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The pattern of gene expression in human CD15+ myeloid progenitor cells

ABSTRACT: We performed a genome-wide analysis of gene expression in primary human CD151 myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes. (Lee S et al. The pattern of gene expression in human CD15+ myeloid progenitor cells. Proc Natl Acad Sci U S A. 98(6):3340-5, 2001; Lee S et al. Gene expression profiles in acute myeloid leukemia with common translocations using SAGE. Proc Natl Acad Sci U S A. 103(4):1030-5, 2006) Human bone-marrow mononuclear cells were isolated from bone marrow with FicollyPaque solution and stored in liquid nitrogen. Cells from three donors were thawed at 37°C, pooled, and used immediately for the isolation of myeloid progenitor cells with CD15 magnetic beads (Dynal, Oslo, Norway). The cells isolated by CD15 magnetic beads were lysed directly with TRIzol reagent for isolation of total RNA. mRNA was purified from 5 mg of total RNA with oligo(dT)25 beads. cDNA was synthesized with a cDNA synthesis kit, and SAGE was performed according to the SAGE protocol with the exceptions described in the original paper. SAGE-tag sequences were collected with the Big-Dye sequencing kit and ABI377 sequencer, and tag sequences were extracted with SAGE 300 software.

ORGANISM(S): Homo sapiens

SUBMITTER: San Ming Wang

PROVIDER: E-GEOD-13007 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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The pattern of gene expression in human CD15+ myeloid progenitor cells.

Lee S S Zhou G G Clark T T Chen J J Rowley J D JD Wang S M SM

Proceedings of the National Academy of Sciences of the United States of America 20010301 6

We performed a genome-wide analysis of gene expression in primary human CD15(+) myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed ...[more]

PMID: 11248080

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Project description:The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment by SAGE analysis. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unabl e to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g., an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modeling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors. SAGE analysis of C. neoformans cells isolated from macrophages The murine macrophage-like cell line J774A.1 was maintained at 37M-BM-0C in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS), 1% nonessential amino acids, 100 M-BM-5g mL-1 penicillin-streptomycin, and 4 mM L-glutamine (Invitrogen). Cryptococcus cells were opsonized with monoclonal antibody 18B7 against capsule (1 M-BM-5g mL-1), and macrophages were treated with recombinant mouse gamma interferon (IFN-gamma) (50 U mL-1) and lipopolysaccharide (LPS) (0.3 M-BM-5g mL-1) prior to co-incubation at a multiplicity of infection (MOI) of 1:1. Macrophages were inoculated with H99 cells and washed after 1 h of inoculation to remove unattached, extracellular fungal cells. After 6 h of incubation, sterile, ice-cold distilled H2O was applied to each well to lyse the macrophages, and the fungal cells (4 x 107) were harvested by centrifugation. H99 control cells were prepared by growth under the same condition but without macrophages, and 108 cell s were harvested. SAGE library construction, sequencing and analysis SAGE library construction, sequencing and analysis were as previously described (Hu et al., 2007; Hu et al., 2008; Steen et al., 2002; Steen et al., 2003). Briefly, RNA was isolated from lyophilized cells by vortexing with glass beads (3.0 mm, acid-washed and RNase-free) for 15 min in 15 mL of TRIZOL extraction buffer (Invitrogen, Carlsbad, CA, USA). The mixture was incubated for 15 min at room temperature and total RNA was isolated according to the manufacturer's instructions. Total RNA was used directly for SAGE library construction as described by Velculescu et al. (1995) using the I-Long SAGE kit (Invitrogen). The tagging enzyme for cDNA digestion was NlaIII and 29 PCR cycles were performed to amplify the ditags during library construction. Colonies were screened by PCR (M13F and M13R primers) to assess the average clone insert size and the percentage of recombinants. Clones from the libraries were sequenced by BigDye primer cycle sequencing on an ABI PRISM 3700 DNA analy zer. Sequence chromatograms were processed using PHRED (Ewing et al., 1998), and vector sequence was detected using Cross_match (Gordon et al., 1998). 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The control (fungal cells alone) and cryptococcal-macrophage interaction SAGE datasets were deposited to the NCBI under accession number GSM986206 and GSM986207 respectively.

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The pattern of gene expression in human CD15+ myeloid progenitor cells

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The pattern of gene expression in human CD15+ myeloid progenitor cells.

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