Abstract
In our previous report, we described the structural organizations of glycophorin A and B genes (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623). During the course of isolation of these genomic clones, we also obtained genomic clones encoding a novel glycophorin. This novel glycophorin, termed glycophorin E (GPE), has a similar genomic structure to that of the GPB gene, and its nucleotide sequence is almost identical to that of the GPB gene. These sequences include a region downstream of an Alu repeat sequence, which has been suggested to be a site for homologous recombination in the GPB gene during or after gene duplication. However, the predicted GPE amino acid sequence specifies blood group M, in contrast to GPB which carries blood group N. Polymerase chain reaction was employed to analyze the transcript of this gene, and its cDNA sequence revealed that the novel glycophorin gene encodes 78 amino acids, including a 19-residue leader peptide. Comparison of genomic and complementary DNAs demonstrates that this gene consists of four exons, and point mutations at sites corresponding to the 5'-splicing sites of intron 3 and intron 4 of the GPA gene lead to the joining of the exon 2 to potential exon 5. Interestingly, an insertion of 24 nucleotides coding for eight amino acid residues in-frame was found in exon 5. The predicted amino acid sequence within this exon indicates that it has a hydrophobic character, suggesting the possible expression of GPE as a membrane protein. Northern blot analysis demonstrated that this novel glycophorin gene is expressed in an erythroid-specific manner and coordinately down-regulated together with GPA and GPB genes by a tumor-promoting phorbol ester. During evolution, this gene might have derived from an ancestral gene common to the GPB gene by gene duplication and subsequent nucleotide substitutions, and constitutes a member of a gene family with GPA and GPB genes.
Highlights
During the course of isolation of these genomic clones, we obtained genomic clones encoding a novel glycophorin
Ah repeat sequence, which has been suggested to be a site for homologous recombination in the GPB gene during or after gene duplication
Polymerase chain reaction was employed to analyze the transcript of this gene, and its cDNA sequence revealed that the novel glycophorin gene encodes 78 amino acids, including a 19-residue leader peptide
Summary
Cell Culture-Human erythroleukemic cell line K562 and human promyelocytic cell line HL-60 were grown in suspension culture in RPM1 1640 medium supplemented with 2 mM glutamine and 10%. NuSieve GTG agarose gel (FMC Corp.), and amplified fragments were analyzed by Southern blot [19] hybridization using GPB cDNA (HGpB-1; Ref. 7) and INS oligonucleotide, corresponding to a 24-bp insertion sequence found in the exon encoding a potential transmembrane domain in the novel glycophorin gene (see below), as probes. Colonies on the replica plates were incubated at 37 “C for 2 h and subjected to colony hybridization using an INS oligonucleotide probe according to the method described previously [22]. Genomic DNAs in Bluescript were sequenced using synthetic oligonucleotides as primers and complementary DNAs in pUC13 were sequenced using Ml3 universal and reverse primers Sequences obtained by these methods were analyzed by Beckman Microgenie software. 0.16 to 1.8 kb and 0.24 to 9.5 kb
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