SELECTIVE AMPLIFICATION OF ADDITIONAL MEMBERS OF THE ADP-RIBOSYLATIONFACTOR (ARF) FAMILY - CLONING OF ADDITIONAL HUMAN AND DROSOPHILA ARF-LIKE GENES

The ADP-ribosylation factor (ARF) family is one of four subfamilies of the RAs superfamily of low molecular weight GTP-binding proteins (G p roteins). Highly degenerate oligonucleotides encoding two conserved re gions were used in a PCR reaction to amplify cDNAs encoding each of th e known ARF proteins and eight addition cDNA fragments encoding previo usly unreported human members of the ARF family. Additional sequences were obtained from yeast or fly libraries by using this technique. The se oligonucleotides specifically amplify members of the ARF family but not the structurally related G protein alpha subunits or members of t he other three subfamilies of the RAS superfamily. Fragments obtained by PCR were used to obtain full-length sequences encoding highly homol ogous ARF-like (ARL) gene products from human and Drosophila melanogas ter libraries, termed ARL2 and Arl84F, respectively. The encoded prote ins are each 184 amino acids long and am 76% identical, with 40-45% id entity to human ARF1 and Drosophila arf-like (arl) proteins. These gen es appear to be generally expressed in human tissues and during Drosop hila development. The purified human ARL2 protein differed in several biochemical properties from human ARF proteins, including the complete absence of ARF activity. Thus, the ARF family of low molecular weight GTP-binding proteins includes at least 15 distinct but structurally c onserved members, including both the functionally conserved ARF protei ns and the functionally disparate ARL proteins. The latter proteins cu rrently comprise two distinct gene products in Drosophila (arl and ARL 84F) and one in man (ARL2).