Molecular analysis of tissue-specific position effects in the transformed line, MM-50, of Drosophila melanogaster

The transformed line, MM-50 was produced by transferring a normal Drosophila Adh (alcohol dehydrogenase) gene into an Adh-negative mutant strain. In this line, the inserted Adh gene displays an abnormal pattern of tissue-specific expression. The ADH activity is limited to a small region of the medial midgut and no other larval tissues express any detectable ADH. The molecular basis for the abnormal expression pattern was investigated. A 14 kb genomic clone from the transformed line containing the Adh insert was isolated. Restriction map analysis revealed that it contains the complete Adh gene, 3.2 kb in length with about 3.0 kb of 5' and 7.8 kb of 3' flanking sequences. Various deletion constructs were made using the restriction sites present on the genomic clone to localize the silencer elements in the flanking region using a somatic transformation assay. A silencer element(s) located upstream of the Adh gene is found within a 1.7 kb fragment that acts in both orientations and at a distance of at least 2.42 kb from the larval Adh promoter. Sequence analysis revealed that the entire fragment is 1773 bp, of which the region from 1-1180 is the MM-50 silencer region while the region from 1181-1773 consists of P element sequences from the transposon vector. There are no long open reading frames present in the silencer region suggesting that it is a noncoding DNA region. A 20-bp sequence (17/20 nucleotides matching) having a high T/C content is repeated within the silencer region. Within each of these homologous regions there is an internal homology consisting of a repeat of the sequence CTCTC. The entire silencer region, including the homologous sequences, have no homology with the silencer regions found in another Adh transformed line, GC-1. Interestingly, however, the repeated sequence contains a consensus motif, CCTCTC, for silencer elements found in rat collagen II gene and several other genes, including the human [beta]-interferon ([beta]-IFN) gene. Another interesting finding is that the MM-50 consensus sequence is homologous to a protein binding site in the negative element of human [beta]-IFN gene. A homologous motif is also found in the human c-myc promoter region, close to a region found to have an inhibitory activity on transcription.