Isolation and characterization of genomic sequences involved in the regulation of the human reduced folate carrier gene (RFC1)

Maokai Gong, Kenneth H. Cowan, Jean Gudas, Jeffrey A. Moscow

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Decreased reduced folate carrier (RFC) activity has been associated with MTX resistance in experimental models of transportmediated MTX resistance, and has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain any one of four distinct 5' untranslated regions (UTRs). We cloned a human genomic DNA fragment upstream from the RFC1 translation start site and determined the nucleotide sequence of a 4.8 kb region that contained the exons corresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quantitative RT-PCR assay using primers specific for each of the 5' UTRs to amplify RNA transcripts containing each of the RFC1 5' exons, and found evidence for differential transcription of RFC1 noncoding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also found that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S transition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of expression of exons 1b and 1c of the RFC1 gene. These studies therefore identify regulatory regions of RFC1 promoters and potential models of regulation in which these regions may exert control.

Original languageEnglish (US)
Pages (from-to)21-31
Number of pages11
JournalGene
Volume233
Issue number1-2
DOIs
StatePublished - Jun 11 1999

Fingerprint

Reduced Folate Carrier Protein
Exons
5' Untranslated Regions
RNA
Genes
Untranslated Regions
Nucleic Acid Regulatory Sequences
Cell Cycle
Theoretical Models
Gene Expression
Polymerase Chain Reaction
DNA
Proteins

Keywords

  • Cell cycle regulation
  • Leucovorin
  • Methotrexate
  • Trimetrexate

ASJC Scopus subject areas

  • Genetics

Cite this

Isolation and characterization of genomic sequences involved in the regulation of the human reduced folate carrier gene (RFC1). / Gong, Maokai; Cowan, Kenneth H.; Gudas, Jean; Moscow, Jeffrey A.

In: Gene, Vol. 233, No. 1-2, 11.06.1999, p. 21-31.

Research output: Contribution to journalArticle

@article{1ffa80a15298418cab5e3ae6de6c6553,
title = "Isolation and characterization of genomic sequences involved in the regulation of the human reduced folate carrier gene (RFC1)",
abstract = "Decreased reduced folate carrier (RFC) activity has been associated with MTX resistance in experimental models of transportmediated MTX resistance, and has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain any one of four distinct 5' untranslated regions (UTRs). We cloned a human genomic DNA fragment upstream from the RFC1 translation start site and determined the nucleotide sequence of a 4.8 kb region that contained the exons corresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quantitative RT-PCR assay using primers specific for each of the 5' UTRs to amplify RNA transcripts containing each of the RFC1 5' exons, and found evidence for differential transcription of RFC1 noncoding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also found that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S transition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of expression of exons 1b and 1c of the RFC1 gene. These studies therefore identify regulatory regions of RFC1 promoters and potential models of regulation in which these regions may exert control.",
keywords = "Cell cycle regulation, Leucovorin, Methotrexate, Trimetrexate",
author = "Maokai Gong and Cowan, {Kenneth H.} and Jean Gudas and Moscow, {Jeffrey A.}",
year = "1999",
month = "6",
day = "11",
doi = "10.1016/S0378-1119(99)00166-3",
language = "English (US)",
volume = "233",
pages = "21--31",
journal = "Gene",
issn = "0378-1119",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Isolation and characterization of genomic sequences involved in the regulation of the human reduced folate carrier gene (RFC1)

AU - Gong, Maokai

AU - Cowan, Kenneth H.

AU - Gudas, Jean

AU - Moscow, Jeffrey A.

PY - 1999/6/11

Y1 - 1999/6/11

N2 - Decreased reduced folate carrier (RFC) activity has been associated with MTX resistance in experimental models of transportmediated MTX resistance, and has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain any one of four distinct 5' untranslated regions (UTRs). We cloned a human genomic DNA fragment upstream from the RFC1 translation start site and determined the nucleotide sequence of a 4.8 kb region that contained the exons corresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quantitative RT-PCR assay using primers specific for each of the 5' UTRs to amplify RNA transcripts containing each of the RFC1 5' exons, and found evidence for differential transcription of RFC1 noncoding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also found that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S transition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of expression of exons 1b and 1c of the RFC1 gene. These studies therefore identify regulatory regions of RFC1 promoters and potential models of regulation in which these regions may exert control.

AB - Decreased reduced folate carrier (RFC) activity has been associated with MTX resistance in experimental models of transportmediated MTX resistance, and has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain any one of four distinct 5' untranslated regions (UTRs). We cloned a human genomic DNA fragment upstream from the RFC1 translation start site and determined the nucleotide sequence of a 4.8 kb region that contained the exons corresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quantitative RT-PCR assay using primers specific for each of the 5' UTRs to amplify RNA transcripts containing each of the RFC1 5' exons, and found evidence for differential transcription of RFC1 noncoding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also found that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S transition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of expression of exons 1b and 1c of the RFC1 gene. These studies therefore identify regulatory regions of RFC1 promoters and potential models of regulation in which these regions may exert control.

KW - Cell cycle regulation

KW - Leucovorin

KW - Methotrexate

KW - Trimetrexate

UR - http://www.scopus.com/inward/record.url?scp=0032807628&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032807628&partnerID=8YFLogxK

U2 - 10.1016/S0378-1119(99)00166-3

DO - 10.1016/S0378-1119(99)00166-3

M3 - Article

C2 - 10375617

AN - SCOPUS:0032807628

VL - 233

SP - 21

EP - 31

JO - Gene

JF - Gene

SN - 0378-1119

IS - 1-2

ER -