Effect of cell proliferation on levels and diversity of poly(A)-containing mRNA

Getz, Michael J.; Elder, Paula K.; Benz, Edmund W.; Stephens, R. E.; Moses, Harold L.

doi:10.1016/0092-8674(76)90025-8

Cited by 104 publications

(57 citation statements)

References 23 publications

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“…To test whether we could distinguish a very abundant class of Rna in polyadenylylated RNA prepared from immunoprecipitated polysomes [termed "Immpt poly(A)+ RNA"], we prepared 3H-labeled cDNA to Immpt poly(A)+ RNA and analyzed its kinetics of hybridization in RNA excess. This method of analysis has been used to define the frequency and sequence complexity components of total cytoplasmic (25)(26)(27), polysomal (28,29), and nuclear (27, 30) RNA populations. As can be seen in RNA was then hybridized to Immpt cDNA-cellulose to an Rot value of 2.5 X 10-2 mol-sec/liter.…”

Section: Resultsmentioning

confidence: 99%

Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver

Strair

Yap

Shafritz

1977

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

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A new procedure is described for purification of rat liver albumin m A. First a population of RNA molecules is enriched for albumin mRNA by immunoprecipitation of polysomes containing albumin nascent chains. Polyadenylylated RNA is prepared from immunoprecipitates, transcribed into complementary DNA, and shown to be enriched severalfold for a particular RNA frequency component. This enriched RNA component is then purified by molecular hybridization to a limited Rot value (product of RNA concentration and incubation time), under conditions in which only the most abundant sequence component is annealed. Potentially, this procedure can be employed for the purification of a wide variety of mRNAs present in lesser amounts in the cell.The isolated RNA appears to be a single frequency component by hybridization to complementary DNA transcribed from itself. This RNA is a 17S species and represents 5-8% of total cytoplasmic polyadenylylated RNA. In vitro translation of the purified RNA has shown that it codes for a single polypeptide that can be identified immunologically as albumin and migrates with rat serum albumin on sodium dodecyl sulfate/polyacrylamide gels. This albumin mRNA was determined to be essentially pure by comparing its kinetics of hybridization to those obtained with rabbit a + P globin mRNA and its DNA complement. The sequence complexity of purified rat albumin mRNA corresponds to 5.9 X 105 daltons. Various procedures have been employed for purification of eukaryotic mRNAs. Affinity chromatography with either oligo(dT)-cellulose or poly(U)-Sepharose has been most successful for separating mRNAs containing a 3'-poly(A) segment from the bulk of ribosomal and other RNA components (1, 2). However, this procedure cannot be used to separate specific polyadenylylated mRNAs from each other, and a portion of eukaryotic mRNA does not contain a 3'-poly(A) segment (3-7). Other commonly used methods for purification of eukaryotic mRNAs have been based either on a unique size for a specific mRNA-e.g., a and ,B globin mRNA(8), histone mRNA (3,9, 10), immunoglobin light chain mRNA (11)(12)(13)(14), ovalbumin mRNA (15), etc.-or an unusual nucleotide composition-e.g., silk fibroin mRNA (16). For purification of mRNAs that are average sized and are present as only a small portion of total cellular mRNA, the method of immunoprecipitation of polysomes containing nascent polypeptide chains for a specific protein is the only specific procedure available at the present time. Immunoprecipitation can enrich a polysome preparation for a specific mRNA by 5-to 10-fold (18-22). However, in order to obtain intact mRNA with good biological activity, the antibody must be highly purified to remove ribonuclease activity. Furthermore, large amounts of antibody are needed to generate sufficient amounts of material for use in molecular studies. In the present study, we have combined the techniques of imThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereb...

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Section: Resultsmentioning

confidence: 99%

Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver

Strair

Yap

Shafritz

1977

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

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“…Furthermore, studies have shown that poly(A)-synthesizing polymerases tend to be highly active in S phase of the cell cycle and are overexpressed in certain forms of cancer [31]. The implications could mean that there is potential for greater levels of polyadenylated mRNA in rapidly dividing cells, or simply that more mRNA is being produced by the cell during S phase.…”

Section: Introductionmentioning

confidence: 99%

Molecular recognition of single‐stranded RNA: Neomycin binding to poly(A)

Gray

Kumar

et al. 2009

FEBS Letters

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Edited by Hans EklundKeywords: RNA recognition Aminoglycoside Neomycin Poly(A) DNA RNA a b s t r a c t Poly(A) is a relevant sequence in cell biology due to its importance in mRNA stability and translation initiation. Neomycin is an aminoglycoside antibiotic that is well known for its ability to target various nucleic acid structures. Here it is reported that neomycin is capable of binding tightly to a single-stranded oligonucleotide (A 30 ) with a K d in the micromolar range. CD melting experiments support complex formation and indicate a melting temperature of 47°C. The poly(A) duplex, which melts at 44°C (pH 5.5), was observed to melt at 61°C in the presence of neomycin, suggesting a strong stabilization of the duplex by the neomycin.

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“…The growth rates of all experimental cultures were determined both before and 20 h after stimulation by measuring the rate of [3H]thymidine incorporation as previously described (13). Cell fractionation and purification of polysomal RNA have been described previously (13 (hybridization buffer is 5 x SSC containing 50% formamide, 250 ,ug of sonicated herring sperm DNA per ml, 50~Lg of polyadenylic acid per ml, 1 x Denhardt solution [0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin], 0.1% sodium dodecyl sulfate). The appropriate 32P-labeled probe (0.5 ,ug), nick translated by using a commercially available kit (New England Nuclear Corp., Boston, Mass.…”

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confidence: 99%

Structure and expression of mouse VL30 genes.

Hodgson¹,

Elder²,

Ono³

et al. 1983

Mol. Cell. Biol.

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DNA sequencing and blot hybridization analyses have been used to study the structure of a mouse VL30 gene and the molecular nature of VL30-related RNA which is induced upon the stimulation of cultured AKR mouse embryo cells with defined peptide growth factors. An integrated mouse VL30 gene was found to contain identical 601-base-pair long terminal repeats (LTRs) which were themselves terminated in short inverted repeats. The entire VL30 gene was flanked by a 4-base-pair direct repeat of cellular DNA. Thus, VL30 genes are structurally analogous to integrated forms of retrovirus proviruses and certain other classes of mobile genetic elements. The LTR sequence was found to contain putative promoter and polyadenylation signals and generally exhibited little sequence homology to murine leukemia virus proviral LTRs. Certain short regions of sequence conservation, however, were evident, including the inverted terminal repeat, LTR-adjacent regions corresponding to origins of murine leukemia virus proviral DNA synthesis, and a 36-base-pair direct repeat bearing homology to the 72-base-pair direct repeat (enhancer sequence) of the murine leukemia virusrelated Moloney sarcoma virus. Upon mitogenic stimulation of quiescent cells with epidermal growth factor and insulin, a major 5.5-kilobase VL30-specific RNA complementary to both LTR and non-LTR sequences was rapidly induced. We conclude that a complete VL30 gene(s) is highly regulated by peptide growth factor binding to specific membrane receptors in these cells.Mouse VL30 elements are a family of dispersed 5-to 6-kilobase (kb) DNA sequences present in 100 to 200 similar copies per genome (19,21). The predominant transcription product is a 30S polyadenylated RNA which is expressed in a variety of cell types (2, 6, 17). VL30 RNA encodes no known polypeptides but is copackaged with high efficiency into the virions of replication-competent retroviruses (35) and can be transmitted to heterologous cell types in culture (33). It is not known whether VL30 elements can replicate independently of helper retroviruses.VL30 sequences appear to have arisen in rodent genomes before Mus speciation, as evidenced by their presence in all tested representatives of the genus Mus as well as in rats (1, 5). However, considerable locus polymorphism can be distinguished in different laboratory strains and feral individuals. We have previously determined that one such polymorphism reflects the differing chromosomal location of a complete or nearly complete VL30 element (5). Therefore, In the present study, we have examined the nucleotide sequence of the terminally redundant 2221 on May 9, 2018 by guest

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Effect of cell proliferation on levels and diversity of poly(A)-containing mRNA

Cited by 104 publications

References 23 publications

Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver

Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver

Molecular recognition of single‐stranded RNA: Neomycin binding to poly(A)

Structure and expression of mouse VL30 genes.

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