Global analysis of gene expression in cells of the immune system II. Cell-free translation products and high-density filter hybridization data

Frey, J. R.; Nguyen, Catherine; Houlgatte, Rémi; Jordan, Bertrand; Auffray, Charles; Fol, Bruno; Kuhn, Lotte; Meyer, Stefan; Hartmann, Klaus-Ulrich; Coleclough, Christopher; Lefkovits, Ivan

doi:10.1002/1522-2683(20000701)21:13<2694::aid-elps2694>3.0.co;2-e

Cited by 8 publications

(23 citation statements)

References 25 publications

(8 reference statements)

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“…We have acquired knowledge on nucleic acids and protein functions while developing proteomic/genomic approaches that allow the identification of the genetic inheritance and the modulation of gene expression (9)(10)(11). Since there is no definite time point characterizing gene expression, we have chosen 18 h of IFN-γ macrophage activation since proteomic and biological studies are usually done at this time.…”

Section: Discussionmentioning

confidence: 99%

“…Since this library was not normalized it contains several redundant clones. Nucleotide sequences from all the clones were established and are available to the scientific community upon request (9)(10)(11).…”

Section: Analysis Of Gene Expressionmentioning

confidence: 99%

“…Nylon sheets with 1536 clones in duplicate were prepared as already described (10). Briefly, the robot first merged four 96-well microtiter plates into a single 386-well plate, and each of the 386-well plates was spotted on the nylon sheet in duplicate.…”

Section: Analysis Of Gene Expressionmentioning

confidence: 99%

“…The gene profiling technology adopted for the present study (9)(10)(11) using an array constructed from 1536 individual cDNA clones allowed us to study the mentioned defined portion of expressed genes. The choice of the library is of high relevance to the problem under study since in our library there are mainly immunologically relevant gene probes originating from an immunologically active organ, i.e., the fetal thymus.…”

Section: Introductionmentioning

confidence: 99%

“…The choice of the library is of high relevance to the problem under study since in our library there are mainly immunologically relevant gene probes originating from an immunologically active organ, i.e., the fetal thymus. Not only can changes be detected at the quantitative and qualitative level of gene expression, but it is also possible to identify them by recognizing their protein product since the expressed sequence tags have been established for the individual entities of the cDNA library used for the evaluation of a given mRNA sample by hybridization, and protein products have been analyzed on 2D-SDS gels (10). By using gene expression profiling to evaluate the mRNA content of IFN-γ-activated macrophages we attempted to provide detailed information about up-or down-regulated gene products to be in turn linked to the modulation of biological macrophage functions.…”

Section: Introductionmentioning

confidence: 99%

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Gene expression in IFN-gamma-activated murine macrophages

Pereira

Modolell

Frey³

et al. 2004

Braz J Med Biol Res

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Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-γ activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up-or down-regulated by IFN-γ in A/J (267 and 266 genes, respectively, up-and down-regulated) or BALB/c (297 and 58 genes, respectively, up-and downregulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-γ-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-γ-activated macrophages of resistant mice.

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Section: Discussionmentioning

confidence: 99%

Section: Analysis Of Gene Expressionmentioning

confidence: 99%

Section: Analysis Of Gene Expressionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Gene expression in IFN-gamma-activated murine macrophages

Pereira

Modolell

Frey³

et al. 2004

Braz J Med Biol Res

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Global analysis of gene expression in cells of the immune system I. Analytical limitations in obtaining sequence information on polypeptides in two-dimensional gel spots

Lefkovits¹,

Frey²

2000

Electrophoresis

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We have outlined various aspects and limitations of the collective analysis of protein species of a cell (lymphocyte). We have indicated research directions that, in to our opinion, deserve more attention. We have evaluated mainly the approach used in our laboratory and we recognize that a bulk of important research on the interface of proteomics and genomics remains to be dealt with. It is of great value that we can proceed in our quest by trial and error. But as much as the human genome initiative was not implemented by trial and error, but by formulating new technological approaches, we hope that our approach can be incorporated in the mainstream of proteomics. We need several integrating research directions, some of which are outlined in this communication, namely the use of ordered cDNA libraries, cell-free expression systems, high density filter hybridization, identification of two-dimensional (2-D) gel spots in terms of their amino acid composition through biosynthetic labeling and identification of restriction sites in the corresponding coding sequences. In the accompanying paper the cDNA ordered library approach will be described in some detail.

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Proteomic analysis of rare molecular species of translated polypeptides from a mouse fetal thymus cDNA library

Lefkovits¹,

Frey²

2001

Proteomics

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Proteomic patterns from an ordered cDNA library of mouse fetal thymus origin of an overall complexity of 1536 clones are described. Patterns have been analyzed at 96, 12, or 8 clones in a mixture, or as individual clones. Clones yield in some instances a single spot, in other cases a complex cluster or family of spots is formed. The determination of the clonal address (a six-digit number, indicating the interception of three pooling dimensions) by inspection of pools of 8, 12 or 16 clones is a reliable approach; nevertheless a complete proof consists in retrieving the clone and then submitting to transcription, translation and proteomic analysis. The spot clusters are meaningful clone identifiers; cluster components (families of polypeptides) are characteristic of individual clones and are independent of clones coexisting (and being co-expressed) in a given pool. A 'cluster' originates from a single cloned message and might be due to post-translational modification (offered by the reticuleocyte machinery) or as a result of programmed degradation. Thirteen clones or families of clonal products are shown, and the heterogeneity of the 'appearance' of clones is documented. In about half, the assignment of a clonal polypeptide product to a storage well position has failed; this might be due to a variety of considerations elaborated herein. The arguments are presented, that analysis of abundant proteins of a cell (activated lymphocyte) comprises 'classical proteomics', but for the analysis of the rare molecular species of proteins, Poissonian approaches of replicable material have to be used.

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Global analysis of gene expression in cells of the immune system II. Cell-free translation products and high-density filter hybridization data

Cited by 8 publications

References 25 publications

Gene expression in IFN-gamma-activated murine macrophages

Gene expression in IFN-gamma-activated murine macrophages

Global analysis of gene expression in cells of the immune system I. Analytical limitations in obtaining sequence information on polypeptides in two-dimensional gel spots

Proteomic analysis of rare molecular species of translated polypeptides from a mouse fetal thymus cDNA library

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