By comparison of the amino acid sequence of argininosuccinate lyase to other proteins in available data banks we noted that it was highly homologous to the major soluble protein in avian lenses, delta crystallin. Preliminary data show that these proteins share antigenic epitopes and that in duck lenses delta crystallin may have argininosuccinate lyase enzyme activity. We propose to demonstrate that the delta-2-crystallin gene in avian species is in fact the gene that encodes argininosuccinate lyase. We propose to clone and sequence the human argininosuccinate lyase gene and compare its structure to the avian delta crystallin genes (preliminary data shows that they are in fact very similar if not identical). A cDNA clone will be isolated from a lambda gtll library prepared from the RNA derived from a non lens tissue such as kidney and its sequence determined. These data will reveal whether one of the previously sequenced delta crystallin genes does encode argininosuccinate lyase. We will also attempt to show that this cDNA will in fact encode active argininosuccinate lyase by expression in argininosuccinate lyase deficient yeast. We will purify the delta crystallin protein(s) from the duck lens, analyze its enzymatic properties and obtain some amino acid sequence data in an attempt to prove that both delta crystallin genes are expressed in the duck lens. A cDNA library will be prepared from RNA isolated from duck lenses and screened with the chicken delta crystallin genes. These cDNA molecules will be sequenced and tested to determine if they are capable of encoding argininosuccinate lyase. The data made available from these studies provide an enormous amount of infomation regarding the amino acid residues in argininosuccinate lyase that are required for enzymatic activity. We will prepare """"""""chimeric proteins"""""""" in which part of the protein is derived from the active protein (argininosuccinate lyase) and part from the inactive isomer (delta crystallin). Such proteins can be synthesized by splicing pieces of the cDNA molecules together at common restriction sites. This approach will be complemented with site directed mutagenesis studies to define the role of individual amino acids.
