LEWIS 1950; BAKER 1968; SPOFFORD 1976). In this report, we provide examples of the classic spreading effect with 1n(3R)ryP’”’j6 and T ( 3 ; 4 ) r ~ P ’ ” ~ ~ where we note that the severity of the position effects diminish with increasing distance from the rearrangement break, and then cease to have effect. TARTOF, HOBBS and JONES (1984) demonstrated that the heterochromatic sequence at a euchromatic-heterochromatic junction is not itself sufficient to bring about variegated expression of an adjacent euchromatic gene. Rather, they suggest that the initiating sequence may be located within the heterochromatin contiguous with the junction, but at some distance from the junction. This argument implies that only certain heterochromatic sequences are capable of bringing about the variegated expression of a euchromatic gene. They define such sequences as initiator sequences that are capable of initiating the heterochromatic state. Impressed by the spreading effect, they propose that heterochromatinization would proceed to include a DNA segment that might encompass several genes, until a “terminator” sequence is reached, at which point heterochromatinization is terminated. Much of our work is consistent with this model, but one glaring exception must be noted. The snake locus should have responded to the 1n(33R)ry5‘ rearrangement with a position effect, at least as strong as seen with Zn(3R)ryP””’36 and T ( 3 ; 4 ) ~ y P ’ ” ~ ~ . The rosy, snake and piccolo genes doubtless function in different tissues and at different times during development. Yet, these loci are able to respond to heterochro-

838 S . H. CLARK AND A. CHOVNICK

matic rearrangements with classical spreading effects (e.g., Zn(3R)ryPS1”j6 and T ( 3 ; 4 ) r ~ P ’ ” ~ ~ ) . Our inability to score any effect of I n ( 3 R ) ~ y ~ ~ on snake locus expression, while the more distinct piccolo locus exhibits a strong position effect, represents a mystery. Perhaps the initiator-terminator model is overly simplistic.

Euchromatic position effects in transformants us. classical genetic studies: The present study, as well as our earlier report (DANIELS et al. 1986), documents the high relative frequencies of euchromatic position effect seen in l ( 3 ) S l Z and rosy locus transformants. We suggest that most of the other reported instances of transformant position effects (reviewed in DANIELS et al. 1986) are euchromatic in nature. In contrast, we note the relative paucity of such effects recorded in the classical genetic literature. Perhaps the classic definition of a gene, based on noncomplementation of mutant phenotypes coupled with relatively crude recombination mapping, served to obscure such position effects. The two position-effect mutations discussed above, the X-ray- induced famb allele and the spontaneous wDzL mutation, were correctly identified only through elaborate fine structure mapping and careful cytogenic analysis, on the one hand, and molecular characterization, on the other. Perhaps there are other euchromatic position-effect mutants to be found among the array of radiation-induced and spontaneous mutations of any given gene.

This investigation was supported by research grant NP-491 from the American Cancer Society to S.H.C. and grant GM-09886 from the United States Public Health Service to A.C.

LITERATURE CITED

ANDERSON, K., and C. NUSSLEIN-VOLHARD. 1984 Information for the dorso-ventral pattern of the Drosophila embryo is stored as maternal mRNA. Nature 311: 223-227.

BAKER, W. K., 1968

BENDER, W., P. SPIERER and D. S. HOGNESS, 1983

Position effect variegation. Adv. Genet. 14 133-169.

Chromosomal walking and jumping to isolate DNA from the bithorax complex and the Ace and rosy loci in Drosophila melanogaster. J. Mol. Biol. 168: 17-33.

Evidence that two mutations, dz‘ and z l , affecting synapsis-dependent genetic behavior of white are transcriptional regulatory mutations. Cell 40:

CHOVNICK, A., W. GELBART, M. MCCARRON, B. OSMOND, E. P. M. CANDIW and D. L. BAILLIE, 1976 Organization of the rosy locus in Drosophila melanogaster: evidence for a control element adjacent to the xanthine dehydrogenase structural element. Genetics 84: 233-255.

CHOVNICK, A., M. MCCARRON, S. H. CLARK, A. J. HILLIKER and C. A. RUSHLOW, 1980 Structural and functional organization of a gene in Drosophila melanogaster. pp. 3-23. In: Development and Neurobiology of Drosophila, Edited by 0. SIDDIQI, P. BABU, L. M. HALL and J. C. HALL. Plenum, New York.

CHOVNICK, A., M. MCCARRON, A. HILLIKER, I. O’DONNELL, W. GELBART and S. CLARK.

BINGHAM, P. M. and Z. ZACHAR, 1985

8 19-825.

1977 1021.

Gene organization in Drosophila. Cold Spring Harbor Symp. Quant. Biol. 4 2 1011-

CLARK, S. H. and A. CHOVNICK, 1985 A selective screen for transposable element mobilization of Drosphila melanogaster. Environ. Mutagen. 7: 439-499.

CLARK, S. H., S. B. DANIELS, C. A. RUSHLOW, A. J. HILLIKER and A. CHOVNICK, 1984 Tissue-


specific and pretranslational character of variants of the rosy locus control element in Droso- phila melanogaster. Genetics 108: 953-968.

CLARK, S. H., M. MCCARRON, C. LOVE and A. CHOVNICK, 1986 On the identification of the rosy locus DNA in Drosophila melanoguster: intragenic recombination mapping of mutations associated with insertions and deletions. Genetics 112: 755-767.

COTI?, B., W. BENDER, D. CURTIS and A. CHOVNICK, 1986 Molecular mapping of the rosy locus in Drosphila melanoguster. Genetics 114: 769-783.

CRAIG, E. A., T. D. INGOLIA and L. J. MANSEAU, 1983 Expression of Drosophila heat-shock cognate genes and development. Dev. Biol. 99: 418-426.

DANIELS, S. B., M. MCCARRON, C. LOVE and A. CHOVNICK, 1985 Dysgenesis-induced instability of rosy locus transformation in Drosophila melunogaster: analysis of excision events and the selective recovery of control element deletions. Genetics 109: 95-1 17.

DANIELS, S. B., M. MCCARRON, C. LOVE, S. H. CLARK and A. CHOVNICK, 1986 The underlying bases of gene expression differences in stable transformants of the rosy locus in Drosophila melanogaster. Genetics 113: 265-286.

DECICCO, D. V. and A. C. SPRADLING, 1984 Localization of a &-acting element responsible for the developmentally regulated amplification of Drosophila chorion genes. Cell 3 8 45-54.

DRETZEN, G., M. BELLARD, P. SASSONE-CORSI and P. CHAMBON, 1981 A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal. Biochem. 112: 295-298.

GAUSZ, J., L. M. C. HALL, A. SPIERER and P. SPIERER, 1986 Molecular genetics of the rosy-ace region in Drosophila melanogaster. Genetics 112 65-78.

HAZELRIGG, T., R. LEVIS and G. M. RUBIN, 1984 Transformation of white locus DNA in Dro- sophila: dosage compensation, zeste interaction and position effects. Cell 3 6 469-481.

HENIKOFF, S., 1979 Position effects and variegation enhancers in an autosomal region of Drosoph- ila melanogaster. Genetics 93: 105-1 15.

HILLIKER, A. J., S. H. CLARK, A. CHOVNICK and W. M. GELBART, 1980 Cytogenetic analysis of the chromosomal region immediately adjacent to the rosy locus in Drosophila melanogaster. Genetics 9 5 95-1 10.

The cytogenetics of a recessive visible mutant associated with a deficiency adjacent to the Notch locus in Drosophila melanoguster. Genetics 85: 497- 506.

LEFEVRE, G., JR., 1971 Cytological information regarding mutants listed in LINDSLEY and GRELL

LEVIS, R., T. HAZELRICG and G. M. RUBIN, 1985 Effects of genomic position on the expression

LEVIS, R. and G. M. RUBIN, 1982 The unstable dzL mutation of Drosophila is caused by a 13

LEWIS, E. B., 1950 The phenomenon of position effect. Adv. Genet. 3: 73-115. LINDSLEY, D. L. and E. H. GRELL, 1968 Genetic variations of Drosophila melanogaster. Carnegie

MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

MCCARRON, M., J. O'DONNELL, A. CHOVNICK, B. S. BHULLAR, J. HEWITT and E. P. M. CANDIDO, 1979 Organization of the rosy locus in Drosophila melanogaster: further evidence in support of a &-acting control element adjacent to the xanthine dehydrogenase structural element. Genetics 91: 275-293.

Genetic transformation of Drosophila with transposable

KEPPY, D. 0. and W. J. WELSHONS, 1977

1968. Drosophila Inform. Serv. 4 6 40.

of transduced copies of the white gene of Drosophila. Science 229 558-561.

kilobase insertion that is imprecisely excised in phenotypic revertants. Cell 3 0 543-550.

Inst. Wash. Publ. 627.

RUBIN, G. M. and A. C. SPRADLING, 1982 element vectors. Science 218: 348-353.


RUBIN, G. M. and A. C. SPRADLING, 1983 Vectors for P element-mediated gene transfer in

RUSHLOW, C. A., W. BENDER and A. CHOVNICK, 1984 Studies on the mechanism of heterochro-

RUSHLOW, C. A. and A. CHOVNICK, 1984

Drosophila. Nucleic Acids Res. 11: 6341-6351.

matic position effect at the rosy locus of Drosophila melanogaster. Genetics 108: 603-61 5.

Heterochromatic position effect at the rosy locus of Drosophila melanogaster: cytological, genetic and biochemical characterization. Genetics 108:

SCHALET, A., R. P. KERNAGHAN and A. CHOVNICK, 1964 Structural and phenotypic definition of the rosy cistron in Drosophila melanogaster. Genetics 50: 1261-1 268.

SCHOLNICK, S. B., B. A. MORGAN and J. HIRSCH, 1983 The cloned dopa decarboxylase gene is developmentally regulated when reintegrated into the Drosophila genome. Cell 34: 37-45.

SCOTT, M. P., A. J. WEINER, T. I. HAZELRIGG, B. A. POLISHY, V. PIRROTTA, F. SCALENGHE and T. C. KAUFMAN, 1983 The molecular organization of the Antennupedia locus of Drosophila. Cell 35: 763-776.

SPOFFORD, J. B., 1976 Position effect variegation in Drosophila. pp. 955-1018. In: Genetics and Biology of Drosophila, Vol. IC, Edited by M. ASHBURNER and E. NOVITSKI. Academic Press, New York.

The effect of chromosomal position on the expression

A structural basis for variegated position effects.

Intragenic deletions and salivary band relationships in

Suppression of the facet-strawberry position effect

Enhancement and suppression of a euchromatic

Regulation of white locus expression: the structure of

Communicating editor: A. SPRADLINC

589-602.

SPRADLINC, A. C. and G. M. RUBIN, 1983

TARTOF, K. D., C. HOBBS and M. JONES, 1984

WELSHONS, W. J. and D. 0. KEPPY, 1975

WELSHONS, W. J. and H. J. WELSHONS, 1985

WELSHONS, W. J. and H. J. WELSHONS, 1986

ZACHAR, Z. and P. M. BINGHAM, 1982

of the Drosophila xanthine dehydrogenase gene. Cell 3 4 47-57.

Cell 37: 869-878.

Drosophila. Genetics 80: 143-1 55.

in Drosophila by lesions adjacent to Notch. Genetics 110: 465-477.

position effect at Notch in Drosophila. Genetics 113: 337-354.

mutant alleles at the white locus of Drosophila melanogaster. Cell 3 0 529-541.

studies of normal and position-affected ...storrs, connecticut 06268 manuscript received march 17,...

Documents