Drosophila chorion gene - PNAS

Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3029-3033, May 1988 Developmental Biology

Temporal regulation in development: Negative and positive cis regulators dictate the precise timing of expression of a Drosophila chorion gene BRIAN D. MARIANI*, JAIRAM R. LINGAPPA*, AND FOTIS C.

KAFATOS*t#

*Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138; and

tInstitute of Molecular Biology and Biotechnology, Research Center of Crete and Department of Biology, University of Crete, Heraclio 711 10, Crete, Greece Contributed by Fotis C. Kafatos, December 31, 1987

ABSTRACT We have used germ-line transformation to dissect the cis regulatory elements responsible for the transcriptional control of an internally marked Drosophila chorion gene (s15-P) during development. A 73-base-pair segment of the proximal 5'-flanking DNA contains sequences essential for the tissue-specific expression and the precise "late" temporal regulation of that gene. A substitute s36-1 segment of similar location can provide the tissue-specific function and imparts an early temporal regulation characteristic of gene s36-1. Within the regulatory DNA of sl5-P, at least three adjacent elements are recognizable: an essential operationally positive element (TCACGT) that is shared by s36-1 and other chorion genes, irrespective of temporal specificity; a second positive element that is required for the normal late expression of slS-P; and, farthest upstream, a negative element that represses precocious expression during the early choriogenic stages.

chorion gene expression and in addition reveal that the precise temporal specificity of this gene is under the control of closely linked positive and negative regulatory elements in the proximal 5'-flanking DNA.

MATERIALS AND METHODS Marking of the siS-1 Gene and Vector Construction. To create the gene hereafter named sJ5-P, the sJ5-1 chorion gene was marked by the insertion of a 288-bp Hae II fragment of an Antheraea polyphemus (silkmoth) chorion cDNA clone, pc408 (12), into the Hae II site 14 codons upstream from the carboxyl terminus of sl5-1 (13). The Hae II fragment is composed entirely of silkmoth chorion protein coding sequence. The insertion preserves the translational reading frame of both pc408 and slS-J and leaves intact the 5'- and 3'-untranslated sequences of s15-1 mRNA. Pelement transformation plasmids were constructed by ligating the sJ5-P gene and flanking DNA into the polylinker of the Carnegie 20 Drosophila transformation vector (pCar2O; ref. 14). Unless otherwise indicated, the 5' limit of the inserted sl5-P DNA or its mutant derivatives (see below) is the Bgl II site at position -814 (from the adenosine of the cap site), and the 3' limit is the Sal I site at position + 778, or 185 bp downstream from the poly(A) addition site. Throughout this report, numbering refers to the terminal nucleotide that is identical to the slS-1 sequence, regardless of origin (e.g., provided by chorion DNA or by a ligated linker). The original full-length construct was generated by Klenow fragment filling of the Bgl II site, followed by Sal I digestion and ligation of the Bgl II-Sal I fragment into the Hpa I and Sal I sites in the pCar2O polylinker. All constructs were oriented with the 3'-flanking DNA of the sJS-P gene adjacent to the y+ gene in pCar2O (Fig. 1). In Vitro Mutagenesis. Internal deletions were generated within the 5'-flanking DNA of s15-P by ligating, at EcoRIlinkered endpoints, appropriately "matched" 5'- and 3'deleted fragments, selected from respective BAL-31 deletion libraries (details to be published elsewhere). Xba I (at position - 386)-Sal I fragments containing these internal deletions were exchanged for the intact Xba I-Sal I fragment of the full-length sl5-P construct described above. A 6-bp clustered substitution was generated by the use of a synthetic 36-mer primer of sJ5-P sequence, centered on the TCACGT element at positions -60 to -55, which was replaced by the mutant sequence GATGTG. This primer was hybridized to an M13-derived template (15) containing a single-stranded Xba I-Kpn I fragment from sJS-P and was used to direct the synthesis of a double-stranded molecule. After bacterial transformation, a mutated phage was selected by sequence analysis, and its Xba I-Kpn I fragment was excised and exchanged for the corresponding fragment in the full-length slS-P construct. The unidirectional, 5' deletion to

In Drosophila melanogaster, the proteinaceous eggshell (chorion), which is a multilayered protective egg covering, is constructed during the final stages of oogenesis (1-3). Crucial to its successful morphogenesis and function (4) is the programmed, sequential synthesis of the various chorion proteins during the 5- to 6-hr choriogenic period (oogenic stages 11-14). The genes encoding these proteins are regulated, being expressed exclusively in the "1000 follicle cells that surround each maturing oocyte during overlapping "early" or "late" periods that are characteristic for each gene (5, 6). For example, transcripts of the very late gene known as sJS-J are detectable in stage 13 and are particularly abundant in stage 14 follicles; although low levels of transient and nontranslated transcripts are seen occasionally during prechoriogenic stages, they are totally absent from stages 11 and 12 (5). The sJS-1 gene is found at chromosomal locus 66D12-15, clustered with three additional chorion genes that also have late but nonidentical temporal specificities (5, 7). We have been investigating the cis-acting elements responsible for the detailed developmental regulation of sl5-1 expression by using in vitro mutagenesis of the 5'-flanking region of the gene, followed by P-element-mediated transformation (8, 9) and assay of in vivo gene expression (10). With the exception of some quantitative regulators, it appears that the cis regulatory elements dictating developmental specificity are found upstream and closely associated with the gene (11). In the present report, we analyze by in vitro mutagenesis the cis regulation of an s5-1 gene, marked with a unique DNA insertion and transformed in isolation of the other three chorion genes that normally flank it. The results identify a 6-base-pair (bp) element as essential for The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

tTo whom reprint requests should be addressed at Harvard University. 3029

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Proc. Natl. Acad. Sci. USA 85

(1988)

Extraction and Blot-Hybridization Analysis. Analysis 1 kb RI RRNA of the pattern of expression of the slS-P gene was carried out UIm as follows. Ovaries from conditioned transformant females SI BE were dissected-in ice-cold Ephrussi-Beadle Ringer's solution, and individual egg chambers (follicles) were staged as KI -, _ ~ ~ ~ - H described by King (1). Total nucleic acid was extracted from XI 120 to 150 staged follicles by using the urea/NaDodSO4 778 + -386 -814 +s15-P method (5). Tissue samples were homogenized in 100 ,ul of a ;-138 solution of 7 M urea, 2% (wt/vol) NaDodSO4, 0.01 M B/S FTris HC1 (pH 8.0), 0.001 M EDTA, and 0.35 M NaCl and RI were extracted sequentially with phenol, phenol/chloroA -420 -J form/isoamyl alcohol [24:24:1 (vol/vol)], and chloroform nd1.. a followed by ethanol precipitation. Total nucleic acid was \~~~~.n ~ ~~~~~ resuspended in H20, glyoxylated (17), and fractionated on 1.6% agarose gels in 0.01 M sodium phosphate (pH 7.2). A pCar20O total of 8.0 gg was loaded per lane, and gels were stained with ethidium bromide (0.5 ttg/ml) to verify uniformity of loading. In some experiments, 4.0 pug of poly(A) + RNA was analyzed. Gels were blotted to Biodyne (Pall, Irvine, CA) 1- 9 10 1t 12 13 14 1-9 10 11 12 13 14 nylon filters in 10 x SSC (1 x SSC = 0.15 M NaCl/0.015 M s36-1 sodium citrate, pH 7.0) overnight, and the nucleic acids were of Uo _ crosslinked to the filter by ultraviolet irradiation (18). Hybridizations used 32P-labeled, T3-derived RNA or Klenow fragment-extended DNA probes at 2 x 106 cpm/ml, in a p s15 W solution of 0.5 M sodium phosphate (pH 7.2), 5% (wt/vol) NaDodSO4, and 1% bovine serum albumin (Sigma) for ii5 p 24-48 hr at 72-750C (RNA probes) or 680C (DNA probes; from ref. 18). To detect only sJS-P transcripts, RNA adapted U. probes were used, derived from the T3 promoter of a Bluescribe plasmid (Stratagene, San Diego, CA) containing a s15-1 202-bp Sac I fragment from the A. polyphemus pc408 insert. The same filter was subsequently hybridized with an M13derived primer-extended DNA probe derived from the siS-1 gene. Filters were washed in 0.04 M sodium phosphate, pH A-138 B/S 7.2/1% NaDodSO4 at 72-750C (RNA) or 680C (DNA) and exposed to Kodak XAR-5 x-ray film with an intensifier FIG. 1. Temporal expression of endogenou Is and transformed screen. chorion genes. (Upper) Diagram indicating the four genes of the autosomal chorion cluster, the chorion DNA regions present in the RESULTS pCar2OB/S (B/S) and the pCar2O -138 (A -138) transposons,

SI

s18-1

s15-1

s16-1

s19-1

.

-

and the DNA contributed by the pCar2O vector. Chorion genes are diagrammed as solid boxes, the A.- polyphemus DINA insert in slS-P as a hatched box, and the P-element ends as an open box with an arrowhead. Arrows indicate the direction of transcBption, and numbers are relative to the cap site. SI, Sal I; BII, XI, Xba I; KI, Kpn I. (Lower) Temporal spew cificity of chorion transcripts. Follicles of the indicated stages (abo ye the lanes) were dissected from transformant lines bearing the B/S or A -138 transposon, and equal amounts of total RNAs we re blot hybridized. The same filter was sequentially hybridized with probes specific for s36-1 (endogenous early chorion gene), the A. polyphemus insert (transformed late slS-P gene), and sIS-1 (endoigenous and transformed late chorion genes). Note that the B/S anid A -138 transposons are expressed with completely normal temr poral specificity.

bp - 138 was generated in an analogous ma]nner: a clustered 4ac mer substitution was generated by using a s ynthetic primer with an EcoRI linker sequence (CG yGAATtCCG) at T at ragpositions -148 to - 139, and the EcoRI-' a I sCfragAll conment was introduced in the polylinker of I structs were sequenced in the region of the mutation or the deletion breakpoint prior to use. Generation of Transformant Lines. Drosoj Dhila transformation was performed essentially as. described 1(8, 9), by injecting 30- to 60-min-old embryos of the cn;r y42 strain with a mixture of DNA consisting of vector (0.4 mg/ml) and helper P-element plasmid p1125.7wp (0.1 mg/ml) (16). Go adults were backcrossed to the parental strain anid individual ry' G1 transformants were again backcrossed pprior to the establishment of homozygous lines. After Soutthern analysis of genomic DNA, lines containing only single, lunique P-element inserts were selected for study.

p~al AllpCar2t

Deletion Analysis of Minimal 5'-Flanking DNA Requirements for Specific Expression. The constructs used in the present

study

were

based

on

s15-P, i.e., on an s5-1 chorion

gene marked by inserting 288 bp of silkmoth DNA, and were designed for optimal analysis of temporal regulation. Transcripts of sl5-P were definitively identified by using as hybridization probe the inserted silkmoth DNA, which does not crosshybridize with endogenous Drosophila sequences. Subsequently, use of an sJS-1 probe permitted simultaneous detection and, therefore, direct temporal comparison of transcripts from the endogenous slS-I and the transformed slS-P genes, which differed in size because of the silkmoth DNA insert. As all of our constructs included the sJS-J structural gene and the first 185 bp of 3'-flanking DNA, we did not consider regulatory elements that might exist within these regions. Complementary fusion constructs have shown that the 5'-flanking DNA alone is sufficient to confer on a reporter Adh gene slS-1-like specificity, although the quantitative level of expression is abnormally low (11). Thus, any intragenic or proximal 3'-flanking elements serving sex, tissue, or temporal specificity would have to be redundant and were neglected in the present study. The starting point of our analysis was a maximal sJS-P transposon, pCar2OB/S (abbreviated B/S), which had 814 bp of 5'-flanking DNA, extending to within 50 bp of the end of the preceding s18-1 gene (13). As reported (ref. 10 and cf. refs. 19 and 20) and as demonstrated in Fig. 1, the chorion gene within that construct was expressed with complete developmental specificity and showed very limited quantitative sensitivity to chromosomal position effects (11). Its

Proc. Natl. Acad. Sci. USA 85 (1988)

Developmental Biology: Mariani et al. transcripts, like those of the endogenous siS-1 gene, began to accumulate in stage 13 and were maximally abundant in stage 14; they were absent from early choriogenic follicles and from other female or male tissues (data not shown). Follicular transcripts of slS-P and s5-1 were of comparable abundance (11), when allowance was made for gene dosage differences resulting from the 60- to 80-fold amplification of the endogenous sJS-1 gene (7). Thus, the isolated unamplified slS-P gene was expressed normally when introduced at random chromosomal locations. To define the minimal 5'-flanking DNA required for normal sl5-P expression, a unidirectional series of deletions from the 5' end of the maximal construct were tested. Fig. 1 shows that one such construct, deletion -138 (pCar20 A -138, abbreviated A -138), which removes all but 138 bp of 5'-flanking DNA, still permitted normal expression, albeit at a somewhat reduced level. Fig. 2 shows that extending the deletion to position -45 (A -45) eliminated the normal sl5-P transcripts (detection limit, 1/500 of the B/S level). Similarly, sJS-P transcripts were suppressed in two internal deletions, A - 189/ -45 and A - 137/ -45, which restore the DNA from the upstream endpoint to position - 814 (Fig. 2). Barely detectable traces of higher molecular weight, nonspecific transcripts may originate from the nearby P-element promoter. These results emphasize the importance of the proximal 5'-flanking DNA and indicate that the DNA between positions -814 and -137 cannot substitute for essential regulators located between positions -137 and -45. An Essential Cis Regulatory Element for s15-P Expression. A prominent sequence feature within the essential segment from position - 137 to - 45 is the hexanucleotide TCACGT (positions -60 to -55). It is present in approximately the same location in all other chorion genes sequenced to date in the genus Drosophila, but not in nonchorion genes (ref. 13 and unpublished results), and is also found in most of the chorion genes of silkmoths (21). To assess the functional significance of this highly conserved element, we replaced TCACGT with the hexanucleotide GATGTG, which maximized the purine-pyrimidine transversions. Outside this cluster of substitutions, the construct was identical to the strongly expressed pCar20B/S construct, yet it showed no significant transcription from the s15-P promoter (Fig. 2). The results identified TCACGT as an essential element for the expression of chorion gene s5-1. Similarly, clustered substitution of TCACGT in a silkmoth chorion promoter abolished the activity of that promoter in transgenic flies (22). Internal Deletion Analysis of the Regulatory DNA of s1S-P. A series of internal deletions of B/S revealed the existence of at least two additional, temporally regulating domains within the -137 to -45 segment of sJ5-P. In these experiments, we maintained the upstream deletion endpoint at position -189 and progressively restored DNA by moving up the downstream -45 endpoint. In this manner we assessed the function of progressively longer DNA sequences in association with the essential TCACGT element at positions -60 to -55. Limited experiments with deletions downstream of position - 137 suggested that the DNA between positions -189 and - 137 was relatively unimportant (J. Grinblat, personal communication). Fig. 3 shows blot hybridization of RNAs from staged transformant follicles for each of the position - 189 deletions. As compared to the null A - 189/ -45 deletion, the first construct A - 189/ - 69 restored 24 bp upstream of position -45, including the TCACGT hexamer. The restored DNA sequence resulted in a dramatic effect: the sJ5-P promoter was reactivated but behaved as a developmentally early-like promoter rather than late promoter. Transcripts appeared at stage 11, were most abundant at stage 12, decreased in quantity at stage 13, and almost disappeared by stage 14-

3031

TCACGT

-814

200 I

Io

-150

50 1 -45

-100 1

+1

-45

-189

I WfIj -137 -45

row

GATGTG

~A

r .1

Total B/S

A-138

1:1 1:51:101:50 1:1

poly A+ cn; ry 1

A-45 2 3 4

LS-60/-55 2 3 4

1

sI5-P E i s15-1

S

B/S i:1

s15-P

1

a - 137/-45 a -189/-45 2 3 41 2 3 4

f

s 5-1

FIG. 2. Inactivity of the s)5-P promoter in a linker-scanner mutation (LS-60/-55) and three deletions lacking the TCACGT element. (Upper) Diagram of the constructs examined. (Lower) Filters bearing 4.0 Zg of total or poly(A)+ (at least 10-fold enriched) ovarian RNAs were hybridized with a probe corresponding to the A. polyphemus insert, to detect transcripts derived from the transposon, and subsequently with a probe that recognizes s5-1 transcripts. As controls, a dilution series (as indicated above lanes) of RNA from wild-type B/S transformants was analyzed as well as RNA from A -138 and from the untransformed cn;ry parental strain. Four transformed lines were analyzed for each transposon (as indicated above lanes) and showed no bona fide sl5-P transcripts. Trace amounts of high molecular weight RNAs were detected from some transposons [ca. 0.1 to 0.5% of the level of slS-P transcripts in B/S, considering the poly(A) selection], probably originating in the P-element promoter. the stage at which mRNAs from the wild-type s5-1 or slS-P genes were most prevalent. The second deletion in the series, A - 189/ - 80, restored an additional 11 bp upstream of position - 69 and resulted in a strikingly different temporal pattern of expression: transcripts again appeared early, at stage 11, and increased in abundance at stage 12, but they continued to accumulate thereafter and became most abundant at stage 14, when they disappeared from the previous deletion. In short, within the A - 189/ -80 construct the slS-P promoter behaved as both an early and late promoter. The third and fourth deletions progressively decreased the early expression, while maintaining the late expression intact. Addition of 9 bp upstream of the position - 80 breakpoint (A -189/-89) repressed expression at stage 11;

Developmental Biology: Mariani et al.

3032 -814

-200

-150

,I -69

1/

-80 -8 r9w

1 -189

-89 I

-1 89

-130

s36-1

-58

ram

1

-189

4,1//

-50 +1 II r3

-100

-189

Proc. Natl. Acad. Sci. USA 85 (1988)

-814

-200

roe-

*

'

-150 1 -3 -

+I

-45

137

- 45

-98 .

1- 9 1-9 10 11

1 2 13 1 4

10

11

12

13

14

1-9 10 11 12 13 14

q3

.i._.

B/S

s36-1

s15- P A -189/-69

A-189/-80

;. Ci

s

U._

15-1

A- 189/-89

A-189/-98 1'1

s15- P

s15- 1

FIG. 3. Temporal disruptions of SI-P expression resulting from internal deletions. (Upper) Diagram of the constructs examined. Solid box represents the essential TCACGT element. (Lower) On the left, stage-specific SI-P transcripts are presented, except that in the wild-type (B/S) s36-1 transcripts are also shown for comparison. Developmental stages are indicated above the lanes. Note that the A 189/-69 deletion prevents the normal late activation and simultaneously permits abnormal early expression of the gene; the other deletions allow late activation and progressively confine the early expression. On the right, the siS-1 transcripts were detected on the same filter and serve as internal controls; slight variations in intensity at stage 13 are due to unavoidable staging variability.

FIG. 4. A 73-bp fragment of the early s36-1 gene restores ovarian expression and converts the lS5-P gene to early temporal specificity when inserted within the null A - 137/ -45 deletion (cf. Fig. 2). (Upper) Diagram of the construct. (Lower) Transcripts were detected by hybridization of the same filter with an s36-1 probe (Top) and a mixture of slI-P and Sl5-1 probes (Middle and Bottom). Developmental stages are indicated above lanes.

missing sJS-J DNA. Evidently, it provided the follicular activation function associated with TCACGT but neither repressed precocious expression nor activated specifically late expression. That segment behaved as an early rather than a late regulatory region, consistent with its origin.

-

transcripts appeared only at stage 12, and increased in abundance through stages 13 and 14. In the next construct, A -189/-98, an additional 9 bp were restored and repressed the appearance of transcripts at stage 12 as well as 11. However, a slight early character (relative to wild-type sJS-J or slS-P expression) was retained, as shown by a slightly higher than normal prevalence of the transcripts at stage 13. It would appear that sequences just upstream of position -98 were still required for attainment of normal temporal specificity. With a A 291/-118 deletion the ratio of stage 13 to stage 14 transcripts was completely normal (data not shown). These results indicate that the DNA from positions -46 to -118 includes at least three regulatory elements: the essential TCACGT hexanucleotide, a latestage activator, and an early-stage repressor (see Discussion). -

A Short Segment from the s36-1 Gene Restores Tissue-

Specific Expression of slS-P and Alters Its Temporal Programming. When a position -130 to 57 fragment of s36-1 was inserted in lieu of the missing DNA within the null 137/-45 deletion, follicular expression of slS-P was restored, but its temporal specificity was shifted to the early stages 11-13, identical to that of s36-1 (Fig. 4). That 74-bp segment included a TCACGT element (at positions 69 to 64) but showed no other obvious sequence similarity to the -

-

-

-

DISCUSSION A fundamental aspect of development is differential gene expression, regulated in space and over time. Because of the availability of methods for DNA modification and transfer into cells or organisms, much has been learned about cis regulators of specific gene expression in appropriate tissues and developmental stages of higher organisms. To our knowledge, this is the first report that identifies regulators involved in detailed temporal programming during development. In the follicles of Drosophila females, the choriogenesis program lasts only 5-6 hr and involves the highly ordered sequential expression of -20 structural genes that are required for proper morphogenesis and function of the eggshell (2-4). In these in vivo studies, we have localized the temporal regulators of a late (sS5-1) chorion gene, which is expressed for just more than 2 hr, and have shown that they consist of countervailing positive and negative elements. Other work in our laboratory with fusion gene constructs had indicated that the developmental regulators of sS5-1 and s36-1 largely reside in the short 5'-flanking DNA region (11). The data presented in this report clearly indicate that multiple regulatory elements of slS-1 are localized within a 73-bp region (positions -46 to -118). The data also indicate that a 74-bp similarly located segment of s36-1 is capable of restoring tissue-specific expression and apparently includes temporal regulator(s) with the early specificity characteristic of s36-1. Although additional experiments are necessary to explore the possibility of redundant regulatory elements and the

Developmental Biology: Mariani et A effects of their spacing, the most straightforward interpretation of the phenotypes of substitution and deletion mutations in the short regulatory DNA of siS-) suggests the existence of at least three regulatory elements. The 35-bp proximal half of that region, from positions -46 to - 80, seems responsible for two operationally distinct positive activations. The TCACGT motif at positions - 60 to - 55, a motif characteristic of chorion genes in flies and moths, appears to be an essential positively acting element, since its removal eliminates the activity of the sJ5-P promoter. Similarly, TCACGT is essential for transcription from moth chorion gene promoters in transgenic flies in either orientation (22). In and of itself, however, TCACGT is not responsible for temporal specificity, since it is present in both early and late genes. By comparison of the results of A - 189/ - 69 and A - 189/ - 80, it appears that a second specifically late-activating element exists, with an upstream border between positions - 80 and - 70. That cis activator cannot function in the absence of TCACGT. We do not know whether it represents the binding site of a trans-acting factor that is separate from but interacts with a TCACGT-associated factor, or whether a single trans-activator binds to two domains, the universal TCACGT element and an upstream temporally specific DNA element. The third farthest upstream cis regulator, which is largely contained between positions - 118 and - 81, seems to act in a negative manner to prevent precocious expression. In its absence, the sJ5-1 gene with its associated activators is revealed to be primed for transcription even during the early choriogenic stages. Therefore, we predict that a factor binds to that region to prevent early expression of siS-) and that the repression imparted by that factor is somehow relieved by late stage 13, perhaps as a result of association of the neighboring late activator with a distinct factor. The negative cis regulator has been dissected into three domains. Addition of only 9 bp (positions - 89 to - 81) to the downstream DNA is sufficient to inhibit expression at stage 11, further addition of 9 bp (positions -98 to - 90) extends the inhibition to stage 12, whereas the most distal portion (position - 118 to - 99) appears to be needed for extending the inhibition into early stage 13. The adjacent, countervailing negative and positive DNA elements appear to be responsible for the remarkable temporal specificity of sJS-J. An attractive feature of such a mechanism is that it may possibly explain the temporal programming of the entire set of chorion genes: differences in affinities of the same trans regulators for corresponding negative and positive cis-acting elements of the various chorion genes could be responsible for the sequential on and off regulation of the genes during choriogenesis. The number, nature, and exact sequence of the cis regulatory elements in s5-1 and other chorion genes must now be defined by additional experiments, by using finer scale substitutions and synthetic constructs in which individual elements are tested in conjunction with unrelated promoters. Furthermore, the predictions concerning trans factors must now be tested by gel retardation and "footprint" analysis, by using follicular cell extracts. Results suggest that countervailing cis-acting elements are responsible for regulated expression in a number of other eukaryotic systems. In yeast, the most notable regulatory system that includes negative regulation of transcription is the "silencing" of the HML and HMR loci by the respective HMLE and HMRE regions (23). In mammals, clear examples include the repression by the ElA adenovirus protein on the activity of simian virus 40 (24) and immunoglobulin heavy chain (25) enhancers, as well as the repression by a cis-acting element of the insulin-1 gene on the simian virus 40

Proc. Natl. Acad. Sci. USA 85 (1988)

3033

enhancer (26). Most analogous to the s5-1 promoter are the regulatory DNAs of human p (27) and a (28) interferon genes, which apparently include positive and negative elements (for discussion, see ref. 28). Thus, the unusually precise temporal regulation of the s5-1 chorion gene appears to use mechanisms similar to those that operate in a wide variety of eukaryotic regulatory phenomena. Note Added in Proof. The s36 numbering assumes an adenosine in position + 1 for a cap site sequence AGCAGT ... (11). Because of a cytidine contributed by the linker, the downstream endpoint of the insertion fragment, shown in Fig. 4, should be - 57 rather than - 58.

We are grateful to D. King and A. Georgi for construction of the LS-60/-55 mutation and characterization of some deletion breakpoints, respectively. We thank J. Grinblat and other members of the Kafatos laboratory for helpful comments and permission to quote unpublished results, and M. Youk-See for artwork, B. Klumpar for photography, and E. Fenerjian for secretarial assistance. B.D.M. was a Fellow of the Jane Coffin Childs Memorial Fund. This work was supported by grants from the National Institutes of Health, the National Science Foundation, and the American Cancer Society. 1. King, R. C. (1970) Ovarian Development in Drosophila melanogaster (Academic, New York). 2. Petri, W. H., Wyman, A. R. & Kafatos, F. C. (1976) Dev. Biol. 49, 185-199. 3. Waring, G. L. & Mahowald, A. P. (1979) Cell 16, 599-607. 4. Margaritis, L. H. (1985) in Comprehensive Insect Physiology Biochemistry and Pharmacology, eds. Kerkut, G. A. & Gilbert, L. F. (Pergamon, Oxford), Vol. 1, pp. 153-230. 5. Griffin-Shea, R., Thireos, G. & Kafatos, F. C. (1982) Dev. Biol. 91, 325-336. 6. Parks, S. & Spradling, A. (1987) Gene Dev. 1, 497-509. 7. Spradling, A. C. (1981) Cell 27, 193-201. 8. Spradling, A. C. & Rubin, G. M. (1982) Science 218, 341-347. 9. Rubin, G. M. & Spradling, A. C. (1982) Science 218, 348-353. 10. Kafatos, F. C., Mitsialis, S. A., Spoerel, N., Mariani, B., Lingappa, J. R. & Delidakis, C. (1985) Cold Spring Harbor Symp. Quant. Biol. 50, 537-547. 11. Romano, C., Bienz-Tadmor, B., Mariani, B. D. & Kafatos, F. C. (1988) EMBO J., in press. 12. Jones, C. W. & Kafatos, F. C. (1982) J. Mol. Evol. 19, 87-103. 13. Wong, Y.-C., Pustell, J., Spoerel, N. & Kafatos, F. C. (1985) Chromosoma 92, 124-135. 14. Rubin, G. M. & Spradling, A. C. (1983) Nucleic Acids Res. 11, 6341-6351. 15. Carter, P., Bedouelle, H. & Winter, G. (1985) Nucleic Acids Res. 13, 4431-4443. 16. Karess, R. E. & Rubin, G. M. (1984) Cell 38, 135-146. 17. McMaster, G. K. & Carmichael, G. C. (1977) Proc. Natl. Acad. Sci. USA 74, 4835-4838. 18. Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995. 19. Kalfayan, B., Levine, J., Orr-Weaver, T., Parks, S., Wakimoto, L., de Cicco, D. & Spradling, A. (1985) Cold Spring

Harbor Symp. Quant. Biol. 50, 527-535.

20. Wakimoto, B. T., Kalfayan, L. J. & Spradling, A. C. (1986) J. Mol. Biol. 187, 33-45. 21. Spoerel, N., Nguyen, H. T. & Kafatos, F. C. (1986) J. Mol. Biol. 190, 23-25. 22. Mitsialis, S. A., Spoerel, N., Leviten, M. & Kafatos, F. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7987-7991. 23. Brand, A. H., Breeden, L., Abraham, J., Sternglanz, R. & Nasmyth, K. (1985) Cell 41, 41-48. 24. Borrelli, E., Hen, R. & Chambon, P. (1984) Nature (London) 312, 608-612. 25. Hen, R., Borrelli, E. & Chambon, P. (1985) Science 230, 1391-1394. 26. Laimins, L., Holmgren-Konig, M. & Khoury, G. (1986) Proc. Natl. Acad. Sci. USA 83, 3151-3155. 27. Goodbourn, S., Burstein, H. & Maniatis, T. (1986) Cell 45, 601-610. 28. Kuhl, D., de la Fuente, J., Chaturvedi, M., Parimoo, S., Ryals, J., Meyer, F. & Weissmann, C. (1987) Cell 50, 1057-1069.