Northern hybridization: rapid and simple electrophoretic conditions

Pellé, Roger; Murphy, Noel B.

doi:10.1093/nar/21.11.2783

Cited by 97 publications

(53 citation statements)

References 3 publications

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“…Cells were lysed mechanically using the FastPrep machine (Bio101; Q-biogene), and RNA was isolated by the RNeasy mini kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. Total RNA was quantified by spectrophotometric analysis ( ϭ 260 nm), and 5 g of RNA of each preparation was loaded onto a 1% agarose gel and separated in 10 mM sodium phosphate buffer as described previously (34). RNA was transferred to a positively charged nylon membrane (Boehringer Mannheim) by capillary blotting as described by Sambrook et al (37).…”

Section: Methodsmentioning

confidence: 99%

Global Virulence Regulation in Staphylococcus aureus : Pinpointing the Roles of ClpP and ClpX in the sar/agr Regulatory Network

2005

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Staphylococcus aureus causes infections ranging from superficial wound infections to life-threatening systemic infections. Essential for S. aureus pathogenicity are a number of cell-wall-associated and secreted proteins that are controlled by a complex regulatory network involving the quorum-sensing agr locus and a large set of transcription factors belonging to the Sar family. Recently, we revealed a new layer of regulation by showing that mutants lacking the ClpXP protease produce reduced amounts of several extracellular virulence factors and that, independently of ClpP, ClpX is required for transcription of spa, encoding Protein A. Here we find that the independent effect of ClpX is not general for other cell wall proteins, as expression of fibronectin-and fibrinogen-binding proteins was increased in the absence of either ClpX or ClpP. To assess the roles of ClpX and ClpP within the sar/agr regulatory network, deletions in clpX and clpP were combined with mutations in these genes. Interestingly, the derepression of spa transcription normally observed in an agrnegative strain was abolished in cells devoid of ClpX, and apparently ClpX modulates both SarS-dependent and SarS-independent control of spa expression, perhaps through the Sar family member Rot. Examination of expression of a single secreted protein, the SspA serine protease, revealed that ClpXP, similar to agr, is required for growth phase-dependent transcriptional induction of sspa. Intriguingly, induction was restored by the concomitant inactivation of Rot. We hypothesize that RNAIII accumulating in the postexponential phase may target Rot for degradation by ClpXP, leading to derepression of sspA.

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Section: Methodsmentioning

confidence: 99%

Global Virulence Regulation in Staphylococcus aureus : Pinpointing the Roles of ClpP and ClpX in the sar/agr Regulatory Network

2005

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“…Northern Analysis-Northern blot was performed as described previously (32). Briefly, 10 g of total RNA was fractionated in a formaldehyde-denaturing agarose gel and transferred onto Hybond nylon filters (Duralon; Stratagene, La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

TRIM8/GERP RING Finger Protein Interacts with SOCS-1

Toniato¹,

Chen

Losman

et al. 2002

Journal of Biological Chemistry

102

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Members of the suppressor of cytokine signaling (SOCS) family of signaling molecules regulate the activation of cytokine signaling. Experimental evidence indicates that SOCS expression is induced by cytokines and pro-inflammatory stimuli and is controlled at both the transcriptional and post-transcriptional levels. SOCS proteins are unstable and seem to be rapidly degraded by proteasomal pathways. However, the mechanisms by which SOCS protein levels are regulated remain unclear. Here, we show that TRIM8/GERP, a RING finger protein, interacts with SOCS-1 in vitro and in vivo. TRIM8/GERP, previously identified as a new member of the family of proteins containing a tripartite motif (TRIM), is a 551-amino acid RING finger protein conserved across species. TRIM8/GERP expression can be induced by interferon-␥ in epithelial and lymphoid cells. Coexpression of TRIM8/GERP with SOCS-1 decreases SOCS-1 protein stability and levels. Functionally, expression of TRIM8/GERP decreases the repression of interferon-␥ signaling mediated by SOCS-1. These data suggest that TRIM8/GERP may be a regulator of SOCS-1 function.Cytokines control many different cellular functions, including proliferation, differentiation, and gene expression (1, 2). Moreover, they participate in the pathophysiology of viral infections, play a central role in the development of the hematopoietic system, and have been implicated in the pathogenesis of autoimmune diseases (3, 4). The biological response of a cell to cytokines involves a complex network of signal transduction machinery. Signaling is initiated by the oligomerization of cognate cytokine receptors expressed on the surface of target cells (5), which triggers the activation of members of the JAK 1 family of protein-tyrosine kinases that constitutively associate with the cytokine receptor. Subsequently, JAKs can phosphorylate tyrosine residues present in the cytoplasmic regions of the receptors. These phosphorylated tyrosines then act as docking sites for signaling molecules, such as members of the STAT family of transcription factors (6).The intensity and duration of cytokine signaling seems to be regulated by several mechanisms. It is now known that at least three different classes of negative regulators contribute to cytokine inhibition: (i) the SH2-containing protein-tyrosine phosphatases 1 and 2 (7, 8), (ii) the protein inhibitors of activated STATs, and (iii) the suppressor of cytokine signaling (SOCS protein) (9 -13). Experimental data suggest that (i) SOCS genes are induced by cytokines; (ii) SOCS molecules can inhibit cytokine signaling by binding to downstream signaling molecules such as JAK kinases (14 -16); (iii) deregulated expression of SOCS proteins perturbs cytokine-related cellular proliferation and hematopoietic differentiation in murine tissues (17). Mice lacking SOCS-1 die shortly after birth from hepatic necrosis (18 -20). Data suggest that this lethality is due, at least in part, to hypersensitivity to IFN-␥ signaling. SOCS-2-deficient mice seem healthy after birth but develop a...

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“…Total RNA was isolated from cell samples incubated for 0, 5, 20, or 45 min at 38.5°C by using an RNeasy Mini kit (Qiagen) as described previously (48). RNA gel electrophoresis, blotting, and hybridization were performed as described previously (36,48). The clpP-and dnaK-specific probes were obtained by PCR by using primers p9 (5Ј-CAAATTCTATCATTGCC-3Ј) and p10 (5Ј-GAGCGATTAGAATTATCA GCAAGG-3Ј) and primers p11 (5Ј-CTGCTGAAAGCTACCTTGGCG-3Ј) and p12 (5Ј-CAGCTGGTTGATTATCAGCGG-3Ј), respectively.…”

Section: Methodsmentioning

confidence: 99%

ClpE from Lactococcus lactis Promotes Repression of CtsR-Dependent Gene Expression

et al. 2003

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The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpPdependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.

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Northern hybridization: rapid and simple electrophoretic conditions

Cited by 97 publications

References 3 publications

Global Virulence Regulation in Staphylococcus aureus : Pinpointing the Roles of ClpP and ClpX in the sar/agr Regulatory Network

Global Virulence Regulation in Staphylococcus aureus : Pinpointing the Roles of ClpP and ClpX in the sar/agr Regulatory Network

TRIM8/GERP RING Finger Protein Interacts with SOCS-1

ClpE from Lactococcus lactis Promotes Repression of CtsR-Dependent Gene Expression

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