Detection of myosin heavy chain mRNA during myogenesis in tissue culture by in vitro and in situ hybridization

John, Huw A.; Patrinou-Georgoulas, Meropi; Jones, Kenneth W.

doi:10.1016/0092-8674(77)90126-x

Cited by 72 publications

(19 citation statements)

References 34 publications

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“…We also speculate that some miRNAs could combine with target messenger RNAs in the nucleolus to be exported as ''presuppressed'' mRNAs, a somatic cell analogy with masked maternal mRNAs in oocytes. Consistent with this idea, there is evidence that the nucleolus is involved in mRNA nucleocytoplasmic transport (Schneiter et al 1995;Thomsen et al 2008), and some mRNAs have been shown to associate with the nucleolus (John et al 1977;Bond and Wold 1993;Bártová et al 2008;Kim et al 2009). …”

Section: Discussionsupporting

confidence: 58%

MicroRNAs with a nucleolar location

Politz¹,

Hogan²,

Pederson³

2009

RNA

169

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There is increasing evidence that noncoding RNAs play a functional role in the nucleus. We previously reported that the microRNA (miRNA), miR-206, is concentrated in the nucleolus of rat myoblasts, as well as in the cytoplasm as expected. Here we have extended this finding. We show by cell/nuclear fractionation followed by microarray analysis that a number of miRNAs can be detected within the nucleolus of rat myoblasts, some of which are significantly concentrated there. Pronounced nucleolar localization is a specific phenomenon since other miRNAs are present at only very low levels in the nucleolus and occur at much higher levels in the nucleoplasm and/or the cytoplasm. We have further characterized a subset of these miRNAs using RT-qPCR and in situ hybridization, and the results suggest that some miRNAs are present in the nucleolus in precursor form while others are present as mature species. Furthermore, we have found that these miRNAs are clustered in specific sites within the nucleolus that correspond to the classical granular component. One of these miRNAs is completely homologous to a portion of a snoRNA, suggesting that it may be processed from it. In contrast, the other nucleolar-concentrated miRNAs do not show homology with any annotated rat snoRNAs and thus appear to be present in the nucleolus for other reasons, such as modification/processing, or to play roles in the late stages of ribosome biosynthesis or in nonribosomal functions that have recently been ascribed to the granular component of the nucleolus.

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Section: Discussionsupporting

confidence: 58%

MicroRNAs with a nucleolar location

Politz¹,

Hogan²,

Pederson³

2009

RNA

169

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“…Myoblast and CEF cultures were infected with wt PRA at a multiplicity of 1 to 5 and 1 to 10, respectively. The cells were washed twice with Tris-buffered saline (0.15 M-sodium chloride, 0.02 M-Tris-HCi pH 7.5), fixed for 30 min at 0 °C in 1.5 ml ethanol : acetic acid (3 : 1) (v/v) and kept at -20 °C until hybridization was carried out (John et al, 1977). The fixative was then decanted, and the dishes were incubated in 0.2 M-HC1 for 20 min at 20 °C.…”

Section: Analysis Of Rnamentioning

confidence: 99%

Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA

Tanaka¹,

Hara

Park

et al. 1992

Journal of General Virology

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We re-examined the generally accepted concept that replication of Rous sarcoma virus (RSV) requires host DNA synthesis. We used terminally differentiated chicken myotubes as the host because chromosomal DNA replication is completely abolished by the natural differentiation process. Southern blot analysis detected unintegrated viral DNA in both the nucleus and cytoplasm of infected myotubes. This indicated that reverse transcription of the infecting viral RNA and transport of the newly synthesized viral DNA into the nucleus proceeded normally in myotubes. However, restriction enzyme digestion of high Mr DNA prepared from infected myotubes produced none of the fragments specific for RSV, indicating that the viral DNA had failed to integrate into the myotube chromosomal DNA. In these infected myotubes, viral RNA was detected by in situ hybridization. Northern blot analysis showed the presence of all three RSV mRNAs (38S, 28S and 21S). The amount of these viral RNAs in infected myotubes was comparable with that found in infected fibroblasts. We conclude that host DNA synthesis is required for RSV integration, but, in contrast to the generally accepted concept, viral DNA integration is not an absolute requirement for transcription of the RSV genome.

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“…In some studies of this type total polyadenylylated RNA has been detected by using poly(U) probes (2)(3)(4). Other approaches have used complementary RNA or DNA reverse transcripts as probes either for viral sequences (5)(6)(7) or for specific messages within individual cells (8)(9)(10). More recently, a purified recombinant DNA clone has been used to detect specific cellular sequences (11).…”

mentioning

confidence: 99%

Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.

Singer¹,

Ward²

1982

Proc. Natl. Acad. Sci. U.S.A.

258

109

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The chicken muscle tissue culture system has been used for visualizing actin gene expression after in situ hybridization. Cell differentiation is morphologically distinguishable in this system as the myoblasts fuse into myotubes. This differentiation involves the production of large amounts of actin required for myofibrils. The presence of actin mRNA has been observed in cells preserved with ethanol and paraformaldehyde by hybridizing a recombinant plasmid into which a biotinated analog ofdUTP was incorporated by nick-translation. The biotin was then detected by using an anti-biotin antibody and a rhodamine-conjugated second antibody. Alternatively, avidin conjugated to rhodamine or avidin complexed to biotinated peroxidase has been used for mRNA detection. The procedure described preserved morphological detail yet is compatible with hybridization conditions and reveals the disposition of actin mRNA during gene expression.The method of in situ hybridization has been a powerful tool for the localization of specific sequences linearly arranged on a chromosome (1). This approach has been modified for the detection of RNA transcripts within individual cells. In some studies of this type total polyadenylylated RNA has been detected by using poly(U) probes (2-4). Other approaches have used complementary RNA or DNA reverse transcripts as probes either for viral sequences (5-7) or for specific messages within individual cells (8-10). More recently, a purified recombinant DNA clone has been used to detect specific cellular sequences (11). All ofthese approaches have used radiolabeled probes and the in situ hybridization has been detected by autoradiography. The inherent lack of resolution in tritium-labeled probes due to the track of the decay particle and the thickness of the emulsion makes the approach undesirable for precise morphological work. Recently, a number of workers have attempted the use of nucleic acid probes conjugated with fluorescent molecules for the detection of in situ hybridizations (12)(13)(14)(15) The investigation of gene expression by using recombinant DNA methods focuses on the molecular aspects of cell differentiation without consideration of the diversity of morphology inherent in the cell population containing these molecules. Because differentiation is often defined on the basis of cell morphology, we have attempted to develop a method relating cellular structure to the molecular aspects ofgene expression. This method requires high-resolution in situ hybridization and preservation of morphological detail. This work reports the initial results we have obtained by using the muscle tissue culture system for the detection of actin gene expression in situ and a recombinant actin plasmid labeled with biotin. This culture system is well suited to the investigation because both the morphology of cell differentiation (fusion of myoblasts into a syncytial myotube) and gene expression during this fusion process (e.g., the actin gene) have been well defined (18-22). MATERIALS AND METHODSPreparat...

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Detection of myosin heavy chain mRNA during myogenesis in tissue culture by in vitro and in situ hybridization

Cited by 72 publications

References 34 publications

MicroRNAs with a nucleolar location

MicroRNAs with a nucleolar location

Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA

Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.

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