Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani

A key molecular target in antileishmanial therapy

Aditi Das, Arindam Dasgupta, Shalini Sharma, Monidipa Ghosh, Tanushri Sengupta, Santu Bandopadhyay, Hemanta K. Majumder

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5′-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at ∼132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

Original languageEnglish (US)
Pages (from-to)1844-1851
Number of pages8
JournalNucleic Acids Research
Volume29
Issue number9
StatePublished - May 1 2001
Externally publishedYes

Fingerprint

Leishmania donovani
Type II DNA Topoisomerase
Genes
Immune Sera
Parasites
Proteins
Crithidia
Trans-Splicing
Trypanosoma
Genomic Library
Therapeutics
Cell Extracts
Glutathione Transferase
Gene Library
DNA Sequence Analysis
Introns
Open Reading Frames
Amino Acid Sequence
Exons
Chromosomes

ASJC Scopus subject areas

  • Genetics

Cite this

Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani : A key molecular target in antileishmanial therapy. / Das, Aditi; Dasgupta, Arindam; Sharma, Shalini; Ghosh, Monidipa; Sengupta, Tanushri; Bandopadhyay, Santu; Majumder, Hemanta K.

In: Nucleic Acids Research, Vol. 29, No. 9, 01.05.2001, p. 1844-1851.

Research output: Contribution to journalArticle

Das, A, Dasgupta, A, Sharma, S, Ghosh, M, Sengupta, T, Bandopadhyay, S & Majumder, HK 2001, 'Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani: A key molecular target in antileishmanial therapy', Nucleic Acids Research, vol. 29, no. 9, pp. 1844-1851.
Das, Aditi ; Dasgupta, Arindam ; Sharma, Shalini ; Ghosh, Monidipa ; Sengupta, Tanushri ; Bandopadhyay, Santu ; Majumder, Hemanta K. / Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani : A key molecular target in antileishmanial therapy. In: Nucleic Acids Research. 2001 ; Vol. 29, No. 9. pp. 1844-1851.
@article{7bb94816da7a436d85e251deec619a11,
title = "Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani: A key molecular target in antileishmanial therapy",
abstract = "The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68{\%}, respectively. LdTOP2 shares a much lower identity of 32{\%} with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5′-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at ∼132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.",
author = "Aditi Das and Arindam Dasgupta and Shalini Sharma and Monidipa Ghosh and Tanushri Sengupta and Santu Bandopadhyay and Majumder, {Hemanta K.}",
year = "2001",
month = "5",
day = "1",
language = "English (US)",
volume = "29",
pages = "1844--1851",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "9",

}

TY - JOUR

T1 - Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani

T2 - A key molecular target in antileishmanial therapy

AU - Das, Aditi

AU - Dasgupta, Arindam

AU - Sharma, Shalini

AU - Ghosh, Monidipa

AU - Sengupta, Tanushri

AU - Bandopadhyay, Santu

AU - Majumder, Hemanta K.

PY - 2001/5/1

Y1 - 2001/5/1

N2 - The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5′-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at ∼132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

AB - The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5′-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at ∼132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

UR - http://www.scopus.com/inward/record.url?scp=0035339610&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035339610&partnerID=8YFLogxK

M3 - Article

VL - 29

SP - 1844

EP - 1851

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 9

ER -