To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important An approach proposed toward this end involves the synthesis of ≈5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H O, Hutchinson, C A, III, Pfannkoch, C & Venter, J C (2003) Proc Natl Acad Sci USA 100, 15440–15445] The major current impediment to the success of this tactic is the difficulty of building the ≈5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp “synthons” with low error frequency by automated PCR-based gene synthesis By parallel processing, these synthons are efficiently joined into multisynthon ≈5-kb segments by using only three endonucleases and “ligation by selection” These large segments can be subsequently assembled into very long sequences by conventional cloning We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli

https://www.pnas.org/content/pnas/101/44/15573.full.pdf

Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster

https://faculty.washington.edu/gelb/2006/mehlin2006_MolBiochemParasitol_vol148no1p144-160.pdf

Heterologous expression of proteins from Plasmodium falciparum: Results from 1000 genes

The malaria parasite's chloroquine resistance transporter (CRT) is an integral membrane protein localized to the parasite's acidic digestive vacuole. The function of CRT is not known and the protein was originally described as a transporter simply because it possesses 10 transmembrane domains. In wild-type (chloroquine-sensitive) parasites, chloroquine accumulates to high concentrations within the digestive vacuole and it is through interactions in this compartment that it exerts its antimalarial effect. Mutations in CRT can cause a decreased intravacuolar concentration of chloroquine and thereby confer chloroquine resistance. However, the mechanism by which they do so is not understood. In this paper we present the results of a detailed bioinformatic analysis that reveals that CRT is a member of a previously undefined family of proteins, falling within the drug/metabolite transporter superfamily. Comparisons between CRT and other members of the superfamily provide insight into the possible role of the protein and into the significance of the mutations associated with the chloroquine resistance phenotype. The protein is predicted to function as a dimer and to be oriented with its termini in the parasite cytosol. The key chloroquine-resistance-conferring mutation (K76T) is localized in a region of the protein implicated in substrate selectivity. The mutation is predicted to alter the selectivity of the protein such that it is able to transport the cationic (protonated) form of chloroquine down its steep concentration gradient, out of the acidic vacuole, and therefore away from its site of action.

/pdf/the-malaria-parasite-s-chloroquine-resistance-transporter-is-3hbzke3m7a.pdf

The Malaria Parasite's Chloroquine Resistance Transporter is a Member of the Drug/Metabolite Transporter Superfamily

Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra-erythrocytic parasite's digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil-reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2005.04556.x

Hanbang Zhang

Papers

Analysis of the antimalarial drug resistance protein Pfcrt expressed in yeast.