Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei

Thomas E. Allen, Buddy Ullman

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a Mr = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21-23 % amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P. falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into Sφ606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin.

Original languageEnglish (US)
Pages (from-to)5431-5438
Number of pages8
JournalNucleic Acids Research
Volume21
Issue number23
StatePublished - Nov 25 1993

Fingerprint

Hypoxanthine Phosphoribosyltransferase
Trypanosoma brucei brucei
Cloning
Organism Cloning
Enzymes
Genes
Gene
Nucleotides
Amino acids
African Trypanosomiasis
Affinity chromatography
Parasitic Diseases
Trypanosomiasis
Sequence Analysis
Amino Acid Sequence
Chromatography
Clone
Homogeneity
Escherichia coli
Escherichia Coli

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei. / Allen, Thomas E.; Ullman, Buddy.

In: Nucleic Acids Research, Vol. 21, No. 23, 25.11.1993, p. 5431-5438.

Research output: Contribution to journalArticle

@article{88f60f6030264a5282c8a6ce9460e010,
title = "Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei",
abstract = "The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a Mr = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21-23 {\%} amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P. falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into Sφ606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin.",
author = "Allen, {Thomas E.} and Buddy Ullman",
year = "1993",
month = "11",
day = "25",
language = "English (US)",
volume = "21",
pages = "5431--5438",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "23",

}

TY - JOUR

T1 - Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei

AU - Allen, Thomas E.

AU - Ullman, Buddy

PY - 1993/11/25

Y1 - 1993/11/25

N2 - The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a Mr = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21-23 % amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P. falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into Sφ606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin.

AB - The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a Mr = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21-23 % amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P. falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into Sφ606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin.

UR - http://www.scopus.com/inward/record.url?scp=0027438369&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027438369&partnerID=8YFLogxK

M3 - Article

VL - 21

SP - 5431

EP - 5438

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 23

ER -