Studies of DNA Amplification in the Hydroxyurea-Resistant Leishmania

• Main Authors: Lye Lon-fye, 賴榮輝
• Other Authors: K. E. Su
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/36712340964554057480

博士 === 國立臺灣大學 === 微生物研究所 === 87 === Tropical diseases such as leishmaniasis, malaria, trypanosomiasis, toxoplasmosis and amoebiasis continue to plaque the world, resulting in considerable morbidity and mortality, especially in the third world countries. These diseases are caused by a group of protozoa, which have over the years undergone evolutionary adaptation to live often intracellularly in a parasitic way of life. So well adapted have they become that they recognize the right hosts and cells to parasitize, yet at the same time they manage to escape from recognition and destruction of the host defense mechanisms. The control of these parasitic diseases often proved difficult because any effective treatment aimed at parasites will also likely have harmful effects on host. Leishmaniasis is one such example. Leishmania can live extracellularly as promastigotes in sand fly vector and intracellularly as amastigotes in the macrophages of the mammalian host. Promastigotes not only infect macrophage exclusively as their host cells but also survive, differentiate and multiply in their phagolysosomes- the very intracellular site where other microorganisms are usually killed. The refore any effective drug treatment against Leishmania will probably be harmful to the host cells. The aim of my study is to identify unique differences at the molecular and structural levels of certain essential gene(s)between the parasite and the host so that designing of specific chemotherapeutic agents for effective inhibition of the gene can be achieved, thus creating an ideal situation for specific elimination of the parasites without hurting the host. Ribonucleotide reductase gene is one such gene we choose for this purpose. In the first place the enzyme ribonucleotide reductase is the key enzyme for the transformation of ribonucleotides to the deoxyribonucleotides essential for DNA synthesis. It likely played an important part in the evolutionary transition from "RNA world" to the "DNA world". Secondly, this gene has been well exploited in mammalian cells, bacteria, clams, viruses and yeast in sequence and in some cases the three dimensional structures, but not in Leishmania and thirdly, we have evidence by Southern hybridization that this gene in Leishmania is different sufficiently enough from that of its mammalian hosts. A ribonucleotide reductase M2 subunit gene is cloned and sequenced from the extrac hromosomal amplified DNA of a hydroxyurea-resistant variant of Leishmania mexicana amazonensis. In terms of nucleotide and amino acid sequences, the M2 gene is more similar to eukaryotic cells than to that of bacteria and virus. The M2 gene, even though it is only a subunit of a ribonucleotide reductase gene complex, it is able to confer resistance to a high concentration of hydroxyurea when transfected into wild-type cells.An extrachromosomal circular DNA of approximately 50 kb in size is amplified in the hydroxyurea-resistant variant of L. m. amazonensis. The amplicon carries the M2 gene of ribonucleotide reductase as part of the resistant gene to hydroxyurea. The amplicon is unstable. It disappears rapidly as shown in pulse field gradient electrophoresis gels after reversion of the cells for 80 days and this loss of amplified DNA is accompanied by the rapid loss resistance to hydroxyurea at the same time period. The amplicon is not hybridized to specific probes from any of the four regions of DNA amplification of Leishmania previously reported. This region of amplification thus appears to be a new region of DNA amplification in Leishmania. It is designated as ''U'' region to differentiate it from the others.