The gain-of-function chinese hamster ovary mutant LEC11B expresses one of two chinese hamster FUT6 genes due to the loss of a negative regulatory factor

Aimin Zhang, Barry Potvin, Ari Zaiman, Wei Chen, Ravindra Kumar, Laurie Phillips, Pamela Stanley

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Abstract

The LEC11 Chinese hamster ovary (CHO) gain-offunction mutant expresses an α(1,3)fucosyltransferase (α(1,3)Fuc-T) activity that generates the Le(X), sialyl-Le(X), and VIM-2 glycan determinants and has been extensively used for studies of E-selectin ligand specificity. In order to identify regulatory mechanisms that control α(1,3)Fuc-T expression in mammals, mechanisms of FUT gene expression were investigated in LEC11 cells and two new, independent mutants, LEC11A and LEC11B. Northern and ribonuclease protection analyses, using probes that span the coding region of a cloned CHO FUT gene, detected transcripts in each LEC11 mutant but not in CHO cells or other gain-of-function CHO mutants that express a different α(1,3)Fuc-T activity. Coding region sequence analysis and α(1,3)Fuc-T acceptor specificity comparisons with recombinant human Fuc-TV and FucTVI showed that the cloned FUT gene is orthologous to the human FUT6 gene. Southern analyses identified two closely related FUT6 genes in the Chinese hamster, whose evolutionary relationships are discussed. The blots showed that rearrangements had occurred in LEC11A and LEC11 genomic DNA, consistent with a cis mechanism of FUT6 gene activation in these mutants. By contrast, somatic cell hybrid analyses revealed that LEC11B cells express FUT6 gene transcripts due to the loss of a trans-acting, negative regulatory factor. Sequencing of reverse transcriptase-polymerase chain reaction products identified unique 5'- and 3'-untranslated region sequences in FUT6 gene transcripts from each LEC11 mutant. Northern and Southern analyses with gene- specific probes showed that LEC11A cells express only the cgFUT6A gene (where cg is Cricetulus griseus), whereas LEC11 and LEC11B cells express only the cgFUT6B gene. In LEC11A x LEC11B hybrid cells, the cgFUT6A gene was predominantly expressed, as predicted if a trans-acting negative regulatory factor functions to suppress cgFUT6B gene expression in CHO cells. This factor is predicted to be a cell type-specific regulator of FUT6 gene expression in mammals.

Original languageEnglish (US)
Pages (from-to)10439-10450
Number of pages12
JournalJournal of Biological Chemistry
Volume274
Issue number15
DOIs
StatePublished - Apr 9 1999

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Cricetulus
Ovary
Genes
Gene expression
Hybrid Cells
galactoside 3-fucosyltransferase
Mammals
Gene Expression
E-Selectin
5' Untranslated Regions
3' Untranslated Regions
Regulator Genes
Ribonucleases
Polymerase chain reaction
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Transcriptional Activation
Reaction products
Polysaccharides
Sequence Analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

The gain-of-function chinese hamster ovary mutant LEC11B expresses one of two chinese hamster FUT6 genes due to the loss of a negative regulatory factor. / Zhang, Aimin; Potvin, Barry; Zaiman, Ari; Chen, Wei; Kumar, Ravindra; Phillips, Laurie; Stanley, Pamela.

In: Journal of Biological Chemistry, Vol. 274, No. 15, 09.04.1999, p. 10439-10450.

Research output: Contribution to journalArticle

Zhang, Aimin ; Potvin, Barry ; Zaiman, Ari ; Chen, Wei ; Kumar, Ravindra ; Phillips, Laurie ; Stanley, Pamela. / The gain-of-function chinese hamster ovary mutant LEC11B expresses one of two chinese hamster FUT6 genes due to the loss of a negative regulatory factor. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 15. pp. 10439-10450.
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abstract = "The LEC11 Chinese hamster ovary (CHO) gain-offunction mutant expresses an α(1,3)fucosyltransferase (α(1,3)Fuc-T) activity that generates the Le(X), sialyl-Le(X), and VIM-2 glycan determinants and has been extensively used for studies of E-selectin ligand specificity. In order to identify regulatory mechanisms that control α(1,3)Fuc-T expression in mammals, mechanisms of FUT gene expression were investigated in LEC11 cells and two new, independent mutants, LEC11A and LEC11B. Northern and ribonuclease protection analyses, using probes that span the coding region of a cloned CHO FUT gene, detected transcripts in each LEC11 mutant but not in CHO cells or other gain-of-function CHO mutants that express a different α(1,3)Fuc-T activity. Coding region sequence analysis and α(1,3)Fuc-T acceptor specificity comparisons with recombinant human Fuc-TV and FucTVI showed that the cloned FUT gene is orthologous to the human FUT6 gene. Southern analyses identified two closely related FUT6 genes in the Chinese hamster, whose evolutionary relationships are discussed. The blots showed that rearrangements had occurred in LEC11A and LEC11 genomic DNA, consistent with a cis mechanism of FUT6 gene activation in these mutants. By contrast, somatic cell hybrid analyses revealed that LEC11B cells express FUT6 gene transcripts due to the loss of a trans-acting, negative regulatory factor. Sequencing of reverse transcriptase-polymerase chain reaction products identified unique 5'- and 3'-untranslated region sequences in FUT6 gene transcripts from each LEC11 mutant. Northern and Southern analyses with gene- specific probes showed that LEC11A cells express only the cgFUT6A gene (where cg is Cricetulus griseus), whereas LEC11 and LEC11B cells express only the cgFUT6B gene. In LEC11A x LEC11B hybrid cells, the cgFUT6A gene was predominantly expressed, as predicted if a trans-acting negative regulatory factor functions to suppress cgFUT6B gene expression in CHO cells. This factor is predicted to be a cell type-specific regulator of FUT6 gene expression in mammals.",
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T1 - The gain-of-function chinese hamster ovary mutant LEC11B expresses one of two chinese hamster FUT6 genes due to the loss of a negative regulatory factor

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AU - Chen, Wei

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AU - Stanley, Pamela

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