Identification by In Vitro Translation and Northern Blot Analysis of Heat Shock mRNAs Isolated from Wheat Seeds Exposed to Different Temperatures During Ripening

ABSTRACT The alternative sigma factor σB of the food pathogen Bacillus cereus is activated upon stress exposure and plays a role in the adaptive response of vegetative cells. This study describes the identification of σB-dependent genes in B. cereus. Two-dimensional gel electrophoresis was performed with protein extracts from a σB-overproducing B. cereus strain. Nine protein spots, which were absent from the negative control, were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry or N-terminal sequencing. The σB-dependent expression of the corresponding genes was confirmed by Northern blot analysis with RNA isolated from B. cereus ATCC 14579 and its sigB null mutant. Northern blot analysis also revealed that six other genes were part of σB-dependent operons. The proteins that are predicted to be encoded by the σB-dependent genes include an intracellular protease, a Mg2+ transporter, and a thiamine biosynthesis protein (ThiG). Highly conserved promoter sites were found to precede all σB-dependent genes, with the exception of thiG. By searching the B. cereus genome for this conserved promoter sequence, five more candidate σB-dependent genes were identified. Northern blot analysis and in vitro transcription experiments with a reconstituted B. cereus σB-RNA polymerase holoenzyme confirmed the σB dependency of two of these genes and strongly suggested that two other genes, encoding an oligopeptide-binding OppA-like protein and subunit II of the cytochrome d ubiquinol oxidase, are also σB dependent. In conclusion, σB of B. cereus not only regulates genes directly involved in the stress response but may also control specific metabolic rearrangements.

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Constitutive expression of steel factor gene by human stromal cells

Blood ◽

10.1182/blood.v82.3.771.771 ◽

1993 ◽

Vol 82 (3) ◽

pp. 771-783 ◽

Cited By ~ 96

Author(s):

MC Heinrich ◽

DC Dooley ◽

AC Freed ◽

L Band ◽

ME Hoatlin ◽

...

Keyword(s):

Gene Expression ◽

Northern Blot ◽

Stromal Cells ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Hematopoietic Progenitor Cell ◽

Steel Factor ◽

Membrane Bound ◽

Human Stromal Cells

Abstract Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

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Something completely different?

Human & Experimental Toxicology ◽

10.1177/096032719601500616 ◽

1996 ◽

Vol 15 (6) ◽

pp. 543-544 ◽

Cited By ~ 1

Author(s):

James P Hardwick ◽

Liping Chen

Keyword(s):

Rat Brain ◽

Northern Blot ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Sequence Similarity ◽

Amino Acid Sequence Similarity ◽

Liver And Kidney ◽

High Level ◽

The Brain

Three cDNAs clones, designated 4f-8, 4f-34 and 4f- 41, coding for three new forms of cytochrome P450 belonging to the CYP4F subfamily were isolated from an untreated rat brain cDNA library. CDNA 4f- 8, 4f-34 and 4f-41 coded for proteins of 522, 526 and 537 amino acids, respectively, and their amino acid sequence similarity to CYP4F ranged from 71 to 80%. These new P450s were thus named CYP4F4, 4F5 and 4F6, respectively. Northern blot analysis revealed that the expression levels of these forms of P450 in the brain were somewhat low and that similar forms of subfamily 4F P450 were expressed in liver and kidney at a relatively high level compared with brain. No CYP4A expression was detected by Northern blot analysis in untreated rat brain mRNA. All three clones were in vitro- translatable using a reticulocyte lysate system. These results show that multiple forms of subfamily 4F P450 exist in the brain and that the subfamily 4F P450 may be one of the major forms of P450 in the brain.

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Constitutive expression of steel factor gene by human stromal cells

Blood ◽

10.1182/blood.v82.3.771.bloodjournal823771 ◽

1993 ◽

Vol 82 (3) ◽

pp. 771-783 ◽

Cited By ~ 1

Author(s):

MC Heinrich ◽

DC Dooley ◽

AC Freed ◽

L Band ◽

ME Hoatlin ◽

...

Keyword(s):

Gene Expression ◽

Northern Blot ◽

Stromal Cells ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Hematopoietic Progenitor Cell ◽

Steel Factor ◽

Membrane Bound ◽

Human Stromal Cells

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

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Northern Blot Analysis of microRNAs and Other Small RNAs in Plants

Methods in Molecular Biology - Plant MicroRNAs ◽

10.1007/978-1-4939-9042-9_9 ◽

2019 ◽

pp. 121-129 ◽

Cited By ~ 1

Author(s):

Carlos De la Rosa ◽

José Luis Reyes

Keyword(s):

Northern Blot ◽

Blot Analysis ◽

Small Rnas ◽

Northern Blot Analysis

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Application of biotinylated oligonucleotide probes to the detection of pituitary hormone mRNA using Northern blot analysis, in situ hybridization at the light- and electron-microscope levels

The Histochemical Journal ◽

10.1007/bf00188075 ◽

1994 ◽

Vol 26 (10) ◽

pp. 771-777 ◽

Cited By ~ 1

Author(s):

Akira Matsuno ◽

Akira Teramoto ◽

Susumu Takekoshi ◽

Hirotoshi Utsunomiya ◽

Yoshitaka Ohsugi ◽

...

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Northern Blot ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Pituitary Hormone ◽

Oligonucleotide Probes

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IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

International Journal of Inflammation ◽

10.1155/2012/714704 ◽

2012 ◽

Vol 2012 ◽

pp. 1-4 ◽

Cited By ~ 1

Author(s):

Roni M. Shtein ◽

Susan G. Elner ◽

Zong-Mei Bian ◽

Victor M. Elner

Keyword(s):

Gene Expression ◽

Northern Blot ◽

Stromal Cells ◽

Blot Analysis ◽

Time Course ◽

Northern Blot Analysis ◽

Statistical Significance ◽

Dependent Manner ◽

Corneal Inflammation ◽

Chemotactic Protein

Purpose. To determine time course of effect of lipopolysaccharide (LPS) on production of interleukin-8 (IL-8) and monocyte chemotactic protein (MCP) by cultured human corneal stromal cells.Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student'st-test.Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05) after corneal stromal cell stimulation with LPS.Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

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