MOLECULAR-CLONING OF RAS AND RAP GENES FROM ENTAMOEBA-HISTOLYTICA

To better understand growth regulation in the protozoan parasite Entam oeba histolytica, ameba genes homologous to the ras oncogene and rap ( Klev-1) anti-oncogene were cloned. Two putative ameba ras genes (Ehras 1 and Ehras2) were identified, which contain 205 and 203 amino acid (a a) open reading frames (ORFs), respectively. The Ehras1 ORF shows an 9 1% positional identity with that of Ehras2, a 55% identity with Dictyo stelium discoideum (Dd) ras, and a 47% identity with human (Hs) ras. T wo ameba rap genes (Ehrap1 and Ehrap2) were identified, both of which contain 184-aa ORFs. The Ehrap1 ORF shows a 93% positional identity wi th that of Ehrap2, a 60% identity with Dd rap, a 61% identity with Hs Krev-1, and a 45% identity with that of Ehras1 Conserved aa in each am eba ras and rap ORF include GTP-binding sites, effector site, site of ADP-ribosylation by Pseudomonas exoenzyme S, and COOH-terminus CAAX. A s all Xs = Leu or Phe, ameba ras and rap proteins may be gerenylgereny lated and not farnesylated. Both ras and rap genes are transcribed by trophozoites. A Single 21-kDa ameba ras protein reacts with the rat Y1 3-259 anti-ras monoclonal antibody, which is located on the cytosolic side of the plasma membrane. These are the first ras and rap genes ide ntified from a protozoan parasite.