cis-acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes.

The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between -211 and -43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from -133 to -48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region.     

Induction and repression of the Drosophila Sgs-3 glue gene are mediated by distinct sequences in the proximal promoter.

The normal developmental expression of the Drosophila salivary gland secretion protein gene Sgs-3 requires the interaction of a distal and proximal regulatory element. A deletion/replacement analysis of the proximal promoter in stably transformed lines shows that induction of an Sgs-3/Adh fusion gene is normal if sequences from +10 to -50 are replaced by those of the hsp70 gene. Sequences between -98 and -50 are necessary for this expression but there is internal redundancy within this region as two distinct upstream sequences of 18 and 22 bp respectively are sufficient for stage- and tissue-specific expression, albeit at reduced levels. A point mutation at -53 eliminates the ecdysone-mediated repression of the Sgs-3 promoter at pupariation. We report mosaicisms of expression within the salivary gland for a number of stably transformed lines.     

Regulatory sequences of the Sgs-4 gene of Drosophila melanogaster analysed by P element-mediated transformation

The X chromosomally located allele Sgs-4 ^c for a larval secretion protein of Drosophila melanogaster is normally expressed in female larvae of the strain Oregon R and is hyperexpressed in male larvae exhibiting dosage compensation; the allele Sgs-4 ^d in the strain Samarkand is weakly expressed and is not hyperexpressed in male larvae showing a dosage effect. P element-mediated transformation of upstream DNA sequences from both alleles combined with Sgs-4 ^d coding and downstream sequences was performed to localize sequences which are responsible for the level of gene expression and for hyperexpression of Sgs-4 ^c in male larvae. Our results demonstrate that weak expression and dosage effect are inherited with the upstream region from –1 to –838. This Samarkand fragment differs from the homologous Oregon R region only by a C to T transiion at –344 which lies within an assumed binding sequence for the ecdysone receptor complex of dyad base symmetry. Replacing the Samarkand upstream region from –1 to –838 by the Oregon R region restores normal Sgs-4 expression and dosage compensation. Hyperexpression in male larvae displays high sensitivity to position effect and is nearly completely inhibited in one transformed line under heterozygous conditions. The integration of an Sgs-4 ^d transposon into a weak spot of polytene chromosome 2L results in a decrease in gene expression. The GTT- and GT-rich regions at –1.2 and –2.0 kb do not obviously influence Sgs-4 expression but possibly play a role in induction of stage-specific chromosome puffing.     

Noncoordinate expression of Drosophila glue genes: Sgs-4 is expressed at many stages and in two different tissues.

The glue genes of Drosophila melanogaster comprise a family of genes expressed at high levels in the salivary glands of late third instar larvae in response to the insect hormone ecdysone. We present evidence that, in contrast to the other glue genes, Sgs-4 is turned on throughout Drosophila development and is not expressed exclusively in the larval salivary glands. Larvae transformed with an Sgs-4/Adh (alcohol dehydrogenase) hybrid gene exhibit Sgs-4-directed Adh expression in the larval proventriculus as well as in the salivary glands as early as the first instar. Sgs-4-specific RNA can be detected at very low levels during all stages of development. During late third instar, levels of Sgs-4 RNA in the salivary glands increase several-thousand-fold, thereby accounting for the large amounts of Sgs-4 protein present in the glue produced by the salivary glands. This pattern of expression is unique to the Sgs-4 gene. While expression of several of the other glue genes can be detected in embryos and early larvae, they appear to be expressed neither throughout development nor in the larval proventriculus. Appearance of the glue gene RNAs in mid third instar salivary glands is noncoordinate, even for the chromosomally clustered genes Sgs-3, Sgs-7, and Sgs-8.     

Developmental regulation by an enhancer from the Sgs-4 gene of Drosophila

A cis acting regulatory region has previously been identified 300-500 bp upstream of the Drosophila glue protein gene, Sgs-4. The functional capabilities of this region have now been examined by fusing it to the Drosophila Adh gene and determining the pattern of expression from the fused construct after transformation. The results show that the Sgs-4 sequences between −150 and −568 are able to direct Adh expression in late third-instar salivary glands, the appropriate tissue and timing for Sgs-4 expression. In addition, the Sgs-4 sequence elevates Adh expression in the anterior midgut and fat body, despite the fact that Sgs-4 is not normally expressed there. All three regulatory activities, tissue specificity, timing and enhancement, show the positional flexibility of enhancer elements. In addition, the Sgs-4 and Adh regulatory elements combine to direct expression in novel spatial/temporal combinations in which neither would normally be expressed.     

DNA sequences, gene regulation and modular protein evolution in the Drosophila 68C glue gene cluster

The 68C locus of the Drosophila melanogaster polytene chromosomes contains the structural genes for three glue polypeptides (sgs-3, sgs-7 and sgs-8) synthesized in the larval salivary glands during the third larval instar. When the messenger RNAs for the glue polypeptides are being synthesized, the locus is puffed; the puff regresses in response to the steroid hormone ecdysterone. The three 68C glue mRNAs are coded in a gene cluster of less than 5000 base-pairs, and are expressed co-ordinately. In the experiments described here we show that the coordinate expression of these RNAs does not result from amplification of the puff DNA, nor is it associated with puff DNA rearrangement. We also report the nucleotide sequence of 6751 base-pairs of genomic DNA that includes the entire gene cluster, and describe coding and non-coding sequences with possible regulatory roles. In addition, we deduce the amino acid sequences of the primary translation products of the glue mRNAs, and show that the glue proteins form a diverged gene family. The members of the family all contain an amino-terminal hydrophobic block of amino acids, which is absent in the mature, secreted glue proteins, and a cysteine-rich carboxy-terminal module. sgs-3 differs from sgs-7 and sgs-8 by containing a third module between the other two, comprised largely of tandem repeats of the five amino acids Pro-Thr-Thr-Thr-Lys.     

The Drosophila fork head factor directly controls larval salivary gland-specific expression of the glue protein gene Sgs3.

The Drosophila Fork head protein participates in salivary gland formation, since salivary glands are missing in fork head embryos. Here we show that the fork head encoded protein binds to an upstream regulatory region of the larval salivary gland glue protein gene Sgs3. Mobility shift assay in the presence of an anti-Fork head antibody demonstrated that the Fork head factor interacts with the TGTTTGC box shown to be involved in tissue-specific Sgs3 expression. Experiments employing a set of oligonucleotide competitors revealed that Fork head binding was prevented by the same single base substitutions that were previously shown to interfere with the TGTTTGC element function in vivo. Furthermore, the anti-Fork head antibody bound to >60 sites of polytene chromosomes, including the puffs of all Sgs genes and Fork head protein was detected in the nuclei of salivary glands of larvae of all examined stages. These data provide experimental evidence for the hypothesis that the protein encoded by the fork head gene is required initially for salivary gland formation and is utilized subsequently in the control of larval genes specifically expressed in this organ.     

Investigation of cis-acting sequences regulating expression of the gene encoding yolk protein 3 in Drosophila melanogaster.

Summary The regulatory sequences leading to the ovarian and fat body expression of yolk proteins 1 and 2 (YP1 and 2) of Drosophila melanogaster have been characterised in some detail. These genes (yp1 and yp2) share many enhancer elements, and some important regulatory sequences lie within the coding regions. We have begun to investigate the cis-regulation of the gene encoding yolk protein 3 (yp3). We describe a system for P element transformation using the complete and unaltered yp3 gene rather than reporter genes and describe sequences conferring correct expression in the ovary and carcass.     

An expandable gene that encodes a Drosophila glue protein is not expressed in variants lacking remote upstream sequences

The Drosophila melanogaster gene Sgs4 encodes one of the glue polypeptides, sgs-4, synthesized in the larval salivary gland. We have examined the structure and expression of Sgs4 in five strains that produce abundant amounts of sgs-4 and its mRNA and in four that do not. The nonproducers include three Japanese strains that accumulate trace amounts of mRNA and one strain, BER-1, that contains no detectable Sgs4 RNA. Sgs4 carries a tandem array of repeated 21 bp elements within its coding sequence. The number of elements per array varies, causing considerable differences in the lengths of Sgs4 and its mRNA among the strains. These differences in length are not correlated with differences in mRNA abundance; rather, the low or zero abundance in nonproducers correlates with the loss of DNA upstream from the gene. The Japanese nonproducers carry a 52 bp deletion 305 bp upstream from the 5′ end of Sgs4, and BER-1 carries a 95 bp deletion 392 bp upstream. Curiously, each deletion encompasses one or more of the salivary-gland-specific DNAase I-hypersensitive sites which are known to flank the Sgs4 gene.