Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani

Keith Wilson, Frank R. Collart, Eliezer Huberman, James R. Stringer, Buddy Ullman

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

A mutant (MPA100) strain of Leishmania donovania was generated from a wild type (D1700) population by virtue of its ability to survive the selective pressure of gradually increasing concentrations of mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase (IMPDH) activity. Comparative growth experiments revealed that the MPA100 strain was 100-fold more resistant to MPA toxicity and cross-resistant to ribavarin, another inhibitor of IMPDH. A direct comparison of IMPDH levels in D1700 and MPA100 cells showed that the latter expressed at least 20-fold higher enzyme activity. In order to evaluate the mechanism by which MPA100 cells overexpressed IMPDH, the leishmanial gene encoding IMPDH was isolated from a genomic library in EMBL3 by cross-hybridization to a mouse IMPDH cDNA, and a 2.3-kilobase EcoRV-PstI fragment was subcloned into a Bluescript vector and sequenced. The EcoRV-PstI fragment contained an open reading frame of 514 amino acids that encompassed the entire leishmanial IMPDH coding sequence. The predicted amino acid sequence showed a 52.5% identity with that of the corresponding human IMPDH. The codon usage of the leishmanial IMPDH gene reflected a strong bias toward codons containing either G or C in the wobble position. The EcoRV-PstI fragment hybridized to a 3.0-kilobase mRNA that was expressed at 10-20-fold greater levels in the MPA100 cells. Using the EcoRV-PstI fragment as a probe, the increased amount of IMPDH activity and IMPDH mRNA in the MPA100 cells could be attributed to an approximately 10-20-fold amplification of the leishmanial IMPDH gene.

Original languageEnglish (US)
Pages (from-to)1665-1671
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number3
StatePublished - Jan 25 1991

Fingerprint

IMP Dehydrogenase
Leishmania donovani
Cloning
Molecular Cloning
Amplification
Genes
Mycophenolic Acid
Codon
Calymmatobacterium
Amino Acids
Messenger RNA
Gene encoding
Genomic Library
Leishmania
Enzyme activity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Wilson, K., Collart, F. R., Huberman, E., Stringer, J. R., & Ullman, B. (1991). Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani. Journal of Biological Chemistry, 266(3), 1665-1671.

Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani. / Wilson, Keith; Collart, Frank R.; Huberman, Eliezer; Stringer, James R.; Ullman, Buddy.

In: Journal of Biological Chemistry, Vol. 266, No. 3, 25.01.1991, p. 1665-1671.

Research output: Contribution to journalArticle

Wilson, K, Collart, FR, Huberman, E, Stringer, JR & Ullman, B 1991, 'Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani', Journal of Biological Chemistry, vol. 266, no. 3, pp. 1665-1671.
Wilson K, Collart FR, Huberman E, Stringer JR, Ullman B. Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani. Journal of Biological Chemistry. 1991 Jan 25;266(3):1665-1671.
Wilson, Keith ; Collart, Frank R. ; Huberman, Eliezer ; Stringer, James R. ; Ullman, Buddy. / Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 3. pp. 1665-1671.
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