Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani

Thomas E. Allen, Ho Yon Hwang, Armando Jardim, Robert Olafson, Buddy Ullman

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

The gene encoding the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani has been cloned and sequenced. The hgprt open reading frame encoded a polypeptide of 211 amino acids that exhibited 3 regions of significant homology with other eukaryotic HGPRTs and a C-terminal tripeptide compatible with a glycosomal targeting signal. Northern blot analysis of L. donovani RNA revealed two hgprt transcripts, a 1.9-kb mRNA and a 1.7-kb transcript. The expression of the 1.7-kb hgprt mRNA and the activity of HGPRT enzyme were both augmented approx. 5-fold in parasites incubated in the absence of purines. Southern blots of genomic DNA indicated only a single hgprt locus within the L. donovani genome. Overexpression of L. donovani hgprt in E. coli complemented genetic deficiencies in hypoxanthine and guanine phosphoribosylating activities and yielded abundant quantities of enzymatically active HGPRT. The recombinant HGPRT was purified to homogeneity and recognized hypoxanthine, guanine and allopurinol, but not adenine or xanthine, as substrates. The hgprt clone and pure HGPRT protein provide essential reagents for validating HGPRT as a therapeutic target for the treatment of leishmaniasis and other diseases of parasitic origin.

Original languageEnglish (US)
Pages (from-to)133-143
Number of pages11
JournalMolecular and Biochemical Parasitology
Volume73
Issue number1-2
DOIs
StatePublished - 1995

Fingerprint

Hypoxanthine Phosphoribosyltransferase
Leishmania donovani
Organism Cloning
Hypoxanthine
Guanine
Purines
Messenger RNA
Parasitic Diseases
Allopurinol
Xanthine
Leishmaniasis
Adenine
Enzymes
Southern Blotting
Northern Blotting
Open Reading Frames
Parasites
Clone Cells
Genome
RNA

Keywords

  • Drug development
  • Expression
  • Hypoxanthine-guanine phosphoribosyltransferase
  • Leishmania
  • Molecular cloning

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani. / Allen, Thomas E.; Hwang, Ho Yon; Jardim, Armando; Olafson, Robert; Ullman, Buddy.

In: Molecular and Biochemical Parasitology, Vol. 73, No. 1-2, 1995, p. 133-143.

Research output: Contribution to journalArticle

Allen, Thomas E. ; Hwang, Ho Yon ; Jardim, Armando ; Olafson, Robert ; Ullman, Buddy. / Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani. In: Molecular and Biochemical Parasitology. 1995 ; Vol. 73, No. 1-2. pp. 133-143.
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