Differential expression of mRNAs for alpha- and beta-tubulin during differentiation of the parasitic protozoan Leishmania mexicana.

Fong, Dunne; Wallach, Michael; Keithly, Jan S.; Melera, Peter W.; Chang, Kwang-Poo

doi:10.1073/pnas.81.18.5782

Cited by 48 publications

(22 citation statements)

References 36 publications

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“…4H) increased in abundance in intracellular amastigotes but not in axenic amastigotes. A similar pattern of differentially regulated ␤-tubulin transcript sizes has been reported for Leishmania amazonensis (30). In summary, three constitutively expressed RNAs (␣-tubulin, LmGT1, LmGT3), three RNAs that are down-regulated in amastigotes (LmGT2, PFR-1, ␤-tubulin 2.4-kb transcript), and two RNAs that are up-regulated in amastigotes (cysteine protease-2 and ␤-tubulin 2.8-kb transcript) are correctly regulated in axenic amastigotes.…”

Section: Regulation Of Lmgt Gene Expression In Promastigotes Andsupporting

confidence: 77%

Differential Regulation of Multiple Glucose Transporter Genes in Leishmania mexicana

Burchmore

Landfear

1998

Journal of Biological Chemistry

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We have studied the structure and expression of glucose transporter genes in the parasitic protozoan Leishmania mexicana. Three distinct glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, are encoded by single copy genes that are clustered together at a single locus. Quantitation of Northern blots reveals that LmGT2 mRNA is present at ϳ15-fold higher level in promastigotes, the insect stage of the parasite life cycle, compared with amastigotes, the intracellular stage of the life cycle that lives within the mammalian host. In contrast, LmGT1 and LmGT3 mRNAs are expressed at similar levels in both life cycle stages. Transcription of the LmGT genes in promastigotes and axenically cultured amastigotes occurs at similar levels, as measured by nuclear run-on transcription. Consequently, the ϳ15-fold up-regulation of LmGT2 mRNA levels in promastigotes compared with amastigotes must be controlled at the post-transcriptional level. Measurement of LmGT2 RNA decay in promastigotes and axenic amastigotes treated with actinomycin D suggests that differential mRNA stability may play a role in regulating glucose transporter mRNA levels in the two life cycle stages.

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Section: Regulation Of Lmgt Gene Expression In Promastigotes Andsupporting

confidence: 77%

Differential Regulation of Multiple Glucose Transporter Genes in Leishmania mexicana

Burchmore

Landfear

1998

Journal of Biological Chemistry

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“…As expected, the tubulin amino acid sequences from L. tarentolae are highly similar to the tubulin sequences from other Leishmania species and with other organisms. Leishmania species possess a single mRNA for α-tubulin (Fong et al, 1984;Burchmore and Landfear, 1998), while multiple β-tubulin mRNAs have been observed in this parasite. Three distinct β-tubulin mRNAs have been detected in L. amazonensis (Fong et al, 1984) and L. major (Coulson et al, 1996), while multiple β-tubulin mRNAs were also found in L. mexicana (Burchmore and Landfear, 1998).…”

Section: Sequences Of L Tarentolae α-And β-Tubulin Genes and Comparimentioning

confidence: 98%

“…Leishmania species possess a single mRNA for α-tubulin (Fong et al, 1984;Burchmore and Landfear, 1998), while multiple β-tubulin mRNAs have been observed in this parasite. Three distinct β-tubulin mRNAs have been detected in L. amazonensis (Fong et al, 1984) and L. major (Coulson et al, 1996), while multiple β-tubulin mRNAs were also found in L. mexicana (Burchmore and Landfear, 1998). The identity between amino acid sequences for L. tarentolae agr;-tubulin and α-tubulins from L. donovani, L. major and L. infantum present in available databases is 98% or greater, while the identity between the L. tarentolae β-tubulin sequence reported here and the various β-tubulin sequences (from L. infantum, L. brazilensis, L. major, L. mexicana, and L. amazonensis) is at least 96%.…”

Section: Sequences Of L Tarentolae α-And β-Tubulin Genes and Comparimentioning

confidence: 98%

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Yakovich

Ragone

Alfonzo

et al. 2006

Experimental Parasitology

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Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, Leishmania amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of α-and β-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly-disassembly cycle in 1% overall recovery based on total cellular protein. L. tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae α-and β-tubulin genes were 99.6% and 99.4% identical to the corresponding amino acid sequences from the L. major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.

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“…Restriction enzyme digests of human placental DNA (a generous gift from Peter W. Melera, Sloan-Kettering Institute) were subjected to electrophoresis through 0.8% agarose gel and transferred to nitrocellulose filters by the method of Southern (26), and hybridized with nick-translated Pst/Eco insert fragment from the clone pCB-1 (27 room temperature (2x NaCl/Cit with 0.5% NaDodSO4) and 680C (0.lx NaCl/Cit with 0.5% NaDodSO4).…”

mentioning

confidence: 99%

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

Fong¹,

Calhoun²,

Hsieh³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonudeotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this done encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with w880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome.Proteinases are present in all forms of living organisms. The events of general and limited proteolyses have been attributed to actions of these enzymes. In general proteolysis, proteinases digest nutrient proteins and participate in the turnover of cellular proteins, whereas in limited proteolysis, proteinases modify substrate proteins and alter the properties and physiological functions of these proteins. Thus, limited proteolysis can play regulatory roles during cell growth and differentiation (1,2).Based on the amino acid residues at their active sites, proteinases are classified into serine, cysteine, aspartate, and metalloproteinases. Members of the cysteine proteinase family have wide phylogenetic distribution, and examples include papain from the papaya plant and cathepsin B from mammalian tissues. Cathepsin B is a lysosomal enzyme that is functional during intracellular protein catabolism and may also be involved in the proteolytic processing of protein precursors such as proinsulin (3)(4)(5). Cathepsin B has been implicated in several disease states including muscular dystrophy (6), rheumatoid arthritis (7), and tumor metastasis (8-11). We and others have demonstrated that cathepsin B-like proteinases may also be involved during differentiation of the cellular slime mold Dictyostelium discoideum (12, 13).To further investigate the role of cathepsin B in cellular functions, we have selected the molecular genetic approach. Here we report our results on the isolation and characterization of a cDNA clone for human cathepsin B.MATERIALS AND METHODS Synthesis of Oligodeoxyribonucleotides. A mixture of 17-nucleotide-long oligonucleotides encoding a portion of the rat liver cathepsin B protein sequence was synthesized by using a solid-phase phosphotriester method (14, 15). For amino acids specified by more than one codon, all possible nucleotides were inserted at the ambiguous positions. The synthesis was performed with a Vega Model 280 Automated Polynucleotide Synthesizer by a...

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Differential expression of mRNAs for alpha- and beta-tubulin during differentiation of the parasitic protozoan Leishmania mexicana.

Cited by 48 publications

References 36 publications

Differential Regulation of Multiple Glucose Transporter Genes in Leishmania mexicana

Differential Regulation of Multiple Glucose Transporter Genes in Leishmania mexicana

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

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