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Comparative Study
, 10 (10), 1546-60

Cloning and Functional Analysis of cDNAs With Open Reading Frames for 300 Previously Undefined Genes Expressed in CD34+ Hematopoietic Stem/Progenitor Cells

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Comparative Study

Cloning and Functional Analysis of cDNAs With Open Reading Frames for 300 Previously Undefined Genes Expressed in CD34+ Hematopoietic Stem/Progenitor Cells

Q H Zhang et al. Genome Res.

Abstract

Three hundred cDNAs containing putatively entire open reading frames (ORFs) for previously undefined genes were obtained from CD34+ hematopoietic stem/progenitor cells (HSPCs), based on EST cataloging, clone sequencing, in silico cloning, and rapid amplification of cDNA ends (RACE). The cDNA sizes ranged from 360 to 3496 bp and their ORFs coded for peptides of 58-752 amino acids. Public database search indicated that 225 cDNAs exhibited sequence similarities to genes identified across a variety of species. Homology analysis led to the recognition of 50 basic structural motifs/domains among these cDNAs. Genomic exon-intron organization could be established in 243 genes by integration of cDNA data with genome sequence information. Interestingly, a new gene named as HSPC070 on 3p was found to share a sequence of 105bp in 3' UTR with RAF gene in reversed transcription orientation. Chromosomal localizations were obtained using electronic mapping for 192 genes and with radiation hybrid (RH) for 38 genes. Macroarray technique was applied to screen the gene expression patterns in five hematopoietic cell lines (NB4, HL60, U937, K562, and Jurkat) and a number of genes with differential expression were found. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the function of genes involved in hematopoietic development and differentiation.

Figures

Figure 1
Figure 1
Homology comparison of ORFs contained in our cDNAs to known genes from different model organisms. The horizonal blocks represent numbers of the ORFs bearing homology to genes in a given species; different colors indicate the degree of homology. Each number listed at right indicates the total number of our ORFs having homologous genes in that organism. (B) Bacteria; (Y) yeast; (C) C. elegans; (D) Drosophila; (A) Arabidopsis; (M) mammals not including primates.
Figure 2
Figure 2
(A) Alternative splicing present in lysophospholipase gene transcripts as long (L, accession no. AF077198) and short (S, accession no. AF077199) forms. The numbers indicate the amino acid positions of deduced proteins. Note that the ORF is maintained in the alternatively spliced S isoform. (B) Overlapping of HSPC070 (accession no. AF161555) and RAF genes located on opposite DNA strands at the same locus. Both genes are mapped to the same region on chromosome 3p25. The comparison of sequences between cDNAs and genomic DNA has allowed the exon–intron structure of both genes to be established, with exons represented by boxes and their numbers indicated. Note that a stretch of 105 bp is shared by the 3′ UTRs of both genes. Arrows indicate the orientations of transcription.
Figure 3
Figure 3
Chromosome localization of 230 previously undefined genes by applying both STS searching and radiation hybrid (those marked with #). Detailed mapping information can be obtained from http://www.chgc.sh.cn
Figure 4
Figure 4
Regression analysis of the cDNA array results (A,B) and Northern blot analysis of three cDNAs (C). (A) The scatterplot of detected signal intensity for duplicate spots on the same membrane. (B) Scatterplot of detected signal intensity for the corresponding dots in two membranes with independent tests from RNA of same cell origin. All signals are normalized by using GAPDH gene as internal control. The figures were made with Microsoft Excel spread sheet and the correlation line was indicated. (C) Northern blot analysis of HSPC070, ZNF254, and HSPC135. (Top) HSPC070 with a ubiquitous tissue expression pattern. GAPDH or 28S/18S ribosomal RNAs were used as sample loading control. (Bottom) Expression of ZNF254 and HSPC135 in hematopoietic cell lines, with GAPDH as control.

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