Changes in the Frequency and Diversity of Messenger RNA Populations in the Course of Myogenic Differentiation

Leibovitch, Marie-Pierre; Leibovitch, Serge A.; Harel, J; Kruh, Jacques

doi:10.1111/j.1432-1033.1979.tb13117.x

Cited by 31 publications

(22 citation statements)

References 27 publications

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“…We do not know the significance of this phenomenon. The existence of singlestranded DNA in preparations of eukaryotic DNA has been reported (23); the structures we observe may correspond to a priori single-stranded DNA. Between 75% and 85% formamide, "bubbles" of denaturation of the type shown in Figs. 1 and 2, appeared within the spread DNA.…”

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confidence: 93%

“…These short sections may correspond mainly to the DNA found within the clusters. On the other hand, 30% of the fragments by number (64% of the measured DNA length) fall into a wide range of lengths of between 3.5 Aum to more than 40 pmm (10-125 kpb); of these, most fall into a more narrow range of 3.5-9.5 pum or [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] kbp. This size class includes the sections of the DNA seen outside the clusters.…”

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confidence: 99%

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Systematic punctuation of eukaryotic DNA by A+T-rich sequences.

Moreau¹,

Matyash-Smirniaguina²,

Scherrer³

1981

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

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Isodenaturation of avian and mammalian DNA of Mr > 2.5 x 108 in 85% (vol/vol) formamide led to the observation by electron microscopy of A+T-rich zones ("AT-rich linkers") of 300-3000 base pairs which aresdistributed over the entire genome in a characteristic pattern. Linkers of mean length 800 base pairs are found either isolated or in clusters of about four to six linkers of more heterogeneous size separated on average by 2500 base pairs (in duck DNA). In between clusters, single linkers segment the DNA at distances of 10 to more than 100 kilobase pairs, with a majority in the range of (3,4) chromosomes of higher organisms.The significance of the large excess of transcribed and untranscribed genomic DNA, compared to the complexity of the actual translated mRNA, is enigmatic, although the final confirmation of the existence of precursors to mRNA allows at least a tentative interpretation of the existence of transcribed nongenic DNA within the eukaryotic transcriptons (see discussion and references in ref. 5). Light satellite DNA (6-10) is an example of nontranscribed (9), probably structural, DNA. Further A+T-rich segments disseminated in eukaryotic DNA (11) might play an important role in DNA structure and function (see model for precursor mRNA in ref. 12) beyond their enrichment at the centromere (10) of metaphase chromosomes. A+T-rich DNA sequences have been found in bacterial promoter/operator regions (13, 14) and in gene-proximal eukaryotic spacers (15). Such sequences are possible targets of A+T-specific drugs such as distamycin or bromodeoxyuridine, which interfere, respectively, with transcription (16) and specific phases of differentiation (17).We thus set out to study the distribution and characteristics of A+T-rich regions in segments of eukaryotic DNA longer than the putative transcriptional units defined by the size of the transcription products (for discussion, see ref. 5).METHODS DNA Preparation. Erythroblasts were selected from the blood of anemic Pekin ducks and were washed in 10 mM Tris-HCl, pH 6.9/10 mM KCV3 mM MgCl2/1 mM MnCl2/ 0.25 M sucrose. The cells were lysed with 0.4% Cemusol NP-6 in washing buffer (pH 7.4) in a precision-bore, hydraulically driven Dounce homogenizer. Nuclei were washed with a double detergent solution (0.5% Tween/0.15% deoxycholate) for 10 sec, diluted with 10 vol of washing buffer, resuspended in 10 mM Tris-HCl, pH 7.4/150 mM NaCl, lysed by 0.5% NaDodSO4, gently transferred to a dialysis bag, and incubated overnight at 18°C with proteinase K (50 pLg/ml) in 5mM EDTA. Protein was removed by extraction in slow-motion roller bottles (18) first with saturated phenol, then with phenoVchloroform/ isoamyl alcohol, 1:1:0.02 (vol/vol), and finally with chloroform. DNA was then dialyzed against 10 mM NaCl/50 mM Tris HCV 1 mM WEDTA, treated with pancreatic RNase (1 mg/i50 ml for 3 hr at 20°C), extracted with satured phenol, and dialyzed exhaustively against Tris-HCl, pH 7.8/100 mM NaCVl1 mM EDTA. Rat and mouse liver cell suspensions were treated similarly.Electron M...

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confidence: 93%

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confidence: 99%

Systematic punctuation of eukaryotic DNA by A+T-rich sequences.

Moreau¹,

Matyash-Smirniaguina²,

Scherrer³

1981

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

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“…Cell types are distinguishable by characteristic differences in their prevalent species of mRNA (1,2), and the processes of cellular differentiation (3,4), embryogenesis (5), and oncogenesis (6) may all involve shifts in both complexity and frequency distribution of mRNAs. The mode of regulation of mRNA complexity and abundance, however, is not clear.…”

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confidence: 99%

Developmental shifts in frequency distribution of polysomal mRNA and their posttranscriptional regulation in the sea urchin embryo.

Shepherd¹,

Nemer²

1980

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

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The frequency distributions of polyadenylylated RNAs from the polysomes of sea urchin blastulae and gastrulae were estimated from their kinetics of hybridization with complementary DNA. Developmental decreases in complexity were observed among abundant, intermediate, and rare frequency classes. The class of highest abundance in the blastula polysomes had a complexity of 5.6 X 104 nucleotides and contained about 30 mRNA species, which divided into subsets according to developmental fate. Studies with purified DNA complementary to this abundant class revealed that five of these mRNA species remained abundant in the gastrula, wherein each comprised 2% of the polyadenylylated RNA in the polysomes.Approximately 5 species decreased to a nearly rare frequency and2o were absent or at the limits of detection in polyadenylylated RNA of gastrula polysomes. These distinctly different developmental fates suggest distinct modes of regulation of mRNA concentration for different subsets. Focusing on the small number of abundant blastula mRNAs, we ascertained that those which were absent from gastrula polysomes were nevertheless represented in the gastrula nuclear RNA. Therefore, the appearance of abundant mRNA species in polysomes can be regulated by posttranscriptional processes.Cell types are distinguishable by characteristic differences in their prevalent species of mRNA (1, 2), and the processes of cellular differentiation (3, 4), embryogenesis (5), and oncogenesis (6) may all involve shifts in both complexity and frequency distribution of mRNAs. The mode of regulation of mRNA complexity and abundance, however, is not clear. The decrease in the number of different mRNA species during sea urchin development (5) appears to be attributable to posttranscriptional processes rather than to a block on transcription. This conclusion follows from the essentially constant complexity and sequence representation of nuclear RNA (7) and the persistence of complex-class mRNA sequences in nuclear RNA long after they have ceased to be present in the polysomes (8). However, prevalent mRNA sequences have not been shown conclusively to be present in nuclear RNA. of cells that do not express these genes. For example, the induction of ovalbumin synthesis involves turning on ovalbumin gene transcription (9, 10), whereas globin mRNA sequences have been reported variously absent from (11) and present in (12) nuclear RNA of cells not engaged in globin synthesis. Hence, an unequivocal demonstration of the persistence in nuclear RNA of mRNA sequences that shift between abundance and absence is still needed as evidence for a possible posttranscriptional regulation of abundant mRNA species.In the present study we observed changes in the frequency distribution of mRNA species during development from blastula to gastrula. Prominent among these changes was the drastic loss of blastula abundant mRNA species; some 20 out of 30 were no longer detectable in gastrula polysomes. This observation affords us the opportunity to test whether or not the prev...

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“…The bHLH proteins somehow lead to activation of the retinoblastoma protein (RB), which in turn titrates E2F factors to effect cell cycle exit upon terminal differentiation. These transcriptional complexes then induce a cascade of gene activation and repression events in which as many as 20,000 genes are differentially regulated (4,14).…”

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Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

Coonrod

Horwitz

2000

Molecular and Cellular Biology

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Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.Satellite cells are a lineage derived from somites that reside under the basement membrane of the myofiber and are responsible for replenishing skeletal muscle during growth in the postnatal period and in response to exercise and injury in the adult animal (24). As muscles hypertrophy, the satellite cells divide and fuse in order to increase the complement of myonuclei in myofibers. Transplant studies in chick embryos indicate that satellite cells are unable to take part in muscle embryogenesis, suggesting that they serve a specialized function as a presumptive skeletal muscle stem cell (3). Satellite cells are required to both proliferate and terminally differentiate, but invariably the proportion of satellite cells in a muscle remains constant, independent of both the age of the animal and the size of the muscle, and returns to this fixed value at the conclusion of muscle regeneration following injury (23).Understanding the molecular mechanisms responsible for the switch between proliferation and differentiation in satellite cells could be a key to understanding skeletal muscle regeneration in response to disease and trauma. However, relatively little is known about the mechanisms at work to ensure maintenance of the satellite cell population. Most of this knowledge comes from embryological studies of mesoderm differentiation in somites or the developing limb bud that have been extrapolated to tissue culture models of satellite myoblast differentiation. As with embryonic myogenesis, expression of MyoD or t...

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Changes in the Frequency and Diversity of Messenger RNA Populations in the Course of Myogenic Differentiation

Cited by 31 publications

References 27 publications

Systematic punctuation of eukaryotic DNA by A+T-rich sequences.

Systematic punctuation of eukaryotic DNA by A+T-rich sequences.

Developmental shifts in frequency distribution of polysomal mRNA and their posttranscriptional regulation in the sea urchin embryo.

Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

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