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Fig. 1. Molecular characterisation of isogenic knobby and knobless clones. (A) Schematic describing the preparation of isogenic clones used in this study. Rosette or mAbBC6 selected cultures were cloned by limiting dilution. Clones that share the same knobby phenotype and express the same PfEMP1 variant are boxed together. The K– clones expressing type 41 PfEMP1 variant were selected from clones originally cloned from rosetting parasites. See Table 1. (B) Schematic of the chromosome 2 breakpoints in the K– clones used in this study. The positions of the breakpoints upstream of the kahrp (PFB0100c) gene are indicated, with the distance from the telomere (located on left as a black semi-circle) indicated in kilobase pairs. All genes located to the left of the breakpoint are deleted. (C) Ethidium bromide staining of size-fractionated chromosomes shows that approximately 100 kilobase pairs is deleted from chromosome 2 in the K– clones. One from each pair of clones (name and knobby phenotype indicated) is shown here. (D) Northern blot analysis indicates specific transcription of var types 41 and 6 in the cloned parasites. Hybridisation with kahrp and EF-1
probes confirms the ethidium bromide (EtBr) staining indicating equal loading of RNA and that the kahrp gene is not transcribed in K– clones. Transcript sizes are indicated in kilobases (kb). (E) Scanning electron micrograph of a K+ IE adhering to a HDMEC monolayer. Note the numerous knobs (k) on the IE plasma membrane. Bar, 1 µm. (F) Scanning electron micrograph of a K– IE adhering to a HDMEC monolayer illustrating the irregular shape of the IE and the absence of knobs on the plasma membrane. Bar, 1 µm.
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