Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity Than 32P-Based Hybridizations

Englerblum, G.; Meier, Markus; Frank, Joseph J.; Müller, Günther

doi:10.1006/abio.1993.1189

Cited by 402 publications

(211 citation statements)

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“…Total cellular RNA was separated on 5% formaldehyde agarose gels and transferred to a Hybond Nϩ nylon membrane (Amersham). Hybridization and high-stringency washing were performed as described previously (20). The probe was a 1,250-bp fragment of the CXR-CT cDNA which encompasses the region encoding amino acids 49 to 448.…”

Section: Methodsmentioning

confidence: 99%

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Tognon

Kirk

Passmore

et al. 1998

Molecular and Cellular Biology

221

207

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As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H-and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester-and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol-and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerolenriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.

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Section: Methodsmentioning

confidence: 99%

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Tognon

Kirk

Passmore

et al. 1998

Molecular and Cellular Biology

221

207

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“…RNA was capillary blotted onto Hybond-N ϩ using 1 M NH 4 -acetate and UV-crosslinked. Hybridization to DIG-labelled riboprobes, washing and detection were according to Engler-Blum et al (1993).…”

Section: Northern Analysismentioning

confidence: 99%

Brown‐midrib maize (bm1) – a mutation affecting the cinnamyl alcohol dehydrogenase gene

et al. 1998

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SummaryBrown-midrib (bm) mutants of maize have modified lignin of reddish-brown colour. Although four independent bm loci are known, only one of the mutant genes has been previously identified. We report here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin. Both the total lignin content and the structure of the polymer are altered by the mutation. We further describe the isolation and characterisation of the maize CAD cDNA and mapping of the CAD gene. CAD maps very closely to the known location of bm1 and co-segregates with the bm1 locus in two independent recombinant inbred populations. These data strongly support the premise that maize bm1 directly affects expression of the CAD gene.

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“…Expression of specific mRNA for RANTES was first analysed using a non-radioactive Northern blot technique as previously described (Engler-Blum et al, 1993). Briefly, 10 µg of total RNA was separated by electrophoresis using a 1.2% agarose formaldehyde gel, subsequently transferred to a positively charged nylon membrane and cross-linked by UV light.…”

Section: Northern Blot Analysismentioning

confidence: 99%

The chemokine RANTES is secreted by human melanoma cells and is associated with enhanced tumour formation in nude mice

et al. 1999

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Modulation of tumour cell growth by tumour-infiltrating leucocytes is of high importance for the biological behaviour of malignant neoplasms. In melanoma, tumour-associated macrophages (TAM) and tumour-infiltrating lymphocytes (TIL) are of particular interest as inhibitors or enhancers of cell growth. Recruitment of leucocytes from the peripheral blood into the tumour site is mediated predominantly by chemotaxins, particularly by the group of chemokines. The aim of this study was to identify peptides released by human melanoma cells with monocyte chemotactic properties. To assure the presence of biologically active mediators, biochemical purification and biological characterization of peptides was based on a detection system dependent on bioactive, monocyte chemotactic activity in vitro. Cell culture supernatants of melanoma cells were fractioned by heparin–sepharose followed by preparative reversed-phase HPLC steps to enrich monocyte chemotactic activity in one single band on a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel. These purified fractions were shown to react with RANTES-specific antibodies in an enzyme-linked immunosorbent assay (ELISA) as well as in Western blot analysis. Amino acid sequencing of the N-terminal protein fragment confirmed 100% homology to the RANTES protein. Further analysis showed that four out of eight melanoma cell lines constitutively expressed and secreted the β-chemokine RANTES as detected by ELISA. The amount of RANTES protein secreted (up to 50 ng ml −1 ) was about 5–50 times higher than interleukin 8 (IL-8), determined in the same supernatant samples. Tumour necrosis factor α (TNF-α), not, however, IL-2, interferon-γ (IFN-γ), or α-melanocyte-stimulating hormone (α-MSH) was able to up-regulate RANTES and interleukin 8 secretion. Furthermore, higher levels of RANTES secretion in vitro were associated with increased tumour formation upon S . C . injection of six human melanoma cell lines in nude mice. Our data provide evidence that a subset of melanoma cells express mRNA and secrete RANTES protein which may be partly responsible for the recruitment of monocytes, T-cells and dendritic cells into the tumours. However, transplantation experiments in nude mice suggest that effects of RANTES may also benefit tumour progression. Further studies are needed to dissect the underlying mechanisms. © 1999 Cancer Research Campaign

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Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity Than 32P-Based Hybridizations

Cited by 402 publications

References 0 publications

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

Brown‐midrib maize (bm1) – a mutation affecting the cinnamyl alcohol dehydrogenase gene

The chemokine RANTES is secreted by human melanoma cells and is associated with enhanced tumour formation in nude mice

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