Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning - PubMed (original) (raw)

. 1998 Jul 7;95(14):8175-80.

doi: 10.1073/pnas.95.14.8175.

G Fu, J S Wu, Q H Zhang, J Zhou, L X Kan, Q H Huang, K L He, B W Gu, Z G Han, Y Shen, J Gu, Y P Yu, S H Xu, Y X Wang, S J Chen, Z Chen

Affiliations

Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

M Mao et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34(+) HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34(+) cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2, 603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5' ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.

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Figures

Figure 1

Figure 1

(A) Silico cloning of the full-length cDNA of human MPPB gene. CBDAOH03 and CBDAOE04 are two clones from the CD34+ cell cDNA library. Thirty overlapping dbESTs, as indicated by arrows for their positions with respect to the cDNA structure (top bar), were assembled into a contig containing an ORF (the striated section of the bar). (B) mRNA isoforms for the human SC2 gene. The hSC2 spliced variant was identified from a clone of the CD34+ cell cDNA library whereas hSC2 sequence was obtained by dbEST assembly.

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