A kinetic estimation of base sequence complexity of nuclear poly(A)-containing RNA in mouse friend cells

Getz, Michael J.; Birnie, G.D.; Young, Bryan D.; MacPhail, Elizabeth; Paul, John

doi:10.1016/0092-8674(75)90118-x

Cited by 152 publications

(64 citation statements)

References 28 publications

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“…We determined that 0.22% of the total nuclear RNA is poly(A) by weight, representing 17.5% of the total poly(A) content of a rat liver cell (Table 1). This high level of nuclear steady-state poly-(A), when compared to the nuclear messenger sequence content, is consistent with the result that only a fraction of total nuclear poly(A)-containing RNA is transported to the cytoplasm [14][15][16]371. Certainly, the major part of nuclear RNA consists of mRNA-unrelated sequences, such as rRNA and its precursors.…”

Section: Discussionsupporting

confidence: 66%

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Frequency Distribution of Messenger Sequences within Polysomal mRNA and Nuclear RNA from Rat Liver

Sippel

Hynes

Groner

et al. 1977

European Journal of Biochemistry

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DNA complementary to polysomal poly(A)-containing mRNA (cDNA) of male rat liver was used to study the diversity of messenger sequences in the nucleus and in polysomes.1. Hybridization of cDNA against an excess of its own polysomal mRNA template revealed that about 10000 different mRNA species are expressed in the liver tissue. They are distributed in a wide frequency range derived from approximately 0.5 % of the total genome.2. Hybridization of the cDNA against total nuclear RNA shows that messenger sequences comprise less than 1 of the mass of total nuclear RNA. Messenger sequences have a different frequency distribution in nucleus and cytoplasm.3. In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.Different rates of synthesis of a specific protein are mediated by parallel changes of the functional level of the corresponding mRNA in a variety of systems [l -51. Mechanisms responsible for altering mRNA concentrations are not well understood, since our knowledge of nuclear processes in the biogenesis of eucaryotic mRNA is limited. Molecular hybridization techniques have brought some insight into the relationship between nuclear and cytoplasmic RNA. It has recently been shown that nuclear RNA is up to 10 times as complex as polysomal mRNA [6-121. Poly(A)-containing nuclear RNA, generally assumed to be the RNA pool from which most cytoplasmic mRNA molecules are derived (for review see [13]), was found to have a significantly higher complexity than polysomal mRNA in a variety of cell types [14-161. From the observed reductions in RNA sequence complexity and RNA size (see accompanying paper), it is clear that only a minor subset of sequences in Abbreviations. cDNA, DNA copy complementary to mRNA; hnRNA, polydisperse high-molecular-weight nuclear RNA ; AMV, avian myeloblastosis virus. rot value, concentration of RNA (as mol phosphate/l) times time of hybridization (in s); rof1p, rot at which half of hybridizable cDNA is hybridized.Enzymes. Staphylococcal nuclease, nucleate 3'-oligonucleotidohydrolase (EC 3.1.4.7) ; reverse transcriptase, RNA-dependent DNA polymerase, deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase (EC 2.7.7.7); DNase I, deoxyribonucleate oligonucleotidohydrolase (EC 3.1.4.5).nuclear RNA leaves the nucleus to become active polysomal mRNA. Nuclear polyadenylation is certainly not a sufficient prerequisite for an RNA sequence to be selected for transport into the cytoplasm.Very little information exists on the frequency distribution of messenger sequences within total nuclear RNA. To obtain more information we have performed a comparative examination of the kinetics of hybridization of nuclear RNA and polysoma1 mRNA to complementary DNA synthesized from poly(A)-containing mRNA. We have found that a different frequency distribution of ...

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Section: Discussionsupporting

confidence: 66%

“…2A), indicates that the cDNA represents the diversity of its template mRNA population. Additional arguments supporting this have also been given by others [30,31,14,24].…”

Section: Isolation Of Mrna and Reverse Transcription Into Complementamentioning

confidence: 67%

Frequency Distribution of Messenger Sequences within Polysomal mRNA and Nuclear RNA from Rat Liver

Sippel

Hynes

Groner

et al. 1977

European Journal of Biochemistry

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“…However, the lysis procedure had been devised to purify polysomes which tested by centrifugation in a CsCl gradient, appear to be free of nuclear RNPs (Spohr et al,34) Nevertheless, even if we allow for a contamination of 10% of poly(A)+ -mRNA by poly(A)+ nuclear RNA, this would contribute a maximum of 10% of cDNA sequences of nuclear origin (probably less, since on a weight basis, poly(A)+ nuclear RNA is reverse-transcribed less than poly(A)+ -mRNA).The complex component of polyadenylated nuclear RNA is about 5 times more complex than poly(A)+ -mRNA (18) and would therefore hybridize with its cDNA at a Rot i of 30 in our conditions. In the experiment of Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The sequence complexity of nuclear RNA has also been measured (sea urchin (14), drosophila cells (15), rat tissues (16), mouse brain (17), uninduced Friend cells (18,19). It has usually been found to be 5-10 times higher than the complexity of polysomal mRNA.…”

Section: Introductionmentioning

confidence: 99%

Frequency distribution of mRNA and pre-mRNA in growing and differentiated Friend cells

Mauron¹,

Spohr²

1978

Nucl Acids Res

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The frequency distribution of poly(A)+-mRNA in growing and in differentiated Friend cells has been measured by mRNA-cDNA hybridization and their differences established by heterologous hybridization of mRNA of one type and cDNA of the other. It was shown that induction of Friend cells involves an increase in abundance of a small number of mRNAs, while no specific pattern of messenger disappearance could be detected. The frequency distribution of pre-mRNA was determined by hybridizing nuclear RNA with the cDNA probes complementary to mRNA. In uninduced Friend cells, it was shown that most precursor messenger sequences are present at a single frequency of about 3 molecules per nucleus, independently of their final frequency in polysomal mRNA. In induced Friend cells, the frequency distribution of premRNA is more heterogeneous and correlated to some extent with the corresponding mRNA frequency distribution. INTRODUCTIONThe control of gene expression in eukaryotic cells still defies a general explanation, partly because of the multiplicity of levels at which molecular regulation devices may operate. In order to appreciate the importance of these various levels of control, it would be important to know which sets of genes are transcribed into nuclear RNA and into messenger RNA in a given cell type.

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“…Acid-precipitable radioactivity released to the medium was determined by precipitation of total and released RNA in ice-cold 10 % (w/v) trichloroacetic acid, followed by filtration through glass fibre (GF/C, Whatman) filter circles, and extensive washing with 10 % trichloroacetic acid containing 1 % sodium pyrophosphate. For other experiments, the nuclei were removed (1000 x g, , , for 5 min at 4 "C), and RNA was recovered from the supernatant fluid by repeated extraction with phenol/chloroform followed by precipitation in 70% ethanol [24]. Poly(A)-rich RNA was isolated by two cycles of chromatography on oligo(dT)-cellulose (Collaborative Research); bound RNA was eluted with distilled water and desalted by filtration through Sephadex G-50 equilibrated with distilled water [25].…”

Section: Isoldion and Assay Of R N A Released To The Mediummentioning

confidence: 99%

Isolation and Purification of Rat Hepatoma Nuclei Active in the Transport of Messenger RNA in vitro

Jacobs

Birnie

1982

Eur J Biochem

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1. A variety of procedures for obtaining metabolically active nuclei from rat hepatoma tissue-culture (HTC) cells has been explored. All gave preparations which were, to a greater or lesser extent, contaminated with cytoplasmic RNA, which appeared to be absorbed non-specifically during the lysis of the cells.2. A medium containing spermidine, monovalent and divalent cations, and cytosol has bccn defined in which the integrity of the isolated nuclei was maintained during incubation in virro.3. The release of steady-state-labelled and pulse-labelled RNA from HTC cell nuclei during incubation in this medium has been studied. The transport of both was dependent on ATP and cytosol, but was distinguishable by the kinetics and by the fact that only the transport of pulse-labelled RNA appeared to involve hydrolysis of ATP. Transported RNA included ribosomal R N A and messenger-likc RNA as judged by criteria of size, oligo(dT)-cellulose binding, and organisation into ribonucleoproteins. A large proportion of the cytoplasmic R N A which adhered to the nuclei was also released during incubation in v i m .4. Although the poly(A)-rich RNA transported in v i m was contaminated with poly(A)-rich RNA sequences arising from non-specific leakage of nuclear RNA and from the release of nucleus-adherent cytoplasmic RNA, the extent of this contamination was comparatively small (10"; and 10-15% by weight respcctively). Thc transportcd poly(A)-rich RNA has been shown to consist of a complex mixture of sequences at heterogeneous abundanccs, resembling polysomal poly(A)-rich mRNA from HTC cells.5. The pattern of relative abundanccs of the sequences in transported poly(A)-rich RNA was intermediate betwieen that of polysomal and nuclear poly(A)-rich RNAs, and indicated that some degree of sequence-spccific selection of processing, or transport, or both, can be maintained in isolated nuclei. Sequence-specific selcction in vitro at the level of individual poly(A)-rich RNA sequences was detected using cloned cDNAs for poly(A)-rich polysomal RNA sequences.

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A kinetic estimation of base sequence complexity of nuclear poly(A)-containing RNA in mouse friend cells

Cited by 152 publications

References 28 publications

Frequency Distribution of Messenger Sequences within Polysomal mRNA and Nuclear RNA from Rat Liver

Frequency Distribution of Messenger Sequences within Polysomal mRNA and Nuclear RNA from Rat Liver

Frequency distribution of mRNA and pre-mRNA in growing and differentiated Friend cells

Isolation and Purification of Rat Hepatoma Nuclei Active in the Transport of Messenger RNA in vitro

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