Conservation of genetic linkage between heat shock protein 100 and glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei and Trypanosoma cruzi.
The experiments described in this paper were designed to try and isolate a recombinant DNA clone encoding a Trypanosoma cruzi homologue of the Trypanosoma brucei glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) gene. Despite the ready biochemical detection of phospholipase C activities that hydrolyse GPI-anchors of cell surface proteins in T. cruzi, it did not prove possible to isolate any recombinant DNA clones using the T. brucei gpi-plc gene as a probe. On determining the DNA sequence to the 5' side of the gpi-plc gene it was found to be adjacent to a gene that encodes a 100 kDa heat shock protein (HSP100). To investigate whether this linkage between the hspl00 and gpi-plc genes was conserved in T. cruzi, a probe derived from the T. brucei hsp100 gene was used to isolate T. cruzi genomic clones. These were partially sequenced and shown to contain an hsp100 gene. Restriction enzyme fragments located to the 3' side of the T. cruzi hsp100 gene were then sequenced and found to contain a gene that encodes a polypeptide (TcPLC1) that has 46% amino acid sequence identity with the T. brucei GPI-PLC including most of the key residues involved in inositol binding and the catalytic histidine. A recombinant form of TcPLC1 was produced and shown to possess phospholipase C activity towards a GPI-substrate. Thus, the hsp100 and gpi-plc genes are adjacent in T. brucei and this linkage is conserved in T. cruzi. This observation has been used to facilitate the isolation of a clone encoding a T. cruzi phospholipase C gene.
Department of Biochemistry, University of Cambridge, UK.
M B
MB
Redpath
N
N
Carnall
H
H
Webb
M
M
Courel
A
A
Amorim
M L
ML
Güther
M L
ML
Cardoso de Almeida
M
M
Carrington
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D017125:Genes, Protozoan; D008040:Genetic Linkage; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase; D006360:Heat-Shock Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics*; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics*; D010738:Type C Phospholipases / Q000235:genetics*
D015342:DNA Probes; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase