Plasmodium yoelii: cloning and characterization of the gene encoding for the mitochondrial heat shock protein 60.
Heat shock proteins are a highly conserved group of proteins required for the correct folding, transport, and degradation of other proteins in vivo. The Hsp70, Hsp90, and Hsp60 families are among the most widely studied families. Hsp60 is found in eubacteria, mitochondria, and chloroplasts, where, in cooperation with Hsp10, it participates in protein folding and translocation of proteins to the organelles. We have cloned and characterized the Hsp60 gene of Plasmodium yoelii (PyHsp60). PyHsp60 is a single-copy gene, located on chromosome 9, 10, or 11. The PyHsp60 cDNA sequence showed an open reading frame of 1737 nucleotides that codes for a polypeptide of 579 amino acids, with 93% amino acid identity to Plasmodium-falciparum Hsp60 (PfHsp60). Cloning and sequencing of a genomic PCR clone showed the presence of a 201-bp intron, located 141 bp downstream of the ATG codon. A single, heat-inducible, 2.3-kb transcript was detected in Northern blots of RNA isolated from blood stage parasites. Mouse antisera raised against a DNA vaccine vector that expresses PyHsp60 recognized sporozoites and liver- and blood-stage parasites by indirect fluorescent antibody test (IFAT). By Western blot, these antisera reacted with the mycobacterial Hsp65 and recognized a protein of approximately 65 kDa in P. yoelii sporozoites and P. falciparum blood stages. These results show that PyHsp60 and PfHsp60 genes are homologous and that of the PyHsp60 gene encodes a heat-inducible, intracellular protein that is expressed in several of the developmental stages of P. yoelii.
Sanchez
G I
GI
Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland, 21205, USA. sanchezg@nmripo.nmri.nnmc.navy.mil
Carucci
D J
DJ
Sacci
J
J
Resau
J H
JH
Rogers
W O
WO
Kumar
N
N
Hoffman
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010967:Plasmodium yoelii / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016053:RNA, Protozoan / Q000737:chemistry; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D018834:Chaperonin 60; D016054:DNA, Protozoan; D016053:RNA, Protozoan