Genetica 85: 185-203, 1992. 0 1992 Kluwer Academic Publishers.

Printed

in the Netherlands.

The suppressor of forked locus in Drosophila melanogaster: genetic and molecular analyses C. L. Fitch*, L. Girton & J. R. Girton Department of Zoology and Genetics, Iowa State University, Ames, 50010, USA * Present address: Howard Hughes Medical Institute, University of Washington, SL-15, Seattle, WA 98195, USA Received 1 October 1991

Accepted 16 January 1992

Key words: Drosophila melanogaster, trans-acting transcriptional pression, enhancement

modification,

transposable elements, sup-

Abstract The suppressor of forked, m(f) locus is one of a class of loci in Drosophila whose mutant alleles are trans-acting allele-specific modifiers of transposable element-insertion mutations at other loci. Mutations of su(f) suppress gypsy insert alleles of forked and enhance the copia insert allele white apricot. Our investigations of su(f) include genetic and molecular analyses of 19 alleles to determine the numbers and types of genetic functions present at the locus. Our results suggest the m(f) locus contains multiple genetic functions. There are two distinct modifier functions and two vital functions. One modifier function is specific for enhancement and the other for suppression. One vital function is required for normal ecdysterone production in the third larval instar, the other is not. We present a restriction map of the su(f) genomic region and the results of an RFLP analysis of several su(f) alleles.

Introduction A better understanding of the genetic mechanisms regulating the transcriptional activity of retrovirallike transposable elements and how these mechanisms relate to those controlling developmental gene activity in Drosophila is a major focus of this study. Trans-acting allele-specific modifier mutations which increase (enhance) or decrease (suppress) the severity of the phenotypes of transposable element-induced mutations provide valuable systems for studying these questions. Modifiable mutations contain transposable elements belonging to the copia-like family, which are retrovirus-like transposable elements (Parkhurst & Corcus, 1987). These elements are polymerase II transcription units containing transcriptional regulatory sequences in their terminal repeated sequences (LTRs). The temporal and spatial specificity of transposable element activity indicates these se-

quences are controlled by developmental regulatory genes. The transcriptional activities of an inserted element may interact or interfere with the surrounding locus, producing a mutant phenotype (Levis et al., 1982; Kubli, 1986, Rutledge et al., 1988). This effect is modulated (increased or decreased) by trans-acting modifier mutations. An increased understanding of the genetic functions of modifier genes will provide a foundation of information which will help to elucidate how transposable element activity is regulated during development. A genetic analysis of a modifier gene is in fact an analysis of a three way genetic interaction between the modifier, the target locus and the transposable element. A major goal of such investigations is to define the number and types of genetic functions at each of these and to determine how these functions interact. Analyses of the transposable elements in modifiable target alleles suggests the interaction of

186 elements and modifiers occurs at the level of transcription. The gypsy element inserted in the forked’ allele reduces the amount but not the size of the forked transcript, suggesting that the element insertion alters the amount but not the function of the forked product (Parkhurst & Corces, 1986a, 1986b). Alleles of the suppressor of Hairy wing [su(Hw)] and m(f), which suppress forked, restore the levels of forked transcripts to 50 to 100% of the normal levels (Parkhurst & Corces, 1985; Kubli, 1986). Neither modifier mutation affects the transcription of the wild type forked locus, indicating they are specifically affecting the element. Curiously, su(Hw) and m(f) mutations have opposite effects on gypsy transcription. The m(f) alleles increase the amount of gypsy transcript, and su(Hw) alleles reduce it. It is not understood how these two opposite effects on gypsy have the same effect on forked (Kubli, 1986). The mutant phenotype of the white upricot mutation results from the premature termination of white transcripts at the copia 3’ LTR. Approximately 95 % of the white transcripts are prematurely terminated (Zachar et al., 1987). The percentage of full length transcripts is increased by alleles of suppressor of white apricot [su(w”)] and decreased by alleles of enhancer of white apricot [en(@)] and su(f) (Kubli, 1986). Analysis of partial revertants of the w” allele in which portions of the coding sequences of the copia element have been deleted indicate that sequences within copia, but not in the LTR, are required for the interaction with modifiers (Mount et al., 1988). The presence of a copia LTR alone is not sufficient. These and other results suggest that targets of modifier genes are transposable elements and that they most often act in some way to alter the transcription and expression of that transposable element (Rutledge et al., 1988). The details of the interaction are not known, but understanding them will give valuable information about the regulation of the transcriptional process. This study focuses on one modifier locus, the suppressor of forked [su(f)] locus. The first suff) mutation was discovered as a trans-acting suppressor offorked (Whittinghill, 1937, 1938). The locus maps to position 65.9 on the X chromosome in the l3 heterochromatin region. The su(f) locus has been shown to be genetically complex and to have multiple functions. These include vital functions, one of which is associated with the production of ecdyster-

one, and modifier functions, suppression and enhancement. Many of the effects of mutations on these functions have been described previously, but these reports usually focused on individual su(f) alleles or individual functions. A goal of this study is to present a detailed genetic analysis for all of these functions. Alleles of su(f) suppress certain alleles at the forked, lozenge, cut, and bithorax loci and enhance certain alleles at the lozenge, white, and Minute(3)P loci (Green, 1955, 1956; Schalet & Lefevre, 1976; Girton et al., 1986). As described above, the m(f) locus also contain vital functions whose action is not restricted to transposable element insertion target alleles. One vital function is mutated in the temperature-sensitive (ts) lethal su(f)rS726 and su(f) rs67galleles (Dudick et al., 1974; Russell, 1974). Individuals homo- or hemizygous for these alleles survive and exhibit a suppressed forked phenotype at 21”-25 “C and are lethal at 29”-30°C. The lethality is not associated with a particular genetic background or any transposable element insert mutations. Exposures of individuals homo- or hemizygous for su(f)rS726 to sublethal restrictive temperature pulses during different developmental stages produces localized patches of cell death in the imaginal discs. These result in pattern abnormalities in adult structures, including leg and head duplications (Russell et al., 1977; Girton & Russell, 1980, 1981) and triplications (Girton, 1981, 1983). The su(f)ls6’g and SU(~)‘~‘~~ alleles also block ecdysterone production during the third larval instar. This blockage suppresses the transcription of glue protein genes (Hansson et al., 198 1; Hansson & Lambertsson, 1984). A second vital su(f) function is mutated in the su(f)ph allele. Individuals homo- or hemizygous for su(f)pb survive only at low (16”-18°C) temperatures and have a suppressed forked phenotype (Schalet & Lefevre, 1976). Individuals with su(f)pb/su(f) rS67g or su(f)pb/su(f) ts726 genotypes survive at all temperatures and have a suppressed forked phenotype. The complementation of some but not all functions is characteristic of alleles of a complex locus in Drosophila. Numerous alleles of su(f) are known (Lindsley & Zimm, 1990), but most previous analyses of su(f) functions have focused on only one or two alleles and one or two phenotypes. In the present report, various genetic analyses of a collection of 19 su(f) alleles were

187 done to investigate the number and types of genetic functions at this locus. The purpose for studying many different alleles is to better understand the complicated genetic functions of this locus. The genetic results are accompanied by a genomic map of the region. It was generated as a result of a restriction fragment length polymorphism (RFLP) study of these alleles. Based on these genetic and preliminary molecular studies, further evidence for separable modifier functions within the su(f) locus is provided.

Materials

and methods

Culture techniques and stocks

Stocks were maintained and crosses performed on a standard media consisting of cornmeal, sugar, agar, mold inhibitor, and live yeast. All cultures were maintained at 25 “C unless otherwise specified. Stocks and alleles used in all crosses were either generated in our lab or obtained from Dr. W. Welshons, from the Department of Genetics Stock Center, University of California Davis, or from the Mid-America Drosophila Stock Center at Bowling Green State University. The alleles of su(f) are referred to by their synonyms. Following are the new designations (Lindsley & Zimm, 1990) for su(f) alleles discussed in this paper: su(f)* = su(f)‘; su(fJ3 = su(f)D13; su(fJ4 = su(“f)R918;

su(f)6 = sU(fp; su(fy = su(f)fq

su(f)" =

su(f)tSTz6; su(f)‘j = su(f)“a$ su(f)?2 = su(f) vE738; and su(f) 28 = su(f) Ms252. Additional su(f) alleles discussed in this paper are being pre-

sented for the first time. All other alleles are described in either Lindsley and Grell(l968) or Lindsley and Zimm (1985, 1987, and 1990). Induction

of su( f) mutations

New su(f) mutations were induced using either a Gl or G2 screen. In a typical Gl screen males with a y* wa f l/Y genotype were mutagenized with EMS according to the procedures of Lewis and Bather (1968). The males were fed a solution of 0.025 M EMS in a 1% sucrose solution. Groups of 10 treated males were mated to females with a genotype of y2 wa et6 f1 Df(l)VE738/FM7. The Df(ljVE738 includes the su(f) locus. Gl progeny with a geno-

type of y2 wa f1 (*J/y2 wa et6 f1 Df(l)VE738 were collected, where (*) represents a potential new su(f) mutation. Strains containing the new allele were established from individuals exhibiting a visible su(f) phenotype. In a typical G2 screen Gl females with a genotype of y2 wa f’ (*)/FM7 were singly mated to three y2 wa et6 f’ Df(l)VE738/BSY males. The G2 progeny with a y2 wa f’ (*j/y2 wa CP f’ Df(ljVE738 genotype were examined for a su(f) phenotype. Potential lethal or sterile alleles were recovered from y2 wa f1 (*)/BSY siblings. The Gl and G2 screens were designed to recover visible, viable alleles. The G2 screen was also designed to recover lethal alleles. Potential alleles were tested for allelism in complementation tests with known su(f) alleles. Alleles used in these studies include su(f)+ (from the Oregon R wild type strain), su(f) ‘, SU(f)EMS5, su(f)EMSI1, su(f)EMs’5, SU(f)‘nadts, su(fP’, su(f) K5, su(f)Ph (= SU(f)R918), SU(f)3a su(f) MS252 (= SU(f)hd252), SU(f)hd7’, su,+ su(f)t-, su(f)8=, SU(f)‘B”$ sLl(fJg4, su(f)D’-1’, and su(f) 126. Analysis of the su( f) phenotype

The phenotypic effects produced by different su(f) alleles were measured in individuals reared at 29 ‘C, 25 ‘C, or 21 “C. Individuals were collected, aged for three to five days after eclosion, and then examined at 10X to 25X using a Zeiss dissecting microscope. The strength of wa enhancement was measured by visual determination of the eye color. Each eye was graded for color on a scale of 1 to 5. Control individuals with a wa su(f)+ genotype (no enhancement) were assigned a value of 1, and control individuals with a genotype of w74c28 (a null allele of white) were assigned a value of 5. The degree of wa enhancement was measured as the decrease in eye color relative to the controls. Once a sample of individuals with a certain genotype had been scored the degree of difference between the average eye color value (x) in the sample and the control (1) was calculated. This difference represents the amount of enhancement caused by the su(f) genotype. This difference was divided by the possible difference and expressed as a percentage: (x-1)/(5-1) * 100 = % enhancement. Two calculations of the strength of suppression of forked were made. The suppression of forked penetrance was measured by determining the re-

188 duction in the average number of bristles showing a forked phenotype. The phenotypes of twelve thoracic bristles (anterior and posterior scutellar, anterior post alar, posterior supra-alar, and the anterior and posterior dorso-central) were determined for each individual. In some experiments each bristle was simply scored as being forked or not forked. Any visible expression of the forked phenotype was counted as forked. The mean number of bristles showing forked in a sample of m(f) individuals represents the amount of forked penetrance which was not suppressed. This mean was subtracted from the mean number of bristles showing forked in the control genotype (m(f)+). The difference represents the amount of suppression by m(f). This difference was divided by the control number of bristles showing forked and the result was expressed as a percentage: (control-x)/(control) * 100 = % suppression of forked penetrance. As described in the Results, the control number was not always exactly 12, asforked’ penetrance varied slightly at different temperatures. Three sets of control individuals were used, one for each temperature. The strength of suppression of forked expression was measured by determining the change in the average severity of the forked phenotype in the bristles showing forked. The normal expression of forked’ varies in the different bristles in the set. The smaller bristles have a lesser expression and are more often completely suppressed. To prevent different strengths of suppression offorked penetrance from influencing our measure offorked expression, we calculated the average change in forked using only the four scutellar bristles on each fly. These bristles showed similar expression and penetrance in the control individuals. In another set of experiments the bristles were scored for the severity of expression of forked on a scale of 1 (not forked) to 5 (extreme expression). For each m(f) genotype the mean severity offorked expression in scutellar bristles showing forked was calculated. This mean represents the amount of forked expression which was not suppressed. Subtracting this mean from that of the controls gives a measure of the amount of suppression. This difference was divided by the possible difference and the result was expressed as a percentage: (control-x)/(control-1) * 100 = % suppression of expression.

In situ hybridization

to polytene chromosomes

Chromosome preparations from the salivary glands of late third instar larvae were made following the procedure described by Engels et al. (1986) with a few modifications. The salivary glands were isolated in 0.75 % NaCl, fixed for 10 set in 45 % acetic acid and squashed in a solution of 1 part 85 % lactic acid, 2 parts H,O, and 3 parts acetic acid. The chromosomes were further flattened under brass weights (-170g) for at least 12 h at room temperature. The slide was frozen in liquid nitrogen, the coverslip was removed and the slide was dehydrated in 95 % ethanol for 10 min and then air dried. After examination the slides were treated in 2X SSC at 65 “C for 30 min and then dehydrated by two 10 min treatments in 70% ethanol and one 10 min treatment in 95 % ethanol. The ethanol was initially heated to 65 “C and allowed to cool during the treatments. Slides were then air dried and stored at 4°C. The DNA used as probes in in situ hybridizations was labeled with digoxigenin-dUTP using the Boehringer Mannheim GeniusTM kit. The labelled DNA concentration was measured on a fluorimeter and then stored at 4 “C. The Genius kit procedure for hybridization of the labelled probe DNA was used. Hybridization was done at 42 “C for 16 h. Following antibody binding and color development the slides were air dried. The chromosomes were examined under a drop of water using phase contrast. Cloning and subcloning of su( f)

A 16.5 kilobase (Kb) DNA fragment cloned into the EMBL4 vector (designated X252.512) was kindly provided by Dr. K. O’Hare. This fragment contains all or part of the m(f) locus. The original fragment was subcloned into smaller fragments which were inserted into plasmid vectors following the procedures of Maniatis et al. (1982). Plasmids containing portions of the original 16.5 Kb fragment identified as pR- l-2 (EcoRI 3.2 Kb fragment), pR-9-42 (EcoRI 5.3 Kb fragment), pX-9-2 (XhoI 4.4 Kb fragment), and pX-3-3 (XhoI 2.7 Kb fragment) were isolated and maintained in the E. coli strain JM83.

189 Southern and Northern analyses

Modifierfinctions,

Genomic DNA was isolated from Drosophila with different su(f) genotypes according to the methods described by Jowett (1986). Genomic DNA was cut with one or more restriction endonucleases, separated on agarose gels, transferred to nitrocellulose membranes and hybridized to probe DNA (Southern 1975). Probe DNA was generated by isolating plasmid DNA from the four subclones. It was linearized and separated on a 1% low EEO agarose (Seakem GTGTM) gel in 1XTAE (4.84 g Tris, 1.14 ml glacial acetic acid, and 2 ml OSM EDTA pH8.0 per liter). The subcloned DNA was cut from the gel and isolated using the BiolOl GENECLEANTM kit. It was then denatured and labelled using 32P dCTP and following the procedures of the Boehringer Mamrheim Random Primed DNA Labeling Kit. Unincorporated nucleotides were removed using a G-50 sephadex spin column. Total RNA was isolated from female larvae (Maniatis 1982). The larvae were either reared continuously at a controlled temperature (2 1 0 or 29 “C) or were reared at 2 1 “C and were shifted to 29 ‘C at the molt between the second and third larval instar. The larvae were harvested and the RNA isolated at 36, 42, or 48 h later. These collection times are prior to the lethal period of the su(f) lethal alleles. [The RNAs were separated by .electrophoresis; blotted, and probed with a DNA copy of the transcribed region of the Sgs-3 gene, (kindly provided by Dr. Elliot Meyerowitz) which hybridizes to a 1.4 Kb Sgs-3 transcript.] The probe DNA was prepared as described above.

Individuals homozygous or hemizygous for y*, wa, and f1 that contained different su(f) alleles were reared at 29 ‘, 25 ‘, or 21 “C and scored for eye and bristle phenotype and for viability. All individuals scored were females containing either two copies of one su(f) allele (homozygous), containing two different alleles (heterozygous or heteroallelic), or containing one allele and a deficiency (hemizygous). The exception was the su(f)pb allele which was scored in males. The strength of enhancement of wa and suppression off’ were recorded for each individual as described in the Materials and methods. The strength of enhancement or suppression by a modifier is, by definition, a measure of change from a control mutant phenotype. Our control individuals were female homozygous for an X chromosome containing y2, wa, fl, and su(f)+. The su(f)+ allele was from the Oregon R wild-type strain. Examples of the eye color and bristle phenotypes given by this genotype are presented in Figure 1. The phenotypes of these control females are not strongly affected by changes in temperature (Table 1). The eye and bristle phenotypes of these individTable types.

1. Suppression

Genotype suy) +/suv)

suv)‘/suv)’ Results The number and types of functions locus

at the su(f)

Two important questions are the number of genetic functions at the su(f) locus and the nature of these functions. The su(f) locus is known to have two general types of genetic functions: modifier (enhancement and suppression) and lethality. To determine whether these are single or multiple functions we measured the range of phenotypes of individuals homozygous or heterozygous for a select set of su(f) alleles. We will consider the results for each function separately below.

+

enhancement and suppression

and enhancement

suv)~.~726

su(jp/Y*

geno-

Description

29 “C

25 “C

21°C

eye color # bristles # females

1.0 12.0 60

1.0 11.5 192

1.0 11.7 221

% enhanced % suppressed # females

48.5 99.2 104

82.5 98.3 75

97.5 95.7 73

0

22.5 87.0 67

42.5 59.0 54

0

47.5 99.1 97

60.0 84.6 72

0

(90.6) 0

% enhanced % suppressed # females suv)“726/

in homozygous

% enhanced % suppressed # females % enhanced % suppressed # males

jo.ot 129)

* reared at 18°C; % enhanced = (mutant control)/4.0 X 100; % suppressed = (control # mutant #)/control # X 100; control = su(n+/su(f)+ at that temperature

190

191 Table 2. Suppression

uals served as a base level, or standard. All enhancement and suppression effects are presented as percent change.

types,

Enhancement of w a

su(f)

The first su(f) phenotype analyzed is the enhancement of white apricot. Individuals homozygous for su(f) I, a nonlethal allele, show moderate enhancement at 29 “C (48.5 %) and stronger enhancement at 21 “C (97.5%) as shown in Table 1. Individuals homozygous for the ts lethal alleles (s~(f)‘~~~~ or su(f) tS67gdie at 29 “C and show moderate enhancement at 25 “C (22.5-47.5%). These alleles also give a stronger enhancement at 21 “C (42.5-60%). Females homozygous for the sops allele die at all temperatures. Males hemizygous for so@ survive at low temperatures and show no enhancement of wa (0.0%). Individuals heterozygous for different su(f) alleles were also measured for strength of enhancement (Table 2). Individuals with a su(f) l/~u(f)~~~*~ or su(f) ‘/k(f) tS67ggenotype survive at all temperatures and have moderate enhancement (34-70%). For both genotypes the enhancement is stronger at lower temperatures. At each temperature the strength of enhancement in these individuals is less than that of homozygous su(f) J individuals and greater than that of homozygous ~u(f)~~~~g or m(f) ts726 individuals. Individuals with a su(f) ‘/ su(f)pb genotype survive and show enhancement at all temperatures. The level of enhancement is less at all temperatures than for the su(f) ‘/su(f) J individuals. Individuals hemizygous for su(f)’ show increased enhancement at all temperatures. Individuals hemizygous for su(f) fS726or su(f) tS67gshow an increase in lethality but only small increases in enhancement. Individuals of su(f)pb/su(f) tS726or su(f)pb/su(f) tS67gphenotypes survive at all temperatures and have variable levels of enhancement. The level of enhancement is low at 29 “C and higher at 2 1 “C. Enhancement in the su(f)pb/su(f) tS726individuals is less than in the su(f) rS7Z6/~u(f)tS726indi-

Fig. 1. The effects of su(f) y” Ma0f 1 m(f)+ individual. bristles of a y2 Ml0 f’ su(f) showing an intermediate intermediate level forked

Genotype

and enhancement

in heterozygous

geno-

Description

29 “C

25 “C

21°C

% enhanced % suppressed # females

44.9 99.2 54

37.9 95.0 26

55.9 67.1 52

% enhanced % suppressed # females

34.5 88.4 113

70.0 94.7 74

69.3 81.6 51

% enhanced % suppressed # females

36.0 98.6 130

56.5 91.3 125

69.5 73.6 84

% enhanced % suppressed # females

0

45.3 96.5 31

47.0 39.7 26

su(f)“67Qu(f)pb

% enhanced % suppressed # females

18.0 98.7 65

36.5 87.8 46

40.8 30.6 57

su(f)‘5726/su(f)ph

% enhanced % suppressed # females

2.0 96.5 112

17.0 72.3 111

27.3 21.0 41

% enhanced % suppressed # females

1.0 13.0 50

6.0 10.0 38

10.0 7.0 59

% enhanced % suppressed # females

0 5.0 50

1.0 2.0 50

9.0 5.0 53

% enhanced % suppressed # females

10.0 9.2 92

7.5 0.0 108

2.5 3.4 55

% enhanced % suppressed # females

0.0 0.8 65

0.0 4.2 52

3.0 5.6 27

su(f)‘/VE738

% enhanced % suppressed # females

78.0 98.2 55

92.0 98.8 33

98.5 99.0 47

su(f)‘S67R/vE738

% enhanced %suppressed # females

‘/m(f)

c567g

su(f)‘/su(f)“7~6

su(f)‘s67KIsu(f)g726

su(f)“678/su(f)

+

su(f)‘~7’6/sll(f)+

su(f)phlsucf)

su(f)‘“7’6/VE738

+

% enhanced % suppressed # females

0

50.0 88.5 34

0

70.5 99.3 56

on w u andf’ at 25 ‘C. A. The eye of a control y’ W” m(f)’ individual. B. The thoracic bristles of a control C. The eye of a yZ U@ f’ su(f) ’ individual showing the enhanced u1° eye color phenotype. D. The thoracic ’ individual showing the suppressed forked bristle phenotype. E. The eye of a y’ w0 f I su(f)rS67g individual level of enhanced rP phenotype. F. The thoracic bristles of a y? IP f’ su(f)‘s67n individual showing an suppression phenotype.

192 viduals. Enhancement in su(f)pb/su(f) rs67gindividuals is low at 29°C and about the same as the su(f) fs67g/s~(f) fs67gindividuals at 25 ’ and 2 1 “C. In all cases enhancement is greater at lower temperatures. In summary, three observations suggest the enhancement of wa function shows a degree of independence from the lethal functions in su(f)fs726, su(f) ts67g, and su(f) pb. First, individuals with ts lethal genotypes reared at intermediate temperatures do not necessarily show strong enhancement; in fact, the reverse is often the case. Second, lethal alleles which complement for lethality do not complement for enhancement. Finally, the lethal effects of su(f)‘q su(f) ts726and su(f)pb are stronger at high temperatures, while the enhancement effects are stronger at low temperatures. There is a complex pattern of partial dominance for enhancement between the different alleles. Considering the enhancement phenotypes of the homozygous and heterozygous individuals, the alleles form a series:

su(f)' >

sll(f)‘S7*6 >

su(f)'s67g > su(f)Pb.

Suppression of forked’

Measuring the strength of suppression of the forked effect of different su(f) alleles is complex. Alleles of su(f) have two primary effects on the forked bristle phenotype. They decrease the penetrance of forked’, that is, reduce the number of bristles which exhibit any forked phenotype. They also decrease the expression of forked’, that is, reduce the severity of the forked phenotype of bristles which exhibit forked. Many su(f) alleles also exhibit a “Minute” effect, reducing the length and thickness of bristles. However, this effect is also seen in individuals who are not expressing the forked’ mutation (data not shown), suggesting that this effect is not related to the suppression of forked]. These three characteristics were measured for individuals with different su(f) genotypes by recording the phenotype (forked vs not forked, degree of forked phenotypic expression, and size) for each bristle in a select set of thoracic bristles, as described in the Materials and methods. To interpret these results, it is necessary to first examine the penetrance and expressivity of forked] in the control individuals. In control individuals the penetrance of the forked’ allele is nearly complete, that is, usually all of the 12 thoracic bristles scored

in our analysis show some degree of forked phenotype. The expression, however, is variable. The smaller thoracic bristles show only partial expression, usually measuring 2 or 3 on a scale of 1 (no forked) to 5 (maximal forked). The larger scutellar bristles show a much more complete penetrance, usually measuring 5. The smaller bristles are also more strongly affected by su(f) mutations, often showing a completely suppressed phenotype in su(f) individuals. The variability in forked’ penetrance between the bristles presents a potential problem in measuring the effects of w(f) alleles on forked’ expression. If two su(f) alleles have different effects on the penetrance of forked’, individuals containing these alleles will have different sets of thoracic bristles expressing the forked phenotype. Our measure of the su(f) suppression effect on forked’ expression is the difference between the average forked expression in the bristles showing forked. If we compare individuals expressing the forked phenotype only in large bristles to controls expressing the forked phenotype in all thoracic bristles, a portion of the difference will be due to the normal difference in forked’ expression in the different sets of bristles. To eliminate this effect we calculated the suppresion effects of m(f) alleles on the forked’ expression using only the four scutellar bristles. The scutellar bristles show similar expression and penetrance. Both the penetrance and the expression of forked’ are greatly reduced in individuals homozygous for several su(f) alleles (Table 1 and Table 2). Considering effects on penetrance first, individuals homozygous for the su(f)’ allele show a strong reduction in forked’ penetrance at all temperatures (90-99%). Suppression is slightly stronger at higher temperatures. Individuals homozygous for su(f) tS67gor su(f) tr726 . show strong suppression at 25 “C and weak suppression at 2 1 “C. The su(f) tS726 allele has a stronger effect than the su(f) ts67gallele at both temperatures. The su(f)Pb/Y individuals show a strong but not complete suppression (90%), stronger than either su(f) ts67~or su(f) rs726individuals reared at low temperature. Individuals with a su(f) l/h(f) rs726 genotype show a strong suppression of forked penetrance at all temperatures (81%-94%). Individuals with su{f) l/su{f)tJQ’ genotypes show strong suppression of forked penetrance at 29 “C (99%) and 25 “C

193 (95%) but weaker suppression at 21 “C (67%). This same pattern is shown by the su(f)‘/s~(f)@ individuals. The suppression is not as strong in any of these genotypes as in the su(f) ‘/su(f) 1 individuals. The weakest suppression of forked’ penetrance occurs in the su(f)zS67g/su(f)pb and su(f)*s726/su(f)pb individuals. These show strong suppression at 29 “C (96-98%) but weak suppression at 21 “C (3021%). There is a complex pattern of partial dominance between the alleles for suppression offorked penetrance. Individuals heterozygous for two alleles, in general, have a phenotype intermediate between that of individuals homozygous for those alleles, although there are exceptions. The su(f)‘/ su(f)pb individuals, for example, when reared at 2 1 “C show weaker suppression of penetrance than either the su(f) ‘/h(f) J or the su(f)Pb/Y individuals. Considering the homozygous and heterozygous genotypes, these alleles can be arranged in an allelit series in terms strength of suppression offorked’ penetrance: su(f) ’ > SU(f)Pb > su(f)‘S7*6 >

mately half the size of wild-type bristles. Individuals with a su(f) l/su(f)z genotype show almost complete suppression of forked at all temperatures, with essentially no reduction in bristle size (89%98%). This indicates that strong suppression of forked expression and penetrance does not automatically lead to a reduction in bristle size. Individuals homozygous for su(f) rS67gor su(f) tS726show a greater reduction in bristle size at 25°C (59% and 26%) than at 21 “C (59% and 80%). Individuals with a m(f) ‘/h(f) rs67gor su(f) */h(f) tS726genotype show greater reduction in size at 29°C (31% and 36%) than at 21 “C (62% and 82%). Females hemizygous for su(f) l show a greater reduction in size at 29 “C (23%) than at 21 “C (43%). The relationship between size reduction and bristle suppression is not absolute. For example, females hemizygous and homozygous for su(f)l have about the same levels of suppression of forked’ penetrance and expression; yet only the hemizygotes show a significant reduction in bristle size.

su(fp7K

The suppression of forked’ expression is much more uniform and less affected by temperature in individuals with homozygous, heterozygous or hemizygous genotypes. Most su(f) genotypes show fairly show fairly strong suppression of forked’ expression (60-70%) at all temperatures, with the exception of m(f) ts67g/su(f) ts726 and su(f) rS67g/ su(f)pb individuals. These genotypes show much weaker suppression of forked’ expression at 2 1 “C. The suppression at this temperature is weaker than the suppression seen in the individuals homozygous for either allele. The suppression of forked’ penetrance and suppression of forked’ expression are clearly related. The genotypes with lower suppression of penetrance also show reduced suppression of expression. These two phenotypes, suppression of forked’ penetrance and suppression of forked’ expression, also show the same temperature dependence, being stronger at 29 “C and weaker at 21 “C.

Bristle size Bristles may be reduced in size to the ‘Minute’ effect of su(f) or may be reduced by extreme expression offorked. As shown in Table 3, the overall bristle length in the control individuals is approxi-

Suppression and enhancement of lozenge

These results suggest that the su(f) locus has discrete enhancement and suppression functions and that these functions are modified in a characteristic way in each allele. If this is correct, then su(f) alleles should suppress and enhance alleles of other loci with the same relative strengths as they show for wa and fJ. To test this we examined the effects of su(f) alleles su(f) ‘, su(f)tS726, su(f) rS67g, su(f) 94, and su(f)+ on modifiable alleles of the lozenge locus. Alleles of the lozenge locus affect

the size, shape, and texture of the eye (Green & Green 1956). Several alleles of lozenge were studied: lz I, l.~~~,l.~~~,and lz 37. The lz J allele contains a gypsy element while the lz34, lz36, and lz37 alleles are designated only as spontaneous origin (Lindsley & Zimm 1990). The lz37 allele has been shown to be enhanced and the lz’ allele suppressed by the su(f)’ allele (Snyder & Smith 1976). It is not known whether a copia element resides in the gene giving rise to the 1z37allele (Dr. M. Green, Universoty of California, Davis, California, personal communication). Studying the effects of several su(f) alleles on enhanceable and suppressible alleles of lz has the advantage that the enhancement and suppression affect the same phenotypes. This test has the disadvantage that the phenotypes given by

194

195 Table 3. Bristle size and forked expression. 29°C Genotype

su(fPv

m(f)

su(f)

‘/su(f)‘S6’~

‘/l/E738

25 “C

21°C

Description

size

SC

size

SC

size

SC

Br phenotype # females

0.47 44

4.4 178

0.44 50

4.5 118

0.49 57

4.5 224

% suppressed # females

0.89 104

70.8 10

0.94 75

71.4 17

0.98 73

71.0 39

% suppressed # females

0

0.91 59

63.4 90

0.59 54

48.7 172

% suppressed females

0

0.26 106

71.4 17

0.80 72

69.0 119

% suppressed # males

0

0

0.46 30

67.8 19

% suppressed # females

0.31 54

70.8 4

0.61 26

71.4 13

0.62 67

60.3 147

% suppressed # females

0.36 113

57.7 112

0.87 74

71.4 39

0.82 51

66.4 90

% suppressed # females

0.89 130

68.8 39

0.78 125

71.4 103

0.77 84

63.5 196

% suppressed # females

0.94 112

67.9 40

0.73 111

60.6 272

0.49 41

35.4 154

% suppressed # females

0

0.69 31

65.7 11

0.65 26

35.7 89

% suppressed # females

0.23 55

0.37 33

71.4 4

0.43 47

71.0 5

% suppressed # females

0

0

0.50 34

67.2 38

% suppressed # females

0

0

0.47 56

71.0 4

70.8 A

SC = mean expression of forked in scutellar bristles; size = relative size of bristles

lozenge alleles are not easily quantified. The control individuals for this study had the genotype lz f’/Y, where the lozenge allele was either lz I, Iz~~, lz36, or lz j7. Individuals of lz 37 su(f) + genotype are shown in Figure 2A. All individuals in this study were raised at 25 ‘. The lozenge eye is ovoid in shape, and the surface has a slightly roughened and glistening appearance, referred to as glossy. The red eye pigment is uniformly distributed. Individuals of the Zz37f’ su(f) j/Y genotype (Fig. 2C) have an eye phenotype which is very ovoid with a surface which is even more glossy than the control. This is a more extreme mutant phenotype and it indicates that su(f) I is a strong

enhancer of lz37. Individuals of the lz37 f l su(f) fS7z6/Ygenotype (Fig. 2B) have an eye phenotype which is slightly more ovoid and slightly more glossy. The su(f) n726 allele is thus a moderate enhancer of 1~~~.The su(f) tS67gallele is also a moderate enhancer of Lz-‘~ (not shown). The relative strengths of these three alleles as enhancer of 1z37 show the same pattern of relative strength as their enhancement of wa. Individuals of the genotype lz’ f' m(f) l/y genotype (Fig. 2F) have an eye phenotype which is less mutant than the control genotype, lz’ fz (Fig. 2D). The eyes are less ovoid and the surface less glossy. This is a relatively strong suppression phenotype.

196

Fig. 3. A Northern analysis of Sgs-3 transcripts in larvae with different m(f) genotypes. Larvae were collected and reared at 21 “C until the molt between the second and third instar when they were shifted to 29 ‘C. At either 36,42, or 48 h after this molt the larvae were collected, total RNA was isolated and probed for the presence of $5-3 transcripts. + = y* w0 su(f)+ larvae, ts67g = y’ wa ~u(f)‘~~~g larvae, su(f) I = y2 w0 f’ su(f) i larvae.

Individuals with the genotype 12’ f' su(f)ts726/y genotype (Fig. 2E) have eyes which are slightly less ovoid and slightly less glossy than the lz, w(f)+ controls. This phenotype indicates su(f)ts726 is a weak to moderate of lz’ (not shown) which is the same pattern of relative strength of suppression for Zz and suppression for f'. None of our alleles showed any enhancement or suppression effects on the 1,~~~or the Zz34alleles. Lethality and glue protein gene suppression The su(f) locus contains two complementing lethal functions (Schalet & Lefevre, 1976). In our analyses this is confirmed by the survival of individuals with su(f)tS67~/s~(f)pb and s~(f)~~~~6/su(f)p~ genotypes at all temperatures. These individuals show varying degrees of enhancement of V and suppression off I, indicating that these alleles do not complement for all m(f) functions. As a further test of the lethal complementation, we examined the effect of these genotypes on glue protein transcription. When individuals with w(f) tS726or su(f)rS6Tq genotypes are reared at the restrictive temperatures dur-

197 ing the third larval instar period the normal premolt rise in ecdysterone does not occur, and the transcription of the glue protein genes is suppressed (Hansson et al., 1981). Lack of ecdysterone is the cause of the glue protein transcript loss as indicated by the fact that glue protein transcripts can be induced by injection of ecdysterone (Hansson & Lambertson, 1983, 1984). We tested whether this phenotype is an effect of the lethal functions or of the enhancer/suppressor functions of the su(f) locus by using Northern analysis to determine whether the glue protein gene transcription is blocked in individuals with different su(f) genotypes. Female larvae were reared continuously at a controlled temperature (2 1’ or 29 “C) or were reared at 2 1 “C and were shifted to 29 “C at the molt between the second and third larval instar. The larvae were harvested and the RNA isolated 36, 42, or 48 h later, The RNAs were isolated and used in Northern analysis. The 1.4 Kb glue protein gene transcript is present at all times in the temperature shift experiment individuals homozygous for su(f) I and su(f) + (Fig. 3). However, the transcript is not present between 36 and 48 h after the molt in temperatureshifted individuals homozygous for su(f) ts726 or s~(f)‘~~‘g. The transcript is present in individuals with su(f)pbfi, su(f)Pb/Df(l)VE738, su(f)“726/ su(f)Pb, su(f)tS6’qsu(f)p4 su(f)‘/su(f)tS”6, su(f)‘/ su(f) ts67g, and su(f) ‘/su(f)pb genotypes reared at

2 1 “C or 29 “C. However, in individuals homozygous for su(f) ts67gor su(f) ts726the transcript is present at 2 1 “C but not at 29 “C. In summary, as shown in Table 4, individuals with a viable su(f) genotype show no suppression of glue protein RNA regardless of whether this genotype shows a strong or a weak enhancement of wa or a strong or a weak suppression off’. Individuals with a lethal su(f)pb genotype also show no suppression of glue protein RNA. Molecular

analysis of su( f)

Cloning, subcloning, and restriction mapping of su( f) alleles: A 16.5 Kb DNA fragment containing all or part of the su(f) locus was isolated by P

element tagging and cloned into the lambda vector EMBL4 by Dr. K. O’Hare (personal communication). The clone, h252.512, was subcloned into pUC plasmid vectors. These subclones were used

Table

4. Northern analysis of glue protein

Genotype

su(“f) ‘Mf) + sucf, ‘/sum’ su(f)r.~7?6/su(f)rs7.76 su(f)

r567+sli(f)

Mf)

‘w

su(f)ph/VE738 su(f)q.w(f) su(f)qsli(f)‘s67~ su(f)‘/su(f)‘.~7~6 su(f)‘/su(f)“67~ S4f)‘/sU~fP

Continuous temperature

Temperature shifts

21°C

29°C

+36

+42

+48

+ +

+ +

+ +

+ +

+ +

-

-

+ ‘~67~

726

transcription.

+ +

+

n/a

n/a

n/a

+

+ + + + + +

n/a n/a n/a n/a n/a nla

n/a n/a nla n/a da n/a

n/a nla nla n/a n/a n/a

+ + + + +

+ = glue protein transcript present; - = glue protein transcript not present

as probes to generate a restriction map of genomic DNA. The genomic map was established by restricting DNA from females with the genotype y2 w” f’ * y+/FM7 with seven different restriction enzymes. DNAs from su(f) Ms252/FM7 females and from FM7fl males were included as controls. Single and double restriction enzyme digests were analyzed for each of the three DNA types, using the restriction enzymes: BamHI, EcoRI, HindIII, PstI, S&I, XbaI, and XhoI. The resulting restriction map is shown in Figure 4. The map includes the 16.5 Kb corresponding to the cloned fragment and an additional 11.1 Kb. The map of the 16.5 Kb genomic region closely matches the map of the cloned fragment (O’Hare, personal communication). Positions that are closer to the centromere are given positive values. A region of homology exists between the two areas underlined with dashed lines in Figure 4. This homology was deduced from the strong cross hybridization of the 4.4 Kb XhoI fragment with the 3.2 Kb EcoRI fragment. Since the su(f) locus is in the l3 heterochromatin of the X chromosome, repeated sequences within this locus are not surprising. Similar cross hybridizing regions were also reported (Lindsley & Zimm, 1990) from -32.0 to -29.4, from - 23.4 to - 19.9, from - 10.2 to - 7.8, from -7.4 to-6.6,from-5.0to-2.0,andfrom+4.9to+19.0. In our map the regions of homology are from the XhoI +6.4 site to the Hind111 +4.35 site and from the EcoRI - 0.2 site to the EcoRI - 3.4 site. There-

198 fore, the strong cross hybridization between these fragments indicates that the regions are highly homologous. The determination of the number and locations of these sequences in the genome by in situ hybridization to salivary gland chromosomes is described below. Restriction fragment length polymorphism study of su( f) alleles: DNA from individuals with different

su(f) alleles were analyzed using Southern hybridization to determine if RFLPs existed. For each allele, DNA was isolated from females heterozygous for the allele and the FM7 balancer chromosome while FM7fl and 6~’ waf 1 su(f) ‘/FM7 DNA served as controls. Of the 19 alleles tested, two, [m(f) MS2x2and su(f) 94] showed RFLPs. The lesion associated with the su(f) Ms252allele is the result of the insertion of a P element at map position + 0.1, also described in Lindsley and Zimm (1990). The other allele in which a RFLP was found, s4”f J94, was isolated by EMS mutagenesis as described in Materials and methods. The RFLP associated with this allele was detected in genomic DNA from homozygous females, digested with BamHI and probed with the 4.4Kb XhoI fragment. This probe hybridizes with equal intensity to two 9.0 Kb wild type fragments, (+ 13.25 to +4.25, and +4.25 to -4.8). When hybridized to DNA from su(fJ9” two bands were observed, a 9.0 Kb and a 7.0 Kb band. The smaller band is due to an additional BamHI site at position + 11.15 (Fig. 4). This new BamHI site is the result of a point mutation, confirmed by additional Southern analyses of mutant DNA digested with EcoRI, SalI, BamHI and EcoRI, and XhoI and HindIII. No differences were detected with these restriction digests, eliminating the possibility of a sizable deletion in this region. Thus, a simple point mutation yielding a new BamHI site at + 11.15 is the best possible explanation for the RFLP. These findings lead to a more thorough study of the phenotype produced by this allele as described below. RFLPs for some m(f) alleles have previously been reported (Lindsley & Zimm, 1990). Our results agree with these findings that alleles su(f) ‘, su(fjD13, su(f)IJb, su(f)‘.s67x, and su(f) n1udr.s have a normal map. The normal restriction map for the other alleles determined in this study have not previously been reported.

In situ hybridization The cloned DNA fragment hybridizes to the su(f) locus. Subcloned fragments were used as probes for in situ hybridization to polytene chromosomes from three different genotypes: Ore R, Dp(l:2)BS, and Dp(l:3)BS. The Ore R served as a wild-type control and the remaining two served as positive test. The duplications contain a wild type copy of the su(f) locus translocated to the terminal region of chromosome 3L (Dp(l:3)) or 2L Dp(l:2)). Two probes were hybridized to chromosomes from each strain. The first was the 1.9 Kb XhoI-Hind111 fragment, containing genomic DNA from the region reported to be transcribed (Lindsley & Zimm, 1990), while the second was the 3.2 Kb EcoRI fragment which contains repeated DNA described above. The purpose of the second probe was to determine whether these repeated sequences are located elsewhere in the Drosophila genome. The first probe hybridized to the base of the X chromosome in all three genotypes. When hybridized to the Dp(l :3)BS chromosomes, it also hybridized to the distal tip of 3L but not to 2L (Fig. 5). When hybridized to chromosomes of the Dp(l:2)Bs strain, it also hybridized to the distal tip of 2L but not to 3L. When the second probe is hybridized to the chromosomes the same pattern of hybridization is observed. The hybridization of this second probe to chromosomes from the Dp(l:3)Bs strain is shown in Figure 5. The hybridization pattern of both probes is consistent with these cloned fragments being part of the su(f) locus. The similar hybridization pattern of the probes with and without the repeated sequence suggests the repeated sequence is unique to the base of the X chromosome. Analysis of su( f) 94

The m(f) 94 allele, generated by EMS mutagenesis, contains the RFLP as described above. To determine if the lesion was responsible for the mutant phenotype, a series of outcrosses and Southern hybridizations were performed. Homozygous su(f) 94 individuals show moderate enhancement and weak forked suppression, while individuals heterozygous for su(f)9J and su(f)‘, su(f)m67g, or su(f)“726 show moderate eye enhancement and moderate bristle of su(f)94 and its suppression. Recombination

199

Fig. 5. The sites of hybridization of su(f) probes to salivary gland chromosomes. Arrows indicate the location of hybridization in each photo. A. Hybridization of the 1.9Kb X/z&Hind111 fragment to chromosomes from Dp(l:3)B’ larvae. B. A higher magnification showing the hybridization to the tip of 3L but not to 2L. C. Hybridization of the 3.2Kb EcoRI fragment to chromosomes from Dp(l:3)B’ larvae. In this photo hybridization to the tip of 3L is shown. D. Hybridization of the 3.2Kb EcoRI fragment to chromosomes from Dp(l:3)BS larvae. In this photo hybridization to the centromere region is shown.

modifiable locus, wU, was performed to test its mutant phenotype. Heterozygous y2 wU f’ safe/+ females were mated to +/Y males and recombinant males selected. Males with a f’ SUCK genotype were recovered and showed a weakly suppressed bristle phenotype and no eye enhancement. One recombinant male was then individually mated to heterozygous y? wUf’ scl(f)+ . y+/FM7 females. The y+ marker is a duplication on the opposite side of the centromere and is tightly linked to su(f). Twenty-four recombinant males of the genotype y’ way su{f)94were selected showing the moderate eye color enhancement (20.0%) and a weak forked bristle suppression (18.3%) phenotype. The return of the moderate eye enhancement shows the mutation to be linked with m(f), that is, sm(f)9Jis an allele of m(f). This was confirmed with complementation tests by using the phenotype of increased bristle suppression in the heterozygous condition with a ts alle. Each of the 24 recombinant males were individually mated to y2 w” f1 su(f)r.5726/FM7 females and the progeny were reared at 29°C. The

wa f1 female progeny with a y’ w f1 su(fj9y” ts726 genotype showed a moderate eye enhancement and moderate bristle suppression. This increased bristle suppression for the heterozygote using the recombined su(f)94 chromosome shows the same level of bristle suppression as that of the original heterozygous individuals. To determine whether the observed RFLP in the m(f) 94 line was associated with the m(f) mutation, DNA was isolated from the y’ Iv” f’ m(f) 9”/FM7 siblings in the 24 crosses described above. These DNA samples were digested with BumHI and analyzed by Southem analysis using the 4.4 Kb XhoI fragment as a probe. The RFLP was detected in each line (data not shown), indicating that the molecular lesion is most likely responsible for the m(f) 94 mutant phenotype. Therefore, among the m(f) alleles tested, all showing varying levels of eye enhancement and bristle suppression, we have found one nonlethal allele with a molecular lesion which appears to be linked to the mutant phenotype.

sL,(f)

200 Discussion The su(f) modifier finctions su( f) lethal finctions

are distinct from the

A major question of the current report concerns the number and types of genetic functions present at the su(f) locus. Previous studies have documented that alleles of this locus produce several distinct phenotypes including: enhancement, suppression, lethality, pattern abnormalities, suppression of ecdysterone, and blockage of glue protein transcription. One possible explanation for these phenotypes is that they result from mutational alterations of a single function. A single su(f) transacting modifier function could produce a variety of phenotypes by interacting with numerous target alleles. Alternatively, the different su(f) phenotypes could result from mutational alterations of several different functions, each function producing a specific phenotype or group of phenotypes. We believe the latter explanation is most likely, and that the su(f) locus in fact contains four genetic functions: enhancement, suppression, lethal-pb and lethal67g. Our results suggest the modifier functions of su(f) are separate from the lethal functions. First, nonlethal su(f) mutant alleles produce strong enhancement and strong suppression phenotypes. The su(f) z allele, for example, produces some of the strongest enhancement and suppression phenotypes yet these individuals show excellent viability and fertility. In addition, the su(f) l allele complements the lethality of known lethal alleles and deficiences of the locus. It does not, however, complement the enhancement or suppression phenotypes of the lethal alleles. For example, individuals with a su(f) I/ su(f) rs726or a su(f) ‘/&(f)ph genotype survive at all temperatures, yet show enhancement and suppression phenotypes. This ability to complement the lethality without complementing the enhancement and suppression suggests these are separate functions. If modification resulted from a sublethal expression of a lethal function then ‘escapees’, individuals with a lethal genotype who survive, should show very strong suppression and enhancement phenotypes. However, individuals with su(f) rs726or su(f) rs67glethal genotypes reared at intermediate temperatures survive and show moderate enhance-

ment and suppression phenotypes. Individuals with a sz@Jpb genotype who survive show little or no w” enhancement and moderate forked suppression. This also suggests the modifier and lethal functions are distinct functions. Finally, the lethal effects of the su(f) ts726and the su(f)pb alleles do not require the presence of transposable element insertion alleles at any other locus. We have recombined these alleles into numerous different genetic backgrounds with and without wa or f without loss of lethality. This suggests that while the vital functions of su(f) may interact with Drosophila genes, they do not interact with the transposable elements present in the modifiable target alleles. The enhancement distinct

and suppression functions

are

Alleles of su(f) have two modifier effects, enhancement and suppression. There are two possible models for these modifier functions of su(f). There may be one genetic function that is responsible for both effects, or there may be separate enhancer and suppressor functions. Several pieces of evidence from a number of studies address this question, First, the mechanism of action of su(f) mutations is different for suppressible vs. enhanceable mutations. The copia insert in the enhanceable wa allele reduces the number of white transcripts by premature termination of transcription at the LTR of the inserted element. Enhancement occurs by an increase in the fraction of terminated transcripts. This suggests that the target of su(f) action is the LTR of the copia element. The suppressible forked alleles contain inserted gypsy elements, and produce a reduced amount of full length forked transcripts. However, truncated forked transcripts are not observed, suggesting that premature termination is not involved. In individuals with m(f) mutations the number of forked transcripts is increased, and the number of gypsy transcripts is altered. There is no evidence that su(f) mutations affect termination at the gypsy LTR. These results suggest that at the molecular level m(f) interacts differently with the transposable elements inserted in suppressible and enhanceable mutations, which is consistent with multiple functions. In our studies we observe that su(f) mutations have different strengths of enhancement and suppression. If there is a single modifier function, we

201 might expect that changes in suppression and enhancement would be correlated, that is, mutations with strong enhancement effects should also have strong suppression effects. This is not what we observe. Different su(f) alleles have strong or weak suppression and have strong or weak enhancement. The lack of correlation of these effects is suggestive of a multiple function model. Further evidence comes from our temperature sensitivity studies. For several alleles we observe a stronger suppression of forked phenotype at lower temperature and a stronger enhancement of white-apricot phenotype at higher temperature. The opposite temperature sensitivities of the two effects is also suggestive of multiple functions. To ensure that our observations of suppression and enhancement were not biased by peculiarities of the forked and white loci, we observed the effects of su(f) mutations on lozenge alleles. We observed the same patterns of activity. Alleles that are strong suppressors offorked are also strong suppressors of lozenge. Alleles that are strong enhancers of whiteapricot are also strong enhancers of lozenge. The patterns of temperature sensitivity are also the same, su(f) alleles with stronger suppression of forked at lower temperature have stronger suppression of lozenge at lower temperature. Alleles with stronger enhancement of white-apricot at higher temperature have stronger enhancement of lozenge at higher temperature. We conclude from these results that there are two so modifier functions, one for suppression and another for enhancement. The fact that known enhanced alleles and suppressed target alleles have different inserted elements (enhanced - copia, suppressed -gypsy) suggests these effects may be studied further, and our hypothesis may be tested, by analyzing the molecular basis of the interaction of so with the different elements. su( f) has two lethal functions Previous reports of individual su(f) lethal alleles suggested that there were two lethal functions in su(f) (Schalet & Lefevre, 1976). This was based on the complementation patterns of the su(f)ph allele with .su(~)“~~R and .YU(~)“~~? All three are strong lethal alleles but individuals with a su(f,ph/ su(fJrS6@, or su(f)Ph/su(f)“726 genotype survive at all temperatures and show a suppressed forked phe-

notype. The classic interpretation of two lethal alleles which complement is that the locus has multiple vital functions. We examined additional aspects of the lethal phenotypes of these alleles to determine whether there were other indications that there are two lethal functions. The su(f)rS67g and su(f) rs726alleles produce cell death and pattern abnormalities in response to sublethal heat treatments. They also enhance the lethal effects of Minute(3) i55. The su(f)pb allele does neither. The su(f)rS6Q and m(f). tr726 alleles block ecdysterone production during the third larval instar. This block affects the activity of a number of genes, including the glue protein genes. Northern analyses confirmed that for one glue protein gene, Sgs-3, transcripts were not present in larvae with either a su(f)Lv6Q or a su(f)tS726 genotype that were shifted to the restrictive temperature. Larvae with a su(f)ph genotype have Sgs-3 transcripts at all temperatures, as do larvae with su(f)tS67g/su(f)ph and SU(~)““~/ su(f)“h genotypes. The su(f)ph lethality apparently does not affect glue protein transcription. There are thus several clear differences in the lethal phenotypes of the su(f)pb and the su(f)tS6@ and SU(~)~~~?~ alleles. Taken together, these observations support the hypothesis that there are two separate lethal functions at the m(f) locus. Individuals with different viable su(f) genotypes were also examined for the presence of Sgs-3 transcripts. Transcripts were found in individuals of all such genotypes, and there were no apparent differences between individuals with genotypes which produced strong enhancement and suppression effects and those with genotypes producing weaker effects. Accepting the lack of Sgs-3 transcript as an indicator of the block of ecdysterone, these results indicate that strong enhancement and suppression effects of su(f) alleles do not require, nor are they necessarily produced by, a block in ecdysterone production during the third instar. This block appears to be only associated with the ts67g-ts726 lethal function. A preliminary molecular study demonstrated that a fragment of DNA, isolated from the base of the X chromosome by P element tagging, contains at least a portion of the m(f) locus. Genomic mapping and RFLP analyses indicate that molecular lesions associated with SK(~)9J and m(f) MS252occur within this region. The molecular lesion associated with su(jJ9j has been shown to be linked with viable

202

Fig. 6. A model for the action of the su(f) gene showing the proposed four functions. 1t, 1, = vital functions, S, = suppression function which interacts with gypsy elements, S2 = enhancement function which interacts with copia elements. The interaction of su(f) with the copia LTR has been shown to be dependent on a function encoded within the copia element (Mount et al., 1988).

su(f) phenotypes. In addition, this DNA hybridizes to copies of su(f) + located on two different translocation chromosomes. It is hoped that additional molecular analyses will soon shed important light on the structure and function of this complex locus, and on the mechanisms of suppression and enhancement. In summary, the results of this and other genetic analyses of the su(f) locus support the hypothesis that there are at least four genetic functions at this locus, Two of these affect transcription of particular transposable elements and are responsible for the modifier phenotypes of su(f) alleles. Further evidence for or against our model of su(f) (Fig. 6) will require additional molecular analysis, particularly

of the structure and biochemical product(s) of this locus.

function of the

Acknowledgements We would like to thank Dr. Kevin O’Hare for the use of the cloned su(f) fragment, Dr. William Welshons for advice on crosses, Jean Welshons for help with stocks, and Franchesca Winandy for help with salivary gland chromosome preparation. This project was partially supported by NSF grant DCB8542374 and is Journal Paper No. 14893 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2978.

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