Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase-thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum

John R. Vásquez, Lisa Goozé, Kami Kim, Jiří Gut, Carolyn Petersen, Richard G. Nelson

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

We have determined the nucleic acid sequences of a gene encoding the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) from bovine and human AIDS isolates of Cryptosporidium parvum. The DHFR-TS gene was isolated from genomic DNA libraries by hybridization with a probe amplified from C. parvum genomic DNA using generic TS primers in the polymerase chain reaction. Genomic Southern and electrophoretic karyotype analyses reveal C. parvum DHFR-TS is a single-copy gene on a 1200-kb chromosome. The DHFR-TS nucleic acid sequence contains no introns and the single 1563-bp open reading frame encodes a 179 residue N-terminal DHFR domain connected by a 55 aminoacid junction peptide to a 287 residue C-terminal Ts domain. The sequences of the DHFR-TS gene from the bovine and human C. parvum isolates differ at two positions in the 5'-flanking sequence and at 38 positions in the coding sequence. These DNA sequence polymorphisms will provide a powerful probe to examine the genotypic diversity and genetic population structure of C. parvum. The two sequences encode identical TS domains which share all except one of the phylogenetically conserved amino acid residues identified among reported TS sequences. The predicted DHFR domain sequences contain nine amino acid differences; these polymorphisms all map to non-active site, surface locations in known DHFR structures. The C. parvum DHFR active site contains novel residues at several positions analogous to those at which point mutations have been shown to produce antifolate resistance in other DHFRs. Thus C. parvum DHFR may be intrinsically resistant to inhibition by some antifolate-DHFR inhibitors which may explain why cryptosporidiosis is refractory to treatment With the clinically common antibacterial and antiprotozoal anlifolates.

Original languageEnglish (US)
Pages (from-to)153-165
Number of pages13
JournalMolecular and Biochemical Parasitology
Volume79
Issue number2
DOIs
StatePublished - Aug 1996

Fingerprint

Folic Acid Antagonists
Cryptosporidium parvum
Genes
Nucleic Acids
Cryptosporidiosis
Amino Acids
Genomic Library
Genetic Structures
5' Flanking Region
thymidylate synthase-dihydrofolate reductase
Karyotype
Gene Library
Point Mutation
Introns
Open Reading Frames
Catalytic Domain
Acquired Immunodeficiency Syndrome
Chromosomes
Polymerase Chain Reaction
Peptides

Keywords

  • Antifolate resistance
  • Cryptosporidium parvum
  • Dihydrofolate reductase
  • Genotypic variation
  • Intraspecific variation
  • Thymidylate synthase

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase-thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum. / Vásquez, John R.; Goozé, Lisa; Kim, Kami; Gut, Jiří; Petersen, Carolyn; Nelson, Richard G.

In: Molecular and Biochemical Parasitology, Vol. 79, No. 2, 08.1996, p. 153-165.

Research output: Contribution to journalArticle

@article{fb921b5e4d534853b699270807ed05c5,
title = "Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase-thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum",
abstract = "We have determined the nucleic acid sequences of a gene encoding the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) from bovine and human AIDS isolates of Cryptosporidium parvum. The DHFR-TS gene was isolated from genomic DNA libraries by hybridization with a probe amplified from C. parvum genomic DNA using generic TS primers in the polymerase chain reaction. Genomic Southern and electrophoretic karyotype analyses reveal C. parvum DHFR-TS is a single-copy gene on a 1200-kb chromosome. The DHFR-TS nucleic acid sequence contains no introns and the single 1563-bp open reading frame encodes a 179 residue N-terminal DHFR domain connected by a 55 aminoacid junction peptide to a 287 residue C-terminal Ts domain. The sequences of the DHFR-TS gene from the bovine and human C. parvum isolates differ at two positions in the 5'-flanking sequence and at 38 positions in the coding sequence. These DNA sequence polymorphisms will provide a powerful probe to examine the genotypic diversity and genetic population structure of C. parvum. The two sequences encode identical TS domains which share all except one of the phylogenetically conserved amino acid residues identified among reported TS sequences. The predicted DHFR domain sequences contain nine amino acid differences; these polymorphisms all map to non-active site, surface locations in known DHFR structures. The C. parvum DHFR active site contains novel residues at several positions analogous to those at which point mutations have been shown to produce antifolate resistance in other DHFRs. Thus C. parvum DHFR may be intrinsically resistant to inhibition by some antifolate-DHFR inhibitors which may explain why cryptosporidiosis is refractory to treatment With the clinically common antibacterial and antiprotozoal anlifolates.",
keywords = "Antifolate resistance, Cryptosporidium parvum, Dihydrofolate reductase, Genotypic variation, Intraspecific variation, Thymidylate synthase",
author = "V{\'a}squez, {John R.} and Lisa Gooz{\'e} and Kami Kim and Jiř{\'i} Gut and Carolyn Petersen and Nelson, {Richard G.}",
year = "1996",
month = "8",
doi = "10.1016/0166-6851(96)02647-3",
language = "English (US)",
volume = "79",
pages = "153--165",
journal = "Molecular and Biochemical Parasitology",
issn = "0166-6851",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase-thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum

AU - Vásquez, John R.

AU - Goozé, Lisa

AU - Kim, Kami

AU - Gut, Jiří

AU - Petersen, Carolyn

AU - Nelson, Richard G.

PY - 1996/8

Y1 - 1996/8

N2 - We have determined the nucleic acid sequences of a gene encoding the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) from bovine and human AIDS isolates of Cryptosporidium parvum. The DHFR-TS gene was isolated from genomic DNA libraries by hybridization with a probe amplified from C. parvum genomic DNA using generic TS primers in the polymerase chain reaction. Genomic Southern and electrophoretic karyotype analyses reveal C. parvum DHFR-TS is a single-copy gene on a 1200-kb chromosome. The DHFR-TS nucleic acid sequence contains no introns and the single 1563-bp open reading frame encodes a 179 residue N-terminal DHFR domain connected by a 55 aminoacid junction peptide to a 287 residue C-terminal Ts domain. The sequences of the DHFR-TS gene from the bovine and human C. parvum isolates differ at two positions in the 5'-flanking sequence and at 38 positions in the coding sequence. These DNA sequence polymorphisms will provide a powerful probe to examine the genotypic diversity and genetic population structure of C. parvum. The two sequences encode identical TS domains which share all except one of the phylogenetically conserved amino acid residues identified among reported TS sequences. The predicted DHFR domain sequences contain nine amino acid differences; these polymorphisms all map to non-active site, surface locations in known DHFR structures. The C. parvum DHFR active site contains novel residues at several positions analogous to those at which point mutations have been shown to produce antifolate resistance in other DHFRs. Thus C. parvum DHFR may be intrinsically resistant to inhibition by some antifolate-DHFR inhibitors which may explain why cryptosporidiosis is refractory to treatment With the clinically common antibacterial and antiprotozoal anlifolates.

AB - We have determined the nucleic acid sequences of a gene encoding the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) from bovine and human AIDS isolates of Cryptosporidium parvum. The DHFR-TS gene was isolated from genomic DNA libraries by hybridization with a probe amplified from C. parvum genomic DNA using generic TS primers in the polymerase chain reaction. Genomic Southern and electrophoretic karyotype analyses reveal C. parvum DHFR-TS is a single-copy gene on a 1200-kb chromosome. The DHFR-TS nucleic acid sequence contains no introns and the single 1563-bp open reading frame encodes a 179 residue N-terminal DHFR domain connected by a 55 aminoacid junction peptide to a 287 residue C-terminal Ts domain. The sequences of the DHFR-TS gene from the bovine and human C. parvum isolates differ at two positions in the 5'-flanking sequence and at 38 positions in the coding sequence. These DNA sequence polymorphisms will provide a powerful probe to examine the genotypic diversity and genetic population structure of C. parvum. The two sequences encode identical TS domains which share all except one of the phylogenetically conserved amino acid residues identified among reported TS sequences. The predicted DHFR domain sequences contain nine amino acid differences; these polymorphisms all map to non-active site, surface locations in known DHFR structures. The C. parvum DHFR active site contains novel residues at several positions analogous to those at which point mutations have been shown to produce antifolate resistance in other DHFRs. Thus C. parvum DHFR may be intrinsically resistant to inhibition by some antifolate-DHFR inhibitors which may explain why cryptosporidiosis is refractory to treatment With the clinically common antibacterial and antiprotozoal anlifolates.

KW - Antifolate resistance

KW - Cryptosporidium parvum

KW - Dihydrofolate reductase

KW - Genotypic variation

KW - Intraspecific variation

KW - Thymidylate synthase

UR - http://www.scopus.com/inward/record.url?scp=0030221884&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030221884&partnerID=8YFLogxK

U2 - 10.1016/0166-6851(96)02647-3

DO - 10.1016/0166-6851(96)02647-3

M3 - Article

C2 - 8855552

AN - SCOPUS:0030221884

VL - 79

SP - 153

EP - 165

JO - Molecular and Biochemical Parasitology

JF - Molecular and Biochemical Parasitology

SN - 0166-6851

IS - 2

ER -