Characterization of two cDNAs encoding folate-binding proteins from L1210 murine leukemia cells: Increased expression associated with a genomic rearrangement

Kevin E. Brigle, Eric H. Westin, Micah T. Houghton, I. David Goldman

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Abstract

L1210 murine leukemic cells grown under conditions of continuous low folate concentrations acquire increased levels of a high affinity/low capacity folate-binding protein (FBP). Using an oligonucleotide probe complementary to the human FBP, we have cloned and sequenced two murine FBP cDNAs isolated from a library constructed using a L1210 subline adapted for growth on low levels of 5-formyltetrahydrofolate. The encoding proteins, designated FBP1 and FBP2, have predicted Mr values of 29,415 and 28,821, respectively. These proteins share significant sequence identity with each other (70%) and with the deduced sequences of the human- and bovine-encoded FBPs (68-79%). Southern blot analysis of the low folate-adapted cell line revealed that, while neither of the two FBP-encoding genes was amplified, the FBP1 genomic locus had undergone rearrangement. On Northern blot analysis, this rearrangement was reflected in the enhanced expression (>100-fold) of a FBP1-specific transcript which was 200 nucleotides shorter than the corresponding L1210 parental transcript. The increased expression of this transcript coincided with the increased expression of a membrane protein (Mr= 38,000) which could be affinity-labeled with a N-hydroxysuccinimide ester of [3H]folate. Accordingly, the FBP1 transcript appears to encode the high affinity/ low capacity FBP. Compared to parental L1210 cells, expression of the FBP2-encoding transcript was unchanged in this cell line and, while the exact nature of the protein is unclear, FBP2 may represent a fetal form of the FBP.

Original languageEnglish (US)
Pages (from-to)17243-17249
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number26
StatePublished - Sep 15 1991
Externally publishedYes

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Leukemia L1210
Folic Acid
Carrier Proteins
Complementary DNA
Cells
Cell Line
Proteins
Gene encoding
Leucovorin
Oligonucleotide Probes
Southern Blotting
Northern Blotting
Libraries
Membrane Proteins
Esters
Nucleotides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of two cDNAs encoding folate-binding proteins from L1210 murine leukemia cells : Increased expression associated with a genomic rearrangement. / Brigle, Kevin E.; Westin, Eric H.; Houghton, Micah T.; Goldman, I. David.

In: Journal of Biological Chemistry, Vol. 266, No. 26, 15.09.1991, p. 17243-17249.

Research output: Contribution to journalArticle

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abstract = "L1210 murine leukemic cells grown under conditions of continuous low folate concentrations acquire increased levels of a high affinity/low capacity folate-binding protein (FBP). Using an oligonucleotide probe complementary to the human FBP, we have cloned and sequenced two murine FBP cDNAs isolated from a library constructed using a L1210 subline adapted for growth on low levels of 5-formyltetrahydrofolate. The encoding proteins, designated FBP1 and FBP2, have predicted Mr values of 29,415 and 28,821, respectively. These proteins share significant sequence identity with each other (70{\%}) and with the deduced sequences of the human- and bovine-encoded FBPs (68-79{\%}). Southern blot analysis of the low folate-adapted cell line revealed that, while neither of the two FBP-encoding genes was amplified, the FBP1 genomic locus had undergone rearrangement. On Northern blot analysis, this rearrangement was reflected in the enhanced expression (>100-fold) of a FBP1-specific transcript which was 200 nucleotides shorter than the corresponding L1210 parental transcript. The increased expression of this transcript coincided with the increased expression of a membrane protein (Mr= 38,000) which could be affinity-labeled with a N-hydroxysuccinimide ester of [3H]folate. Accordingly, the FBP1 transcript appears to encode the high affinity/ low capacity FBP. Compared to parental L1210 cells, expression of the FBP2-encoding transcript was unchanged in this cell line and, while the exact nature of the protein is unclear, FBP2 may represent a fetal form of the FBP.",
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N2 - L1210 murine leukemic cells grown under conditions of continuous low folate concentrations acquire increased levels of a high affinity/low capacity folate-binding protein (FBP). Using an oligonucleotide probe complementary to the human FBP, we have cloned and sequenced two murine FBP cDNAs isolated from a library constructed using a L1210 subline adapted for growth on low levels of 5-formyltetrahydrofolate. The encoding proteins, designated FBP1 and FBP2, have predicted Mr values of 29,415 and 28,821, respectively. These proteins share significant sequence identity with each other (70%) and with the deduced sequences of the human- and bovine-encoded FBPs (68-79%). Southern blot analysis of the low folate-adapted cell line revealed that, while neither of the two FBP-encoding genes was amplified, the FBP1 genomic locus had undergone rearrangement. On Northern blot analysis, this rearrangement was reflected in the enhanced expression (>100-fold) of a FBP1-specific transcript which was 200 nucleotides shorter than the corresponding L1210 parental transcript. The increased expression of this transcript coincided with the increased expression of a membrane protein (Mr= 38,000) which could be affinity-labeled with a N-hydroxysuccinimide ester of [3H]folate. Accordingly, the FBP1 transcript appears to encode the high affinity/ low capacity FBP. Compared to parental L1210 cells, expression of the FBP2-encoding transcript was unchanged in this cell line and, while the exact nature of the protein is unclear, FBP2 may represent a fetal form of the FBP.

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