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The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

Webb H, Carnall N, Vanhamme L, Rolin S, Van Den Abbeele J, Welburn S, Pays E, Carrington M - J. Cell Biol. (1997)

Bottom Line: To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC mutant trypanosome has been generated by targeted gene deletion.Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation.This phenotype is partially alleviated when the mutant is modified to express low levels of GPI-PLC.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cambridge University, United Kingdom.

ABSTRACT
In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an 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 mutant is modified to express low levels of GPI-PLC.

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Related in: MedlinePlus

Structure and restriction enzyme maps of the  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. (a) Deletion of the PLC gene; structure of genome shows the  sites of polyadenylation (pA)  for the PLC gene and the  main sites of mini-exon addition (ME) for both the PLC  gene and the downstream  gene, gene 3. These were  mapped by RNase protection  analysis (data not shown).  Boxes represent coding sequences. K* (KpnI) and X*  (XbaI) mark the positions of  restriction sites introduced  into genomic clones by site-directed mutagenesis. Constructs shows the PLC elimination constructs in which the PLC gene has been replaced by a chimeric expression cassette. The two constructs differ in the presence  of either the long or short version of an RFLP in the intergenic region. The BglI (Bg) sites were used to linearize constructs before electroporation. Desired recombination product shows the genome after targeting. (b) PLC replacement. Structure of genome shows a representative section of the T. brucei tubulin gene cluster. Construct shows the PLC replacement construct, based on a PstI to PstI fragment that includes two complete α tubulin genes separated by one entire β tubulin gene. BamHI sites were used in Southern analysis of  transformed cell lines. The other restriction sites were involved in construction: BstEII (Bs), ClaI (C), HindIII (H), MluI (M), NcoI (N),  StuI (St).
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Figure 1: Structure and restriction enzyme maps of the 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. (a) Deletion of the PLC gene; structure of genome shows the sites of polyadenylation (pA) for the PLC gene and the main sites of mini-exon addition (ME) for both the PLC gene and the downstream gene, gene 3. These were mapped by RNase protection analysis (data not shown). Boxes represent coding sequences. K* (KpnI) and X* (XbaI) mark the positions of restriction sites introduced into genomic clones by site-directed mutagenesis. Constructs shows the PLC elimination constructs in which the PLC gene has been replaced by a chimeric expression cassette. The two constructs differ in the presence of either the long or short version of an RFLP in the intergenic region. The BglI (Bg) sites were used to linearize constructs before electroporation. Desired recombination product shows the genome after targeting. (b) PLC replacement. Structure of genome shows a representative section of the T. brucei tubulin gene cluster. Construct shows the PLC replacement construct, based on a PstI to PstI fragment that includes two complete α tubulin genes separated by one entire β tubulin gene. BamHI sites were used in Southern analysis of transformed cell lines. The other restriction sites were involved in construction: BstEII (Bs), ClaI (C), HindIII (H), MluI (M), NcoI (N), StuI (St).

Mentions: The gene encoding GPI-PLC (PLC) and flanking DNA was subcloned from λBS2 (Carrington et al., 1989) and λBS3 as a 9.4-kb EcoRI–SalI fragment. This plasmid is referred to as p1172. An XbaI site was introduced downstream of the GPI-PLC gene, after the polyadenylation sites (see Fig. 1 a) by site-directed mutagenesis to give p1172x. The ILTar 1 serodeme (Miller and Turner, 1981) is heterozygous for an insertion type RFLP in the intergenic region upstream of the GPI-PLC gene (see Fig. 1 a). Since the constructs were originally designed to target this trypanosome line, both alleles were cloned as PCR products, and a KpnI site was introduced into each by site directed mutagenesis (see Fig. 1 a). These were subcloned back into p1172x as a ClaI–BstEII fragment, in place of the endogenous ClaI–BstEII fragment, to give two constructs, p1172kxL and p1172kxS containing the long and short RFLP allele, respectively. The trypanosome line actually used for the gene deletion (see below) is homozygous for the long RFLP.


The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

Webb H, Carnall N, Vanhamme L, Rolin S, Van Den Abbeele J, Welburn S, Pays E, Carrington M - J. Cell Biol. (1997)

Structure and restriction enzyme maps of the  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. (a) Deletion of the PLC gene; structure of genome shows the  sites of polyadenylation (pA)  for the PLC gene and the  main sites of mini-exon addition (ME) for both the PLC  gene and the downstream  gene, gene 3. These were  mapped by RNase protection  analysis (data not shown).  Boxes represent coding sequences. K* (KpnI) and X*  (XbaI) mark the positions of  restriction sites introduced  into genomic clones by site-directed mutagenesis. Constructs shows the PLC elimination constructs in which the PLC gene has been replaced by a chimeric expression cassette. The two constructs differ in the presence  of either the long or short version of an RFLP in the intergenic region. The BglI (Bg) sites were used to linearize constructs before electroporation. Desired recombination product shows the genome after targeting. (b) PLC replacement. Structure of genome shows a representative section of the T. brucei tubulin gene cluster. Construct shows the PLC replacement construct, based on a PstI to PstI fragment that includes two complete α tubulin genes separated by one entire β tubulin gene. BamHI sites were used in Southern analysis of  transformed cell lines. The other restriction sites were involved in construction: BstEII (Bs), ClaI (C), HindIII (H), MluI (M), NcoI (N),  StuI (St).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139819&req=5

Figure 1: Structure and restriction enzyme maps of the 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. (a) Deletion of the PLC gene; structure of genome shows the sites of polyadenylation (pA) for the PLC gene and the main sites of mini-exon addition (ME) for both the PLC gene and the downstream gene, gene 3. These were mapped by RNase protection analysis (data not shown). Boxes represent coding sequences. K* (KpnI) and X* (XbaI) mark the positions of restriction sites introduced into genomic clones by site-directed mutagenesis. Constructs shows the PLC elimination constructs in which the PLC gene has been replaced by a chimeric expression cassette. The two constructs differ in the presence of either the long or short version of an RFLP in the intergenic region. The BglI (Bg) sites were used to linearize constructs before electroporation. Desired recombination product shows the genome after targeting. (b) PLC replacement. Structure of genome shows a representative section of the T. brucei tubulin gene cluster. Construct shows the PLC replacement construct, based on a PstI to PstI fragment that includes two complete α tubulin genes separated by one entire β tubulin gene. BamHI sites were used in Southern analysis of transformed cell lines. The other restriction sites were involved in construction: BstEII (Bs), ClaI (C), HindIII (H), MluI (M), NcoI (N), StuI (St).
Mentions: The gene encoding GPI-PLC (PLC) and flanking DNA was subcloned from λBS2 (Carrington et al., 1989) and λBS3 as a 9.4-kb EcoRI–SalI fragment. This plasmid is referred to as p1172. An XbaI site was introduced downstream of the GPI-PLC gene, after the polyadenylation sites (see Fig. 1 a) by site-directed mutagenesis to give p1172x. The ILTar 1 serodeme (Miller and Turner, 1981) is heterozygous for an insertion type RFLP in the intergenic region upstream of the GPI-PLC gene (see Fig. 1 a). Since the constructs were originally designed to target this trypanosome line, both alleles were cloned as PCR products, and a KpnI site was introduced into each by site directed mutagenesis (see Fig. 1 a). These were subcloned back into p1172x as a ClaI–BstEII fragment, in place of the endogenous ClaI–BstEII fragment, to give two constructs, p1172kxL and p1172kxS containing the long and short RFLP allele, respectively. The trypanosome line actually used for the gene deletion (see below) is homozygous for the long RFLP.

Bottom Line: To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC mutant trypanosome has been generated by targeted gene deletion.Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation.This phenotype is partially alleviated when the mutant is modified to express low levels of GPI-PLC.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cambridge University, United Kingdom.

ABSTRACT
In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an 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 mutant is modified to express low levels of GPI-PLC.

Show MeSH
Related in: MedlinePlus