Nonradioactive Detection of Differentially Expressed Genes Using Complex RNA or DNA Hybridization Probes

Ross, Ralf; Ross, Xiao-Lan; Rueger, Barbara; Laengin, T.; Reske‐Kunz, Angelika B.

doi:10.2144/99261pf01

Cited by 19 publications

(14 citation statements)

References 8 publications

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“…Genespecific overgo probes based on variable sequences within the T-box-encoded DNA-binding domains were used to screen the CHORI-302 BAC library according to established protocols (Ross et al 1999). Tbx2/3 probe, 5′-CAA CGA CAT CAT GAA GCT TCC CTA CTG TCA C TT CCG CAC CTA-3′, Tbx4/5 probe, 5′-CAG CGA GAA CAA CAA GTT TGA ACT GAA GAA GAC GTG TTT CAG-3′.…”

Section: Screening Of the Bac Library And Clone Sequencingmentioning

confidence: 99%

Conservation of linkage and evolution of developmental function within the Tbx2/3/4/5 subfamily of T-box genes: implications for the origin of vertebrate limbs

et al. 2008

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T-box genes encode a family of DNA-binding transcription factors implicated in numerous developmental processes in all metazoans. The Tbx2/3/4/5 subfamily genes are especially interesting because of their key roles in the evolution of vertebrate appendages, eyes, and the heart, and, like the Hox genes, the longevity of their chromosomal linkage. A BAC library derived from the single male amphioxus (Branchiostoma floridae) used to sequence the amphioxus genome was screened for AmphiTbx2/3 and AmphiTbx4/5, yielding two independent clones containing both genes. Using comparative expression, genomic linkage, and phylogenetic analyses, we have reconstructed the evolutionary histories of these members of the T-box gene family. We find that the Tbx2-Tbx4 and Tbx3-Tbx5 gene pairs have maintained tight linkage in most animal lineages since their birth by tandem duplication, long before the divergence of protostomes and deuterostomes (e.g., arthropods and vertebrates) at least 600 million years ago, and possibly Dev Genes Evol (Electronic supplementary material The online version of this article (

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Section: Screening Of the Bac Library And Clone Sequencingmentioning

confidence: 99%

Conservation of linkage and evolution of developmental function within the Tbx2/3/4/5 subfamily of T-box genes: implications for the origin of vertebrate limbs

et al. 2008

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“…The library was screened by hybridization with a probe containing the mouse CCL22 open reading frame (ORF) (9). Hybridization reactions and signal detection were performed as reported previously (33).…”

Section: Isolation and Characterization Of A Murine Genomic Ccl22 Clonementioning

confidence: 99%

Differential Regulation of CCL22 Gene Expression in Murine Dendritic Cells and B Cells

Ghadially

Ross

Kerst

et al. 2005

The Journal of Immunology

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The activated T cell-attracting CC chemokine CCL22 is expressed by stimulated B cells and mature dendritic cells (DC). We have cloned and sequenced the complete mouse gene, including 4 kb of the 5′-flanking promoter region, and detected two distinct sites for initiation of transcription by 5′-RACE. Reporter gene assays indicate that the promoter reflects the specificity of the endogenous gene. Within the proximal promoter region, we identified potential binding sites for NF-κB, Ikaros, and a putative GC box. All three regions bind proteins. The NF-κB site was shown to specifically bind NF-κB subunits p50 and p65 from nuclear extracts of LPS-stimulated B cells, B cell line A20/2J, TNF-α-stimulated bone marrow-derived DC, and DC line XS106. Furthermore, promoter activity was affected by targeted mutagenesis of the NF-κB site and transactivation with p50 and p65. The region harboring the putative Ikaros site contributes to promoter activity, but the binding protein does not belong to the Ikaros family. The GC box was shown to specifically bind Sp1 using extracts from LPS-stimulated B cells and A20/2J but not from DC and DC line XS106. Additionally, Sp1 transactivated the promoter in A20/2J but not in XS106 cells, and mutation of the Sp1 site diminished transactivation. Furthermore, binding of the protein complex at the GC box is required for NF-κB activity, and the spatial alignment of the binding sites is of critical importance for promoter activity. Thus, identical and distinct proteins contribute to expression of CCL22 in DC and B cells.

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“…Probe hFas-UTR was used to identify genomic restriction fragments containing 3Ј parts of the gene. Hybridization reactions and signal detection were performed as reported (24). PAC clones selected following library screening were used for DNA preparation (NucleoBond Plasmid kit; Clontech Laboratories, Palo Alto, CA).…”

Section: Isolation and Characterization Of Human Genomic Fascin Clonesmentioning

confidence: 99%

The Human Fascin Gene Promoter Is Highly Active in Mature Dendritic Cells Due to a Stage-Specific Enhancer

Bros

Ross

Pautz

et al. 2003

The Journal of Immunology

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Dendritic cells (DC), regarded as the most efficient APCs of the immune system, are capable of activating naive T cells. Thus, DC are primary targets in immunotherapy. However, little is known about gene regulation in DC, and for efficient transcriptional targeting of human DC, a suitable promoter is still missing. Recently, we successfully used the promoter of the murine actin-bundling protein fascin to transcriptionally target DC by DNA vaccination in mice. In this study, we report on isolation of the human fascin promoter and characterization of its regulatory elements. The actively expressed gene was distinguished from a conserved inactive genomic locus and a continuous region of 14 kb covering the gene and 3 kb of 5′-flanking sequences was subcloned, sequenced, and analyzed for regulatory elements. Regulatory sequences were found solely in the 5′-flanking promoter region. The promoter exerted robust activity in DC and a fascin-positive neuronal cell line, but not in the fascin-negative cells tested. Notably, promoter activity in DC markedly increased with maturation of DC. By progressive 5′ deletion, we identified a core promoter region, harboring a putative GC box, a composite cAMP responsive element/AP-1 binding site and a TATA box. By internal deletion, we demonstrated functional importance of either regulatory element. Furthermore, we identified a more distal stage-specific enhancer region also containing silencer elements. Taken together, the human fascin promoter allows for transcriptional targeting of mature DC and represents a promising tool for immunotherapy. To our knowledge, this study reports for the first time on promoter activity in human monocyte-derived DC.

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Nonradioactive Detection of Differentially Expressed Genes Using Complex RNA or DNA Hybridization Probes

Cited by 19 publications

References 8 publications

Conservation of linkage and evolution of developmental function within the Tbx2/3/4/5 subfamily of T-box genes: implications for the origin of vertebrate limbs

Conservation of linkage and evolution of developmental function within the Tbx2/3/4/5 subfamily of T-box genes: implications for the origin of vertebrate limbs

Differential Regulation of CCL22 Gene Expression in Murine Dendritic Cells and B Cells

The Human Fascin Gene Promoter Is Highly Active in Mature Dendritic Cells Due to a Stage-Specific Enhancer

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