MOLECULAR-CLONING AND CHARACTERIZATION OF A RAS-RELATED GENE OF RAN TC4/SPI1 SUBFAMILY IN GIARDIA-LAMBLIA/

The significance of Ras-like proteins in the protozoa is relatively un explored. In this report, a gene encoding a Ras-like nuclear (Ran) pro tein was identified in Giardia lamblia by a polymerase chain reaction- based cloning strategy. The sequence analyses suggest that the gene wa s intronless, and had short 5'-untranslated leader sequences in the co rresponding mRNA up to -2, -4, or -29 bases upstream of the first init iation codon. The full-length cDNA sequence predicted a protein compri sing 226 amino acids, in which the highly conserved functional motifs of the Ras superfamily were all preserved. This protein showed 52% ide ntity to human TC4 and 50% identity to yeast Spi1 proteins, suggesting that it is closely related to the Ran proteins, and it was therefore designated gRan. gRan produced from recombinant Escherichia coli exhib ited GTP binding activity by an overlay assay. In good agreement with the predicted size of gRan, a 27-kDa protein was identified in a lysat e of G. lamblia by Western blotting using antiserum raised against rec ombinant gRan. The protein was further localized in both nuclei of G. lamblia by immunofluorescence staining. Recombinant gRan exhibited low affinity for GTP with a K-d value of 16.8 mu M. The affinity was enha nced to a K-d value of 2.2 mu M in the presence of 10 mM Mg2+. The int rinsic GTPase activity of gRan was observed only in the presence of 10 mM Mg2+ and had an estimated K-m of 5.6 mu M and a K-cat of 0.33/h. T hese observations demonstrate the presence of Ras-like proteins in the most primitive eukaryotic cells, G. lamblia, and infer that the Ran p rotein may play a functional role in the nuclei of this organism.