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 expression in human bone marrow and develop strategies to isolate novel genes. Forty-eight random DNAs from total human bone marrow were subjected to single-pass DNA sequence analysis to determine a limited complexity of mRNAs expressed in the bone marrow. Overall, 8 cDNAs (17%) showed no similarity to known sequences. Information from DNA sequence analysis was used to develop a differential prescreen to subtract unwanted cDNAs and to enrich for unknown cDNAs. Forty-eight cDNAs that were negative with a complex probe were subject to single-pass DNA sequence determination. Of these prescreened cDNAs, the number of unknown sequences increased to 23 (48%). Unknown cDNAs were also characterized by RNA expression 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. Interestingly, 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 revealed a significant similarity to a serine/threonine-protein kinase at the amino acid level (56% identity for 123 amino acids) and may represent a previously unknown kinase. Differential screening techniques coupled with single-pass cDNA sequence analysis may prove to be a powerful and simple technique to examine developmental gene expression.
Orr
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SL
Division of Biology, California Institute of Technology, Pasadena 91125.
Hughes
T P
TP
Sawyers
C L
CL
Kato
R M
RM
Quan
S G
SG
Williams
S P
SP
Witte
O N
ON
Hood
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001853:Bone Marrow / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis*; D005260:Female; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007938:Leukemia; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013552:Swine; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D017346:Protein Serine-Threonine Kinases