Expression of D930015E06Rik during erythroid terminal differentiation
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Erythroid terminal differentiation involves differentiation of nucleated precursor cells (erythroblasts) into enucleated, biconcave red blood cells. Previous studies on erythroid terminal differentiation performed in our lab have identified a novel cDNA in erythroblasts, designated Late Erythroblast 3 that shares 99% homology to D930015E06Rik. D930015E06Rik is a gene predicted to exist on mouse chromosome 3 and shares little homology with previously described murine genes. The function and cellular location of D930015E06Rik are unknown. Genes of unknown function homologous to D930015E06Rik have been predicted in the genomes of other species, including humans. Multiple clones spanning the coding region of D930015E06Rik were isolated from erythroblasts and sequenced. Sequence alignment of the experimentally compiled D930015E06Rik sequence with the D930015E06Rik sequence in Genbank initially demonstrated eight potential discrepancies. Re-sequencing experiments resolved seven of the discrepancies, and identified one point of difference between the predicted and experimentally deduced sequence of D930015E06Rik that resulted in an amino acid substitution at position 563(S563A) . In addition, evidence for alternate splicing of exon 29 of D930015E06Rik was demonstrated. A curated amino acid sequence of D930015E06Rik was thus deduced. Overlap-extension PCR was used to synthesize the D930015E06Rik coding region; to be used further for cloning into a mammalian expression vector. The long term goal being development of a positive control in order to 1) examine the effect of overexpression of D930015E06Rik in erythroblasts and 2) to standardize the detection of the D930015E06Rik protein on western blots and by immunofluorescence.