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Plasmodium falciparum strains bearing quadruple mutations of dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) at codons 51, 59, 108, and 164 are highly resistant to pyrimethamine (PYR), a diaminopyrimidine, but sensitive to WR99210 (WR), a cycloguanil analog, suggesting different enzyme-inhibitor binding interactions. A combination of these inhibitors to delay the onset of antifolate resistance is proposed. Using error-prone PCR, libraries of random mutants of wild-type PfDHFR and PfDHFR-TS were generated and used to transform Escherichia coli, and transformants were then selected for PYR or WR resistance. Mutants highly resistant to either PYR or WR were also generated from libraries obtained from further random mutagenesis of quadruple mutants (QM) with mutations in PfDHFR or PfDHFR-TS. For reversion mutants carrying altered residues I51N, N108S, and L164I, a further mutation of D54N was required to achieve resistance against WR, but these mutants regained sensitivity to PYR. When a combination of PYR and WR was used, fewer resistant mutants were generated from both mutant libraries using the QM gene templates. The effectiveness of the drug combination in reducing the appearance of resistance mutations is likely due to conflicting requirements for mutations conferring resistance to the two drugs. Thus, a combination of inhibitors from these two drug classes should be effective in impeding the emergence of P. falciparum resistance to antifolates.

Genetic variation and molecular phylogeny of 22 taxa representing 14 extant species and 3 unidentified taxa of Boesenbergia in Thailand and four outgroup species (Cornukaempferia aurantiflora, Hedychium biflorum, Kaempferia parviflora, and Scaphochlamys rubescens) were examined by sequencing of 3 chloroplast (cp) DNA regions (matK, psbA-trnH and petA-psbJ). Low interspecific genetic divergence (0.25-1.74%) were observed in these investigated taxa. The 50% majority-rule consensus tree constructed from combined chloroplast DNA sequences allocated Boesenbergia in this study into 3 different groups. Using psbA-1F/psbA-3R primers, an insertion of 491 bp was observed in B. petiolata. Restriction analysis of the amplicon (380-410 bp) from the remaining species with Rsa I further differentiated Boesenbergia to 2 groupings; I (B. basispicata, B. longiflora, B. longipes, B. plicata, B.pulcherrima, B. tenuispicata, B. thorelii, B. xiphostachya, Boesenbergia sp.1 and Boesenbergia sp.3; phylogenetic clade A) that possesses a Rsa I restriction site and II (B.curtisii, B. regalis, B. rotunda and Boesenbergia sp.2; phylogenetic clade B and B. siamensis; phylogenetic clade C) that lacks a restriction site of Rsa I. Single nucleotide polymorphism (SNP) and indels found can be unambiguously applied to authenticate specie-origin of all investigated samples and revealed that Boesenbergia sp.1, Boesenbergia sp.2 and B. pulcherrima (Mahidol University, Kanchanaburi), B. cf. pulcherrima1 (Prachuap Khiri Khan) and B. cf. pulcherrima2 (Thong Pha Phum, Kanchanaburi) are B. plicata, B. rotunda and B. pulcherrima, respectively. In addition, molecular data also suggested that Boesenbergia sp.3 should be further differentiated from B. longiflora and regarded as a newly unidentified Boesenbergia species.

The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to control both the position and rate of mutation in degenerated region of the primers. The primers are integrated into newly synthesized plasmid DNA by primer extension reaction using Pfu DNA polymerase. After plasmid DNA template encoding wild-type sequence is eliminated from the reaction by DpnI digestion, the pool of mutagenized plasmids can then be used directly in screening procedures.

A simple and effective system has been developed from which a number of Plasmodium falciparum dihydrofolate reductase (pfDHFR) mutants conferring resistance to antifolates were randomly generated and characterized. The system exploited error-prone PCR to generate random mutations in the pfDHFR. Using the synthetic gene encoding for wild-type and quadruple mutant (N51I+C59R+S108N+I164L) pfDHFRs as templates, mutants resistant to pyrimethamine (Pyr), m-Cl analogue of Pyr (SO3) and WR99210 were selected by bacterial complementation system in which the endogenous DHFR activity of bacterial host cells, but not of Plasmodium, is selectively inhibited by trimethoprim (Tmp). Mutants conferring resistance to antimalarial antifolates were selected under the condition that inhibited the growth of the wild-type pfDHFR. All obtained Pyr resistant mutants possessed S108 mutation, in combination with common mutations of N51I, C59R and I164L previously found in the field. New Pyr resistant mutants with novel mutations (K27T, N121D, N144K and V213E) not found in the field were also identified. Exposure of the randomly mutated pfDHFR libraries to WR99210 or SO3 resulted in selection of novel single and multiple mutants including D54N, F58L and a combination of C50R, K181R, T219P and K227E, which exhibited 2- to over 2000-fold increase in resistance against antifolates. Kinetic analysis of these mutants suggested that apart from the active site residues that are crucial for DHFR activity, residues remote from the binding pocket also play essential roles in substrate and inhibitor binding.

As an approach to targeted repression of genes of interest, we describe the development of human estrogen receptor (ER) alpha-KRAB repressor domain chimeras that are potent ligand-dependent repressors of the transcription of estrogen response element (ERE)-containing promoters and analyze their mechanisms of action. Repression by the KRAB domain was dominant over transactivation mediated by ER AF1 and AF2. An ERE and an ER ligand (estrogen or antiestrogen) were required for repression. Studies with several promoters and cell lines demonstrated that the presence of EREs, rather than the capacity for estrogen induction, determines the potential for repression of a gene by the KRAB-ERalpha-KRAB (KERK) chimera. A single consensus ERE was sufficient for repression, but the KERK chimera was unable to suppress transcription from the imperfect ERE in the native pS2 promoter. We recently reported mutations that enhance binding of a steroid receptor DNA-binding domain to the ERE. Introducing these mutations into wild-type ER enhanced transactivation from the pS2 ERE. Insertion of these mutations into KERK created the novel repressor KERK-3M, which is a potent repressor of both ER-induced and basal transcription on a promoter containing the pS2 ERE. These modified ER-KRAB chimeras should prove useful as new tools for the functional analysis and repression of ER-regulated genes.