THE CLONING AND CHARACTERIZATION OF THE HUMAN TRANSCOBALAMIN-II GENE

Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (c obalamin; Cbl) and facilitates the cellular uptake of the vitamin by r eceptor-mediated endocytosis. In genetic disorders that are characteri zed by congenital deficiency of TCII, intracellular Cbl deficiency occ urs, resulting in an early onset of megaloblastic anemia that is somet imes 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 cont ains nine exons and eight introns, with a polyadenylation signal seque nce located 509 bp downstream from the termination codon and a transcr iption initiation site beginning 158 bp upstream from the ATG translat ion start site. The 5' flanking DNA does not have a TATA or CCAAT regu latory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5'-CCCC-3' tetramers. T his 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-ric h sequence that binds the SP1 protein is located 356 nucleotides upstr eam from the first of the series of CCCC tetramers. Although this GC s equence is at an unusual location with respect to the CAP site, a 507- bp fragment containing this GC box drives the chloramphenicol acetyltr ansferase (CAT) reporter gene after transient transfection into NIH 3T 3 cells. No CAT activity was observed when a 420-bp fragment lacking t his GC box but containing the ETF-binding domains was similarly transf ected into this cell line. One consensus and two atypical motifs for t he c-myc ligand are located downstream and upstream, respectively, of the GC box, and this could explain the elevated plasma TCII observed i n some patients with multiple myeloma, as the c-myc product is overexp ressed in some myeloma cells. Restriction endonuclease digestion of ge nomic DNA from eight normal subjects with Tag I, Hinfl, Msp I, and Bgl I identified three patterns of restriction fragment length polymorphi sm (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 a n ancestral gene. This characterization of the TCII gene and the RFLP should facilitate the identification of the mutation(s) responsible fo r the genetic abnormalities of TCII expression. (C) 1995 by The Americ an Society of Hematology.