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The cloning and characterization of the human transcobalamin II gene
A Regec, EV Quadros, O Platica and SP Rothenberg
Division of Hematology/Oncology, State University of New York (SUNY)-
Health Science Center, Brooklyn 11203, USA.
Transcobalamin II (TCII) is a plasma protein that binds vitamin B12
(cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by
receptor-mediated endocytosis. In genetic disorders that are characterized
by congenital deficiency of TCII, intracellular Cbl deficiency occurs,
resulting in an early onset of megaloblastic anemia that is sometimes
accompanied by a neurologic disorder. To define the genetic basis for TCII
deficiency, we have cloned and characterized the human gene that encodes
this protein. The gene spans a minimum of 18 kbp and contains nine exons
and eight introns, with a polyadenylation signal sequence located 509 bp
downstream from the termination codon and a transcription initiation site
beginning 158 bp upstream from the ATG translation start site. The 5'
flanking DNA does not have a TATA or CCAAT regulatory element, but a
34-nucleotide stretch beginning just upstream of the CAP site contains four
tandemly organized 5'-CCCC-3' tetramers. This sequence is a motif for a
trans-active transcription factor (ETF) that regulates expression of the
epidermal growth factor receptor gene (EGFR), which also lacks TATA and
CCAAT regulatory elements. A GC-rich sequence that binds the SP1 protein is
located 356 nucleotides upstream from the first of the series of CCCC
tetramers. Although this GC sequence is at an unusual location with respect
to the CAP site, a 507-bp fragment containing this GC box drives the
chloramphenicol acetyltransferase (CAT) reporter gene after transient
transfection into NIH 3T3 cells. No CAT activity was observed when a 420-bp
fragment lacking this GC box but containing the ETF-binding domains was
similarly transfected into this cell line. One consensus and two atypical
motifs for the c-myc ligand are located downstream and upstream,
respectively, of the GC box, and this could explain the elevated plasma
TCII observed in some patients with multiple myeloma, as the c-myc product
is overexpressed in some myeloma cells. Restriction endonuclease digestion
of genomic DNA from eight normal subjects with Taq I, Hinfl, Msp I, and Bgl
I identified three patterns of restriction fragment length polymorphism
(RFLP). A number of the exon/intron splice junctions of human TCII, TCI,
and IF genes are located in homologous regions of these proteins, providing
evidence that these genes have evolved by duplication of an ancestral gene.
This characterization of the TCII gene and the RFLP should facilitate the
identification of the mutation(s) responsible for the genetic abnormalities
of TCII expression.
Volume 85,
Issue 10,
pp. 2711-2719,
05/15/1995
Copyright © 1995 by The American Society of Hematology
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