Larval salivary gland secretion proteins in Drosophila structural analysis of the Sgs-5 gene

The structure of the Drosophila melanogaster salivary gland secretion gene Sgs-5 has been determined by DNA sequence analysis of cloned genomic DNA. This developmentally and tissue-specific gene is a member of the third instar intermolt gene set and is under control of the insect molting hormone ecdysterone. RNA protection experiments show that the RNA coding region of Sgs-5 contains 769 nucleotides and is divided into three exons by two small introns. The protein-coding region appears to begin after a short untranslated RNA leader (33 nucleotides) and to result in a protein of 163 amino acids. The first 18 amino acids give the amino-terminal end the highly hydrophobic nature characteristic of a signal peptide.     

Genetic analysis of the larval secretion gene Sgs-4 and its regulatory chromosome sites in Drosophila melanogaster

Larval salivary gland secretion from seven wild-type stocks of Drosophila melanogaster was electrophoretically analyzed. Considerable variability occurs in the X-chromosomally coded secretion protein 4, both qualitatively, as expressed by differences in electrophoretic mobilities, and quantitatively as seen by its relative amount in the secretion. Drosophila stocks with normal amounts of protein 4 show approximately 80–90% dosage compensation in the males, whereas in two stocks with lower amounts of protein 4 there is no indication of dosage compensation. — Genetic analysis showed that the properties of secretion protein 4 and the level of expression of the Sgs-4 gene are controlled by the X-chromosome. Recombination experiments indicate that the stock-specific characteristics of protein 4 are properties of the structural gene Sgs-4 itself or of a chromosome region immediately adjacent to Sgs-4. One recombinant (R + 79), manifesting an intermediate level of dosage compensation, indicates that a chromosome segment closely distal to Sg-4 is responsible for the regulation of the gene and for dosage compensation in particular. Accordingly, Sgs-4 must be transcribed from distal to proximal. Its position on the genetic map is 3.6. Two stocks, Hikone-R and Kochi-R, which were originally described as 0-mutants produce very low amounts of a specific secretion protein, 4 h, as revealed by a transvection effect and also by fluorography of overloaded gels.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

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.     

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.     

Nucleolus-organizing regions in salivary gland chromosomes of Drosophila melanogaster

Summary The nucleolus of the salivary gland nucleus of Drosophila melanogaster is formed by nucleolus-organizing regions which exist in the heterochromatin of the sex chromosomes. This interpretation is supported by the discovery of a series of induced chromosomal alterations involving transfer of nucleolus-forming regions to euchromatic sections of the chromosomes.     

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.     

The secretory proteins of the larval salivary gland of Drosophila melanogaster

The gene for a major salivary gland secretion protein (Sgs-1) in Drosophila melanogaster has been mapped to chromosome 2 between dp (13.0) and cl (16.5). In the late third instar larva, a puff forms in this region. This puff (25 B) regresses as the ecdysteroid concentration increases prior to puparium formation. Quantitative analysis of the secretory protein 1, showed that, when present in extra dose, region 25 B results in a significant elevation in its relative amount. This suggests that the structural gene for this protein is localized in this region and that its synthesis is directly correlated to the activity of the 25 B puff.     

Rapidly labeled proteins on the salivary gland chromosomes of Drosophila melanogaster

Experiments on short-term and pulse-chase labeling of chromosome proteins of the salivary glands of Drosophila melanogaster show unique patterns of label in the vicinity of chromosome puffs. A high turnover rate is indicated for these nonhistone proteins, which appear to form a fibrous sheath around the chromosomes. Acrylamide gel analyses of the chromosomal proteins that are quickly labeled, comparing compositions at different stages of development with compositions after heat shock, show that all are different and dependent on which chromosomal puffs are active and producing messenger RNA. The necessity for a continuous and rapid interchange of protein between the nucleus and cytoplasm is indicated, and it appears that regulation of gene activity must be related to this dynamic state of protein exchange. From the technical standpoint, it has been found that scanning electron microscopy (SEM) is especially useful for observing silver grains on opaque autoradiographs. It appears also that SEM will prove useful in a variety of studies of chromosome structure.     

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.