Vikki M. Marshall

Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant

Insights into the role of ankyrin-1 (ANK-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an N-ethyl-N-nitrosourea (ENU)-induced Ank-1-null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero and most die perinatally, indicating that Ank-1 plays a nonredundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein and a marked reduction in beta-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture and severely altered hemorheologic properties. Heterozygous mutant mice, which have wild-type levels of ANK-1 and spectrin in their RBC membranes and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance.

Integral membrane protein located in the apical complex of Plasmodium falciparum

We describe the cloning of a novel antigen of Plasmodium falciparum which contains a hydrophobic domain typical of an integral membrane protein. This antigen is designated apical membrane antigen 1 because it appears to be located in the apical complex. Apical membrane antigen 1 appears to be transported to the merozoite surface near the time of schizont rupture.

Platelets Kill Intraerythrocytic Malarial Parasites and Mediate Survival to Infection

Platelets play a critical role in the pathogenesis of malarial infections by encouraging the sequestration of infected red blood cells within the cerebral vasculature. But platelets also have well-established roles in innate protection against microbial infections. We found that purified human platelets killed Plasmodium falciparum parasites cultured in red blood cells. Inhibition of platelet function by aspirin and other platelet inhibitors abrogated the lethal effect human platelets exert on P. falciparum parasites. Likewise, platelet-deficient and aspirin-treated mice were more susceptible to death during erythrocytic infection with Plasmodium chabaudi. Both mouse and human platelets bind malarial-infected red cells and kill the parasite within. These results indicate a protective function for platelets in the early stages of erythrocytic infection distinct from their role in cerebral malaria.

MOLECULAR VARIATION IN A NOVEL POLYMORPHIC ANTIGEN ASSOCIATED WITH PLASMODIUM FALCIPARUM MEROZOITES

A cDNA clone encoding part of a novel polymorphic merozoite antigen from Plasmodium falciparum was isolated by screening a cDNA library with human immune serum from Papua New Guinea. Immunofluorescence microscopy and immunoblotting with affinity-purified antibodies recognized a highly polymorphic antigen, Ag956, present in schizonts and merozoites. Biosynthetic labeling and immunoprecipitation experiments demonstrated that Ag956 is proteolytically cleaved during merozoite maturation. The complete genomic sequence of Ag956 from the D10 clone of P. falciparum isolate FC27 encodes a secreted protein of calculated molecular mass 43,243 that is very hydrophilic and contains a region of unusual heptad repeats of the general structure AXXAXXX. This antigen has been named the secreted polymorphic antigen associated with merozoites (SPAM). The sequence of a second SPAM allele from the 3D7 clone of isolate NF54 reveals that the alanine heptad repeats and the hydrophilic C-terminal half of the protein are conserved. Variation among SPAM alleles is the result of deletions and amino acid substitutions in non-repetitive sequences within and flanking the alanine heptad-repeat domain. Heptad repeats in which the a and d position contain hydrophobic residues generate amphipathic alpha-helices which give rise to helical bundles or coiled-coil structures in proteins. Thus, SPAM is the first example of a P. falciparum antigen in which a repetitive sequence has features characteristic of a well-defined structural element.

Close linkage of three merozoite surface protein genes on chromosome 2 of Plasmodium falciparum

We have analysed a 10.5 kb region of chromosome 2 in Plasmodium falciparum that encompasses the coding region of four genes. Three genes are arranged in a head-to-tail orientation and encode the merozoite surface proteins MSP2 and MSP4 as well as a previously unreported sequence that encodes a polypeptide with the characteristics of a merozoite surface protein, now designated MSP5. The fourth gene, asl, is arranged in a tail-to-tail orientation with msp2 and has homology with prokaryotic and eukaryotic genes encoding adenylosuccinate lyase (ASL), an enzyme involved in purine biosynthesis and salvage. The genes, arranged in the order msp4, msp5, msp2 and asl, are separated by intergenic distances of 1021, 1017 and 722 bp, respectively. msp4 and msp5 are clearly related genes, each being composed of 2 exons and encoding proteins of identical length. Both msp4 and msp5 encode proteins that contain hydrophobic signal sequences, apparent glycosylphosphatidylinositol (GPI) attachment signals and a single epidermal growth factor-like (EGF-like) domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. It appears that one of these genes arose as a result of a relatively ancient gene duplication event and both genes have subsequently diverged considerably. This study shows that msp5 is transcribed in asexual stages and its encoded product is a 40 kDa protein that appears to be located on the merozoite surface as determined by immunofluorescence assays.

Allelic variants of the Plasmodium falciparum merozoite surface antigen 2 (MSA-2) in a geographically restricted area of Irian Jaya

Blood samples were collected from 12 residents of 4 villages in the Oksibil area of Irian Jaya. Eleven patients were positive for Plasmodium falciparum infection as evidenced by successful amplification of the MSA-2 gene by the polymerase chain reaction. Two patients showed evidence of infection by 2 strains of Plasmodium falciparum. All MSA-2 genes were completely sequenced and all could be assigned to one of the two major allelic families of MSA-2, however all MSA-2 gene sequences differed from previously described alleles. Five new allelic forms were identified, one of which was present in 8 of the 11 patients. Within small natural populations of P. falciparum, it appears that variation in MSA-2 approximates that seen world-wide. All samples were also analysed by hybridisation of amplified DNA to family specific probes and all samples hybridised to known probes. Our results demonstrate that there is a degree of microheterogeneity of MSA-2 that is undetectable by hybridisation studies alone.

Temporal expression of an H2-linked locus in host response to mouse malaria.

Abstract The action of host genes in response to malarial infection is complex. Two mouse loci, Char1, and Char2, have previously been shown to control peak parasitemia and host survival. Recent analysis of host response to mouse malaria has demonstrated that the action of several loci is time dependent. Char1 and Char2 act prior to peak parasitemia. Analysis of additional crosses revealed significant linkage to Chromosome 17 on the day following peak parasitemia. This H2-linked locus acts late in infection and is therefore crucial in clearing parasites from the circulation. The cloning of this gene will lead to a greater understanding of the host-parasite interaction, and the kinetics of host gene expression during an immune response.

Mice that are congenic for the char2 locus are susceptible to malaria.

ABSTRACT A major advance has been made towards the positional cloning of char2 (a quantitative trait locus encoding resistance to Plasmodium chabaudi malaria). Mice congenic for the locus have been used to fine map the gene and to prove that char2 plays a significant role in the outcome of malarial infection, independently of other resistance loci.

Constructing Contigs from Large‐Insert Clones

This unit describes three approaches that are widely used to define alignments between overlapping clones bearing large-insert genomic DNA and to generate extensive contiguous overlapping sets of clones (contigs). The three approaches are sequence-tagged site (STS) content mapping, repetitive-element hybridization fingerprinting, and Alu-PCR fingerprinting. Methods for isolating the necessary BAC DNA suitable for automated fluorescent sequencing and generating new STS markers are discussed in support protocols. An alternate protocol presents repetitive-element hybridization fingerprinting to detect overlaps and build contigs with full-genomic YAC libraries.

Improvements in allelic discrimination of microsatellite markers using denaturing polyacrylamide gel electrophoresis

Abstract. Poor resolution, retarded progress of DNA through gels, and variable sizing of DNA fragments between and within gels hinder accurate genotyping of some simple sequence length polymorphism (SSLP) markers with the Perkin Elmer Applied Biosystems 377 Sequenator. These problems are similar to renaturation related problems observed in DNA sequencing gels. PCR products especially susceptible to these problems are shown to have higher melting temperatures (Tm) than others. Gels containing increased concentrations of denaturants allow greater accuracy in allelic discrimination. This is especially beneficial where quantification is necessary.