scholarly journals Targeting of c-myc and β-globin coding sequences to cytoskeletal-bound polysomes by c-myc 3′ untranslated region

1994 ◽  
Vol 298 (1) ◽  
pp. 143-148 ◽  
Author(s):  
J Hesketh ◽  
G Campbell ◽  
M Piechaczyk ◽  
J M Blanchard
Keyword(s):  
In Situ Hybridization ◽  
Untranslated Region ◽  
Mrna Localization ◽  
Eukaryotic Cells ◽  
Beta Globin ◽  
Globin Mrna ◽  
Directional Information ◽  
Coding Sequences ◽  
Membrane Bound

The influence of the 3′ untranslated region on mRNA localization was investigated by measuring the distribution of myc, beta-globin and hybrid myc-globin mRNAs between free, cytoskeletal-bound and membrane-bound polysomes in cells transfected with either control or chimeric gene constructs. c-myc sequences and beta-globin-coding sequences linked to the myc 3′ untranslated region were present at greatest enrichment in cytoskeletal-bound polysomes. beta-Globin mRNA and myc-coding sequences linked to the beta-globin 3′ untranslated region were recovered largely in the free polysomes. In situ hybridization confirmed that replacement of the c-myc 3′ untranslated region by that of globin caused a relocalization of the mRNA. The results suggest that mRNA localization in differentiated eukaryotic cells depends on a mechanism that involves directional information in the 3′ untranslated region of mRNAs.

In situ hybridization reveals co-expression of embryonic and adult alpha globin genes in the earliest murine erythrocyte progenitors

Development ◽  
1992 ◽  
Vol 116 (4) ◽  
pp. 1041-1049 ◽  
Author(s):  
A. Leder ◽  
A. Kuo ◽  
M.M. Shen ◽  
P. Leder
Keyword(s):  
In Situ Hybridization ◽  
Embryonic Development ◽  
Fetal Liver ◽  
Globin Gene ◽  
Yolk Sac ◽  
Globin Genes ◽  
Globin Mrna ◽  
Mature Adult ◽  
Alpha Globin

Murine erythropoiesis begins with the formation of primitive red blood cells in the blood islands of the embryonic yolk sac on day 7.5 of gestation. By analogy to human erythropoiesis, it has been thought that there is a gradual switch from the exclusive expression of the embryonic alpha-like globin (zeta) to the mature adult form (alpha) in these early mouse cells. We have used in situ hybridization to assess expression of these two globin genes during embryonic development. In contrast to what might have been expected, we find that there is simultaneous expression of both zeta and alpha genes from the very onset of erythropoiesis in the yolk sac. At no time could we detect expression of embryonic zeta globin mRNA without concomitant expression of adult alpha globin mRNA. Indeed, adult alpha transcripts exceed those of embryonic zeta in the earliest red cell precursors. Moreover, the pattern of hybridization reveals co-expression of both genes within the same cells. Even in the fetal liver, which supersedes the yolk sac as the major site of murine fetal erythropoiesis, there is a brief co-expression of zeta and alpha genes followed by the exclusive expression of the adult alpha genes. These data indicate an important difference in hematopoietic ontogeny between mouse and that of human, where zeta expression precedes that of alpha. In addition to resolving the embryonic expression of these globin genes, our results suggest that the embryonic alpha-like globin gene zeta may be physiologically redundant, even during the earliest stages of embryonic development.

Sheep 5HT2A receptors: partial cloning of the coding sequence and mRNA localization by in situ hybridization in the ewe hypothalamus

1999 ◽  
Vol 295 (2) ◽  
pp. 231-239 ◽  
Author(s):  
J. Pelletier ◽  
Colette Auzan ◽  
Agnès Daveau ◽  
Eric Clauser ◽  
Philippe Chemineau
Keyword(s):  
In Situ Hybridization ◽  
Mrna Localization ◽  
Coding Sequence

Localization of low abundance DNA sequences in tissue sections by in situ hybridization

1986 ◽  
Vol 81 (1) ◽  
pp. 143-162 ◽  
Author(s):  
C.W. Lo
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequences ◽  
Detection System ◽  
Mouse Tissue ◽  
Globin Genes ◽  
Beta Globin ◽  
Tissue Sections ◽  
Specific Hybridization ◽  
Hybridization Procedure

Specific DNA sequences were loaclized in the nuclei of paraffin-embedded mouse tissue sections with in situ hybridization using a biotinylated globin probe in conjunction with a streptavidin-alkaline phosphatase detection system. Globin inserts were clearly detected in the tissue sections of transgenic mice containing 1000, 120 or 5 copies of the exogenously introduced beta-globin genes. In addition, specific hybridization signal was also obtained for the endogenous complement of beta-globin genes in the tissue sections of normal mice. These results demonstrate that this hybridization procedure is very sensitive and should be useful for characterizing the distribution of low abundance DNA sequences in cells and tissue sections.

scholarly journals Renal kallikrein mRNA localization by in situ hybridization

1989 ◽  
Vol 35 (6) ◽  
pp. 1324-1329 ◽  
Author(s):  
William Xiong ◽  
Lee Chao ◽  
Julie Chao
Keyword(s):  
In Situ Hybridization ◽  
Mrna Localization ◽  
Renal Kallikrein

scholarly journals Effects of digitonin on the intracellular content of rat hepatocytes: implications for its use in the study of intralobular heterogeneity.

1993 ◽  
Vol 41 (7) ◽  
pp. 991-1001 ◽  
Author(s):  
L Racine ◽  
J Y Scoazec ◽  
A Moreau ◽  
D Bernuau ◽  
G Feldmann
Keyword(s):  
In Situ Hybridization ◽  
Rat Hepatocytes ◽  
Cell Suspensions ◽  
Intracellular Content ◽  
Membrane Bound ◽  
Collagenase Perfusion ◽  
Beta Hydroxybutyrate ◽  
Membrane Bound Enzymes ◽  
Nadph Dehydrogenase

Anterograde or retrograde perfusion of rat liver with digitonin selectively permeabilizes the periportal or the perivenous zone of the hepatic lobule. Digitonin perfusion is used to analyze the effluents released by permeabilized hepatocytes or, combined with collagenase perfusion, to obtain cell suspensions enriched in either periportal or perivenous hepatocytes. Despite the wide use of digitonin to study lobular heterogeneity, its affects on rat hepatocytes are not well documented. We therefore analyzed the effects of digitonin perfusion on the intracellular content of rat hepatocytes by combining electron microscopy, histoenzymology, immunohistochemistry, and in situ hybridization. At the concentration currently used for the study of lobular heterogeneity, digitonin perfusion induced a marked cytosolic clarification of permeabilized hepatocytes, while most organelles except mitochondria were well preserved. In the digitonin-altered zones, there was no histochemical detection of non-membrane-bound enzymes (lactate dehydrogenase, glutamate dehydrogenase), whereas membrane-bound enzymes (succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, NADPH dehydrogenase, glucose-6-phosphatase) were still detected. Immunohistochemistry and in situ hybridization revealed significant amounts of several plasma proteins (albumin, alpha 2-macroglobulin, alpha 1-inhibitor 3, alpha 1-acid glycoprotein) and their respective mRNAs in digitonin-permeabilized hepatocytes. The demonstration that digitonin-permeabilized hepatocytes retain many intracellular constituents shows that biochemical analysis of cellular effluents released from digitonin-permeabilized hepatocytes must be interpreted with caution and that the apparent characteristics of cell suspensions obtained by the digitonin-collagenase technique might be significantly altered by contamination with permeabilized hepatocytes from the opposite zone.

scholarly journals Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.

1997 ◽  
Vol 17 (5) ◽  
pp. 2698-2707 ◽  
Author(s):  
N M Yeilding ◽  
W M Lee
Keyword(s):  
Myogenic Differentiation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
C2c12 Cells ◽  
Regulatory Function ◽  
Mrna Levels ◽  
Beta Globin ◽  
Globin Mrna ◽  
Exon 2 ◽  
Coding Sequences

Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the human beta-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences from myc exon 2 or exon 3 abolished downregulation of myc mRNA during myogenic differentiation, while deletion of introns or sequences in the 5' or 3' untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary for myc mRNA downregulation. Fusion of coding sequences from either myc exon 2 or 3 to beta-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion of myc 3' UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements in myc exons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in either myc exon 2 or myc exon 3 are sufficient to confer downregulation onto beta-globin mRNA, but neither element alone was sufficient for myc mRNA downregulation, suggesting that some feature of beta-globin mRNA may potentiate the regulatory properties of myc exons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of either myc exon 2 or myc exon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that two myc regulatory elements, one exon 2 and one in exon 3, are required for myc mRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated by myc exons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event.

scholarly journals Determinants that contribute to cytoplasmic stability of human c-fos and beta-globin mRNAs are located at several sites in each mRNA.

1988 ◽  
Vol 8 (8) ◽  
pp. 3244-3250 ◽  
Author(s):  
K S Kabnick ◽  
D E Housman
Keyword(s):  
Half Life ◽  
Untranslated Region ◽  
Calf Serum ◽  
Beta Globin ◽  
Mrna Synthesis ◽  
Globin Mrna ◽  
Mrna Species ◽  
Quiescent Cells ◽  
The Stability ◽  
Mrna Half Life

We have analyzed the contributions to cytoplasmic stability in an mRNA species with a very short half-life (human c-fos) and an mRNA species with a very long half-life (human beta-globin). When the human c-fos promoter was used to drive the expression of human c-fos, beta-globin, and chimeric DNAs between c-fos and beta-globin in transfected cells, a pulse of mRNA synthesis was obtained following induction of transcription by refeeding quiescent cells with medium containing 15% calf serum. The mRNA half-life was determined by using Northern (RNA) blot analysis of mRNAs prepared at various times following the pulse of transcription. Under these conditions human c-fos mRNA exhibited a half-life of 6.6 min and human beta-globin mRNA exhibited a half-life of 17.5 h. Replacement of the 3' end of the c-fos mRNA with the 3' end of the beta-globin mRNA increased the half-life of the resultant RNA from 6.6 to 34 min. The reciprocal chimera had a half-life of 34.6 min compared with the 17.5-h half-life of beta-globin mRNA. These results suggest that sequences which make a major contribution to mRNA stability reside in the 3' end of either or both molecules. A chimera in which the 5' untranslated region of globin was replaced by part of the 5' untranslated region of fos led to destabilization of the encoded mRNA. This construct produced an mRNA with a half-life of 6.8 h instead of the 17.5-h half-life of globin. This result suggests that additional determinants of stability reside in the 5' end of these mRNA molecules. Substitution of part of the 5' untranslated region of fos by the 5' untranslated region of beta-globin yielded an mRNA with stability similar to fos mRNA. These results suggest that interactions among sequences within each mRNA contribute to the stability of the respective molecules.

scholarly journals Characterization of Germ Cell-Specific Expression of the Orphan Nuclear Receptor, Germ Cell Nuclear Factor*

Endocrinology ◽  
1997 ◽  
Vol 138 (10) ◽  
pp. 4364-4372 ◽  
Author(s):  
Deborah Katz ◽  
Craig Niederberger ◽  
Gayle R. Slaughter ◽  
Austin J. Cooney
Keyword(s):  
In Situ Hybridization ◽  
Germ Cell ◽  
Nuclear Receptors ◽  
Untranslated Region ◽  
Northern Analysis ◽  
Nuclear Factor ◽  
Wild Type ◽  
Complementary Dna ◽  
Germ Cell Nuclear Factor

Abstract Nuclear receptors, such as those for androgens, estrogens, and progesterones, control many reproductive processes. Proteins with structures similar to these receptors, but for which ligands have not yet been identified, have been termed orphan nuclear receptors. One of these orphans, germ cell nuclear factor (GCNF), has been shown to be germ cell specific in the adult and, therefore, may also participate in the regulation of reproductive functions. In this paper, we examine more closely the expression patterns of GCNF in germ cells to begin to define spatio-temporal domains of its activity. In situ hybridization showed that GCNF messenger RNA (mRNA) is lacking in the testis of hypogonadal mutant mice, which lack developed spermatids, but is present in the wild-type testis. Thus, GCNF is, indeed, germ cell specific in the adult male. Quantitation of the specific in situ hybridization signal in wild-type testis reveals that GCNF mRNA is most abundant in stage VII round spermatids. Similarly, Northern analysis and specific in situ hybridization show that GCNF expression first occurs in testis of 20-day-old mice, when round spermatids first emerge. Therefore, in the male, GCNF expression occurs postmeiotically and may participate in the morphological changes of the maturing spermatids. In contrast, female expression of GCNF is shown in growing oocytes that have not completed the first meiotic division. Thus, GCNF in the female is expressed before the completion of meiosis. Finally, the nature of the two different mRNAs that hybridize to the GCNF complementary DNA was studied. Although both messages contain the DNA binding domain, only the larger message is recognized by a probe from the extreme 3′ untranslated region. In situ hybridization with these differential probes demonstrates that both messages are present in growing oocytes. In addition, the coding region and portions of the 3′ untranslated region of the GCNF complementary DNA are conserved in the rat.

scholarly journals IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

1974 ◽  
Vol 63 (2) ◽  
pp. 402-413 ◽  
Author(s):  
P. R. Harrison ◽  
D. Conkie ◽  
N. Affara ◽  
J. Paul
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Fetal Liver ◽  
Liver Cells ◽  
Mrna Levels ◽  
Messenger Rnas ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Fetal Liver Cells

Globin mRNA levels in 11–15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled DNA copy (cDNA) of adult globin messenger RNAs (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 11th day of development to 80–85% by days 13–15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin mRNAs begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition. The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin mRNAs has been confirmed by comparing the hybridization in solution of globin cDNA to cytoplasmic RNA extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.

Sign in / Sign up

Export Citation Format

Share Document