REGULATION OF ARGININE KINASE IN DROSOPHILA MELANOGASTER

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A single gene encodes cytoplasmic and mitochondrial arginine kinase in Drosophila melanogaster. A molecular characterization of this gene and the transcripts made from it is proposed as an initial step in understanding how these alternative products are produced and regulated. Transcripts of a conserved active site sequence of chick creatine kinase will be used to screen a Drosophila cDNA library enriched for muscle specific transcripts. The identity of these clones will be verified by in situ hybridization to salivary gland polytene chromosomes, immunoprecipitation of in vitro translated hybrid selected mRNA, and comparison of the cDNA sequence to that of the homologous enzyme from chick, rabbit, rat and electric ray. Clones which hybridize to the proper chromosomal locus and have demonstrable sequence similarity to creatine kinase will be used to isolate the corresponding sequences from a Drosophila genomic library. The orientation of transcription will be determined by hybridization of strand specific transcripts of cDNA to "Northern" blots of late pupal mRNA. Subclones near the 5' end of the transcription unit will be used to map the 5' transcription start site by S1 mapping and primer extension analysis. Two different transcripts are expected, each with a unique start site. Comparison of the restriction maps for the corresponding two classes of cDNA clones and the genomic clones will provide the data for inferring the alternative splice sites necessary to produce the alternative transcripts. From the sequence of the cDNA clones the amino acid sequence can be inferred for the two forms of arginine kinase, including the anticipated leader sequence of the mitochondrial form.