[Subtractive cDNA cloning and analysis of murine erythroid terminal differentiation related factor]

Cloning and identification of cDNA related to erythroid terminal differentiation factor (MEDRF).

cDNA related to erythroid terminal differentiation from the Friend virus anemia (FVA) infected splenic erythroblasts of BALB/c mice were performed by using subtractive hybridization combined with PCR technique. The splenic proerythroblasts isolated were cultured in the presence of erythropoietin for 36 hrs. Subtractive cDNA clones of differentially expressed in the 36 hrs erythroblasts (sub cDNA-36) but absent in the uncultured proerythroblasts were observed. The sub cDNA-36 was then used for construction of subtractive cDNA library using the Bluescript-SK(+) phage vector system, and differentially screened by 32P-labelled PCR generated probes. The positive clones were analyzed by nucleotide sequencing.

The results indicated that a 472 bp cDNA fragment which contained a 309 bp reading frame from 51 to 359 coding 102 amino acids was identified and it has been accepted by GenBank as a new cDNA sequence that without comparable homology of existing sequences (accession number: AA114369). Northern blot analysis demonstrated that it was differentially expressed in the 36 hrs cultured intermediate-late stages of erythroblasts.

The newly found cDNA, which expresses specifically in intermediate-late stages of erythroblasts, not in stages of proerythroblasts, may be a new gene related to murine erythroid terminal differentiation.