Selective amplification of additional members of the ADP-ribosylation factor (ARF) family: cloning of additional human and Drosophila ARF-like genes.

摘要

The ADP-ribosylation factor (ARF) family is one of four subfamilies of the RAS superfamily of low molecular weight GTP-binding proteins (G proteins). Highly degenerate oligonucleotides encoding two conserved regions were used in a PCR reaction to amplify cDNAs encoding each of the known ARF proteins and eight additional cDNA fragments encoding previously unreported human members of the ARF family. Additional sequences were obtained from yeast or fly libraries by using this technique. These oligonucleotides specifically amplify members of the ARF family but not the structurally related G protein alpha subunits or members of the other three subfamilies of the RAS superfamily. Fragments obtained by PCR were used to obtain full-length sequences encoding highly homologous ARF-like (ARL) gene products from human and Drosophila melanogaster libraries, termed ARL2 and Ar184F, respectively. The encoded proteins are each 184 amino acids long and are 76% identical, with 40-45% identity to human ARF1 and Drosophila arf-like (arl) proteins. These genes appear to be generally expressed in human tissues and during Drosophila 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 conserved members, including both the functionally conserved ARF proteins and the functionally disparate ARL proteins. The latter proteins currently comprise two distinct gene products in Drosophila (arl and ARL84F) and one in man (ARL2).