The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing.

The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.

Characterization of a Drosophila cDNA clone that encodes a 252-amino acid non-muscle tropomyosin isoform.

We report here the isolation and DNA sequence of a cDNA clone encoding a 252-amino acid non-muscle or cytoskeletal tropomyosin (cTm) isoform from Drosophila. The Drosophila cTm shows considerable homology with vertebrate cTm throughout the middle portion of the molecule. The amino-terminal end of the molecule, however, shows less homology and contains five more amino acids than the equine platelet and human tropomyosins. There is also a proline at position 6 in the Drosophila protein. The carboxyl-terminal 27 amino acids also show little homology with vertebrate non-muscle tropomyosins. This is a region of the molecule that shows considerably diversity among other Drosophila tropomyosins and vertebrate tropomyosins. A comparison of the DNA sequence of the cTm cDNA and a previously reported muscle tropomyosin II cDNA sequence shows regions of identical DNA sequence alternating with regions of nonidentical sequence, suggesting that both mRNAs are produced by alternate splicing of the same gene.

Nucleotide sequence of a cDNA clone encoding a Drosophila muscle tropomyosin II isoform.

A cDNA clone was sequenced that contains the entire coding region for the muscle tropomyosin II isoform from Drosophila. The cDNA clone is 1253 nucleotides (nt) long and contains an 88-nt 5'-leader sequence and a 310-nt 3'-untranslated sequence. The muscle tropomyosin II isoform consists of 285 amino acids and is 60% homologous with the previously reported muscle tropomyosin I isoform Drosophila and 55% homologous with rabbit muscle tropomyosin.

Requirement of protein synthesis for the induction of ribonucleoside diphosphate reductase mRNA in Escherichia coli.

An RNA-DNA hybridization assay was used to quantitate the ribonucleoside diphosphate reductase mRNA synthesis (nrd mRNA) to show that gene expression was dependent on protein synthesis. The increased nrd mRNA synthesis induced by inhibition of DNA synthesis was eliminated by simultaneous inhibition of protein synthesis. It was further found that protein synthesis is required not only initially but continuously during DNA inhibition for increased expression of nrd mRNA synthesis.

Characterization of the mRNA coding for ribonucleoside diphosphate reductase in Escherichia coli.

Total Escherichia coli RNA was separated by electrophoresis on methyl mercury agarose gels, transferred to diazobenzyloxymethyl-paper, and hybridized to various DNA probes containing different segments of the nrd genes to determine the organization of these genes. A 3.2-kilobase polycistronic mRNA transcript which hybridizes to both the nrdA and nrdB genes indicated that the nrdA and nrdB genes are organized in an operon. The polycistronic transcript contained the nrdA gene at the 5' end and the nrdB gene at the 3' end. The size of the polycistronic mRNA was sufficient to code for the 80,000-molecular-weight B1 protein and the 40,000-molecular-weight B2 protein. The results also indicated that the nrdA and nrdB genes are the only genes in E. coli that code for ribonucleoside diphosphate reductase. Two smaller RNA species that hybridized to nrd DNA were observed and probably overlap with the 3.2-kilobase nrd mRNA.

Regulation of ribonucleoside diphosphate reductase mRNA synthesis in Escherichia coli.

A RNA-DNA hybridization assay for ribonucleoside diphosphate reductase (RDP reductase) mRNA was used to determine whether control of RDP reductase synthesis in Escherichia coli is at the level of RNA transcription. The correlation observed between the level of RDP reductase enzymatic activity and the rate of RDP reductase mRNA synthesis suggested that the control is at the level of RNA transcription. No increase in RDP reductase enzymatic activity or RDP reductase mRNA was observed during the first 15 min after removal of thymine from a thymine-requiring culture. Thereafter, the rate of RDP reductase mRNA synthesis increased linearly for approximately 75 min before beginning to level off. The addition of thymine to a culture starved for thymine resulted in a decreasing rate of RDP reductase mRNA synthesis. However, 30 min of growth in the presence of thymine was required before the rate of RDP reductase mRNA synthesis dropped to the rate observed in an exponentially growing culture. Inhibition of DNA synthesis caused by shifting a culture of a polC mutant or a dnaB mutant to nonpermissive growth conditions resulted in an increase in the rate of RDP reductase mRNA synthesis similar to that observed for a thymine-starved culture.