Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major.
This paper describes the cloning of a Golgi-associated GTP-binding protein homologue from Leishmania major. The gene was isolated using degenerate oligonucleotides to conserved sequences amongst the small GTP-binding proteins in a polymerase chain reaction on genomic DNA of the L. major cloned line V121. The reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. A full length copy of the gene was isolated from a lambda gt10 V121 genomic library and sequenced. At the amino acid level the gene showed highest similarity to the human/rat rab1 A gene and the mouse/yeast YPT gene and was named LmYPT. The LmYPT gene was present as a single copy gene in both the L. major and L. donovani genomes. Karyotype analysis localized the LmYPT gene to chromosome band 18 in V121, but it was located on a larger chromosome in the different L. major isolate L119. The LmYPT gene was transcribed as a 3.9-kb transcript in both the promastigote and amastigote forms of the parasite. Western blot analysis, using a polyclonal rabbit antiserum raised against an Escherichia coli expressed portion of the molecule, identified a doublet at 20 and 23 kDa in total promastigote protein. Immunoelectron microscopy in combination with peroxidase staining localized the LmYPT molecule to the Leishmania Golgi apparatus.
Cappai
R
R
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Osborn
A H
AH
Gleeson
P A
PA
Handman
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D017125:Genes, Protozoan; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D018320:Leishmania major / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D019204:GTP-Binding Proteins