IDENTIFICATION OF A NOVEL DNA-SEQUENCE DIFFERENTIALLY EXPRESSED BETWEEN NORMAL HUMAN CD34(-CELLS()CD38(HI) AND CD34(+)CD38(LO) MARROW)

We have applied the recently developed differential display method to extend the molecular characterization of the less mature CD34(+)CD38(l o) bone marrow progenitors in comparison with the CD34(+)CD38(hi) cell s to better understand their functional differences. Immunomagnetic en richment of CD34(+) cells followed by flow cytometry was used to isola te CD34(+)CD38(lo) and CD34(+)CD38(hi) cells from human organ donor bo ne marrow, A limited set of the poly A+ RNA sequences present in these two cell populations was amplified by a combination of reverse transc ription with an anchored oligo dT-based primer and polymerase chain re action with the same oligo dT primer and arbitrary decamers. A radioac tive tracer allowed these sequences to be displayed as a series of ban ds on a denaturing polyacrylamide gel, Eight bands were chosen that ap peared in multiple displays to represent gene sequences differentially expressed between CD34(+)CD38(hi) and CD34(+)CD38(lo) cells. Comparis on of the sequences with public DNA sequence databases available ident ified one sequence as myeloperoxidase. Two other clones matched sequen ce fragments of unknown function, whereas the remaining five are novel sequences not present in existing databases, The relative level of ex pression of all of the sequences was tested by an independent reverse transcriptase-polymerase chain reaction with sequence-specific oligonu cleotide primers. The lower level of myeloperoxidase mRNA in CD34(+)CD 38(lo) cells was confirmed, as was the higher expression of the novel sequence 345. Sequence 345 expression is highest in CD34(+)CD38(-) cel ls and decreases with increased CD38 expression, It is expressed in ne gligible amounts in hematopoietic cell lines and other sources of huma n tissue, suggesting it may have a functional role in normal hematopoi esis. (C) 1995 by The American Society of Hematology.