Adenosine‐rich sequences in rapidly hybridizing messenger‐like RNA and their possible significance for reiterated base sequences in eukaryotic DNA

Scherrer, Klaus

doi:10.1016/0014-5793(71)80565-3

Cited by 16 publications

(4 citation statements)

References 21 publications

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“…Finally, amDNA complementary to globin mRNA did not form hybrids with the second predominant mRNA species (12 S) from duck erythroblasts (26). This result argues that the annealing of amDNA to mIRNA does not reflect some sequence common to all messengers; e.g., most mRNAs, as well as mlRNA, contain oligo(A) (4,27) and poly(A) sequences (28,29). To insure that these particular nonspecific sequences did not lead to erroneous conclusions, we labeled the amDNA only with dC and dG.…”

Section: Methodsmentioning

confidence: 95%

“…It seems surprising, however, that such breaks could remain "hidden" upon treatment with hot phenol. Alternatively, it is not excluded that smaller, already processed pre-mRNA molecules formed large networks by cross-hybridization of poly(A) (28,29) or oligo(A) (4,27) with oligo (U) (41) segments; such a structure would break down in (CH3)2S0. We are aware that (CH3)2S0 may have effects other than simply rupturing hydrogen bonds e.g., it is a mild oxidant and may break RNA (E. Jacob, personal communication).…”

Section: Methodsmentioning

confidence: 99%

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Demonstration of Globin Messenger Sequences in Giant Nuclear Precursors of Messenger RNA of Avian Erythroblasts

Imaizumi

Diggelmann

Scherrer

1973

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

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151

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Highly purified globin mRNA from ducks was copied with RNA-directed DNA polymerase from avian myeloblastosis virus into anti-messenger DNA. With excess RNA, more than 90% of this DNA annealed back to its template with a Cot/2 value of 7.5 X 10-4 mol-sec* liter-'; the melting temperature of the hybrid was 860. Giant nuclear RNA fractions with sedimentation coefficients of more than 50 S formed hybrids of almost equal stability at Cot/2 values of 0.05-0.42 mol sec liter-1, indicating amRNA content of 0.3-1.5%. 12S RNA from the same polyribosomes and nuclear giant RNA from HeLa cells did not cross-hybridize. Although a large part of the giant RNA broke down in 99% dimethylsulfoxide gradients, RNA fractions sedimenting faster than 28S rRNA still were found to consist of up to 0.03% globin mRNA sequences. Thus, the mRNA sequences are contained in the covalent structure of giant nuclear precursors, which are termed precursor-mRNA.Since the first observation of giant messenger-like RNA with a molecular weight of more than 3.5 X 106 (miRNA) (1)(2)(3)(4)(5) in the nuclei of animal cells, the function of this class of RNA has been questioned. Indirect evidence first led us to postulate that this RNA was a precursor to mRNA (3,4,6), in analogy to the nucleolar precursor of ribosomal RNA (rRNA) (1,7,8). High Cot hybridization-competition experiments demonstrated base-sequence homology between polyribosomal mRNA and giant nascent mIRNA from HeLa cells (9). Viralspecific RNA in SV40-transformed (10) and polyoma-infected (11) cells are transcribed as giant RNA molecules several times the size of the viral genome.We report here direct evidence that giant nascent mlRNA does indeed contain the same nucleotide sequence as functional mRNA. Globin mRNA from ducks was transcribed by RNA-directed DNA polymerase (12,13) from oncornaviruses into anti-messenger DNA (amDNA). This DNA could be annealed with giant nascent nuclear RNA, forming a hybrid almost as stable as that with the 9S mRNA itself. METHODS9S and 1RS Polyribosomal ImRNA Was Prepared from immature duck erythrocytes as described (14), followed by hot phenol extraction, DNase treatment (5 ug/ml, 30 min, 00), and elimination of DNase by Macaloid adsorption. The 9S preparations thus obtained directed the synthesis of all duck Abbreviations: pre-mRNA, precursor to messenger RNA [replaces the largely synonymous mlRNA (6), HnRNA (5), and D-RNA (1, 2)]; rRNA, ribosomal RNA; pre-rRNA, precursor to rRNA; amDNA, DNA strand copied from mRNA by the RNAdirected DNA polymerase (anti-messenger DNA); Hb, hemoglobin; AMV, avian myeloblastosis virus; Tm, melting tempera- Al for up to 2 days. Hybrid formation was assayed by rinsing the incubation mixture into 200 ul of 30 mM Na-acetate (pH 4.5), 0.15 M NaCl, 1 mM ZnSO4, and 5 ug of denatured salmon-sperm DNA, containing 2 units/ml of partially purified DNase Si (18). Incubation was for 90 min at 370, followed by trichloroacetic acid precipitation and assay for radioactivity. The (Fig. 1). With relatively low concentrations (40-50 MM...

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Section: Methodsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Demonstration of Globin Messenger Sequences in Giant Nuclear Precursors of Messenger RNA of Avian Erythroblasts

Imaizumi

Diggelmann

Scherrer

1973

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

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151

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“…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.…”

mentioning

confidence: 99%

“…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%

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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Control of Gene Expression in Animal Cells: The Cascade Regulation Hypothesis Revisited

Scherrer¹

1974

Control of Gene Expression

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Adenosine‐rich sequences in rapidly hybridizing messenger‐like RNA and their possible significance for reiterated base sequences in eukaryotic DNA

Cited by 16 publications

References 21 publications

Demonstration of Globin Messenger Sequences in Giant Nuclear Precursors of Messenger RNA of Avian Erythroblasts

Demonstration of Globin Messenger Sequences in Giant Nuclear Precursors of Messenger RNA of Avian Erythroblasts

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

Control of Gene Expression in Animal Cells: The Cascade Regulation Hypothesis Revisited

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