Malaria. Malaria has long been recognized as an important parasitic disease of humans, having been described by the early Egyptians in the third millennium B.C.,
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(1) that 1.5 billion persons live in areas of the world where malaria is an endemic disease,
(2) that the number of infected humans exceeds 500,000,000, and,
(3) that 1-3 million persons die each year, including about 1 million children under 5 years old (3,000 per day).
Science: Volume 289, Number 5485 (2000), p. 1763
There are four important species that infect humans, causing malaria;
* P. vivax - benign tertian malaria - accounts for ~43% of cases
* P. falciparum - malignant tertian malaria accounts for ~50% cases
* P. malariae - quartan malaria - accounts for ~7% of cases
* P. ovale - mild tertian malaria - accounts for < 1% of cases.
P. ovale - mild tertian malaria: similar
Knobs promote cell clumping… clogging veins and arteries.P. falciparum - malignant tertian malaria
This is thought to be receptor mediated.
Ookinetes cross the midgut epithelium,
This is thought to be receptor mediated.Plasmodium: Mosquito Life Cycle
Molecules bind receptor sites, block Ookinete transfer.
enters cellReceptor Mediated Transfer
phage gIII gene sequence.Phage Display
…recombinant phage express the sequence as part of the gIII protein.
Insert DNA coding for dodecapeptides (XCX8CX) into T7 genome.
Allow recombinant DNAs to replicate and repackage themselves.
Run T7 phage over the receptor.
A mouse model system has been established in order to provide a clinical vertebrate host.
pBacR: piggyback vector, transposon derived
3xP3-EGFP-SV40: Green fluorescent protein, eye specific promoter
AgCP promoter: mosquito promoter, activated by blood feast.
Signal: peptide sequence that sends protein to the midgut.
SM14: SM1 DNA sequence repeated 4 times, linkedTransgenic Construct
Show that the insert is present.Southern Blot
…till a NotI or BglII site.
3xP3-EGFP-SV40 and SM1 DNA,
Genomic DNA from different transgenic individuals, digested w/ NotI and BglII.
Probes: SM1 DNA (mt rRNA for control) the genome.
Why no male expression?
mt rRNA Control: constant expression gene, shows total RNA level was similar in all lanes.
SM1 expression…after a blood meal.Northern Blot
Further: SM1 was inserted into 4 different genomic regions, all had the same phenotype,
This suggests that the anti-ookinete function wasn’t due to a disrupted gene.
When in a transgene, and subsequently translated, the location of transgene expression in the organism can be assayed.Epitope Tagging
in situ location of transgene expression in the organism can be assayed., “Western”
Genetics location of transgene expression in the organism can be assayed.
…in the news.