Adult flies mutant for doublesex dominant (dsxD) are intermediate in phenotype between males and females. The dsxD mutation acts in the heterozygous state to transform only flies with two X chromosomes into intersexes, XY flies are unaffected by the mutation. Yolkprotein synthesis, which normally occurs in the ovaries and fat bodies of females, but not in males unless stimulated with 20-hydroxy-ecdysone, is reduced. The dsxD fat body synthesizes less yolk proteins throughout adult life, and the gonads rarely make yolk proteins. Using cloned yolk-protein genes as probes for measuring transcript levels we have shown that expression of these genes in dsxD is regulated both transcriptionally and post-transcriptionally. We suggest that the dsxD locus regulates the expression of the yolk-protein genes from within the fat body cells and does not operate by modulating ecdysteroid titres in the adults.

The three yolk polypeptides of Drosophila melanogaster are synthesized by the fat body and the ovarian follicle cells of adult females (Bownes & Hames, 1978; Brennan, Warren & Mahowald, 1980). Each polypeptide is coded for by a single-copy gene located on the X chromosome (Barnett, Paehl, Gergen & Wensink, 1980). Their expression is regulated in isolated female abdomens by juvenile hormone and 20-hydroxy-ecdysone (Jowett & Postlethwait, 1980), and males can be induced to express the yolk-protein genes by high levels of 20-hydroxy-ecdysone (Postlethwait, Bownes & Jowett, 1980; Bownes, 1982). Whether or not the yolk-protein genes are expressed in adults is correlated with the expression of a series of autosomal genes affecting sex determination. Mutations in these genes (intersex, transformer, transformer-2 and doublesex) alter the expression of many genes such that XX flies, which would normally follow a female developmental pathway, can become phenotypic males (pseudomales) or intersexual, and XY flies, which would normally follow a male developmental pathway, can become intersexual (Baker & Ridge, 1980). All intersexual flies have circulating yolk proteins in the haemolymph, and all pseudomales lack yolk proteins regardless of the X and Y chromosome constitution (Postlethwait et al. 1980; Bownes & Nöthiger, 1981).

In our previous studies we found that the quantities of yolk proteins circulating in the haemolymph were low in intersexual flies compared to wild-type females, even in cases where there were two X chromosomes present and thus reduced levels were not due to XY flies having only half the number of genes available for transcription (Bownes & Nöthiger, 1981). We have investigated the expression of the yolk-protein genes in one of the sex mutants, doublesex dominant (dsxD) which when heterozygous causes XX, but not XY, flies to develop an intersexual phenotype, in an attempt to understand the mechanism by which the sex genes regulate the expression of the YP genes.

We have measured yolk-protein synthesis in adults during maturation and the levels of transcripts coding for the yolk proteins during adult development and in various tissues. The results presented in this paper lead us to propose that the dsxD gene affects the expression of the YP genes in the fat body from the time of eclosion and that no feedback mechanisms operate involving circulating yolkprotein levels. We propose that post-transcriptional control mechanisms affect the level of expression of the yolk-protein genes and as a consequence of this the transcripts of the yolk-protein genes are translated less frequently into polypeptides in vivo in dsxD than in wild-type flies.

Maintenance of stocks

Flies were maintained on a standard yeast, cornmeal, sugar, and agar medium at 25 °C. Wild-type control flies were an Oregon R (OrR) strain and the sex mutant used was dsxD as a +/T (1;3) OR60, /TM6μsxQSb e stock (for mutations and symbols see Lindsley & Grell, 1968) which allows identification of dsx° adults by morphological markers. The dsxD allele was selected for these experiments because dsxD affects flies with 2 X chromosomes so there are no problems of reduced numbers of copies of the yolk-protein genes as there are in XY flies and because this stock is healthy and viable. Several populations of dsxD and OrR were maintained.

The mutant flies used were dsxD/ +, XX and are referred to throughout as dsxD adults or intersexes.

Preparation of tissues

When RNA was to be isolated from individual tissues, or when tissues were cultured in vitro, they were dissected into a Ringer’s solution (Chan & Gehring, 1971). These tissues were either cultured in Ringer’s or Grace’s medium (Grace, 1962) or placed in RNA-extraction buffer (see below) and then stored at –20 °C or extracted immediately as appropriate. For preparing various tissues the gut and Malpighian tubules were separated at the joint of the thorax and abdomen and at the genitalia, gonads were separated at the genitalia. The head and thorax were taken intact, and the remainder was the abdominal body walls.

Analysis and quantitation of yolk proteins

Flies were injected with 0·2μl of [35S]methionine and maintained for 4h at 25 °C. The haemolymph of each set (8–10 flies) was then collected into 50μ1 Laemmli buffer (Laemmli, 1970) containing 1% SDS, 0·1% 2-mercaptoethanol, 10% glycerol, and 0·05M-Tris HC1 pH6·8. The flies were also placed in 50 μ Laemmli buffer, vortexed, and the cuticle and insoluble parts removed by centrifugation. The samples were then heated to 90°C for 15 min, 1μl of each sample was T.C.A.-precipitated and the amount of label incorporated was measured by liquid scintillation counting. Equal numbers of counts of each sample (the exact number varied from gel to gel) were loaded onto a 7 % –20 % polyacrylamide gradient slab gel and run overnight (Laemmli, 1970). The gels were then prepared for fluorography using the technique of Bonner & Laskey, 1974.

The resulting autoradiographs were scanned with a densitometer and the areas under the yolk-protein bands were calculated. This method is useful for comparison of yolk-protein synthesis between samples.

In vitro culture of tissues

Tissues were cultured in 20 μl of Ringer’s or Grace’s medium supplemented with 4μl of [35S]methionine for 4h. Cells and medium were separated by centrifugation and either Laemmli buffer or antibody-precipitation buffer was added to each sample. The cells were broken by vortexing and the debris was removed by centrifugation. Samples were either analysed directly as above, or were first precipitated with anti-yolk protein antibody before separation using SDS polyacrylamide gel electrophoresis.

Precipitation with anti-YP antibody

The specificity of the antibody has been described previously (Bownes & Nöthiger, 1981) and the precipitations were carried out in the same way.

Isolation of RNA

RNA was extracted from tissues and whole flies by homogenization in RNA-extraction buffer (2 mM-MgCh, 0· 5 % SDS and 10 mM-Tris/HCl, pH 7· 5). They were then subjected to multiple rounds of extraction against a phenol/chloroform mix (6:1, by volume) by vortexing. The organic layers were re-extracted at least once before being discarded. The final aqueous layer was subjected to ethanol precipitation (2· 5 volumes ethanol plus 0· 1 volume of 3 M-sodium acetate pH6· 0) at – 20 °C overnight. The resulting precipitated nucleic acids were washed in ethanol and again precipitated (with 0· 6 ml ethanol plus 0· 3 ml 04 M-NaCl 0· 0· 1 M-Hepes buffer pH 7· 0) at —20 °C overnight, and dried under vacuum. The resulting RNA was stored as an aqueous stock solution.

Translation of RNA in a cell-free translation system

Samples of RNA, either total RNA samples or polyA+ and poly A fractions separated using an oligo-dT column, were translated in the rabbit reticulocyte cell-free translation system as described by Pelham & Jackson (1976).

Resulting proteins were precipitated with anti-yolk-protein antibody as in Isaac & Bownes (1982).

Preparation of32P-labelled YP-probes

g of an equimolar mixture of pYPl, pYP2, and pYP3, which are plasmids containing one copy of a yolk-protein DNA sequence (details of the plasmids used can be found in Barnett et al. 1980), was nick translated to approximately 107c.p.m./μg DNA (method adapted from Maniatis, Jeffrey & Kleid, 1975). Unincorporated nucleotides were removed using a Sephadex G-50 column.

Measurement of YP-RNA levels

There was some translation of yolk proteins from the polyA fraction in the rabbit reticulocyte lysate cell-free translation system. Although this was minor compared to the polyA+ fraction it was not clear that this would always be the case in experiments, thus we used total extracted nucleic acids for these experiments, quantitating YP-RNA levels.

RNA was separated on formaldehyde. The gels were 1·2 % agarose, 20 mM-MOPS, 5mM-sodium acetate, ImM-EDTA and 2·2M-formaldehyde pH7. The RNA samples were dried down and dissolved in 20μl 50% formamide, 2·2 M-formaldehyde, 20mM-MOPS, 5mM-sodium acetate and ImM-EDTA. Bromophenol blue and Ficoll were added to the samples. The gels were run at 200 V for 1 to 2h and the RNA was transferred by blotting for 12–15 h onto nitrocellulose previously equilibrated with 20×SSC. For dot blots the RNA was spotted directly onto nitrocellulose which has been equilibrated with 20 x SSC and dried under a lamp. The nitrocellulose was then baked for 2 h at 80 °C under vacuum (Thomas, 1980).

After prehybridization for 8–20 h at 37 °C in 50% (vol/vol) formamide, 5 × SSC, 50mM-sodium phosphate pH 6·5, sonicated denatured salmon sperm DNA at 250μg/ml and 1 × Denhardts (0·02 % BSA, 0·02 % Ficoll and 0·02 % P.V.P), the filters were hybridized to the labelled probe in four parts prehybridization buffer and one part 50 % (weight/vol) dextran sulphate for 20 h at 37 °C. The filters were washed with four changes of 2 × SSC, 0·1 % SDS for 5 mins each at room temp followed by two changes 0·1 × SSC, 04 % SDS for 15 min at 50 °C, and then exposed to X-ray film for varying lengths of time at —70°C. Some RNA is lost in the transfer method since, when similar quantities of RNA were either spotted directly onto nitrocellulose or transferred to it by blotting and the filters hybridized in the same probe and exposed to X-ray film for the same period of time, the signal was much stronger from the spots. Consequently we used the dot blot method for RNA quantitation in these experiments.

By dotting variable amounts of RNA onto nitrocellulose and carrying out hybridizations and autoradiography we found that if we measured the area x density of the resulting spot this was proportional to the amount of RNA loaded (Fig. 1) between 2 and 5 μg RNA. We have therefore used this method to measure the amount of YP-RNA in the total RNA samples and we always loaded a 5 μg RNA and a 2·5 μg RNA spot. The results can then be compared between samples though we do not have a direct measure of the precise number of YP-RNA molecules present. This technique does not distinguish hnRNA from mRNA.

Fig. 1.

Increasing amounts of RNA were dotted onto nitrocellulose and hybridized to 32P-labelled pYPl, pYP2 and pYP3. The filter was exposed to X-ray film and the area and density of the dots on the resulting autoradiographs were measured. The graph shows four repeats of this experiment using RNA isolated from different populations of females and hybridized to different probes.

Fig. 1.

Increasing amounts of RNA were dotted onto nitrocellulose and hybridized to 32P-labelled pYPl, pYP2 and pYP3. The filter was exposed to X-ray film and the area and density of the dots on the resulting autoradiographs were measured. The graph shows four repeats of this experiment using RNA isolated from different populations of females and hybridized to different probes.

Hydrolysis of RNA

To remove RNA from samples and discover the background hybridization level to DNA extracted along with the RNA, alkaline hydrolysis was used. 5 μg samples of RNA were placed in 45 μl water and 5 μl 5 N-NaOH. After heating to 60°C for 2h the solutions were neutralized with 250μl of 300mM-NaCl, 60 mM-Tris base and 140mM-HCl, and precipitated with ethanol as described above. Fig. 2 shows the results of a dot hybridization with 5 μg of female RNA before and after hydrolysis. A weak signal remains due to the DNA in the samples. This gives approximately the same level of hybridization as 5 μg of male RNA. Males, therefore, have undetectable levels of YP transcripts and hybridization to the controls of each filter provides a measure of background hybridization to DNA.

Fig. 2.

Dot blot of 5 μg samples of female RNA before and after alkaline hydrolysis.

Fig. 2.

Dot blot of 5 μg samples of female RNA before and after alkaline hydrolysis.

Morphology of dsxD adults

Adults carrying the dsxD mutation are intersexual in phenotype. Exactly which sexual morphological characters are shown varies between individuals. The intersexual flies tend to be similar in size to females rather than males. The sex combs, normally present on the male prothoracic leg but absent from the female prothoracic leg, are poorly formed and the pigmentation of the posterior abdominal segments is much darker and more solid than in a female. The external genitalia are also intermediate between those of males and females (Fig. 3).

Fig. 3.

A–C Genitalia and D–F gonads from wild-type males and females and dsx° intersexual flies. Size bars represent 0·1 mm. A and D, female; B and E, dsxD-, C and F, male.

Fig. 3.

A–C Genitalia and D–F gonads from wild-type males and females and dsx° intersexual flies. Size bars represent 0·1 mm. A and D, female; B and E, dsxD-, C and F, male.

The gonads of males and females are shown in Fig. 3, along with an example from a dsxD adult. The gonads are of an intersexual type, often very rudimentary, but tending to be ‘male-like’ more frequently than ‘female-like’ with structures resembling deformed accessory glands. It is very rare to observe any ovarian cell types but occasionally we saw ‘nurse-cell-like’ cells and one or two flies in over 100 analysed had defective yolky oocytes present.

dsxDflies lack a major site of yolk-protein synthesis

The levels of transcripts coding for the yolk proteins in the ovary as compared to the fat body (body walls) were measured using dot blots (see Materials and Methods). Whole body-wall preparations from the head, thorax and abdomen were used for these experiments. Although this means that there are other cell types present the tissue loss dissecting fat body free of the cell walls would be too great. The results in Table 1 show the distribution of transcripts in two groups of wild-type flies. The contribution of the ovary, once oocytes are developing, ranges from as low as 8 % to as high as 65 % of the total yolk-protein transcripts in a fly. This high variability is not really surprising because the ovarian contribution will depend upon the number of oocytes present in stages 8 to 11 (King, 1970) since only these stages synthesize yolk proteins (Isaac & Bownes, 1982; Brennan, Weiner, Goralski & Mahowald, 1982). The average ovarian contribution of approximately 35 % of the total yolk-protein transcripts agrees well with results obtained by translating polysomal RNA from ovaries and fat bodies in a cell-free translation system (Isaac & Bownes, 1982) and with measurement of yolk-protein poly A+ RNA (Brennan et al. 1982).

Table 1.

Distribution ofyolk-protein (YP) transcripts in the ovaries and fat bodies of wild-type adult flies.

Distribution ofyolk-protein (YP) transcripts in the ovaries and fat bodies of wild-type adult flies.
Distribution ofyolk-protein (YP) transcripts in the ovaries and fat bodies of wild-type adult flies.

We analysed the distribution of yolk-protein transcripts from 72 h-old females in more detail (Fig. 4). The results were: gut and Malpighian tubules 0 %, ovaries 40 %, head and thorax 30 %, and abdominal body wall 30 %. Expression of the yolk-protein genes is clearly tissue limited as well as sex limited.

Fig. 4.

Dot blot of 5μg samples of RNA extracted from various tissues of a single group of flies. (OrR females.)

Fig. 4.

Dot blot of 5μg samples of RNA extracted from various tissues of a single group of flies. (OrR females.)

The very small gonads from dsxP flies give very poor yields of RNA. When transcripts coding for yolk proteins from the gonads were measured they were rarely significantly above the male background. Occasionally we have detected very small amounts of yolk-protein transcripts in these samples, presumably from some of the rare female-like gonads. Clearly dsxD flies lack one of the major sites of yolk-protein synthesis which may account, at least in part, for the reduced levels of the yolk-protein synthesis detected in these flies.

Yolk-protein, synthesis in dsxD and wild-type adults

To establish how similar rates of yolk-protein synthesis are between the fat bodies of wild-type and dsxD flies we labelled the proteins with [35S]methionine for 4–5 h periods at eclosion and at daily intervals thereafter until the adults were 5 days old. The [35S]methionine was injected into the haemolymph of groups of ten flies, and at the end of the labelling period haemolymph was collected. We analysed how much newly-synthesized yolk protein was present in the haemolymph as compared to total newly synthesized haemolymph proteins. This should account for most of the yolk proteins synthesized by the fat body. The yolk proteins synthesized by the follicle cells do not circulate in the haemolymph but pass directly into the oocyte (Srdic et al. 1979). During the 5 h period some of the yolk proteins synthesized in the fat body could be transported into the oocytes of wild-type females, but not dsxD flies which lack ovaries. This experiment may therefore slightly underestimate fat-body synthesis of yolk proteins in the wild-type flies.

It can be seen from Fig. 5 that at every stage there is always considerably less newly-synthesized yolk protein circulating in dsxD compared to wild-type adult haemolymphs. The results of several experiments are listed in Table 2. The proportion of yolk proteins synthesized in the labelling period is variable between groups of flies, but is always strikingly reduced in dsxD. There is another haemolymph polypeptide, marked Hl in Fig. 5, which continues to be synthesized as efficiently in dsxD as in wild-type, suggesting that yolk-protein synthesis is selectively reduced in the dsxD flies.

Table 2.

Newly-synthesized yolk-proteins in the haemoloymph of wild-type ( OrR) and dsxD adults

Newly-synthesized yolk-proteins in the haemoloymph of wild-type ( OrR) and dsxD adults
Newly-synthesized yolk-proteins in the haemoloymph of wild-type ( OrR) and dsxD adults
Fig. 5.

Autoradiograph of newly-synthesized haemolymph proteins separated by gel electrophoresis. Samples were collected from flies of varying ages. YPs, Yolk polypeptides; Hl, haemolymph protein 1; ♀ = female; ⚥ = intersex, dsx°-, h = age in hours.

Fig. 5.

Autoradiograph of newly-synthesized haemolymph proteins separated by gel electrophoresis. Samples were collected from flies of varying ages. YPs, Yolk polypeptides; Hl, haemolymph protein 1; ♀ = female; ⚥ = intersex, dsx°-, h = age in hours.

It was possible that dsxD flies instead of secreting the yolk proteins were retaining them in the fat body. All the tissues of the flies were therefore analysed by SDS polyacrylamide gel electrophoresis and the example in Fig. 6 shows that there is not a large retention of yolk proteins in dsxD tissues.

Fig. 6.

Autoradiograph of newly-synthesized proteins in the tissues of flies at various ages. ♀ = female; ♂ = male; ⚥ = intersex, dsxD-, YPs = yolk polypeptides.

Fig. 6.

Autoradiograph of newly-synthesized proteins in the tissues of flies at various ages. ♀ = female; ♂ = male; ⚥ = intersex, dsxD-, YPs = yolk polypeptides.

There appears to be a genuine reduction in yolk-protein synthesis in the fat body rather than a failure of secretion. Since the low level of yolk-protein synthesis in dsxD is observed from early adult life it presumably does not result from a feedback mechanism operating because there are no ovaries to sequester the yolk proteins and prevent their accumulation in the haemolymph. The lack of ovarian yolk-protein synthesis in dsxD flies does not, therefore, account for the reduced levels of yolk-protein synthesis in the whole flies, synthesis being also reduced in the fat bodies.

Accumulation of transcripts coding for the yolk proteins

To establish whether the reduced levels of yolk-protein-gene expression in dsxD resulted from reduced transcript levels in these adults, we isolated RNA from whole adults at various ages after eclosion and measured the transcripts by hybridization to a mixed probe containing cloned DNA coding for each of the three yolk polypeptides. We used several populations of flies for these experiments so that variability between groups of flies could be taken into consideration. In all, five populations were used for various experiments and the variability in accumulation of yolk-protein transcripts was quite marked between these groups of flies and with the age of the adults. The results of some of these experiments (those using probes of similar specific activity (approximately 1 × 107c.p.m.)) are shown in Fig. 7.

Fig. 7.

Graphs of area x density of dot, as a measure of yolk-protein transcript levels plotted against the age of the flies. Results from three populations are shown. ○ – – – ○, dsxD population 1; ▵ – – –▵, dsxD population 2; dsxD population 3; • – – – •, OrR population 1; ▴ — ▴, OrR population 2; ▪ — ▪, OrR pop ulation 3.

Fig. 7.

Graphs of area x density of dot, as a measure of yolk-protein transcript levels plotted against the age of the flies. Results from three populations are shown. ○ – – – ○, dsxD population 1; ▵ – – –▵, dsxD population 2; dsxD population 3; • – – – •, OrR population 1; ▴ — ▴, OrR population 2; ▪ — ▪, OrR pop ulation 3.

Initially the dsx° flies have considerably fewer transcripts coding for the yolk proteins than wild-type flies. Generally the ratio of dsxP to OrR levels was between 0·01 and 0·3. The fact that levels are low from the beginning of adult life again suggests that there is no feedback mechanism operating to reduce transcription of the yolk-protein genes as the quantities of yolk proteins increase in the haemolymph. After several days, however, this pattern changes and dsxD flies have accumulated yolk-protein transcripts to levels close to the wild-type level and in one population there was several times more than in the wild-type flies (see Fig. 7).

The mechanism bringing about this increased accumulation of transcripts coding for the yolk proteins in mature dsxD flies is not known. The genes could be more actively transcribed, the polyploidy of the fat body cells could increase thus increasing the number of genes available for transcription, or the stability of the RNA could be increased. However, whatever the mechanism used in vivo, these transcripts are not translated efficiently into proteins since actual fat-body synthesis of the yolk proteins does not increase at this time.

The RNA from the dsxD and OrR adults was separated on formamide, formaldehyde gels, transferred to nitrocellulose and hybridized to the cloned yolk-protein genes. The RNA was intact and of the same size in both mutant and wild-type flies (data not shown). The gels were not, however, of a resolution sufficient to distinguish whether the introns had been processed from these transcripts. They are about 70 base pairs in length (Hung & Wensink, 1981). Thus it was possible that much of this RNA was not suitable for translation into proteins. We translated 5 μg samples of the RNA in the rabbit reticulocyte cell-free translation system and found that the RNA from dsxD flies at 96 h (when maximum transcripts were detected) translated just as efficiently as the wild-type RNA; (the ratio of yolk-protein transcripts in OrR:ifcxD was 1:1·15 and the ratio of yolk proteins synthesized in the cell-free translation system was 1: 1·14). A similar pattern of yolk polypeptides was observed when the translation products were precipitated with anti-yolk-protein antibodies and analysed by gel electrophoresis (Fig. 8). Thus there seems to be a mechanism in vivo whereby these accumulated yolk-protein transcripts are not efficiently translated.into proteins, but this does not appear to be due to changes in the mRNA which prevent it from being efficiently translated in vitro.

Fig. 8.

Autoradiograph of yolk-polypeptide synthesis in the rabbit reticulocyte lysate cell-free translation system from 5 μg samples of dsxD and OrR RNA from 96 h, population 1. The translated proteins were antibody precipitated before being separated by gel electrophoresis.

Fig. 8.

Autoradiograph of yolk-polypeptide synthesis in the rabbit reticulocyte lysate cell-free translation system from 5 μg samples of dsxD and OrR RNA from 96 h, population 1. The translated proteins were antibody precipitated before being separated by gel electrophoresis.

As might be predicted from many studies of egg-laying rates in Drosophila, the expression of the yolk-protein genes, which code for the major egg components, is very variable. Studies on the levels of expression of these genes under various experimental conditions must therefore be interpreted with caution, and the variability between groups of flies and with age must always be considered. Despite these problems we can make a number of conclusions from our studies about yolk-protein-gene expression in the intersexual, dsxD, adults. These flies lack one of the major sites of yolk-protein synthesis, the ovary, and the fat body synthesizes and secretes into the haemolymph considerably reduced levels of yolk proteins compared to wild-type females. The reduced protein synthesis is initially reflected in the reduced accumulation of RNA transcripts coding for the yolk proteins, however when the flies are 3–5 days old, transcripts build up in dsxD and sometimes exceed wild-type levels. We therefore propose that the actual expression of the genes is regulated at both transcriptional and post-transcriptional levels.

We need to consider the mechanism by which the mutated dsx gene affects transcription of the yolk-protein genes. One way which might explain both these results and the induction of yolk-protein synthesis in isolated abdomens with ecdysteroids (Jowett & Postlethwait, 1980) would be that the dsx locus modulates titres of steroid hormones in the flies and that females would have a high titre, males a low titre and intersexual flies an intermediate titre. However, experiments measuring ecdysteroid titres in adult males and females detect no such differences. (Handler, 1982; Bownes, Dübendorfer & Redfern, unpublished). These experiments must be extended to analyse haemolymph levels of active 20-hydroxy-ecdysone, and we cannot, therefore, be sure that there are no differences in hormones between males and females. The other aspects of the sexual phenotype, such as bristle patterns and other morphological characteristics affected by the sex genes, are controlled in a cell-autonomous fashion (Baker & Ridge, 1980). It seems likely, therefore, that the dsx® mutation exerts its effect on expression of yolk-protein genes from within the fat-body cells. The normal wild-type allele of dsx would therefore, by its activity or inactivity, lead to the yolk-protein genes being transcribed in female but not in male fat-body cells.

Ecdysteroids are required for the metamorphosis of the fat body, and for synthesis of yolk proteins in newly-emerged females (Jowett & Postlethwait, 1980; Dübendorfer & Eichenberger-Glinz, 1980), but not for continued yolkprotein synthesis in mature females (Bownes, 1982). It is possible that the effect of dsx® on expression of yolk-protein genes occurs at the time of fat-body metamorphosis, and that different (qualitatively or quantitatively) hormone receptors are present in male and female fat-body cells so that there is a differential response to the same levels of hormone. There is no evidence either for or against this idea, and it is entirely possible that the wild-type dsx genes act to regulate the expression of the yolk-protein genes via a pathway totally independent of ecdysteroids and their receptors, and they may well exert their effect throughout adult life rather than just during fat-body development.

At present we can only speculate upon how the wild-type dsx allele can affect the expression of the yolk-protein genes and our experiments give no indication of what is abnormal in dsxD mutants such that they have altered sexual characteristics, which includes altered yolk-protein synthesis. However, now that we know that the yolk-protein genes are regulated by the sex genes we can begin to investigate how this is achieved in vivo. Experiments designed to unravel the relationship between the sex genes, ecdysteroids and the yolk-protein genes are in progress.

This research was supported by the Medical Research Council. We are grateful to Pieter Wensink for providing us with pYPl, pYP2 and pYP3, Judy Chisholm for typing the manuscript and Graham Brown for printing the photographs.

Baker
,
B. S.
&
Ridge
,
K. A.
(
1980
).
Sex and the single cell. I. On the action of major loci affecting sex-determination in Drosophila melanogaster
.
Genetics
94
,
383
423
.
Barnett
,
T.
,
Pachl
,
C.
,
Gergen
,
J. P.
&
Wensink
,
P. C.
(
1980
).
The isolation and characterisation of Drosophila yolk protein genes
.
Cell
21
,
729
738
.
Bonner
,
W. M.
&
Laskey
,
R. A.
(
1974
).
A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels
.
Eur. J. Biochem
46
,
83
88
.
Bownes
,
M.
(
1982
).
The role of 20-hydroxy-ecdysone in yolk polypeptide synthesis by male and female fat bodies of Drosophila melanogaster
.
J. Insect Physiol
.
28
,
317
328
.
Bownes
,
M.
&
Hames
,
B. D.
(
1978
).
Analysis of the yolk proteins in Drosophila melanogaster
.
FEBS Lett
.
96
,
327
330
.
Bownes
,
M.
&
Nöthiger
,
R.
(
1981
).
Sex determining genes and vitellogenin synthesis in Drosophila melanogaster
.
Molec. gen. Genet
.
182
,
222
-
228
. -
Brennan
,
M. D.
,
Warren
,
M.
&
Mahowald
,
A. P.
(
1980
).
Signal peptides and signal peptidase in Drosophila melanogaster
.
J. Cell Biol
.
87
,
516
520
.
Brennan
,
M. D.
,
Weiner
,
A. J.
,
Goralski
,
T. J.
&
Mahowald
,
A. P.
(
1982
).
The follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster
.
Devi Biol
.
89
,
225
236
.
Chan
,
L. N.
&
Gehring
,
W.
(
1971
).
Determination of blastoderm cells in Drosophila melanogaster
.
Proc. natn. Acad. Sci., U.S.A
.
68
,
2217
2221
.
DüBendorfer
,
A.
&
Eichenberger-Glinz
,
S.
(
1980
).
Development and metamorphosis of larval and adult tissues of Drosophila in vitro
.
In Invertebrate Systems In Vitro
(eds
E.
Kurstak
,
K.
Maramorosch
&
A.
Dübendorfer
), pp.
169
185
.
Amsterdam
:
Elsevier/North Holland Biomedical Press
.
Grace
,
T. D. C.
(
1962
).
Establishment of four strains of cells for insect tissues grown in vitro
.
Nature
195
,
788
789
.
Handler
,
A. M.
(
1982
).
Ecdysteroid titers during pupal and adult development in Drosophila melanogaster
.
Devi Biol
.
89
,
225
236
.
Hung
,
M. C.
&
Wensink
,
P.
(
1981
).
The sequence of the Drosophila melanogaster gene for yolk protein 1, Nuc
.
Acids Res
.
23
,
6407
6419
.
Isaac
,
P. G.
&
Bownes
,
M.
(
1982
).
Ovarian and fat body vitellogenin synthesis in Drosophila melanogaster
.
Eur. J. Biochem
.
123
,
527
534
.
Jowett
,
T.
&
Postelthwait
,
J. H.
(
1980
).
The regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxy-ecdysone and a juvenile hormone analogue
.
Devl Biol
.
80
,
225
234
.
King
,
R. C.
(
1970
).
Ovarian Development in Drosophila melanogaster
.
New York
:
Academic Press
.
Laemmli
,
U. K.
(
1970
).
Cleavage of structural proteins during assembly of the head of bacteriophage T4
.
Nature
227
,
680
685
.
Lindsley
,
D. L.
&
Grell
,
E. H.
(
1968
).
Genetic variations of Drosophila melanogaster
.
Carnegie Inst. Wash. Publ. No
.
627
.
Maniatis
,
T.
,
Jeffrey
,
A.
&
Kleid
,
D. G.
(
1975
).
Nucleotide sequence of the rightward operator of phage
.
Proc. natn. Acad. Sci., U.S.A
.
72
,
1184
1188
.
Pelham
,
H. R. B.
&
Jackson
,
R. J.
(
1976
).
An efficient mRNA-dependent translation system from reticulocyte lysates
.
Eur. J. Biochem
.
67
,
247
256
.
Postlethwait
,
J.
,
Bownes
,
M.
&
Jowett
,
T.
(
1980
).
Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster
.
Devl Biol
.
79
,
379
387
.
Srdic
,
Z.
,
Reinhardt
,
C.
,
Beck
,
H.
&
Gloor
,
H.
(
1979
).
Autonomous yolk protein synthesis in ovaries of Drosophila cultured in vivo
.
Wilhelm Roux’ Arch, devl Biol
.
187
,
255
266
.
Thomas
,
P. S.
(
1980
).
Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose
.
Proc. natn. Acad. Sci., U.S.A
.
77
,
5201
5205
.