Structural and Functional Studies of the 68C Glue Protein Gene Cluster of Drosophila melanogaster

Citation

Garfinkel, Mark David (1988) Structural and Functional Studies of the 68C Glue Protein Gene Cluster of Drosophila melanogaster. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/gjtw-k928. https://resolver.caltech.edu/CaltechTHESIS:01222013-140636948

Abstract

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 third instar larval salivary glands. The three 68C glue mRNAs are coded in a gene cluster of less than 5000 base-pairs, and are expressed coordinately under the control of the steroid hormone ecdysterone. Neither amplification nor DNA rearrangement of the locus occurs in the salivary gland. The nucleotide sequence of genomic DNA that includes the entire gene cluster was determined, as were the structures of each of the three glue protein mRNAs. Analysis of the sequences revealed that the three glue proteins form a diverged gene family. Each member of the gene family contains an amino-terminal hydrophobic block of amino acids, which is absent in the mature, secreted glue proteins, and a cysteine-rich carboxyl 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.

Two of the genes Sgs-7 and Sgs-8 are divergently transcribed with 475 base-pairs separating the two 5' ends. A transcriptional fusion gene was constructed by joining the 5' untranslated region of Sgs-7 to the 5' untranslated region of the D. melanogaster Adh gene. A translational fusion gene was constructed by joining the Sgs-8 gene to the Escherichia coli lacZ gene. When the fusion genes are placed in their normal divergently transcribed arrangement and reintroduced into D. melanogaster using P element gene transfer, third instar larval salivary gland expression of both alcohol dehydrogenase activity and β-galactosidase activity was observed. Expression of the two fusion genes requires the l(1)npr-1⁺ gene product, which is known to regulate the 68C glue protein genes, supporting the proposal that this trans-acting factor affects glue protein gene transcription. Normal tissue, stage, and quantity of Sgs-7—Adh fusion gene expression is observed when 211 bp of the 5' flanking sequence is present. An Sgs-7—Adh fusion gene with 92 base-pairs upstream is non-functional. Third instar larval salivary gland expression of the Sgs-8—lacZ fusion gene is observed when 432 base-pairs of the intergenic region are present, while 415 base-pairs of 5' flanking sequence permits normal tissue and stage of expression at levels at least twentyfold reduced. The experiments suggest that a single region functioning bidirectionally, located closer to the Sgs-7 gene, is required for expression of both genes.

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