ISOLATION OF UNKNOWN GENES FROM HUMAN BONE-MARROW BY DIFFERENTIAL SCREENING AND SINGLE-PASS CDNA SEQUENCE DETERMINATION

A cDNA sequencing project was initiated to characterize gene expressio n in human bone marrow and develop strategies to isolate novel genes. Forty eight random cDNAs from total human bone marrow were subjected t o single-pass DNA sequence analysis to determine a limited complexity of mRNAs expressed in the bone marrow. Overall, 8 cDNAs (17%) showed n o similarity to known sequences. Information from DNA sequence analysi s was used to develop a differential prescreen to subtract unwanted cD NAs and to enrich for unknown cDNAs. Forty-eight cDNAs that were negat ive with a complex probe were subject to single-pass DNA sequence dete rmination. Of these prescreened cDNAs, the number of unknown sequences increased to 23 (48%). Unknown cDNAs were also characterized by RNA e xpression analysis using 25 different human leukemic cell lines. Of 13 unknown cDNAs tested, 10 were expressed in all cell types tested and 3 revealed a hematopoietic lineage-restricted expression pattern. Inte restingly, while a total of only 96 bone marrow cDNAs were sequenced, 31 of these cDNAs represent sequences from unknown genes and 12 showed significant similarities to sequences in the data bases. One cDNA rev ealed a significant similarity to a serine/threonine-protein kinase at the amino acid level (56% identity for 123 amino acids) and may repre sent a previously unknown kinase. Differential screening techniques co upled with single-pass cDNA sequence analysis may prove to be a powerf ul and simple technique to examine developmental gene expression.