Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

Landfear, Scott M.; McMahon-Pratt, Diane; Wirth, Dyann F.

doi:10.1128/mcb.3.6.1070

Cited by 111 publications

(63 citation statements)

References 35 publications

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“…The size of this transcript, which arises from splice acceptors within the vector and insert, is also ϳ3 kb. Levels of poly(A) ϩ RNA were normalized for each cell line using the L. enriettii ␣-tubulin gene (23). There is an additional band that hybridizes to the ␣-tubulin probe in the pLdNT2-ALTNEO lane, suggestive of an alternatively spliced transcript, the reason for which is unclear.…”

Section: Molecular Characterization Of Ldnt2 In Wild Type and Fbd5mentioning

confidence: 99%

“…Poly(A) ϩ RNA, prepared from total RNA using the Oligotex mRNA mini kit (Qiagen Inc., Valencia, CA), was subjected to denaturing agarose electrophoresis, transferred to a GeneScreen Plus® hybridization transfer membrane (NEN Life Science Products), and probed with the 2.2-kb HindIII fragment described above. Signals were normalized by hybridization to probes corresponding to Leishmania enriettii ␣-tubulin gene (23).…”

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confidence: 99%

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Cloning of a Novel Inosine-Guanosine Transporter Gene fromLeishmania donovani by Functional Rescue of a Transport-deficient Mutant

Carter¹,

Drew²,

Sánchez³

et al. 2000

Journal of Biological Chemistry

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Purine transport is an indispensable nutritional function for protozoan parasites, since they are incapable of purine biosynthesis and must, therefore, acquire purines from the host milieu. Exploiting a mutant cell line (FBD5) of Leishmania donovani deficient in inosine and guanosine transport activity, the gene encoding this transporter (LdNT2) has been cloned by functional rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrative nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a single transcript of 3 kilobase pairs. Transfection of FBD5 parasites with LdNT2 reestablished their ability to take up inosine and guanosine with a concurrent restoration of sensitivity to the inosine analog formycin B. Kinetic analyses reveal that LdNT2 is highly specific for inosine (K m ‫؍‬ 0.3 M) and guanosine (K m ‫؍‬ 1.7 M) and does not recognize other naturally occurring nucleosides. Expression of LdNT2 cRNA in Xenopus oocytes significantly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These results authenticate genetically and biochemically that LdNT2 is a novel nucleoside transporter with an unusual and strict specificity for inosine and guanosine.Leishmania donovani is a protozoan parasite and the etiologic agent of visceral leishmaniasis, a devastating and invariably fatal disease if untreated. The parasite exhibits an intricate life cycle in which the extracellular, flagellated promastigote exists in the phlebotomine sandfly vector, and the intracellular amastigote resides in the phagolysosome of macrophages and other reticuloendothelial cells of the mammalian host. Drugs are the only defense against visceral leishmaniasis, but the efficacy of these empirically derived agents is compromised both by drug toxicity and resistance (1). Thus, it is increasingly imperative to identify new and unique biochemical targets in the parasite for potential therapeutic exploitation.Among the most conspicuous metabolic differences between parasites and their mammalian hosts is the purine pathway. Whereas animal cells synthesize purine nucleotides de novo, all protozoan parasites are incapable of synthesizing purines and depend upon purine acquisition from their hosts to survive and proliferate (2). Hence, each genus of parasite has evolved a unique complement of purine salvage enzymes in order to scavenge purines from the host milieu (2). The first step in this salvage process involves the translocation of purines across the parasite plasma membrane, a process mediated by membrane permeases. These permeases also initiate the uptake of pyrazolopyrimidine nucleobase and nucleoside analogs of hypoxanthine and inosine that are selectively toxic to Leishmania spp. (3, 4). Thus, purine transporters play vital roles in both purine nutrition and antiparasitic drug targeting i...

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Section: Molecular Characterization Of Ldnt2 In Wild Type and Fbd5mentioning

confidence: 99%

mentioning

confidence: 99%

Cloning of a Novel Inosine-Guanosine Transporter Gene fromLeishmania donovani by Functional Rescue of a Transport-deficient Mutant

Carter¹,

Drew²,

Sánchez³

et al. 2000

Journal of Biological Chemistry

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“…The authors suggested that this number could generate functionally specific tubulin heterogeneity controlled by differential gene expression. A similar study of Leishmania enreittii tubulin genes (18) revealed approximately 15 alpha and 15 beta tubulin gene copies arranged as separate repeated sequences but argued that their restriction site homogeneity demonstrated a high degree of relatedness. Unfortunately, there is a lack of information pertaining to the tubulins found in this group of organisms.…”

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confidence: 99%

Tubulin heterogeneity in the trypanosome Crithidia fasciculata.

Russell¹,

Miller²,

Gull³

1984

Mol. Cell. Biol.

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The interphase cell of Crithidia fasciciulata has three discrete tubulin populations: the subpellicular microtubules, the axonemal microtubules, and the nonpolymerized cytoplasmic pool protein. These three tubulin populations were independently and selectively purified, yielding, in each case, microtubule protein capable of self-assembly. All three preparations polymerized to form ribbons and sheets rather than the more usual microtubular structures. Analyses of the tubulin by two-dimensional polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping indicated that the beta-tubulin complex remained constant regardless of source but that some heterogeneity was present in the alpha subunit. Cytoplasmic pool alpha tubulins (alpha 1/alpha 2) were the only alpha isotypes in the cytoplasm and also formed most of the alpha tubulin species in the pellicular fraction. Flagellar alpha tubulin (alpha 3) was the sole alpha isotype in the flagella; it appeared in small amounts in the pellicular fraction but was completely absent from the cytoplasm. In vitro translation products from polyadenylated RNA from C. fasciciulata were also examined by two-dimensional polyacrylamide gel electrophoresis and possessed a protein corresponding to alpha 1/alpha 2 tubulin but lacked any alpha 3 tubulin. The alpha 3 polypeptide arose from a posttranslational modification of a precursor polypeptide not identifiable by two-dimensional polyacrylamide gel electrophoresis as alpha 3. Peptide mapping data indicated that cytoplasmic alpha tubulin is the most likely precursor. These results demonstrate alpha-tubulin heterogeneity in this organism and also how close the relationship between flagellar and cytoskeletal tubulins can be among lower eucaryotes.Microtubules are present in most eucaryotic cells where they fulfill a diverse range of functions vital to the existence of the cell. Until recently, tubulin research has been conducted almost exclusively on protein isolated from mammalian brain tissue. Although invaluable to our current understanding of the physical properties of the protein, it has been of limited value to our appreciation of the roles of tubulin polypeptides within the cell. What is becoming increasingly obvious is that, despite its conserved nature, tubulin exists as a family of proteins. As the range of organisms from which tubulin has been purified broadens the extent of the diversification of the protein becomes clearer. In this context, eucaryotic microbes are particularly useful since, unlike most metazoan cells, they possess rigorously organized cytoskeletons, often with highly ordered microtubular organelles devoted to specific activities. It is, therefore, easier to examine the microtubular proteins and also to predict the constraints under which the microtubules in such organelles would operate.

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“…All Southern and Northern blots were visualized by autoradiography. The ao-tubulin gene from L. ennettii (33) used to normalize the amount of DNA in preparations from DI700 and VINB1000 cells was provided by Scott Landfear of the Oregon Health Sciences University.…”

mentioning

confidence: 99%

Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

Henderson¹,

Sifri²,

Rodgers³

et al. 1992

Mol. Cell. Biol.

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148

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Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastineresistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, Idmdrl, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINBIOOO cells were resistant and sensitive to all drugs to which VINBlOO0 cells were sensitive. These studies demonstrate that amplification of the ldmdrl gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.

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Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

Cited by 111 publications

References 35 publications

Cloning of a Novel Inosine-Guanosine Transporter Gene fromLeishmania donovani by Functional Rescue of a Transport-deficient Mutant

Cloning of a Novel Inosine-Guanosine Transporter Gene fromLeishmania donovani by Functional Rescue of a Transport-deficient Mutant

Tubulin heterogeneity in the trypanosome Crithidia fasciculata.

Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

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