Structure and Expression of the Actin Gene Family of Drosophila melanogaster

We have isolated the six actin genes of Drosophila melanogaster from a Drosophila genomic DNA library and have compared structural features of the genes by restriction mapping, electron microscopy and DNA sequencing. We found that at least two of the actin genes contain intervening sequences which interrupt the genes at different positions. Several of the genes were shown to be lacking intervening sequences in the analogous positions. This nonconservation of intron position is in striking contrast to the strong conservation of intron positions seen in other gene families. The DNA sequences of the protein coding regions of the genes are highly conserved while the intron and untranslated sequences are not. The primary sequences of all the Drosophila actins resemble mammalian cytoplasmic actins more than mammalian muscle actins. We studied the distribution of actin mRNAs in different developmental stages and in different dissected body parts with the use of gene specific hybridization probes which we isolated from the 3' untranslated portions of the genes. We found that the genes fall into three main categories with respect to their patterns of expression in Drosophila. Trancripts from two of the genes are found throughout Drosophila development. They are expressed at higher levels in ovaries and embryonic cultured cells than in muscle containing tissue and are thought to be cytoplasmic actins. Two others encode thoracic muscle actins. Their transcripts accumulate predominantly in the thoracic regions of the adult where the flight and jump muscles are found. The other two genes are most active in larvae and in adult abdomens. They are thought to encode actins used in the larval, pupal, and adult intersegmental muscles. We studied the structure of the cytoplasmic actin gene, act5C, in detail and found that it encodes at least six different mRNAs. At the 5' end there are two nonhomologous leader exons which are alternately spliced to the remainder of the gene. At the 3' end of the gene, three sites of polyadenylation are used. The 3' variation is the principal cause of the transcript length heterogeneity observed in the transcripts. In whole animal RNA, the two leader exons are expressed with the same pattern through development and with all three polyadenylation sites. There is some developmental variability in the use of the three polyadenylation sites. In order to determine if each exon is preceded by a functional promoter and to identify sequences important for transcription initiation from each exon, we made fusions between act5C promoter fragments and the bacterial chloramphenicol acetyltransferase (CAT) gene and tested these for promoter activity in transient assays in Kc cells. We found that each exon is preceded by a separate, functional promoter. At least two regions of DNA sequences are necessary for optimal expression from exon 1. One of these lies greater than 1.9 kb upstream from the exon 1 cap site. All of the sequences required for exon 2 transcription lie within 450 bases of its cap site. There is evidence from some constructions that transcription initiation from exon 1 may inhibit transcription initiation from ex on 2.