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.     

A transcriptional switch between the Pig-1 and Sgs-4 genes of Drosophila melanogaster.

Pig-1 and Sgs-4 are a pair of closely linked and divergently transcribed Drosophila melanogaster genes, which are both expressed in larval salivary glands but at different times during development. While Sgs-4 is expressed at high levels only at the end of the third instar, Pig-1 exhibits a major peak of expression during late second and early third instar. Thus, Pig-1 expression declines as Sgs-4 expression is induced. In this paper, we show that three adjacent elements located within the short region between these genes can account for the switch from Pig-1 to Sgs-4 expression. A 170-bp segment acts as an enhancer to direct Sgs-4 expression in late-third-instar salivary glands. A 64-bp sequence located just upstream from the enhancer can modify its temporal specificity so that it works throughout the third instar. Expression induced at mid-third instar by a combination of these two elements can be repressed by a negative regulatory sequence located still further upstream. We present evidence suggesting that the changing interactions between these regulatory elements and the Sgs-4 and Pig-1 promoters lead to the correct pattern of expression of the two genes.     

A trans-acting regulatory product necessary for expression of the Drosophila melanogaster 68C glue gene cluster

The mutation I(1)npr-1 is located at cytological location 2B5 on the X chromosome in Drosophila melanogaster. We have found that this mutation causes absence of the normal product of the 2B5 locus and that it has the following phenotypes: the 68C glue puff on the third chromosome does not regress when mutant salivary glands are cultured in the presence of ecdysterone; the three 68C glue protein mRNAs are not synthesized; and a transformed Drosophila strain carrying both a normal resident 68C Sgs-3 gene and an introduced functional Sgs-3 gene with only a few kb of flanking sequences expresses neither Sgs-3 RNA if the I(1)npr-1 mutation is crossed into the stock. Thus the normal product of the I(1)npr11 gene is required for regression of the 68C puff, and the I(1)npr-1 gene product allows expression of the Sgs-3 gene by interacting, either directly or indirectly, with DNA sequences near this glue protein gene.     

Two puff-specific proteins bind within the 2.5 kb upstream region of theDrosophila melanogaster Sgs-4 gene

TheDrosophila nuclear proteins Bj6 and Bx42 characterized previously are detected in a series of developmentally active puffs on salivary gland chromosomes. Here the binding of both proteins at puff 3C11-12 containing the glue protein geneSgs-4 is described in more detail. By deletion analysis we show that both proteins bind within a chromosomal segment containing 17–19 kb of DNA surrounding theSgs-4 gene. They are detectable at this site during the intermoult stages, before the puff regresses in response to the moulting hormone ecdysone. If theSgs-4 gene together with flanking DNA sequences is brought into a different chromosomal position by P element transfer, both proteins are detected at this new location. Both proteins are bound to the chromosome within the range of 2.5 kb DNA upstream of theSgs-4 gene. A strain containing a 52 bp deletion within this region fails to bind Bx42 protein suggesting that the missing DNA, which overlaps a hypersensitive region, may be required for the binding of the Bx42 protein.     

Sequences sufficient for correct regulation of Sgs-3 lie close to or within the gene.

The Drosophila melanogaster 68C chromosomal locus is the site of a prominent polytene chromosome puff that harbors the genes Sgs-3, Sgs-7 and Sgs-8. These genes code for proteins that are part of the salivary glue that Drosophila larvae secrete as a means of fixing themselves to an external substrate for the duration of the pre-pupal and pupal period. The 68C glue genes are regulated by the steroid hormone ecdysterone, with the hormone required for both initiation and cessation of gene expression during the third larval instar. Previous work has defined sequences sufficient for expression of abundant levels of Sgs-3 mRNA at the correct time and in the correct tissue. We show here that sequences sufficient for normal tissue- and stage-specific accumulation of Sgs-3 RNA, but adequate only for low levels of expression, lie within 130 bp of the 5' end of the gene, or within the gene.     

Cis-acting sequences which regulate expression of the Sgs-4 glue protein gene of Drosophila.

The Sgs-4 glue protein gene of Drosophila is expressed only in third-instar larval salivary glands. Previous work suggests that a regulatory region lies 5' and remote to the gene, as indicated by a region of tissue-specific DNase I hypersensitivity and by underproducing mutants with DNA lesions in the hypersensitive region. Here we demonstrate by germ line transformation of cloned fragments containing Sgs-4 that the sequences between 840 bp 5' and 130 bp 3' to the gene are sufficient for Sgs-4 activity. When 5' sequence was removed to -392, activity was eliminated, thereby verifying the existence of essential sequences far upstream. Fragments that are active include, in addition to the capacity for normal levels of expression, three other cis-acting regulatory activities: developmental timing, tissue specificity, and dosage compensation. In contrast, the fragments tested did not specify formation of the puff with which Sgs-4 is normally associated. As shown by chromosomal rearrangements, the region required for puffing is limited to 16-19 kb surrounding the gene.     

Association of a Drosophila transposable element of the roo family with chromosomal deletion breakpoints.

A 9.3 kb transposable element of the roo family has been found inserted 3' to the Sgs-4 glue protein gene of Drosophila. The X chromosome which carries this insert also carries wDZL, a dominant, unstable allele of the white locus caused by the insertion of the 13 kb wDZL element. Three deletions isolated from the wDZL strain have molecular breakpoints 3' to Sgs-4 that are associated with the roo element. Though the deletions eliminate much of the DNA between white and Sgs-4, none of the distal breakpoints fall at or near the wDZL element. The results suggest that this copia-like element, which is structurally similar to an integrated retrovirus, is capable of promoting chromosomal deletions.     

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.