The parasite Plasmodium vivax is a major public health

Size: px
Start display at page:

Download "The parasite Plasmodium vivax is a major public health"

Transcription

1 Pyrimethamine and WR99210 exert opposing selection on dihydrofolate reductase from Plasmodium vivax Michele D. Hastings and Carol Hopkins Sibley* Department of Genome Sciences, University of Washington, Box , Seattle, WA Edited by Louis H. Miller, National Institutes of Health, Rockville, MD, and approved July 18, 2002 (received for review May 16, 2002) Plasmodium vivax is a major public health problem in Asia and South and Central America where it is most prevalent. Until very recently, the parasite has been effectively treated with chloroquine, but resistance to this drug has now been reported in several areas. Affordable alternative treatments for vivax malaria are urgently needed. Pyrimethamine-sulfadoxine is an inhibitor of dihydrofolate reductase (DHFR) that has been widely used to treat chloroquine-resistant Plasmodium falciparum malaria. DHFR inhibitors have not been considered for treatment of vivax malaria, because initial trials showed poor efficacy against P. vivax. P. vivax cannot be grown in culture; the reason for its resistance to DHFR inhibitors is unknown. We show that, like P. falciparum, point mutations in the dhfr gene can cause resistance to pyrimethamine in P. vivax. WR99210 is a novel inhibitor of DHFR, effective even against the most pyrimethamine-resistant P. falciparum strains. We have found that it is also an extremely effective inhibitor of the P. vivax DHFR, and mutations that confer high-level resistance to pyrimethamine render the P. vivax enzyme exquisitely sensitive to WR These data suggest that pyrimethamine and WR99210 would exert opposing selective forces on the P. vivax population. If used in combination, these two drugs could greatly slow the selection of parasites resistant to both drugs. If that is the case, this novel class of DHFR inhibitors could provide effective and affordable treatment for chloroquine- and pyrimethamine-resistant vivax and falciparum malaria for many years to come. The parasite Plasmodium vivax is a major public health problem in Asia and South and Central America where it is most prevalent (1). Until very recently, the parasite has been effectively treated with chloroquine, but resistance to this drug has now been reported in several areas (1 13). Affordable alternative treatments for vivax malaria are urgently needed. Pyrimethamine-sulfadoxine is an inhibitor of dihydrofolate reductase (DHFR) that has been widely used to treat chloroquineresistant Plasmodium falciparum malaria (14). DHFR inhibitors have not been considered for treatment of vivax malaria, because initial trials showed poor efficacy against P. vivax (15, 16). P. vivax cannot be grown in culture; the reason for its resistance to DHFR inhibitors is unknown. DHFR (E.C ) is a key enzyme in the metabolism of all cells. Inhibition of DHFR activity depletes the cellular pool of tetrahydrofolate, a cofactor that is essential for both DNA and protein synthesis, and specific inhibitors have been designed for both prokaryotic and eukaryotic pathogens (17). Pyrimethamine is a specific competitive inhibitor of DHFR from P. falciparum, (18) and with sulfadoxine, is one component of the antimalaria drug, Fansidar. In P. falciparum, extensive field and laboratory studies have shown that resistance to pyrimethamine is caused by point mutations in the dhfr gene (reviewed in refs. 15 and 19 21). However, in contrast to P. falciparum, P. vivax cannot be maintained in culture and so the mechanism of resistance to pyrimethamine has not been established for this species. The dhfr gene from P. vivax was recently cloned, and a number of alternative of the gene were identified (22, 23). Fig. 1 compares the alignment of the dhfr coding regions from P. falciparum and P. vivax. The alignment demonstrates that the polymorphisms observed at positions 58, 117, and 173 of the P. vivax sequence correspond to the changes in codons 59, 108, and 164 that are known to cause pyrimethamine resistance in the P. falciparum enzyme (24, 25). Several of these mutant P. vivax enzymes were purified and showed less sensitivity to inhibition by pyrimethamine in vitro (26, 27). In addition, a recent study showed that polymorphisms in amino acids 57, 58, and 117 are very common in Thailand where pyrimethamine-sulfadoxine has been used extensively, but less prevalent in India and Madagascar where antifolate use has been lower (28). These studies suggested that amino acid substitutions might be responsible for pyrimethamine resistance in P. vivax. Because P. vivax cannot be maintained in culture, there is no way to test reference strains in vitro to set criteria for relative drug sensitivity; drug testing can be accomplished only by short-term treatment of parasites newly isolated from patients. This requirement has severely hampered studies of drug efficacy and has made it difficult to determine the relative importance of point mutations in the P. vivax dhfr gene for antifolate resistance. We have developed a system for expression of DHFR enzymes from pathogens in strains of the budding yeast, Saccharomyces cerevisiae, that lack endogenous DHFR activity (29, 30). This approach allows rapid assessment of the relative sensitivity of heterologous DHFR enzymes to inhibitors, simply by measuring the growth of the yeast in the presence of potential inhibitors of the enzyme. We and others have shown that for a range of of P. falciparum the growth of the yeast strain in the presence of the inhibitor reflects the in vivo drug sensitivity of the DHFR enzyme that is expressed (14, 30 32). This approach has been particularly useful in situations where a novel allele of dhfr is of interest, but only the DNA of the parasite is available (31 34). We have adapted the system to assess the relative sensitivity of various P. vivax DHFR to both approved and investigational DHFR inhibitors. Materials and Methods Strains and Plasmids. The S. cerevisiae yeast strain used, TH5 (Mata ura3 52 leu2 3,112 trp1 tup1 dfr1::ura3), is null for the yeast dfr1 gene and requires supplemental dtmp, adenine, histidine, and methionine for growth (35). The expression vector is a shuttle plasmid that can be propagated in both Escherichia coli and S. cerevisiae. It has a yeast centromere, and so is maintained at approximately one copy per yeast cell, and trp1 and bla genes for selection of transformants in either E. coli or S. cerevisiae (36). We modified the plasmid for expression of the P. vivax dhfr coding sequence: it carries a 600-bp fragment from the region 5 of the yeast dfr1 coding sequence, which acts as the Pvdhfr promoter, and the terminator is a 400-bp fragment from 3 of the yeast dfr1 coding sequence (30). E. coli strain DH5 is This paper was submitted directly (Track II) to the PNAS office. Abbreviation: DHFR, dihydrofolate reductase. *To whom reprint requests should be addressed. cgi doi pnas PNAS October 1, 2002 vol. 99 no

2 Fig. 1. The alignment of amino acid sequences of the P. vivax DHFR and P. falciparum DHFR domains. Amino acids correlated with resistance to pyrimethamine in PfDHFR and the corresponding amino acid in PvDHFR are shown in bold. used for propagation and preparation of the shuttle plasmid. Strains are maintained according to standard lab protocols. Recombinant DHFR Expression. We obtained genomic P. vivax DNA from a line that has been maintained in vivo in Aotus monkeys, a gift from William Collins, Centers for Disease Control and Prevention, Atlanta. We used the published P. vivax dhfr-ts sequence (GenBank accession no. X98123) to design PCR primers for amplification of the Pvdhfr domain, plus 19 downstream nt. In addition, the 5 end of each primer (italicized) was complementary to the sequence of the shuttle plasmid at the desired insertion position to facilitate homologous recombination in the yeast (forward: 5 -CGACGAGCGGATCCATTTAT- ATTTTCTCCTTTTTATGGAGGACCTTTCAGATGTA- TT-3 ; reverse: 5 -CATTTACGATTGTATAGAGTGAATTCG- AAGCCCGTCAACTCCCCCCCCCACCTTGCTGTAAA-3 ). Yeast were transformed by using a high-efficiency lithium acetate protocol (37), then were plated onto medium lacking tryptophan to select for the plasmid and lacking dtmp to select for functional DHFR activity. Construction of Mutants. Site-directed mutagenesis (QuikChange, Stratagene) was used to generate point mutations at codons 50, 57, 58, 117, and 173 of the Pvdhfr coding sequence. The desired point mutation was designed into complementary primers, and PCR was performed to incorporate the mutation into the target DNA sequence. The wild-type Pvdhfr coding sequence on the shuttle plasmid was mutagenized and then transformed into E. coli. The plasmid from 2 4 transformants was isolated and sequenced to ensure that only the desired mutation was present. Sequencing was conducted via fluorescent dye chemistry (Mega- BACE, Amersham Pharmacia Biotech) and analyzed with SE- QUENCHER software (Gene Codes, Ann Arbor, MI). The plasmid with the desired Pvdhfr mutation was then transformed back into TH5 yeast for drug sensitivity assays. Determination of Drug Sensitivity. The pyrimethamine, chlorcycloguanil, and WR99210 were gifts from Jacobus Pharmaceuticals, Princeton, NJ. Drugs were dissolved in DMSO and spread on plates to attain the desired final concentration or dissolved in DMSO for assay of yeast growth in liquid culture as described (29). An IC 50 assay was performed for each allele to obtain a quantitative measure of drug sensitivity. The IC 50 value is defined as the concentration of drug at which the growth of the yeast is inhibited by 50% relative to the untreated control. Transformed yeast were grown overnight in a 96-well culture dish in complete medium (yeast extract peptone dextrose broth) lacking dtmp. The first row of wells served as the control and contained yeast in the presence of the drug solvent (DMSO), but no drug. The next seven rows contained yeast in the presence of increasing concentrations of drug dissolved in DMSO. Four were measured in each 96-well dish, with three columns per allele. The growth of the yeast in each well was measured by reading the optical density at 650 nm after approximately 24 h incubation time. The average reading for each allele at each drug concentration was then used to plot the percent growth relative to the yeast extract peptone dextrose DMSO (no drug) control. The IC 50 value was calculated from the slope and intercept of the line defined by the two data points that bracket 50% relative growth. Comparisons of the IC 50 values of the mutant to the IC 50 value for the wild-type allele for a drug were used to assess the relative resistance level of each allele to the drug. Results and Discussion In P. vivax, the DHFR enzyme comprises the N-terminal domain of a bifunctional protein that also carries the thymidylate syn cgi doi pnas Hastings and Sibley

3 Table 1. List of mutant constructed Table 2. Calculated IC 50 values for each mutant allele Single mutant Double mutant Triple mutant Allele IC 50 M (SD) N Relative resistance N50I N50I S117N S58R S117N I173L F57L S58R S117N S58R S58R I173L S117N S117N I173L I173L Abbreviations are the standard amino acid code. thase enzyme activity (EC ). We amplified the dhfr domain from genomic DNA and cloned the fragment by homologous recombination into a yeast shuttle vector. The vector has a centromere sequence so that the plasmid is stably maintained at about one copy per cell (31, 36). Under these conditions, the expression of the parasite DHFR is just sufficient to maintain cell growth; inhibition of the enzyme is reflected in a proportional decrease in cell growth. Using the wild-type sequence as a baseline, we engineered a set of P. vivax dhfr that reflect polymorphisms that had been observed in the field (22, 23, 28) or changes that correspond to mutations that have been associated with pyrimethamine resistance in P. falciparum (15, 19 21). The that were constructed are listed in Table 1. The response of each yeast strain to pyrimethamine was measured by its relative growth in 0 to M pyrimethamine. We used the IC 50 value (the concentration of drug required to inhibit growth by 50%) to measure the relative sensitivity of the DHFR enzymes, and these data are collected in Table 2 and Fig. 2A. The color-coding of the in Fig. 2 represents the relative sensitivity as a rainbow from least pyrimethamine-resistant (S58R; blue) to most pyrimethamineresistant ( ; red). This analysis showed that the wild-type allele encodes an enzyme that is the most sensitive to pyrimethamine with an IC 50 value of M. However, all of the mutations tested show increased resistance to pyrimethamine. Fig. 2A and Table 2 reveal that there are three discernable groups: with a modest effect, increasing the IC 50 value by less than 6-fold, that increase the IC 50 by approximately 50-fold, and those that are essentially insensitive to the drug. Most striking, a single base pair mutation, resulting in the substitution of serine to asparagine at codon 117, increased the IC 50 value more than 80-fold. Addition of a second mutation, resulting in the substitution of serine to arginine at codon 58, produced an enzyme that was more than 400-fold more resistant to pyrimethamine, and further addition of the isoleucine to leucine mutation at codon 173 made the triple mutant approximately 500-fold more resistant than the wild-type allele. All of these mutations have been found in field samples, and the 58R 117N double mutant allele is present in particularly high frequencies in Southeast Asian isolates (28). These data strongly support the idea that point mutations in the dhfr domain can cause resistance to pyrimethamine in P. vivax and can explain the very rapid selection of resistance seen in this species when pyrimethamine-sulfadoxine was tested in the 1950s (16). Mutations at codons 57, 58, 117, and 173 were found in the 30 isolates whose complete dhfr sequences have been determined. The isolates were from a variety of sites in Asia and South America, but carried the double 58R 117N mutation and one carried the triple mutant 58R 117N 173L genotype (23). In a more extensive study, Imwong and her colleagues (28) used allele-specific methods to identify that carried mutations at codons 57, 58 or 117. They found that samples from Thailand had mutations at one or more of these codons, and the Pyrimethamine Wild type 0.53 (0.11) S58R 0.98 (0.17) I173L 2.1 (1.0) N50I 2.5 (0.5) S58R I173L 3.0 (1.1) F57L 28 (2) 4 52 N50I S117N 30 (13) 5 57 S117N 46 (10) 7 87 S58R S117N 250 (30) S58R S117N I173L 260 (80) S117N I173L 370 (100) Chlorcycloguanil Wild type 0.62 (0.09) S58R 3.2 (0.8) I173L 2.0 (0.1) N50I 2.2 (0.1) S58R I173L 6.2 (1.2) F57L 160 (90) N50I S117N 30 (6) 5 49 S117N 8.5 (1.4) 3 14 S58R S117N 220 (80) S58R S117N I173L 370 (70) S117N I173L 290 (120) WR99210 Wild type 0.38 (0.07) S58R 2.6 (0.1) I173L 0.35 (0.03) N50I 0.36 (0.02) S58R I173L 2.2 (0.1) F57L 0.92 (0.65) N50I S117N 0.07 ( 0.01) S117N 0.03 ( 0.01) S58R S117N 0.47 (0.05) S58R S117N I173L 0.26 (0.04) S117N I173L 0.05 (0.02) Values were determined as shown in Fig. 2. The relative resistance was calculated in comparison to the response of the wild-type allele. majority were double or triple mutants. Pyrimethaminesulfadoxine would almost certainly have poor efficacy in any P. vivax population in which these were prevalent, and even these small samples suggest that mutant may be common in many regions, especially in Southeast Asia. Chlorproguanil is a biguanide prodrug that is metabolized to chlorcycloguanil, another specific inhibitor of the P. falciparum DHFR (38, 39). In P. falciparum, chlorcycloguanil is a more effective inhibitor of DHFR than pyrimethamine, and phase III trials of chlorcycloguanil-dapsone have recently been completed (40). To further define the effect of this set of mutations on the P. vivax DHFR enzyme, we also assayed the sensitivity of each yeast strain to 0 to M chlorcycloguanil. Table 2 and Fig. 2B compile these data. In contrast to the results in P. falciparum (40 42), chlorcycloguanil is not a more effective inhibitor of the P. vivax enzyme than is pyrimethamine; the range of IC 50 values against the set of mutant is the same for both drugs. In P. falciparum, each point mutation that increases resistance to pyrimethamine also increases resistance to chlorcycloguanil by roughly the same degree (42). We observed the same trend when the P. vivax enzymes were assayed. This is apparent from the spectrum of colors in Fig. 2B. There was one exception, however. A single mutation (F57L) produces an enzyme that is about Hastings and Sibley PNAS October 1, 2002 vol. 99 no

4 Fig. 2. Sensitivity of P. vivax DHFR to (A) pyrimethamine, (B) chlorcycloguanil, and (C) WR Drugs were dissolved in DMSO for assay of yeast growth in liquid culture as described in Materials and Methods. 250-fold more resistant to chlorcycloguanil than the wild-type allele, but only about 50-fold more resistant to pyrimethamine. This difference may provide some useful leads for development of drugs specifically targeted to the P. vivax enzyme. WR99210 is a triazine inhibitor of the P. falciparum DHFR. Like chlorcycloguanil, it is cyclized in vivo from its prodrug, PS-15 (43). A series of compounds based on PS-15 are in preclinical development because they are extremely active against the most pyrimethamine-resistant of P. falciparum dhfr, both in vivo and in vitro (42 44). Fig. 2C shows the effectiveness of 0 to M WR99210 against the set of mutant P. vivax, and these IC 50 values are also listed in Table 2. There are two important observations. First, WR99210 is a more potent inhibitor than pyrimethamine or chlorcycloguanil of all of the P. vivax dhfr except S58R, which is equally sensitive to all three drugs. This S58R allele showed the highest level of resistance to WR99210, but it is still only 7-fold more resistant to WR99210 than the wild-type allele. Second, the that are most resistant to pyrimethamine are among the most sensitive to WR The color spectrum in Fig. 2C emphasizes the striking reversal of sensitivity to the two drugs when the IC 50 data are compared. Three (117N 173L, 117N, and 50I 117N) are 5- to 10-fold more sensitive to WR99210 than the wild-type allele. The S117N substitution, which seems to be a critical mutation for the development of high-level pyrimethamine resistance, also appears to render the enzyme particularly sensitive to the action of WR Because P. vivax cannot be grown in culture, there were no in vitro data on the sensitivity of the parasite to pyrimethamine on which to base our study. The yeast system offers one way to compare the relative sensitivity of particular DHFR enzymes to standard drugs and to chlorcycloguanil and WR99210 that are still under development. At the outset, we chose to construct mutants based on the initial DNA sequences published by de Pecoulas and his colleagues (23) and on the additional assumption that the P. falciparum and P. vivax enzymes would be similar in their sensitivity to this set of drugs. In general, that assumption is supported by the data, even for mutations that have not been observed yet in P. vivax isolates. For example, a mutation from asparagine to isoleucine at amino acid 51 is common in P. falciparum isolates from widely divergent geographic locations (45), but has not been reported in the few published studies of P. vivax. However, we constructed the equivalent mutation, N50I in P. vivax and observed that it does confer modest levels of resistance to pyrimethamine and chlorcycloguanil. This finding suggests that studies of field isolates might include methods for detecting this change. Although the sensitivity of the P. falciparum and P. vivax enzymes to pyrimethamine shows considerable similarity, the results of studies with chlorcycloguanil and WR99210 yielded some interesting surprises. First, in laboratory studies of P. falciparum, chlorcycloguanil is more effective against the wild type and canonical set of pyrimethamine-resistant than pyrimethamine (31, 42). Furthermore, in combination with dapsone, chlorproguanil has been shown to be clinically more effective than sulfadoxine-pyrimethamine (38, 40, 41, 46). Thus, it was a surprise that the effectiveness of chlorcycloguanil against the wild type and mutant enzymes of P. vivax was indistinguishable from pyrimethamine. Second, the prodrug of WR99210, PS-15, is highly effective against the most pyrimethamine-resistant of P. falciparum DHFR (42). A crystal structure of the P. falciparum DHFR has not been published, but homology models have been used to further understand the structural differences that underlie the effectiveness of WR99210 (47 49). The 117 position is equivalent to amino acid 108 in P. falciparum, and this position is clearly critical for drug resistance and for the catalytic function of DHFR from P. falciparum and a wide variety of species (21, 50). In P. falciparum, substitution of any amino acid but serine, asparagine, or threonine at amino acid 108 produces an enzyme with little or no activity (51). The fact that WR99210 is about 10-fold more effective against P. vivax DHFR enzymes that carry the 117N mutation is in striking contrast to P. falciparum. When the structures of the P. vivax and P. falciparum DHFR enzymes are solved to sufficient resolution, the data on drug efficacy in mutant DHFR enzymes of both species will be a rich source of information for further development of highly effective inhibitors. This remarkable efficacy of WR99210 raises two exciting possibilities. The fact that WR99210 is most effective against the that show the highest levels of resistance to pyrimethamine strongly suggests that WR99210 would be effective even in areas where these are common. For example, the S117N mutation was found in samples from Thailand (28), and it seems likely that WR99210 would be particularly effective against the parasites from that region. In fact, this observation suggests that pyrimethamine and WR99210 could be used together, forcing the selection of resistance by some mechanism other than the simple point mutations in the dhfr gene. Combining them with yet another drug with a different mechanism of action altogether could greatly retard the selection of resistance. Second, mixed infections of P. vivax and P. falciparum are frequently observed, especially in Southeast Asia (52 54). When chloroquine was still effective against both species, patients with mixed infections were treated effectively with chloroquine alone, because the drug could be expected to clear both infections. The rise of chloroquine-resistant P. falciparum rendered this strategy cgi doi pnas Hastings and Sibley

5 ineffective and forced the use of more expensive alternatives like mefloquine. The PS-15 series is already in preclinical development for P. falciparum treatment (43, 44), and a PS-15 analogue is likely to be combined with dapsone to make an affordable antimalarial drug. Our results suggest that when it is deployed, mixed P. vivax P. falciparum infections would again be sensitive to a single, affordable drug treatment. The life of this formulation could be further prolonged by combination with yet a third drug. One might choose one with an independent mode of action, like amodiaquine or artesunate, or even pyrimethamine to exploit the opposing selection that is exerted by pyrimethamine and WR We thank Dr. Jane Carlton and Dr. William Collins for their gifts of the P. vivax DNA and Jacobus Pharmaceutical for its gifts of pyrimethamine, chlorcycloguanil, and WR This work was supported by Public Health Service Grant AI (to C.H.S.). M.D.H. is supported by a National Science Foundation training fellowship. 1. Mendis, K., Sina, B. J., Marchesini, P. & Carter, R. (2001) Am. J. Trop. Med. Hyg. 64, Barat, L. M. & Bloland, P. B. (1997) Infect. Dis. Clin. North Am. 11, Singh, R. K. (2000) Trans. R. Soc. Trop. Med. Hyg. 94, Taylor, W. R., Widjaja, H., Richie, T. L., Basri, H., Ohrt, C., Tjitra, E., Taufik, E., Jones, T. R., Kain, K. C. & Hoffman, S. L. (2001) Am. J. Trop. Med. Hyg. 64, Nomura, T., Carlton, J. M., Baird, J. K., del Portillo, H. A., Fryauff, D. J., Rathore, D., Fidock, D. A., Su, X., Collins, W. E., McCutchan, T. F., et al. (2001) J. Infect. Dis. 183, Soto, J., Toledo, J., Gutierrez, P., Luzz, M., Llinas, N., Cedeno, N., Dunne, M. & Berman, J. (2001) Am. J. Trop. Med. Hyg. 65, Collins, W. E., Sullivan, J. S., Fryauff, D. J., Kendall, J., Jennings, V., Galland, G. G. & Morris, C. L. (2000) Am. J. Trop. Med. Hyg. 62, Kain, K. C., Shanks, G. D. & Keystone, J. S. (2001) Clin. Infect. Dis. 33, Baird, J. K., Basri, H., Purnomo, Bangs, M. J., Subianto, B., Patchen, L. C. & Hoffman, S. L. (1991) Am. J. Trop. Med. Hyg. 44, Baird, J. K., Sustriayu Nalim, M. F., Basri, H., Masbar, S., Leksana, B., Tjitra, E., Dewi, R. M., Khairani, M. & Wignall, F. S. (1996) Trans. R. Soc. Trop. Med. Hyg. 90, Garg, M., Gopinathan, N., Bodhe, P. & Kshirsagar, N. A. (1995) Trans. R. Soc. Trop. Med. Hyg. 89, Marlar, T., Myat Phone, K., Aye Yu, S., Khaing Khaing, G., Ma, S. & Myint, O. (1995) Trans. R. Soc. Trop. Med. Hyg. 89, Murphy, G. S., Basri, H., Purnomo, Andersen, E. M., Bangs, M. J., Mount, D. L., Gorden, J., Lal, A. A., Purwokusumo, A. R., Harjosuwarno, S., et al. (1993) Lancet 341, Sibley, C. H., Hyde, J. E., Sims, P. F. G., Plowe, C. V., Kublin, J. G., Mberu, E. K., Cowman, A. F., Winstanley, P. A., Watkins, W. M. & Nzila, A. M. (2001) Trends Parasitol. 17, Peters, W. (1998) Adv. Parasitol. 41, Young, M. D. & Burgess, R. W. (1959) Bull. W.H.O. 20, Hitchings, G. S. & Burchall, J. J. (1965) Adv. Enzymol. 27, Peters, W. (1987) Chemotherapy and Drug Resistance in Malaria (Academic, London). 19. Wang, P., Lee, C. S., Bayoumi, R., Djimde, A., Doumbo, O., Swedberg, G., Dao, L. D., Mshinda, H., Tanner, M., Watkins, W. M., et al. (1997) Mol. Biochem. Parasitol. 89, Plowe, C. V., Kublin, J. G. & Doumbo, O. K. (1998) Drug Resistance Updates 1, Hyde, J. E. (1990) Pharmacol. Ther. 48, de Pecoulas, P. E., Basco, L. K., Tahar, R., Ouatas, T. & Mazabraud, A. (1998) Gene 211, de Pecoulas, P. E., Tahar, R., Ouatas, T., Mazabraud, A. & Basco, L. K. (1998) Mol. Biochem. Parasitol. 92, Brobey, R. K. B., Sano, G., Itoh, F., Aso, K., Kimura, M., Mitamura, T. & Horii, T. (1996) Mol. Biochem. Parasitol. 81, Sirawaraporn, W., Sathitkul, T., Sirawaraporn, R., Yuthavong, Y. & Santi, D. V. (1997) Proc. Natl. Acad. Sci. USA 94, Tahar, R., de Pecoulas, P. E., Basco, L. K., Chiadmi, M. & Mazabraud, A. (2001) Mol. Biochem. Parasitol. 113, Leartsakulpanich, U., Imwong, M., Pukrittayakamee, S., White, N. J., Snounou, G., Sirawaraporn, W. & Yuthavong, Y. (2002) Mol. Biochem. Parasitol. 119, Imwong, M., Pukrittakayamee, S., Looareesuwan, S., Pasvol, G., Poirreiz, J., White, N. J. & Snounou, G. (2001) Antimicrob. Agents Chemother. 45, Sibley, C. H., Brophy, V. H., Cheesman, S., Hamilton, K. L., Hankins, E. G., Wooden, J. M. & Kilbey, B. (1997) Methods 13, Wooden, J. M., Hartwell, L. H., Vasquez, B. & Sibley, C. H. (1997) Mol. Biochem. Parasitol. 85, Cortese, J. F. & Plowe, C. V. (1998) Mol. Biochem. Parasitol. 94, Iyer, J. K., Milhous, W. K., Cortese, J. F., Kublin, J. G. & Plowe, C. V. (2001) Lancet 358, Brophy, V. H., Vasquez, J., Nelson, R. G., Forney, J. R., Rosowsky, A. & Sibley, C. H. (2000) Antimicrob. Agents Chemother. 44, Lau, H., Ferlan, J. T., Brophy, V. H., Rosowsky, A. & Sibley, C. H. (2001) Antimicrob. Agents Chemother. 45, Huang, T., Barclay, B. J., Kalman, T. I., VonBorstel, R. C. & Hastings, P. J. (1992) Gene 121, Sikorski, R. S. & Hieter, P. (1989) Genetics 122, Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) J. Bacteriol. 153, Amukoye, E., Winstanley, P. A., Watkins, W. M., Snow, R. W., Hatcher, J., Mosobo, M., Ngumbao, E., Lowe, B., Ton, M., Minyiri, G. & Marsh, K. (1997) Antimicrob. Agents Chemother. 41, Winstanley, P., Watkins, W., Muhia, D., Szwandt, S., Amukoye, E. & Marsh, K. (1997) Trans. R. Soc. Trop. Med. Hyg. 91, Mutabingwa, T. K., Maxwell, C. A., Sia, I. G., Msuya, F. H., Mkongewa, S., Vannithone, S., Curtis, J. & Curtis, C. F. (2001) Trans. R. Soc. Trop. Med. Hyg. 95, Mutabingwa, T., Nzila, A., Mberu, E., Nduati, E., Winstanley, P., Hills, E. & Watkins, W. (2001) Lancet 358, Hankins, E. G., Warhurst, D. C. & Sibley, C. H. (2001) Mol. Biochem. Parasitol. 117, Canfield, C. J., Milhous, W. K., Ager, A. L., Rossan, R. N., Sweeney, T. R., Lewis, N. J. & Jacobus, D. P. (1993) Am. J. Trop. Med. Hyg. 49, Jensen, N. P., Ager, A. L., Bliss, R. A., Canfield, C. J., Kotecka, B. M., Rieckmann, K. H., Terpinski, J. & Jacobus, D. P. (2001) J. Med. Chem. 44, Plowe, C. V., Cortese, J. F., Djimde, A., Nwanyanwu, O. C., Watkins, W. M., Winstanley, P. A., EstradaFranco, J. G., Mollinedo, R. E., Avila, J. C., Cespedes, J. L., et al. (1997) J. Infect. Dis. 176, Nzila-Mounda, A., Mberu, E. K., Sibley, C. H., Plowe, C. V., Winstanley, P. A. & Watkins, W. M. (1998) Antimicrob. Agents Chemother. 42, Rastelli, G., Sirawaraporn, W., Sompornpisut, P., Vilaivan, T., Kamchonwongpaisan, S., Quarrell, R., Lowe, G., Thebtaranonth, Y. & Yuthavong, Y. (2000) Bioorg. Med. Chem. 8, Warhurst, D. C. (1998) Drug Discovery Today 3, Warhurst, D. C. (2002) Sci. Prog. 85, Hyde, J. E. (2002) Microbes Infect. 4, Sirawaraporn, W., Yongkiettrakul, S., Sirawaraporn, R., Yuthavong, Y. & Santi, D. V. (1997) Exp. Parasitol. 87, Singh, B., Cox-Singh, J., Miller, A. O., Abdullah, M. S., Snounou, G. & Rahman, H. A. (1996) Trans. R. Soc. Trop. Med. Hyg. 90, Mehlotra, R. K., Lorry, K., Kastens, W., Miller, S. M., Alpers, M. P., Bockarie, M., Kazura, J. W. & Zimmerman, P. A. (2000) Am. J. Trop. Med. Hyg. 62, Mayxay, M., Pukritrayakamee, S., Chotivanich, K., Imwong, M., Looareesuwan, S. & White, N. J. (2001) Am. J. Trop. Med. Hyg. 65, Hastings and Sibley PNAS October 1, 2002 vol. 99 no

Chapter 9. Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA

Chapter 9. Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA Chapter 9 Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA Q&A Interferons are species specific, so that interferons to be used in humans must be produced in human cells. Can you think

More information

Genetics Lecture Notes 7.03 2005. Lectures 1 2

Genetics Lecture Notes 7.03 2005. Lectures 1 2 Genetics Lecture Notes 7.03 2005 Lectures 1 2 Lecture 1 We will begin this course with the question: What is a gene? This question will take us four lectures to answer because there are actually several

More information

Molecular and Cell Biology Laboratory (BIOL-UA 223) Instructor: Ignatius Tan Phone: 212-998-8295 Office: 764 Brown Email: ignatius.tan@nyu.

Molecular and Cell Biology Laboratory (BIOL-UA 223) Instructor: Ignatius Tan Phone: 212-998-8295 Office: 764 Brown Email: ignatius.tan@nyu. Molecular and Cell Biology Laboratory (BIOL-UA 223) Instructor: Ignatius Tan Phone: 212-998-8295 Office: 764 Brown Email: ignatius.tan@nyu.edu Course Hours: Section 1: Mon: 12:30-3:15 Section 2: Wed: 12:30-3:15

More information

PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI,

PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Supplemental Text/Tables PCR Amplification and Sequencing PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Foster City, CA). Each PCR reaction contained 20 ng genomic

More information

Antimalarial Drugs Clear Resistant Parasites from Partially Immune Hosts

Antimalarial Drugs Clear Resistant Parasites from Partially Immune Hosts ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2001, p. 2897 2901 Vol. 45, No. 10 0066-4804/01/$04.00 0 DOI: 10.1128/AAC.45.10.2897 2901.2001 Copyright 2001, American Society for Microbiology. All Rights

More information

Biotechnology and Recombinant DNA

Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA Recombinant DNA procedures - an overview Biotechnology: The use of microorganisms, cells, or cell components to make a product. Foods, antibiotics, vitamins, enzymes Recombinant

More information

Selection of Drug Combinations for Resistant Malaria

Selection of Drug Combinations for Resistant Malaria Selection of Drug Combinations for Resistant Malaria Dennis E. Kyle, PhD Professor University of South Florida Current Issues with ACTs Artemisinin derivative combined with an existing drug Existing drug

More information

Recombinant DNA Unit Exam

Recombinant DNA Unit Exam Recombinant DNA Unit Exam Question 1 Restriction enzymes are extensively used in molecular biology. Below are the recognition sites of two of these enzymes, BamHI and BclI. a) BamHI, cleaves after the

More information

The Awesome Power of Yeast Genetics: Spontaneous and Induced Mutagenesis and Complementation Analysis using Saccharomyces cerevisiae.

The Awesome Power of Yeast Genetics: Spontaneous and Induced Mutagenesis and Complementation Analysis using Saccharomyces cerevisiae. The Awesome Power of Yeast Genetics: Spontaneous and Induced Mutagenesis and Complementation Analysis using Saccharomyces cerevisiae. Mutations occur as a consequence of normal cellular physiology and

More information

The Need for a PARP in vivo Pharmacodynamic Assay

The Need for a PARP in vivo Pharmacodynamic Assay The Need for a PARP in vivo Pharmacodynamic Assay Jay George, Ph.D., Chief Scientific Officer, Trevigen, Inc., Gaithersburg, MD For further infomation, please contact: William Booth, Ph.D. Tel: +44 (0)1235

More information

Recombinant DNA and Biotechnology

Recombinant DNA and Biotechnology Recombinant DNA and Biotechnology Chapter 18 Lecture Objectives What Is Recombinant DNA? How Are New Genes Inserted into Cells? What Sources of DNA Are Used in Cloning? What Other Tools Are Used to Study

More information

EU Reference Laboratory for E. coli Department of Veterinary Public Health and Food Safety Unit of Foodborne Zoonoses Istituto Superiore di Sanità

EU Reference Laboratory for E. coli Department of Veterinary Public Health and Food Safety Unit of Foodborne Zoonoses Istituto Superiore di Sanità Identification and characterization of Verocytotoxin-producing Escherichia coli (VTEC) by Real Time PCR amplification of the main virulence genes and the genes associated with the serogroups mainly associated

More information

Introduction to Bioinformatics 3. DNA editing and contig assembly

Introduction to Bioinformatics 3. DNA editing and contig assembly Introduction to Bioinformatics 3. DNA editing and contig assembly Benjamin F. Matthews United States Department of Agriculture Soybean Genomics and Improvement Laboratory Beltsville, MD 20708 matthewb@ba.ars.usda.gov

More information

Integrated Protein Services

Integrated Protein Services Integrated Protein Services Custom protein expression & purification Version DC04-0012 Expression strategy The first step in the recombinant protein generation process is to design an appropriate expression

More information

Lecture 13: DNA Technology. DNA Sequencing. DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology

Lecture 13: DNA Technology. DNA Sequencing. DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology Lecture 13: DNA Technology DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology DNA Sequencing determine order of nucleotides in a strand of DNA > bases = A,

More information

Bacillus Subtilis Expression Vectors. Product Information and Instructions November 2005

Bacillus Subtilis Expression Vectors. Product Information and Instructions November 2005 Bacillus Subtilis Expression Vectors Product Information and Instructions November 2005 1 Content 1. Introduction... 3 2. The pht Vectors...4 2.1. Vector Map pht01...4 2.2. Vector Map pht43...5 2.3. Location

More information

Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College

Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Primary Source for figures and content: Eastern Campus Tortora, G.J. Microbiology

More information

June 09, 2009 Random Mutagenesis

June 09, 2009 Random Mutagenesis Why Mutagenesis? Analysis of protein function June 09, 2009 Random Mutagenesis Analysis of protein structure Protein engineering Analysis of structure-function relationship Analysis of the catalytic center

More information

SUPPLEMENTARY FIGURES

SUPPLEMENTARY FIGURES SUPPLEMENTARY FIGURES Fig. S1: Effect of ISO- and TAC-treatments on the biosynthesis of FAS-II elongation products in M. tb H37Ra. LC/MS chromatograms showing a decrease in products with elemental compositions

More information

Common Course Topics Biology 1414: Introduction to Biotechnology I

Common Course Topics Biology 1414: Introduction to Biotechnology I Common Course Topics Biology 1414: Introduction to Biotechnology I Assumptions Students may be enrolled in this course for several reasons; they are enrolled in the Biotechnology Program, they need a science

More information

Transformation. Making Change Happen

Transformation. Making Change Happen Transformation Making Change Happen Genetic Engineering Definition: The alteration of an organism s genetic, or hereditary, material to eliminate undesirable characteristics or to produce desirable new

More information

GENE CLONING AND RECOMBINANT DNA TECHNOLOGY

GENE CLONING AND RECOMBINANT DNA TECHNOLOGY GENE CLONING AND RECOMBINANT DNA TECHNOLOGY What is recombinant DNA? DNA from 2 different sources (often from 2 different species) are combined together in vitro. Recombinant DNA forms the basis of cloning.

More information

restriction enzymes 350 Home R. Ward: Spring 2001

restriction enzymes 350 Home R. Ward: Spring 2001 restriction enzymes 350 Home Restriction Enzymes (endonucleases): molecular scissors that cut DNA Properties of widely used Type II restriction enzymes: recognize a single sequence of bases in dsdna, usually

More information

BCOR101 Midterm II Wednesday, October 26, 2005

BCOR101 Midterm II Wednesday, October 26, 2005 BCOR101 Midterm II Wednesday, October 26, 2005 Name Key Please show all of your work. 1. A donor strain is trp+, pro+, met+ and a recipient strain is trp-, pro-, met-. The donor strain is infected with

More information

Gene mutation and molecular medicine Chapter 15

Gene mutation and molecular medicine Chapter 15 Gene mutation and molecular medicine Chapter 15 Lecture Objectives What Are Mutations? How Are DNA Molecules and Mutations Analyzed? How Do Defective Proteins Lead to Diseases? What DNA Changes Lead to

More information

Description: Molecular Biology Services and DNA Sequencing

Description: Molecular Biology Services and DNA Sequencing Description: Molecular Biology s and DNA Sequencing DNA Sequencing s Single Pass Sequencing Sequence data only, for plasmids or PCR products Plasmid DNA or PCR products Plasmid DNA: 20 100 ng/μl PCR Product:

More information

Introduction To Real Time Quantitative PCR (qpcr)

Introduction To Real Time Quantitative PCR (qpcr) Introduction To Real Time Quantitative PCR (qpcr) SABiosciences, A QIAGEN Company www.sabiosciences.com The Seminar Topics The advantages of qpcr versus conventional PCR Work flow & applications Factors

More information

LECTURE 6 Gene Mutation (Chapter 16.1-16.2)

LECTURE 6 Gene Mutation (Chapter 16.1-16.2) LECTURE 6 Gene Mutation (Chapter 16.1-16.2) 1 Mutation: A permanent change in the genetic material that can be passed from parent to offspring. Mutant (genotype): An organism whose DNA differs from the

More information

Integrated Protein Services

Integrated Protein Services Integrated Protein Services Custom protein expression & purification Last date of revision June 2015 Version DC04-0013 www.iba-lifesciences.com Expression strategy The first step in the recombinant protein

More information

Gene Expression Assays

Gene Expression Assays APPLICATION NOTE TaqMan Gene Expression Assays A mpl i fic ationef ficienc yof TaqMan Gene Expression Assays Assays tested extensively for qpcr efficiency Key factors that affect efficiency Efficiency

More information

From DNA to Protein. Proteins. Chapter 13. Prokaryotes and Eukaryotes. The Path From Genes to Proteins. All proteins consist of polypeptide chains

From DNA to Protein. Proteins. Chapter 13. Prokaryotes and Eukaryotes. The Path From Genes to Proteins. All proteins consist of polypeptide chains Proteins From DNA to Protein Chapter 13 All proteins consist of polypeptide chains A linear sequence of amino acids Each chain corresponds to the nucleotide base sequence of a gene The Path From Genes

More information

Structure and Function of DNA

Structure and Function of DNA Structure and Function of DNA DNA and RNA Structure DNA and RNA are nucleic acids. They consist of chemical units called nucleotides. The nucleotides are joined by a sugar-phosphate backbone. The four

More information

CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA

CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA INTRODUCTION DNA : DNA is deoxyribose nucleic acid. It is made up of a base consisting of sugar, phosphate and one nitrogen base.the

More information

Innovations in Molecular Epidemiology

Innovations in Molecular Epidemiology Innovations in Molecular Epidemiology Molecular Epidemiology Measure current rates of active transmission Determine whether recurrent tuberculosis is attributable to exogenous reinfection Determine whether

More information

Genetics 301 Sample Final Examination Spring 2003

Genetics 301 Sample Final Examination Spring 2003 Genetics 301 Sample Final Examination Spring 2003 50 Multiple Choice Questions-(Choose the best answer) 1. A cross between two true breeding lines one with dark blue flowers and one with bright white flowers

More information

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q5B

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q5B INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE QUALITY OF BIOTECHNOLOGICAL PRODUCTS: ANALYSIS

More information

Troubleshooting the Single-step PCR Site-directed Mutagenesis Procedure Intended to Create a Non-functional rop Gene in the pbr322 Plasmid

Troubleshooting the Single-step PCR Site-directed Mutagenesis Procedure Intended to Create a Non-functional rop Gene in the pbr322 Plasmid Troubleshooting the Single-step PCR Site-directed Mutagenesis Procedure Intended to Create a Non-functional rop Gene in the pbr322 Plasmid Lina Jew Department of Microbiology & Immunology, University of

More information

Essentials of Real Time PCR. About Sequence Detection Chemistries

Essentials of Real Time PCR. About Sequence Detection Chemistries Essentials of Real Time PCR About Real-Time PCR Assays Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected

More information

Data Analysis for Ion Torrent Sequencing

Data Analysis for Ion Torrent Sequencing IFU022 v140202 Research Use Only Instructions For Use Part III Data Analysis for Ion Torrent Sequencing MANUFACTURER: Multiplicom N.V. Galileilaan 18 2845 Niel Belgium Revision date: August 21, 2014 Page

More information

Concluding lesson. Student manual. What kind of protein are you? (Basic)

Concluding lesson. Student manual. What kind of protein are you? (Basic) Concluding lesson Student manual What kind of protein are you? (Basic) Part 1 The hereditary material of an organism is stored in a coded way on the DNA. This code consists of four different nucleotides:

More information

Antibiotic susceptibility

Antibiotic susceptibility Antibiotic susceptibility Antibiotic: natural chemicals produced by bacteria, fungi, actinomycetes, plants or animals, and either inhibits or kills other microbes and/or cells Chemotherapeutic agent: A

More information

HCS604.03 Exercise 1 Dr. Jones Spring 2005. Recombinant DNA (Molecular Cloning) exercise:

HCS604.03 Exercise 1 Dr. Jones Spring 2005. Recombinant DNA (Molecular Cloning) exercise: HCS604.03 Exercise 1 Dr. Jones Spring 2005 Recombinant DNA (Molecular Cloning) exercise: The purpose of this exercise is to learn techniques used to create recombinant DNA or clone genes. You will clone

More information

RNA and Protein Synthesis

RNA and Protein Synthesis Name lass Date RN and Protein Synthesis Information and Heredity Q: How does information fl ow from DN to RN to direct the synthesis of proteins? 13.1 What is RN? WHT I KNOW SMPLE NSWER: RN is a nucleic

More information

International Journal for Parasitology: Drugs and Drug Resistance

International Journal for Parasitology: Drugs and Drug Resistance International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 126 133 Contents lists available at SciVerse ScienceDirect International Journal for Parasitology: Drugs and Drug Resistance journal

More information

Genetic Technology. Name: Class: Date: Multiple Choice Identify the choice that best completes the statement or answers the question.

Genetic Technology. Name: Class: Date: Multiple Choice Identify the choice that best completes the statement or answers the question. Name: Class: Date: Genetic Technology Multiple Choice Identify the choice that best completes the statement or answers the question. 1. An application of using DNA technology to help environmental scientists

More information

Biotechnology. Biotechnology s Laboratories. Lab Name Location Person in Charge Programs Served Courses Served. Biotechnology Department

Biotechnology. Biotechnology s Laboratories. Lab Name Location Person in Charge Programs Served Courses Served. Biotechnology Department Biotechnology s oratories Biotechnology Name Location Person in Charge Programs Served Courses Served General Biology W12-039 Zahra Yassin General Microbiology M12-132 Aisha Echtibi General (Basic Course)

More information

AMES TEST: Bacterial Reverse Mutation Assay

AMES TEST: Bacterial Reverse Mutation Assay AMES TEST: Bacterial Reverse Mutation Assay 1. Introduction The bacteria reversed mutation assay (Ames Test) is used to evaluate the mutagenic properties of test articles. The test uses amino acid-dependent

More information

A and B are not absolutely linked. They could be far enough apart on the chromosome that they assort independently.

A and B are not absolutely linked. They could be far enough apart on the chromosome that they assort independently. Name Section 7.014 Problem Set 5 Please print out this problem set and record your answers on the printed copy. Answers to this problem set are to be turned in to the box outside 68-120 by 5:00pm on Friday

More information

pcas-guide System Validation in Genome Editing

pcas-guide System Validation in Genome Editing pcas-guide System Validation in Genome Editing Tagging HSP60 with HA tag genome editing The latest tool in genome editing CRISPR/Cas9 allows for specific genome disruption and replacement in a flexible

More information

30. Genetics and recombination in bacteria Lecture Outline 11/16/05. The Bacterial Genome and Its Replication The bacterial chromosome

30. Genetics and recombination in bacteria Lecture Outline 11/16/05. The Bacterial Genome and Its Replication The bacterial chromosome 30. Genetics and recombination in bacteria Lecture Outline 11/16/05 Replication in bacteria Types of recombination in bacteria Transduction by phage Conjugation ( mating ) F+ plasmids Hfr s Transformation

More information

Artemether-lumefantrine (four-dose regimen) for treating uncomplicated falciparum malaria (Review)

Artemether-lumefantrine (four-dose regimen) for treating uncomplicated falciparum malaria (Review) Artemether-lumefantrine (four-dose regimen) for treating uncomplicated falciparum malaria (Review) Omari AAA, Gamble C, Garner P This is a reprint of a Cochrane review, prepared and maintained by The Cochrane

More information

CCR Biology - Chapter 9 Practice Test - Summer 2012

CCR Biology - Chapter 9 Practice Test - Summer 2012 Name: Class: Date: CCR Biology - Chapter 9 Practice Test - Summer 2012 Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Genetic engineering is possible

More information

RETRIEVING SEQUENCE INFORMATION. Nucleotide sequence databases. Database search. Sequence alignment and comparison

RETRIEVING SEQUENCE INFORMATION. Nucleotide sequence databases. Database search. Sequence alignment and comparison RETRIEVING SEQUENCE INFORMATION Nucleotide sequence databases Database search Sequence alignment and comparison Biological sequence databases Originally just a storage place for sequences. Currently the

More information

Chapter 8. Summary and Perspectives

Chapter 8. Summary and Perspectives Chapter 8 Summary and Perspectives 131 Chapter 8 Summary Overexpression of the multidrug resistance protein MRP1 confer multidrug resistance (MDR) to cancer cells. The contents of this thesis describe

More information

Bacterial Transformation and Plasmid Purification. Chapter 5: Background

Bacterial Transformation and Plasmid Purification. Chapter 5: Background Bacterial Transformation and Plasmid Purification Chapter 5: Background History of Transformation and Plasmids Bacterial methods of DNA transfer Transformation: when bacteria take up DNA from their environment

More information

Open Access RESEARCH. Artimovich et al. Malar J (2015) 14:387 DOI 10.1186/s12936-015-0860-7. *Correspondence: stakala@medicine.umaryland.

Open Access RESEARCH. Artimovich et al. Malar J (2015) 14:387 DOI 10.1186/s12936-015-0860-7. *Correspondence: stakala@medicine.umaryland. DOI 10.1186/s12936-015-0860-7 RESEARCH Open Access The effect of local variation in malaria transmission on the prevalence of sulfadoxine pyrimethamine resistant haplotypes and selective sweep characteristics

More information

HENIPAVIRUS ANTIBODY ESCAPE SEQUENCING REPORT

HENIPAVIRUS ANTIBODY ESCAPE SEQUENCING REPORT HENIPAVIRUS ANTIBODY ESCAPE SEQUENCING REPORT Kimberly Bishop Lilly 1,2, Truong Luu 1,2, Regina Cer 1,2, and LT Vishwesh Mokashi 1 1 Naval Medical Research Center, NMRC Frederick, 8400 Research Plaza,

More information

Malaria treatment policies and drug efficacy in Haiti from 1955-2012

Malaria treatment policies and drug efficacy in Haiti from 1955-2012 von Fricken et al. Journal of Pharmaceutical Policy and Practice 2013, 6:10 REVIEW Open Access Malaria treatment policies and drug efficacy in Haiti from 1955-2012 Michael E von Fricken 1,2*, Thomas A

More information

Chapter 20: Biotechnology

Chapter 20: Biotechnology Name Period The AP Biology exam has reached into this chapter for essay questions on a regular basis over the past 15 years. Student responses show that biotechnology is a difficult topic. This chapter

More information

Real-Time PCR Vs. Traditional PCR

Real-Time PCR Vs. Traditional PCR Real-Time PCR Vs. Traditional PCR Description This tutorial will discuss the evolution of traditional PCR methods towards the use of Real-Time chemistry and instrumentation for accurate quantitation. Objectives

More information

Presentation Plan. Abbreviations. Before 2005. A Lao technician said in 2000: 9/02/10

Presentation Plan. Abbreviations. Before 2005. A Lao technician said in 2000: 9/02/10 Clinical and Laboratory Assessment of Antimalarial Drug Efficacy in the Lao P.D.R 1. Introduction Presentation Plan 2. Studies conducted before 2 (in-vivo) 3. Studies after 2: - In-vitro study - Molecular

More information

Green Fluorescent Protein (GFP): Genetic Transformation, Synthesis and Purification of the Recombinant Protein

Green Fluorescent Protein (GFP): Genetic Transformation, Synthesis and Purification of the Recombinant Protein Green Fluorescent Protein (GFP): Genetic Transformation, Synthesis and Purification of the Recombinant Protein INTRODUCTION Green Fluorescent Protein (GFP) is a novel protein produced by the bioluminescent

More information

427 J App Pharm Vol. 6; Issue 4: 427-431; October, 2014 Humera et al., 2014

427 J App Pharm Vol. 6; Issue 4: 427-431; October, 2014 Humera et al., 2014 427 J App Pharm Vol. 6; Issue 4: 427-431; October, 2014 Humera et al., 2014 Original Research Article PHARAMCOECONOMIC OF ANTIMALARIAL DRUGS AVAILABLE IN KARACHI, PAKISTAN ABSTRACT: Humera Khatoon*, Hina

More information

Forensic DNA Testing Terminology

Forensic DNA Testing Terminology Forensic DNA Testing Terminology ABI 310 Genetic Analyzer a capillary electrophoresis instrument used by forensic DNA laboratories to separate short tandem repeat (STR) loci on the basis of their size.

More information

Technical Note. Roche Applied Science. No. LC 18/2004. Assay Formats for Use in Real-Time PCR

Technical Note. Roche Applied Science. No. LC 18/2004. Assay Formats for Use in Real-Time PCR Roche Applied Science Technical Note No. LC 18/2004 Purpose of this Note Assay Formats for Use in Real-Time PCR The LightCycler Instrument uses several detection channels to monitor the amplification of

More information

Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual

Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual I. Purpose...1 II. Introduction...1 III. Inhibition of Bacterial Growth Protocol...2 IV. Inhibition of in vitro

More information

they differ from each other when judged in terms of temperature sensitivity,'

they differ from each other when judged in terms of temperature sensitivity,' 72 GENETICS: LACY AND BONNER.PRoc. N. A. S. 11 Pratt, David, and Gunther S. Stent, these PROCEEDINGS, 45, 1507 (1959). 12 The authors gratefully acknowledge the contributions of Dr. A. W. Kimball of the

More information

Basic Concepts of DNA, Proteins, Genes and Genomes

Basic Concepts of DNA, Proteins, Genes and Genomes Basic Concepts of DNA, Proteins, Genes and Genomes Kun-Mao Chao 1,2,3 1 Graduate Institute of Biomedical Electronics and Bioinformatics 2 Department of Computer Science and Information Engineering 3 Graduate

More information

Expression and Purification of Recombinant Protein in bacteria and Yeast. Presented By: Puspa pandey, Mohit sachdeva & Ming yu

Expression and Purification of Recombinant Protein in bacteria and Yeast. Presented By: Puspa pandey, Mohit sachdeva & Ming yu Expression and Purification of Recombinant Protein in bacteria and Yeast Presented By: Puspa pandey, Mohit sachdeva & Ming yu DNA Vectors Molecular carriers which carry fragments of DNA into host cell.

More information

Biotechnology: DNA Technology & Genomics

Biotechnology: DNA Technology & Genomics Chapter 20. Biotechnology: DNA Technology & Genomics 2003-2004 The BIG Questions How can we use our knowledge of DNA to: diagnose disease or defect? cure disease or defect? change/improve organisms? What

More information

Multiple Choice Write the letter that best answers the question or completes the statement on the line provided.

Multiple Choice Write the letter that best answers the question or completes the statement on the line provided. Name lass Date hapter 12 DN and RN hapter Test Multiple hoice Write the letter that best answers the question or completes the statement on the line provided. Pearson Education, Inc. ll rights reserved.

More information

Chapter 20: Biotechnology

Chapter 20: Biotechnology Name Period Chapter 20: Biotechnology The AP Biology exam has reached into this chapter for essay questions on a regular basis over the past 15 years. Student responses show that biotechnology is a difficult

More information

Molecular Biology. Yeast Transformation. Yeast Plasmids. Gene Disruption, tagging. Cloning by Complementation. Epistasis

Molecular Biology. Yeast Transformation. Yeast Plasmids. Gene Disruption, tagging. Cloning by Complementation. Epistasis Molecular Biology Yeast Transformation Yeast Plasmids Gene Disruption, tagging Cloning by Complementation Epistasis Transformation Transformation: introduction of DNA 1978, ca 1000x less efficient than

More information

Chapter 8: Recombinant DNA 2002 by W. H. Freeman and Company Chapter 8: Recombinant DNA 2002 by W. H. Freeman and Company

Chapter 8: Recombinant DNA 2002 by W. H. Freeman and Company Chapter 8: Recombinant DNA 2002 by W. H. Freeman and Company Genetic engineering: humans Gene replacement therapy or gene therapy Many technical and ethical issues implications for gene pool for germ-line gene therapy what traits constitute disease rather than just

More information

Lessons from the Stanford HIV Drug Resistance Database

Lessons from the Stanford HIV Drug Resistance Database 1 Lessons from the Stanford HIV Drug Resistance Database Bob Shafer, MD Department of Medicine and by Courtesy Pathology (Infectious Diseases) Stanford University Outline 2 Goals and rationale for HIVDB

More information

Supplementary Materials for

Supplementary Materials for www.sciencesignaling.org/cgi/content/full/7/339/ra80/dc1 Supplementary Materials for Manipulation of receptor oligomerization as a strategy to inhibit signaling by TNF superfamily members Julia T. Warren,

More information

Difficult DNA Templates Sequencing. Primer Walking Service

Difficult DNA Templates Sequencing. Primer Walking Service Difficult DNA Templates Sequencing Primer Walking Service Result 16/18s (ITS 5.8s) rrna Sequencing Phylogenetic tree 16s rrna Region ITS rrna Region ITS and 26s rrna Region Order and Result Cloning Service

More information

Insilico drug designing. Dinesh Gupta Structural and Computational Biology Group ICGEB

Insilico drug designing. Dinesh Gupta Structural and Computational Biology Group ICGEB Insilico drug designing Dinesh Gupta Structural and Computational Biology Group ICGEB Modern drug discovery process Target identification Target validation Lead identification Lead optimization Preclinical

More information

Treatment for. Malaria: DHA/PQP. Science Day MMV Stakeholders Meeting. Defeating Malaria Together 1

Treatment for. Malaria: DHA/PQP. Science Day MMV Stakeholders Meeting. Defeating Malaria Together 1 ANewOnce-a-day Treatment for Uncomplicated Malaria: DHA/PQP Science Day MMV Stakeholders Meeting Dr. Ambrose Talisuna Former Director Global Access, MMV & Field Coordinator, African Eurartesim Registration

More information

Lab 9: Bacterial Transformation with pglo

Lab 9: Bacterial Transformation with pglo Name: Lab 9: Bacterial Transformation with pglo OBJECTIVES: ο Practice formulating hypotheses, predictions, and experimental design. ο Describe the principles of bacterial transformation. ο Explain the

More information

Total Test Questions: 71 Levels: Grades 10-12 Units of Credit: 1.0 STANDARD 1 STUDENTS WILL INVESTIGATE THE PAST, PRESENT, AND FUTURE APPLICATIONS OF

Total Test Questions: 71 Levels: Grades 10-12 Units of Credit: 1.0 STANDARD 1 STUDENTS WILL INVESTIGATE THE PAST, PRESENT, AND FUTURE APPLICATIONS OF DESCRIPTION Biotechnology is designed to create an awareness of career possibilities in the field of biotechnology. Students are introduced to diagnostic and therapeutic laboratory procedures that support

More information

The Chemical Structure of Ampicillin

The Chemical Structure of Ampicillin Bio 210A Bacterial Transformation-Gene Cloning Purpose To understand the relevance of gene cloning to the biotechnology industry. To understand the definition of transformation, clone, and cloning vector.

More information

Hydroxynitrile lyase (Hnl)

Hydroxynitrile lyase (Hnl) Hydroxynitrile lyase (Hnl) R 1 HCN R 1 OH R 2 C O R 2 C * CN S-selective: Hevea brasiliensis R-selective: Prunus spp. (S)-Hnl of Hevea brasiliensis and (R)-Hnl of Prunus amygdalus Hb_Hnl Type II Hnl intracellular

More information

How many of you have checked out the web site on protein-dna interactions?

How many of you have checked out the web site on protein-dna interactions? How many of you have checked out the web site on protein-dna interactions? Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail. Find and be ready to discuss

More information

Micromyx. Micromyx. A Microbiology Services Company. Lab Services Research - Consulting -

Micromyx. Micromyx. A Microbiology Services Company. Lab Services Research - Consulting - A Microbiology Services Company Lab Services Research - Consulting - Regulatory Company description is a microbiology services company specializing in antiinfective discovery and development for the pharmaceutical,

More information

Genomes and SNPs in Malaria and Sickle Cell Anemia

Genomes and SNPs in Malaria and Sickle Cell Anemia Genomes and SNPs in Malaria and Sickle Cell Anemia Introduction to Genome Browsing with Ensembl Ensembl The vast amount of information in biological databases today demands a way of organising and accessing

More information

Mitochondrial DNA Analysis

Mitochondrial DNA Analysis Mitochondrial DNA Analysis Lineage Markers Lineage markers are passed down from generation to generation without changing Except for rare mutation events They can help determine the lineage (family tree)

More information

Identification of the VTEC serogroups mainly associated with human infections by conventional PCR amplification of O-associated genes

Identification of the VTEC serogroups mainly associated with human infections by conventional PCR amplification of O-associated genes Identification of the VTEC serogroups mainly associated with human infections by conventional PCR amplification of O-associated genes 1. Aim and field of application The present method concerns the identification

More information

European Medicines Agency

European Medicines Agency European Medicines Agency July 1996 CPMP/ICH/139/95 ICH Topic Q 5 B Quality of Biotechnological Products: Analysis of the Expression Construct in Cell Lines Used for Production of r-dna Derived Protein

More information

Genetics Module B, Anchor 3

Genetics Module B, Anchor 3 Genetics Module B, Anchor 3 Key Concepts: - An individual s characteristics are determines by factors that are passed from one parental generation to the next. - During gamete formation, the alleles for

More information

Supplemental Tables and Figure

Supplemental Tables and Figure A bacterial regulatory RNA attenuates virulence, spread and human host cell phagocytosis. Hélène Le Pabic, Noëlla Germain-Amiot, Valérie Bordeau and Brice Felden* Inserm U835-Upres EA2311, Biochimie Pharmaceutique,

More information

A Primer of Genome Science THIRD

A Primer of Genome Science THIRD A Primer of Genome Science THIRD EDITION GREG GIBSON-SPENCER V. MUSE North Carolina State University Sinauer Associates, Inc. Publishers Sunderland, Massachusetts USA Contents Preface xi 1 Genome Projects:

More information

Recombinant DNA Technology

Recombinant DNA Technology Recombinant DNA Technology Dates in the Development of Gene Cloning: 1965 - plasmids 1967 - ligase 1970 - restriction endonucleases 1972 - first experiments in gene splicing 1974 - worldwide moratorium

More information

Trasposable elements: P elements

Trasposable elements: P elements Trasposable elements: P elements In 1938 Marcus Rhodes provided the first genetic description of an unstable mutation, an allele of a gene required for the production of pigment in maize. This instability

More information

Just the Facts: A Basic Introduction to the Science Underlying NCBI Resources

Just the Facts: A Basic Introduction to the Science Underlying NCBI Resources 1 of 8 11/7/2004 11:00 AM National Center for Biotechnology Information About NCBI NCBI at a Glance A Science Primer Human Genome Resources Model Organisms Guide Outreach and Education Databases and Tools

More information

Genetics Test Biology I

Genetics Test Biology I Genetics Test Biology I Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Avery s experiments showed that bacteria are transformed by a. RNA. c. proteins.

More information

Chapter 18: Applications of Immunology

Chapter 18: Applications of Immunology Chapter 18: Applications of Immunology 1. Vaccinations 2. Monoclonal vs Polyclonal Ab 3. Diagnostic Immunology 1. Vaccinations What is Vaccination? A method of inducing artificial immunity by exposing

More information

C1. A gene pool is all of the genes present in a particular population. Each type of gene within a gene pool may exist in one or more alleles.

C1. A gene pool is all of the genes present in a particular population. Each type of gene within a gene pool may exist in one or more alleles. C1. A gene pool is all of the genes present in a particular population. Each type of gene within a gene pool may exist in one or more alleles. The prevalence of an allele within the gene pool is described

More information

STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS

STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS THESIS SUBMITTED FOR THE DEGREB OF DOCTOR OF PHILOSOPHY (SCIENCE) OF THE UNIVERSITY OF CALCUTTA 1996 NRISINHA DE, M.Sc DEPARTMENT OF BIOCHEMISTRY

More information

All your base(s) are belong to us

All your base(s) are belong to us All your base(s) are belong to us The dawn of the high-throughput DNA sequencing era 25C3 Magnus Manske The place Sanger Center, Cambridge, UK Basic biology Level of complexity Genome Single (all chromosomes

More information