Dataset Information

Molecular cloning, chromosomal assignment and tissue-specific expression of a murine alpha(1,2)fucosyltransferase expressed in thymic and epididymal epithelial cells.

ABSTRACT: Terminal Fucalpha(1-2)Galbeta epitopes have been proposed to play significant roles in cell-cell interactions in development, cell adhesion, and malignant transformation. To begin to investigate the regulation and function of alpha(1-2)fucosylated epitopes in an animal model, we have isolated and characterized a mouse genomic DNA segment encoding a protein orthologous to the human H blood group locus alpha(1,2)fucosyltransferase (FUT1). This segment maintains an open reading frame encoding 376 amino acids sharing 75% sequence identity with the enzyme encoded by human FUT1, and 55% sequence identity with the enzyme encoded by the human Secretor blood group locus (FUT2). Expression of the open reading frame in COS-7 cells yields an alpha(1,2)fucosyltransferase activity with a Km of 7.6 mM for phenyl-beta-d-galactoside. Southern blotting and interspecific backcross analyses indicate that this murine locus represents a single copy sequence mapping to a novel locus 2.1 centimorgans from the Klk1 locus, in a region of homology between mouse chromosome 7 and the human FUT1 locus on the long arm of chromosome 19. Mouse FUT1 yields a 2.8 kb mRNA transcript identifiable in many organs, including thymus, lung, stomach, pancreas, small intestine, colon, uterus and epidiymis. Hybridization analyses in situ localize expression of FUT1 transcripts to thymic medullary and epididymal epithelial cells, implying that this gene determines the expression of cell surface Fucalpha(1-2)Galbeta epitopes in these tissues.

SUBMITTER: Domino SE

PROVIDER: S-EPMC1218769 | biostudies-other | 1997 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundThe rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.ResultsThe cDNAs encoding the rat ?1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a ?1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.ConclusionAltogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.

Dataset Information

Molecular cloning, chromosomal assignment and tissue-specific expression of a murine alpha(1,2)fucosyltransferase expressed in thymic and epididymal epithelial cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure