Quantifi cation of mRNA in Neuronal Tissue by Northern Analysis

2003 ◽  
pp. 161-180
Author(s):  
Christine L. Konradi
Keyword(s):  
Northern Analysis ◽  
Neuronal Tissue

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

Alternative Splicing Is Responsible for the Presence of Two Tissue Factor mRNA Species in LPS Stimulated Human Monocytes

1992 ◽  
Vol 67 (02) ◽  
pp. 272-276 ◽  
Author(s):  
C Paul ◽  
E van der Logt ◽  
Pieter H Reitsma ◽  
Rogier M Bertina
Keyword(s):  
Alternative Splicing ◽  
Tissue Factor ◽  
Stop Codon ◽  
Cell Types ◽  
Northern Analysis ◽  
Human Monocytes ◽  
Mrna Species ◽  
Protein Levels ◽  
Intron 1 ◽  
Tf Gene

SummaryAlthough normally absent from the surface of all circulating cell types, tissue factor (TF) can be induced to appear on circulating monocytes by stimulants like bacterial lipopolysaccharide (LPS) and phorbolesters. Northern analysis of RNA isolated from LPS stimulated human monocytes demonstrates the presence of 2.2 kb and 3.1 kb TF mRNA species. The 2.2 kb message codes for the TF protein. As demonstrated by Northern blot analysis with a variety of TF gene probes, the 3.1 kb message arises from an alternative splicing process which fails to remove 955 bp from intron 1. Because of a stop codon in intron 1 no TF protein is produced from the 3.1 kb transcript. This larger transcript should therefore not be taken into account when comparing TF gene transcription and TF protein levels.

scholarly journals Nuclear Gene Dosage Effects Upon the Expression of Maize Mitochondrial Genes

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1711-1721
Author(s):  
Donald L Auger ◽  
Kathleen J Newton ◽  
James A Birchler
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Gene Dosage ◽  
Nuclear Gene ◽  
Eukaryotic Cell ◽  
Mitochondrial Genes ◽  
Northern Analysis ◽  
Α Subunit ◽  
Dosage Effects ◽  
A Genome

Abstract Each mitochondrion possesses a genome that encodes some of its own components. The nucleus encodes most of the mitochondrial proteins, including the polymerases and factors that regulate the expression of mitochondrial genes. Little is known about the number or location of these nuclear factors. B-A translocations were used to create dosage series for 14 different chromosome arms in maize plants with normal cytoplasm. The presence of one or more regulatory factors on a chromosome arm was indicated when variation of its dosage resulted in the alteration in the amount of a mitochondrial transcript. We used quantitative Northern analysis to assay the transcript levels of three mitochondrially encoded components of the cytochrome c oxidase complex (cox1, cox2, and cox3). Data for a nuclearly encoded component (cox5b) and for two mitochondrial genes that are unrelated to cytochrome c oxidase, ATP synthase α-subunit and 18S rRNA, were also determined. Two tissues, embryo and endosperm, were compared and most effects were found to be tissue specific. Significantly, the array of dosage effects upon mitochondrial genes was similar to what had been previously found for nuclear genes. These results support the concept that although mitochondrial genes are prokaryotic in origin, their regulation has been extensively integrated into the eukaryotic cell.

P2u purinoceptor stimulation of surfactant secretion coupled to phosphatidylcholine hydrolysis in type II cells

1994 ◽  
Vol 267 (5) ◽  
pp. L625-L633 ◽  
Author(s):  
L. I. Gobran ◽  
Z. X. Xu ◽  
Z. Lu ◽  
S. A. Rooney
Keyword(s):  
Phospholipase D ◽  
Receptor Gene ◽  
Primary Cultures ◽  
Cell Receptor ◽  
Northern Analysis ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Cell ◽  
Subsequent Activation ◽  
Phosphatidylcholine Secretion

ATP is known to stimulate surfactant phospholipid secretion in type II cells, and there is evidence that this effect is mediated by a P2 purinoceptor. At least five subtypes of the P2 receptor have been reported, but it is not clear which one exists on the type II cell. To determine whether it is the P2u subtype, at which UTP is equipotent with ATP, we have compared the effects of ATP and UTP on phosphatidylcholine secretion and second messenger formation in primary cultures of rat type II cells. ATP and UTP were equally potent in stimulating phosphatidylcholine secretion and phospholipase D activation. The potency order, UTP = ATP > ADP > 2-methylthio-ATP, was the same as that reported for the P2u receptor. UTP stimulated diacylglycerol and phosphatidic acid formation to the same extent as ATP. ATP also increased choline formation. Formation of diacylglycerol was biphasic, and the first peak in response to ATP was previously shown to be associated with inositol trisphosphate formation. Northern analysis showed that the P2u receptor gene was expressed to a greater extent in type II cells than in whole lung. These data suggest that ATP and UTP act via a P2u receptor that is coupled to phosphoinositide-specific phospholipase C with subsequent activation of phospholipase D acting on phosphatidylcholine. ATP has also been reported to act at an additional type II cell receptor coupled to adenylate cyclase. In contrast, UTP did not promote adenosine 3',5'-cyclic monophosphate formation and therefore does not act at that receptor.

scholarly journals Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras.

1997 ◽  
Vol 17 (3) ◽  
pp. 1396-1406 ◽  
Author(s):  
N P Fam ◽  
W T Fan ◽  
Z Wang ◽  
L J Zhang ◽  
H Chen ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Calcium Influx ◽  
Calcium Ionophore ◽  
Mitogen Activated Protein Kinase ◽  
Northern Analysis ◽  
Free Form ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Iq Motif ◽  
Guanine Nucleotide Exchange

Conversion of Ras proteins into an activated GTP-bound state able to bind effector proteins is catalyzed by specific guanine nucleotide exchange factors in response to a large number of extracellular stimuli. Here we report the isolation of mouse cDNAs encoding Ras-GRF2, a multidomain 135-kDa protein containing a COOH-terminal Cdc25-related domain that stimulates release of GDP from Ras but not other GTPases in vitro. Ras-GRF2 bound specifically to immobilized Ras lacking bound nucleotides, suggesting stabilization of the nucleotide-free form of Ras as a mechanism of catalyzing nucleotide exchange. The NH2-terminal region of Ras-GRF2 is predicted to contain features common to various signaling proteins including two pleckstrin homology domains and a Dbl homology region. Ras-GRF2 also contains an IQ motif which was required for its apparent constitutive association with calmodulin in epithelial cells ectopically expressing Ras-GRF2. Transient expression of Ras-GRF2 in kidney epithelial cells stimulated GTP binding by Ras and potentiated calcium ionophore-induced activation of mitogen-activated protein kinase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF2 subcellular localization to change from cytosolic to peripheral, suggesting a possible mechanism for controlling Ras-GRF2 interactions with Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner with minimal intercellular contacts. Northern analysis indicated a 9-kb GRF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be expressed, and RNAs of 12 kb and 2.2 kb were detected in several tissues. Thus, Ras-GRF2 proteins with different domain structures may be widely expressed and couple diverse extracellular signals to Ras activation.

Sequential intrastriatal grafting of allogeneic embryonic dopamine-rich neuronal tissue in adult rats: Will the second graft be rejected?

Neuroscience ◽  
1993 ◽  
Vol 57 (2) ◽  
pp. 261-274 ◽  
Author(s):  
W.-M. Duan ◽  
H. Widner ◽  
A. Bjo¨rklund ◽  
P. Brundin
Keyword(s):  
Adult Rats ◽  
Neuronal Tissue

Prior treatment with heat shock attenuates the stress response in isolated working rat hearts

1995 ◽  
Vol 73 (1-2) ◽  
pp. 31-39
Author(s):  
Todd A. Dakin ◽  
R. William Currie
Keyword(s):  
Heat Shock ◽  
Control Mechanism ◽  
Negative Control ◽  
Northern Analysis ◽  
Moderate Increase ◽  
Flow Rates ◽  
Rat Hearts ◽  
Heart Perfusion ◽  
Paired Control ◽  
Working Heart

We examined the expression of the mRNAs encoding for the inducible heat shock protein (HSP) 71 and the constitutively synthesized HSP73 in control and 24-h post-heat-shocked (post-HS) hearts during isolated working heart perfusion. Paired control and 24-h post-HS rat hearts were perfused in the working heart mode for 1, 2, 3, or 4 h. Aortic and coronary flow rates and heart rates were not different between the control and 24-h post-HS hearts during the perfusion periods. After perfusion, total RN A was extracted and separated by gel electrophoresis. RNA was blotted to membranes, subsequently probed with 32P-labelled cDNA probes for HSP71 and HSP73 transcripts, and autoradiographed. Control hearts showed a sharp increase in transcripts for HSP71 and a more moderate increase in transcripts for HSP73 accumulation during perfusion. However, the increase in HSP71 and HSP73 transcripts in the HS hearts was markedly less than that in the control hearts. This suppression in gene expression in the HS hearts seems to suggest a negative control mechanism regulating transcription of mRNA encoding HSP71 and HSP73.Key words: mRNA, heart, Northern analysis, working heart perfusion, heat shock.

scholarly journals The Basic Helix-Loop-Helix Transcription Factor Cph2 Regulates Hyphal Development in CandidaalbicansPartly via Tec1

2001 ◽  
Vol 21 (19) ◽  
pp. 6418-6428 ◽  
Author(s):  
Shelley Lane ◽  
Song Zhou ◽  
Ting Pan ◽  
Qian Dai ◽  
Haoping Liu
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Binding Sites ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Mitogen Activated Protein Kinase ◽  
Northern Analysis ◽  
Hyphal Development ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix

ABSTRACT Candida albicans undergoes a morphogenetic switch from budding yeast to hyphal growth form in response to a variety of stimuli and growth conditions. Multiple signaling pathways, including a Cph1-mediated mitogen-activated protein kinase pathway and an Efg1-mediated cyclic AMP/protein kinase A pathway, regulate the transition. Here we report the identification of a basic helix-loop-helix transcription factor of the Myc subfamily (Cph2) by its ability to promote pseudohyphal growth inSaccharomyces cerevisiae. Like sterol response element binding protein 1, Cph2 has a Tyr instead of a conserved Arg in the basic DNA binding region. Cph2 regulates hyphal development in C. albicans, ascph2/cph2 mutant strains show medium-specific impairment in hyphal development and in the induction of hypha-specific genes. However, many hypha-specific genes do not have potential Cph2 binding sites in their upstream regions. Interestingly, upstream sequences of all known hypha-specific genes are found to contain potential binding sites for Tec1, a regulator of hyphal development. Northern analysis shows that TEC1 transcription is highest in the medium in which cph2/cph2 displays a defect in hyphal development, and Cph2 is necessary for this transcriptional induction of TEC1. In vitro gel mobility shift experiments show that Cph2 directly binds to the two sterol regulatory element 1-like elements upstream of TEC1. Furthermore, the ectopic expression of TEC1 suppresses the defect ofcph2/cph2 in hyphal development. Therefore, the function of Cph2 in hyphal transcription is mediated, in part, through Tec1. We further show that this function of Cph2 is independent of the Cph1- and Efg1-mediated pathways.

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