Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of Toxoplasma gondii

Alan M. Johnson, Susana Illana, Peter J. McDonald, Takashi Asai

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Toxoplasma gondii expresses high levels of an active nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) with several unique properties. It has been detected as a circulating antigen in mice, making it an ideal candidate for diagnostic tests for toxoplasmosis. A cDNA library constructed from T. gondii poly(A)+ RNA was made in λgt11. One hundred thousand members of this library were immunoscreened with a rabbit polyclonal antibody to the purified NTPase. Six positive clones were subcloned into the plasmid, pGEX-1N, and the inserts were restriction-mapped. All clones had identical partial restriction enzyme maps. One insert was subcloned into M13mp18 and sequencing by the deoxy/dideoxy method showed an NTPase-encoding gene (ntp) fragment of 571 bp. The insert was also purified, radiolabelled, and used to hybridize to Northern blots of tachyzoite RNA and quantitative Southern blots of tachyzoite DNA. Northern blotting revealed that the NTPase mRNA was m great abundance and had a length of about 2800 nucleotides. Southern blotting showed a gene copy number of between one and five, and the possibility that ntp is tandemly repeated over a large length of DNA. The NTPase was expressed as a glutathione S-transferase (EC 2.5.1.18) fusion protein of about 50 kDa, which reacted with polyclonal rabbit antibody on Western blotting.

Original languageEnglish
Pages (from-to)215-220
Number of pages6
JournalGene
Volume85
Issue number1
DOIs
Publication statusPublished - 1989 Dec 21
Externally publishedYes

Fingerprint

N-Glycosyl Hydrolases
Nucleoside-Triphosphatase
Toxoplasma
Organism Cloning
Genes
Southern Blotting
Northern Blotting
Clone Cells
Rabbits
Messenger RNA
Gene Dosage
Antibodies
DNA
Toxoplasmosis
Glutathione Transferase
Gene Library
Routine Diagnostic Tests
Libraries
Plasmids
Nucleotides

Keywords

  • diagnostic antigen
  • expression library
  • fusion protein
  • immunoblots
  • phage λ vectors
  • plasmids pGEX
  • protist
  • Recombinant DNA
  • tachyzoite
  • toxoplasmosis

ASJC Scopus subject areas

  • Genetics

Cite this

Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of Toxoplasma gondii. / Johnson, Alan M.; Illana, Susana; McDonald, Peter J.; Asai, Takashi.

In: Gene, Vol. 85, No. 1, 21.12.1989, p. 215-220.

Research output: Contribution to journalArticle

Johnson, Alan M. ; Illana, Susana ; McDonald, Peter J. ; Asai, Takashi. / Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of Toxoplasma gondii. In: Gene. 1989 ; Vol. 85, No. 1. pp. 215-220.
@article{4b56c4c6a74a4ec6ad967401354c909d,
title = "Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of Toxoplasma gondii",
abstract = "Toxoplasma gondii expresses high levels of an active nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) with several unique properties. It has been detected as a circulating antigen in mice, making it an ideal candidate for diagnostic tests for toxoplasmosis. A cDNA library constructed from T. gondii poly(A)+ RNA was made in λgt11. One hundred thousand members of this library were immunoscreened with a rabbit polyclonal antibody to the purified NTPase. Six positive clones were subcloned into the plasmid, pGEX-1N, and the inserts were restriction-mapped. All clones had identical partial restriction enzyme maps. One insert was subcloned into M13mp18 and sequencing by the deoxy/dideoxy method showed an NTPase-encoding gene (ntp) fragment of 571 bp. The insert was also purified, radiolabelled, and used to hybridize to Northern blots of tachyzoite RNA and quantitative Southern blots of tachyzoite DNA. Northern blotting revealed that the NTPase mRNA was m great abundance and had a length of about 2800 nucleotides. Southern blotting showed a gene copy number of between one and five, and the possibility that ntp is tandemly repeated over a large length of DNA. The NTPase was expressed as a glutathione S-transferase (EC 2.5.1.18) fusion protein of about 50 kDa, which reacted with polyclonal rabbit antibody on Western blotting.",
keywords = "diagnostic antigen, expression library, fusion protein, immunoblots, phage λ vectors, plasmids pGEX, protist, Recombinant DNA, tachyzoite, toxoplasmosis",
author = "Johnson, {Alan M.} and Susana Illana and McDonald, {Peter J.} and Takashi Asai",
year = "1989",
month = "12",
day = "21",
doi = "10.1016/0378-1119(89)90484-8",
language = "English",
volume = "85",
pages = "215--220",
journal = "Gene",
issn = "0378-1119",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of Toxoplasma gondii

AU - Johnson, Alan M.

AU - Illana, Susana

AU - McDonald, Peter J.

AU - Asai, Takashi

PY - 1989/12/21

Y1 - 1989/12/21

N2 - Toxoplasma gondii expresses high levels of an active nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) with several unique properties. It has been detected as a circulating antigen in mice, making it an ideal candidate for diagnostic tests for toxoplasmosis. A cDNA library constructed from T. gondii poly(A)+ RNA was made in λgt11. One hundred thousand members of this library were immunoscreened with a rabbit polyclonal antibody to the purified NTPase. Six positive clones were subcloned into the plasmid, pGEX-1N, and the inserts were restriction-mapped. All clones had identical partial restriction enzyme maps. One insert was subcloned into M13mp18 and sequencing by the deoxy/dideoxy method showed an NTPase-encoding gene (ntp) fragment of 571 bp. The insert was also purified, radiolabelled, and used to hybridize to Northern blots of tachyzoite RNA and quantitative Southern blots of tachyzoite DNA. Northern blotting revealed that the NTPase mRNA was m great abundance and had a length of about 2800 nucleotides. Southern blotting showed a gene copy number of between one and five, and the possibility that ntp is tandemly repeated over a large length of DNA. The NTPase was expressed as a glutathione S-transferase (EC 2.5.1.18) fusion protein of about 50 kDa, which reacted with polyclonal rabbit antibody on Western blotting.

AB - Toxoplasma gondii expresses high levels of an active nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) with several unique properties. It has been detected as a circulating antigen in mice, making it an ideal candidate for diagnostic tests for toxoplasmosis. A cDNA library constructed from T. gondii poly(A)+ RNA was made in λgt11. One hundred thousand members of this library were immunoscreened with a rabbit polyclonal antibody to the purified NTPase. Six positive clones were subcloned into the plasmid, pGEX-1N, and the inserts were restriction-mapped. All clones had identical partial restriction enzyme maps. One insert was subcloned into M13mp18 and sequencing by the deoxy/dideoxy method showed an NTPase-encoding gene (ntp) fragment of 571 bp. The insert was also purified, radiolabelled, and used to hybridize to Northern blots of tachyzoite RNA and quantitative Southern blots of tachyzoite DNA. Northern blotting revealed that the NTPase mRNA was m great abundance and had a length of about 2800 nucleotides. Southern blotting showed a gene copy number of between one and five, and the possibility that ntp is tandemly repeated over a large length of DNA. The NTPase was expressed as a glutathione S-transferase (EC 2.5.1.18) fusion protein of about 50 kDa, which reacted with polyclonal rabbit antibody on Western blotting.

KW - diagnostic antigen

KW - expression library

KW - fusion protein

KW - immunoblots

KW - phage λ vectors

KW - plasmids pGEX

KW - protist

KW - Recombinant DNA

KW - tachyzoite

KW - toxoplasmosis

UR - http://www.scopus.com/inward/record.url?scp=0024845193&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024845193&partnerID=8YFLogxK

U2 - 10.1016/0378-1119(89)90484-8

DO - 10.1016/0378-1119(89)90484-8

M3 - Article

C2 - 2482826

AN - SCOPUS:0024845193

VL - 85

SP - 215

EP - 220

JO - Gene

JF - Gene

SN - 0378-1119

IS - 1

ER -