Hybridization Properties of Dna Sequences Directing the Synthesis of Messenger Rna and Heterogeneous Nuclear Rna

Greenberg, Jay; Perry, Robert P.

doi:10.1083/jcb.50.3.774

Cited by 97 publications

(26 citation statements)

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“…The results also suggest strongly that in each case mRNA is transcribed preferentially from the single-copy fraction of the DNA. This is in agreement with previous work from this and from other laboratories on mRNA sequences in various organisms (20)(21)(22)(23). This result is particularly striking in Triturus, since the majority of the genome consists of repeated DNA.…”

supporting

confidence: 82%

“…3 and 6 strongly suggest that the basic transcription patterns in Xenopus and Triturus ovaries are very similar. In both cases, mRNA is transcribed preferentially from the single-copy fraction of the genome, in agreement with previous results on other systerns (20)(21)(22)(23)(24). Second, the number of different RNA species present in the ovaries of the two organisms is similar.…”

supporting

confidence: 81%

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Analysis of the C-Value Paradox by Molecular Hybridization

Rosbash

Ford

Bishop

1974

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

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Poly(A)-containing RNA was isolated from ovaries of Xenopus laevis laevis and Triturus cristatus carnifex and used as a template for the synthesis of radioactive complementary DNA with RNA-dependent DNA polymerase. When annealed with an excess of homologous DNA, the complementary DNA is rendered double-stranded with kinetics that suggest that the coding sequences are singlecopy in both these organisms. In Triturus, these sequences are distinct from the majority of the genome, which consists of repeated sequences, and distinct from the ribosomal cistrons, which are present in proportion to the increase in C-value relative to the Xenopus genome. Moreover, the number of different poly(A)-containing molecules in the ovary (sequence complexity) is the same in Xenopus and in Triturus.There exists an enormous variation in C-value (DNA content per haploid genome) among eukaryotic organisms (1 (3,4).On the basis of this range of DNA values and cytological evidence from various sources, Callan proposed that chromosomal DNA, and in particular genetically significant DNA, is arranged in tandemly repeated sequences (5). These repeated sequences consist of a "master" sequence and "slave" sequences; the "master" sequence would be subject to mutational and recombinational events while the "slave" sequences would be corrected once per life-cycle to reflect the sequence of the "master" sequence. The formation of extended lampbrush loops during meiotic prophase was proposed as the stage at which this correction process takes place.In washed in Barth X medium. Nucleic acid was prepared by the method of Kirby, with the addition of 0.5% sodium lauryl sulfate to the aqueous phase, as described (7). Highmolecular-weight RNA was prepared by homogenizing the pelleted nucleic acid in 3 M NaOAc (pH 6) to solubilize DNA, transfer RNA, and 5S RNA (8). The RNA precipitate was resuspended in "binding" buffer [0.4 M NaCl, 1 mM EDTA, 0.1% sodium lauryl sulfate, 10 mM Tris * HCl (pH 7.4), 10% glycerol] and passed over oligo(dT)-cellulose (9). The poly-(A)-containing RNA was eluted in "elution" buffer [10 mM Tris * HCl pH 7.4, 0.1% sodium lauryl sulfate], and the RNA spectrum was measured in the Unicam SP800A spectrophotometer. The amount of poly(A) in this eluate was routinely assayed by hybridization with radioactive poly(U), as described (7). By comparing the amount of poly (A) (Fig. 1). About 75% of

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supporting

confidence: 82%

supporting

confidence: 81%

Analysis of the C-Value Paradox by Molecular Hybridization

Rosbash

Ford

Bishop

1974

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

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“…Measurements of Greenberg and Perry (4) suggest that most L-cell mRNA is homologous to nonrepetitive DNA, while a small fraction may be transcribed from repetitive sequences.…”

mentioning

confidence: 99%

Nonrepetitive DNA Sequence Representation in Sea Urchin Embryo Messenger RNA

Goldberg¹,

Galau²,

Britten

et al. 1973

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

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Messenger RNA was prepared from developing sea urchin gastrulae by puromycin release from polyribosomes. Approximately 60% of the total mRNA radioactivity of the postnuclear supernatant was recovered and shown to be free of any other labeled RNA species such as ribosomal and nuclear RNA. The mRNA was examined by hybridization to DNA present in great excess. The mRNA hybridizes almost exclusively with nonrepetitive DNA. Almost all of the messenger RNA molecules of sea urchin gastrulae therefore consist of transcripts from nonrepetitive sequences. It appears that the structural genes expressed at this stage are typically not repeated in the genome and the mRNA does not include recognizable repetitive sequence.The DNA of higher cells is made up of repetitive and nonrepetitive sequences, many of which occur in an interspersed or alternating pattern (1). This pattern of organization is thought to determine the function of the genome and the regulation of genetic activity (2) and it is of considerable interest to establish experimentally the roles of the different sequences. The experiments reported in this paper were designed to determine whether the messenger RNAs of a developing animal system are transcribed from repetitive or nonrepetitive sequences. Previous work in other laboratories has indicated that certain specific structural gene mRNAs are probably transcribed from nonrepetitive DNA sequences, while histone gene sequences are probably repetitive (see ref.3). Measurements of Greenberg and Perry (4) suggest that most L-cell mRNA is homologous to nonrepetitive DNA, while a small fraction may be transcribed from repetitive sequences. MATERIALS AND METHODSGrowth of Embryos and Labeling of RNA. Strongylocentrotus purpuratus embryos were grown at 2 X 104 embryos per ml at 150 in Millipore-filtered sea water containing streptomycin and penicillin (5). They attained the 600-cell gastrula stage with three-pointed spicules by 36 hr. Between 36 and 39 hr they were labeled at 50 ,Ci/ml with [3HJuridine (SchwarzMann, 26 Ci/mmol). 5 ,uCi/ml of Na32PO4 (New England Nuclear Corp., carrier-free) was added for the final 10 min of in- cubation to detect contaminating heterogeneous nuclear RNA (hnRNA).Preparation of Nucleic Acids. DNA was extracted from sea urchin sperm by standard techniques, sheared to segments about 450 nucleotides long by passage through a press at 50,000 psi, and passed over Chelex 100 (Biorad, 200-400 mesh) (6).RNA was extracted from messenger ribonucleoprotein (mRNP) preparations as described in the legend to Fig. 1 (a-c). The RNA was deproteinized with Sevag solution (24:1 chloroform-isoamyl alcohol) and phenol mixed 1:1. The mRNA was precipitated together with sheared sea urchin DNA and stored at -80°in distilled water.mRNA -DNA Hybridization. mRNA and DNA were incubated at 600 (unless otherwise noted) in Kontes microflex vials in 0.12 or 0.41 M PB (PB is phosphate buffer that contains equa-l molar concentrations of Na2HPO4 and NaH2-P04). After incubation all hybridization mixtures were analy...

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“…Studies of the kinetics of hybridization of rapidly labelled polysomal RNA from Xenopus embryos [26] suggest that each molecule contains a rapidly hybridizable segment. The presence of sequences of this kind has also been demonstrated in the polysomal RNA from mouse L cells [27] as well as in poly(A)-containing polysomal RNA of Dictyostelium [28]. Even if allowance is made for the presence of a poly(A) segment and, possibly, of a sequence of the kind just mentioned in the high-molecularweight mRNAs described here, their information content must be large, which suggests that they could be polycistronic or could direct the synthesis of ex-ceptionally large polypeptides.…”

Section: Discussionmentioning

confidence: 93%

Poly(A)‐Containing RNA in Tetrahymena pyriformis

Rodriguez-Pousada

Hayes

1976

European Journal of Biochemistry

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Poly(A)-containing RNAs with sedimentation coefficients in the range 10-45 S and 10-35 S ( M , 0.2-3.3 x lo6 and 0 . 2 -2 . 2~ lo6 approximately) are present in the nucleus and cytoplasm of T. pyriformis respectively. The poly(A) segments present in both nuclear and cytoplasmic poly(A)-containing RNAs have a chain length of 80-150 nucleotides. Significant amounts of highmolecular-weight poly(A)-containing RNAs (35 s, M , 2.2 x 1 0 6 ) are found in the post-polysomal fraction of the cytoplasm (messenger ribonucleoprotein particles?) and also in large polysomes. In order to exclude the possibility that these high-molecular-weight species are products of nonspecific aggregation, RNA preparations were submitted to severe denaturing conditions before analysis and all electrophoreses were carried out in the presence of 95 % (v/v) formamide.The currently accepted model for mRNA synthesis in eukaryotic cells, proposed by Darnel1 and coworkers [l], involves synthesis of large precursor RNAs in the cell nucleus, post-transcriptional addition of poly(adeny1ic acid) sequences to their 3' termini, reduction of the size of the poly(A)-containing RNAs without loss of the poly(A) segments and transfer of the shortened molecules to the cytoplasm. As required by this model, poly(A) sequences have been found in cytoplasmic and nuclear RNA of mammalian cells [2], and of a variety of lower eukaryotic organisms [ 3 -71. We have previously described several species of nonmethylated cytoplasmic RNA in the unicellular eukaryote, Tetrahymena pyriformis [8], and in subsequent work have found that their RNAs contain poly(A) sequences. In this report we describe how poly(A)-containing RNAs isolated from the nucleus, cytoplasm and polysomes of T. pyriformis have been characterised by electrophoretic analysis under strongly denaturing conditions to avoid aggregation [9,10] and by estimation of the lengths of their poly(A) segments. MATERIALS AND METHODS ChemicalsTriethanolamine (analytical grade) and formamide (technical grade) were supplied by Prolabo, France. The latter was purified by fractional distillation in vucuo followed by treatment with Amberlite MB-1 mixed-bed ion-exchange resin (B.D.H., England) according to Pinder et al. [lo]. Acrylamide (Fluka, Switzerland) and N,N'-bisacrylamide (Eastman, Kodak, U.S.A.) were purified by recrystallisation according to Loening [ll]. Cyanogen bromide was supplied by Eastman Kodak, U.S.A., dextran sulfate and Sepharose 4B by Pharmacia, Sweden ; pancreatic RNase, and RNase A by Sigma, U.S.A.; and T1 RNase by Sankyo, Japan. Other products were obtained from Prolabo, France, or Merck, Germany.[S3H]Uridine (spec. act. 20-28 Ci/mmol), and [2-3H]adenine (spec. act. 15 -25 Ci/mmol) were supplied by the Commissariat a 1'Energie Atomique, France. BuffersBuffer 1 = 10 mM Tris-HC1 pH 7.5,lO mM MgClz, 0.1 M NaC1, 5 pg/ml cycloheximide and 40 pg/ml dextran sulfate. Buffer 2 = 10 mM Tris-HC1 pH 7.6, 0.1 M NaCl, 10 mM EDTA, 0.2% sodium dodecyl sulfate. Buffer 3 = 10 mM Tris-HC1 pH 7.5, 0.36 M NaCl. Buffer 4 = 5...

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Hybridization Properties of Dna Sequences Directing the Synthesis of Messenger Rna and Heterogeneous Nuclear Rna

Cited by 97 publications

References 32 publications

Analysis of the C-Value Paradox by Molecular Hybridization

Analysis of the C-Value Paradox by Molecular Hybridization

Nonrepetitive DNA Sequence Representation in Sea Urchin Embryo Messenger RNA

Poly(A)‐Containing RNA in Tetrahymena pyriformis

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