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Biochem Biophys Acta. entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC. African trypanosomes are unusual among protozoan parasites in that they never enter host cells and yet persist for extended periods of time in mammalian blood and tissues. They are able to survive in the host because conserved cell surface polypeptides are shielded from host antibodies by a dense monolayer of a single polypeptide species, the variant surface glycoprotein (VSG).1 The parasites are able to establish a persistent infection by a system of antigenic variation, whereby a single trypanosome stops expressing one VSG gene and starts expressing another antigenically distinct VSG. The VSG is a homodimer, each monomer consisting of an NH2-terminal domain comprising 350 residues and a COOH-terminal domain(s) of 50 to 100 residues (Johnson and Cross, 1979). The structure of the NH2-terminal domain is known in the case of two VSGs (Blum et al., 1993) and shows a high degree of conservation of tertiary structure but not primary sequence. The VSG is anchored in the membrane by the covalent attachment of the COOH-terminal carboxyl to a glycosylphosphatidylinositol (GPI), the structure of which was first solved for the VSG MITat 1.4 (Ferguson et al., 1988). The GPI anchor is added to the VSG within 1 min of the completion of protein synthesis (Ferguson et al., 1986). contains an endogenous phospholipase C (PLC) known as the GPI-PLC (Blow and Overath, 1986; Fox et al., 1986; Hereld et al., 1986) that is capable of hydrolyzing the GPI anchor on the VSG, releasing dimyristyl glycerol (Ferguson et al., 1985). The result of this hydrolysis is to convert the hydrophobic membrane form VSG (mfVSG) to a water soluble VSG (sVSG; Cardoso de Almeida and Turner, 1983), and hypotonic lysis MLN8237 (Alisertib) (Cardoso de Almeida and Turner, 1983) or stress (Voorheis et al., 1982; Rolin et al., 1996) Rabbit Polyclonal to AXL (phospho-Tyr691) MLN8237 (Alisertib) results in the shedding of the VSG from the membrane. This conversion can be detected immunologically as it results in the exposure of the cross-reacting determinant (CRD; Holder and Cross, 1981; Cardoso de Almeida and Turner, 1983), an epitope contained in the residue MLN8237 (Alisertib) of the anchor left attached to the VSG after PLC hydrolysis. Consequently mfVSG is poorly recognized by anti-CRD antibodies when compared with sVSG. As well as the VSG, the majority of the precursors of the GPI anchor are also substrates for the GPI-PLC in vitro (for review of GPI biosynthesis see McConville and Ferguson, 1993). Recently, it has been shown that GPI-PLC can hydrolyze phosphatidylinositol (PI) under certain assay conditions (Btikofer et al., 1996). However the GPI-PLC may play MLN8237 (Alisertib) a role in the regulation of GPI levels (Gther et al., 1994; Gther and Ferguson, 1995) or PI levels (Btikofer et al., 1996) within the cell. However, it is not immediately obvious why a general role in (G)PI metabolism or homeostasis, unlike expression of GPI-PLC and VSG, would be confined to the bloodstream stage of the life cycle. Most of the circumstantial evidence outlined above thus points to a role for the GPI-PLC in the hydrolysis of the GPI anchor of the VSG. To address directly the role of this, a GPI-PLC null mutant (-tubulin gene. The constructs were derived from pJF6 (Clayton et al., 1990). The chloramphenicol acetyl transferase gene was removed by HindIIICBamHI digestion and replaced, in a three-fragment ligation, by a HindIIICEcoRI fragment containing the coding sequence and an EcoRICBglII fragment from the -tubulin gene. The restriction sites were added to the coding sequence using PCR or adaptors. PLC Gene Deletion Constructs, pLN and pSH The gene encoding GPI-PLC (and tubulin loci, the constructs with which they were targeted, and the desired homologous recombination products. None of the constructs contains promoters but rely on polycistronic transcription for selectable marker expression. (gene; structure of genome shows the sites of polyadenylation (pA) for the gene and the main sites of mini-exon addition (gene and the.