Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tubulin biosynthesis was rapidly induced during transformation of the mammalian (amastigote) stage of the kinetoplastid parasite Leishmania donovani to flagellated promastigotes. However, transcription of beta-tubulin genes occurred constitutively, as judged by nascent RNA synthesis in isolated nuclei and Northern blotting of steady-state mRNA. Two mRNA species of 2.2 and 2.4 kb were shared by the two cell-types, while a third 2.6 kb species, constituting about 20% of the total, was present in large amounts in promastigotes. RNase protection experiments demonstrated sequence microheterogeneity in the 5'-untranslated region, the pattern of which was identical in promastigotes and amastigotes. By primer extension assays, heterogeneity in the 5'-terminal cap structure of amastigote beta-tubulin mRNA and differential pausing of reverse transcriptase within the mini-exon leader region were detected. These differences correlated with enhanced translational efficiency of tubulin mRNA from promastigotes in a rabbit reticulocyte lysate system. The results indicate that translational control plays a major role in tubulin induction during L. donovani differentiation.
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PMID:Evidence for translational control of beta-tubulin synthesis during differentiation of Leishmania donovani. 174 47

Resistance to antimitotic chemotherapeutics in pathogenic nematodes, fungi and mammalian cells is closely associated with structural changes in cytoskeletal beta-tubulin. We investigated the possibility of using the well-characterised free-living nematode Caenorhabditis elegans as a model for studying the mechanism of resistance against benzimidazole (BZ) drugs in the parasitic nematode Haemonchus contortus. Functional analysis of a conserved beta-tubulin isotype (tub-1) mutation near GTP-binding domain II, which is linked to BZ resistance, was carried out in C. elegans by heterologous expression of: (1) parasite BZ-sensitive alleles; (2) BZ-resistant alleles; and (3) in vitro mutagenised beta-tubulin gene constructs. The injected heterologous gene constructs were not only stably maintained, but also expressed as shown by reverse transcriptase-polymerase chain reaction analysis. The degree of BZ drug susceptibility of the transformants was assayed and quantified by incubation with both benomyl and thiabendazol. All H. contortus tub-1 constructs, which encoded Phe at position 200, conferred susceptibility to thiabendazole in BZ-resistant C. elegans ben-1 mutants. In contrast, constructs carrying Tyr200 did not alter the BZ drug phenotype. From these experiments we conclude that: (1) C. elegans can be used as an expression host, since injected parasite genes were biologically active; and (2) the single Phe to Tyr mutation at position 200 in beta-tubulin isotype 1 is the cause of BZ resistance in H. contortus.
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PMID:Beta-tubulin genes from the parasitic nematode Haemonchus contortus modulate drug resistance in Caenorhabditis elegans. 787 71

The efficacy of taxol against a wide range of sensitive and refractory solid tumors has prompted extensive investigation into the factors that influence its cytotoxicity. Our preliminary observations indicated that taxol had a superior antitumor effect against human cells (Daudi, K562, 2008, 2008/C13*, 2780 and C70) compared with its effect against rodent cells (WS, WR, NIH3T3, and CHO). Although verapamil, an inhibitor of P-glycoprotein function, markedly increased the efficacy of taxol against the rodent cells (WS, WR, and CHO), the expression of P-glycoprotein was found only at low levels in the WR cells. In addition, levels of the multidrug resistance-associated protein (MRP), as assessed by reverse transcriptase-polymerase chain reaction analysis, were found to be higher in the human than in the rodent cells, although MRP mRNA was not detected by northern blotting. Transport studies indicated that the reduced sensitivity of the rodent cells to taxol was due to decreased intracellular taxol levels and reduced intracellular binding. However, no correlation was found between the intracellular binding of taxol and the intracellular levels of alpha- and beta-tubulin, or the intracellular concentration of polymerized tubulin. These studies were extended further by assessing the binding of taxol to semi-purified microtubule proteins from WS, CHO and 2008/C13* cells in vitro. The microtubule protein preparations from WS, CHO and 2008/C13* cells, which have a 50-fold difference in their sensitivity to taxol, were found to bind equal amounts of radiolabeled taxol, and this binding was inhibited (80%) in the presence of unlabeled taxol. These results lead us to propose the presence in the rodent cells of an alternative taxol transport system that is distinct from the P-glycoprotein and MRP systems.
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PMID:Species-specific differences in taxol transport and cytotoxicity against human and rodent tumor cells. Evidence for an alternate transport system. 857 97

Estramustine (EM), an antimicrotubule agent, is effective against hormone-refractory prostate cancer when used in combination with vinblastine or paclitaxel. To understand the effect of EM on beta-tubulin, a cellular target for this class of drugs, human prostate carcinoma cells (DU-145) were made resistant to EM, and two cell lines were selected at 12- (EM-12) and 15-microMolar (EM-15) concentrations of the drug. These cell lines exhibited 8- to 9-fold resistance to EM and 2- to 4-fold cross-resistance to paclitaxel. Immunofluorescent staining of the cells with beta-tubulin isotype-specific antibodies showed an approximately 6-fold increase in the beta(III)-tubulin levels and moderate increase in overall beta-tubulin levels in EM-resistant cells when compared to DU-145 cells. This increase of beta(III) isotype was confirmed by Western analysis. A reverse transcriptase-PCR assay was also employed using beta-tubulin isotype-specific primers to quantify beta-tubulin isotype RNA. A 4-fold increase in beta(III) and a 3-fold increase in beta(IV alpha) transcript were seen in both EM-resistant cell lines. These results indicate that overexpression of specific beta-tubulin isotypes may play a role in the cellular defense against EM and other antimicrotubule agents.
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PMID:Increase of beta(III)- and beta(IVa)-tubulin isotopes in human prostate carcinoma cells as a result of estramustine resistance. 865 1

The indole-diterpene paxilline is a potent tremorgenic mammalian mycotoxin and a known inhibitor of maxi-K ion channels. The gene cluster responsible for paxilline biosynthesis in Penicillium paxilli was identified by mapping four large plasmid-induced chromosome deletions. The cluster is predicted to lie within a 50 kb region of chromosome Va and to contain 17 genes, including a geranylgeranyl pyrophosphate (GGPP) synthase (paxG), two FAD-dependent monooxygenases (paxM and N), two cytochrome P450 monooxygenases (paxP and Q), a dimethylallyltryptophan (DMAT) synthase (paxD) and two possible transcription factors (paxR and paxS), which contain a Zn(II)2Cys6 DNA-binding motif. Targeted replacement of paxG confirmed that it is essential for paxilline biosynthesis but dispensable for growth. The GGPP for primary metabolism is predicted to be provided by a second GGPP synthase (ggs1) that was cloned, sequenced and mapped to chromosome IV. Semi-quantitative reverse transcriptase-polymerase chain reaction analysis demonstrated that the expression of paxG, paxM and paxP in submerged liquid cultures of P. paxilli increased dramatically with the onset of paxilline biosynthesis. In contrast, the expression of beta-tubulin (tub2) and ggs1 was not induced. This is the first description of the molecular cloning and genetic analysis of an indole-diterpene gene cluster.
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PMID:Molecular cloning and genetic analysis of an indole-diterpene gene cluster from Penicillium paxilli. 1116 15

A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and beta-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders.
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PMID:Expression of neural markers in human umbilical cord blood. 1152 Jan 25

The obligate intracellular protozoan parasite, Toxoplasma gondii exists as 2 life-cycle forms in intermediate hosts. The rapidly dividing tachyzoites responsible for acute disease, present in the first 14 days of infection, give rise to slowly dividing bradyzoites that reside in tissue cysts. Reactivation of disease is associated with conversion of bradyzoites to tachyzoites. A sensitive method for detection and assessment of the number of each life-cycle stage would be useful for following these events. Herein we describe the construction and validation of a plasmid (pSWITCH) containing a polycompetitor construct (SWITCH) for use in competitive reverse transcriptase-PCR (cRT-PCR). pSWITCH contains competitors for SAG2A and LDH2 genes, which are exclusively expressed by tachyzoite and bradyzoite stages respectively, and for beta-tubulin, a gene expressed by both stages. Using cRT-PCR, samples can first be accurately normalized for expression of the housekeeping gene, beta-tubulin and then the relative levels of SAG2A and LDH2 expression compared to follow stage conversion. The abundance of transcripts for other genes of interest can then be followed during this process as demonstrated here for the SAG2-related family of genes. This technique offers a powerful tool for studying the processes involved in tachyzoite and bradyzoite interconversion.
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PMID:Construction and validation of a polycompetitor construct (SWITCH) for use in competitive RT-PCR to assess tachyzoite-bradyzoite interconversion in Toxoplasma gondii. 1171 53

The unicellular green alga Acetabularia acetabulum has proven itself to be a superior model for studies of morphogenesis because of its large size and distinctive polar morphology. The giant cell forms an elongated tube (a stalk of up to 60 mm in length), which at its apical pole makes whorls of hairs, followed by one whorl of gametophores in the shape of a cap. At its basal pole, the cell extends into a rhizoid wherein the single nucleus is positioned. In this study, we have determined the level of specific messenger RNAs in the apical, middle, and basal regions using reverse transcriptase-PCR methodology. Four mRNA classes were distinguished: those that were uniformly distributed (small subunit of Rubisco, actin-1, ADP-glucose, centrin, and alpha- and beta-tubulin), those that expressed apical/basal (calmodulin-4) or basal/apical gradients (calmodulin-2 and a Ran-G protein), and those with development-specific patterns of distribution (mitogen-activated protein kinase, actin-2, and UDP-glucose-epimerase). Restoration of the apical/basal calmodulin-4 mRNA gradient after amputation of the apical region of the cell requires the nucleus and was abolished by cytochalasin D. Accumulation of actin-1 mRNA in the vicinity of the wound set by the amputation needs, likewise, the presence of the nucleus and was also inhibited by cytochalasin. This suggests that actin microfilaments of the cytoskeleton are involved in directed transport and/or anchoring of these mRNAs.
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PMID:Differential messenger RNA gradients in the unicellular alga Acetabularia acetabulum. Role of the cytoskeleton. 1211 94

Recent studies on dendritic cell (DC)-associated genes have been performed using monocyte-derived DCs (MoDCs) in different maturation stages. In our approach, to uncover the novel DC-associated genes and their expression profiles among the different DC subsets, we constructed a subtracted DC-cDNA library from CD1a(+), CD14(+), and CD11c(-) DCs by subtracting the genes shared with T cells, B cells, and monocytes, and we then screened the libraries with the aid of microarray technique. The genes showing remarkable specificity to DCs in the microarray analysis were selected and confirmed by semiquantitative reverse transcriptase-polymerase chain reaction. Our investigations revealed the following: (1) Genes highly expressed in myeloid DCs are those involved in antigen uptake/processing/presentation, cell metamorphosis, or chemotaxis. (2) Most of the genes previously identified in MoDCs, such as TARC, ferritin L-chain, lysosomal acid lipase, alpha- and beta-tubulin, osteopontin (Eta-1), and others, are not markedly expressed in CD11c(-) DCs regardless of their maturation status. On the other hand, specific transcription factors and MHC class II molecules, such as interferon regulatory factor-4 (IRF4) and HLA-DR, are similarly expressed in both DC subsets. (3) CD14(+) DCs retain unique features of tissue DCs, as evidenced by the gene expression profile of "no CCR7 but more CCR1" and "no TARC but abundant MCP1 and Eta-1." (4) The genes for immunoglobulin (Ig) superfamily Z39Ig, CD20-like precursor, glycoprotein NMB (GPNMB), transforming growth factorbeta (TGF-beta)-induced protein (TGFBI), myeloid DAP12-associated lectin (MDL-1), and 6 novel genes are newly identified as being associated with the phenotypic expression of the DC subsets. These identifications provide important molecular information for further functional studies of the DC subsets.
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PMID:Identification of the genes differentially expressed in human dendritic cell subsets by cDNA subtraction and microarray analysis. 1217 96

Each fiber of cotton (Gossypium hirsutum) is a single epidermal cell that rapidly elongates to 2.5 to 3.0 cm from the ovule surface within about 16 d after anthesis. A large number of genes are required for fiber differentiation and development, but so far, little is known about how these genes control and regulate the process of fiber development. To investigate gene expression patterns in fiber, a cDNA, GhTUB1, encoding beta-tubulin was isolated from a cotton fiber cDNA library. The analyses of RNA northern-blot hybridization and reverse transcriptase-polymerase chain reaction demonstrated that GhTUB1 transcripts preferentially accumulated at high levels in fiber, at low levels in ovules at the early stage of cotton boll development, and at very low levels in other tissues of cotton. The corresponding GhTUB1 gene including the promoter region was isolated by screening a cotton genomic DNA library. To demonstrate the specificity of the GhTUB1 promoter, the 5'-flanking region including the promoter and 5'-untranslated region was fused with the beta-glucuronidase reporter gene. The expression of the reporter chimera was examined in a large number of transgenic cotton plants. Histochemical assays demonstrated that GhTUB1::beta-glucuronidase fusion genes were expressed preferentially at high levels in fiber and primary root tip of 1- to 3-d-old seedlings and at low levels in other tissues such as ovule, pollen, seedling cotyledon, and root basal portion. The results suggested that the GhTUB1 gene may play a distinct and required role in fiber development. In addition, the GhTUB1 promoter may have great potential for cotton improvement by genetic engineering.
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PMID:Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fiber. 1237 34


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