Repetitive sequences and hairpin-like structures in giant RNA of pigeon erythroid cells

In giant molecules (>45 S) of HnRNA from pigeon bone marrow and peripheral blood erythroid cells a correlation is demonstrated between the amounts of hairpin-like structures and the sequences transcribed from the DNA repetitions. The same correlation is observed in the >45 S poly(A)⁺ and poly(A)^- subfractions.Abbreviations HnRNA heterogeneous nuclear RNA - poly(A)⁺ RNA RNA molecules containing polyadenylic acid sequences - poly(A)^- RNA RNA molecules which do not contain polyadenylic acid sequences - dsRNA double-stranded RNA - SDS sodium dodecylsulphate     

Concentrations of individual RNA sequences in polyadenylated nuclear and cytoplasmic RNA populations of Drosophila cells.

Steady state concentrations of individual RNA sequences in poly(A) nuclear and cytoplasmic RNA populations of Drosophila Kc cells were determined using cloned cDNA fragments. These cDNAs represent poly(A) RNA sequences of different abundance in the cytoplasm of Kc cells, but their steady state concentrations in poly(A) hnRNA was always lower. Of ten different sequences analysed, eight showed some four-fold lower concentration in hnRNA mRNA, two were underrepresented in hnRNA relative to the others. The obvious clustering of mRNA/hnRNA ratios is discussed in relation to sequence complexity and turnover rates of these RNA populations.     

Hybridization of low molecular weight nuclear RNA with pre-mRNA from erythroid bone marrow cells and rabbit mRNA

Hybridization of labeled low molecular weight (LMW) nuclear RNA's to pre-mRNA from rabbit non-matured erythroid bone marrow cells or globin mRNA from reticulocytes revealed three RNA species having approximately 90, 100 and 160 nucleotides which are were specifically hybridized with purified cytoplasmic globin messenger RNA, while one (100 nucleotides) was also hybridized with rabbit 18S rRNA. The identity of these rabbit RNAs to LMW RNAs described for other animal species, as well as their possible hybridization sites and function are discussed.     

Repetitious and unique sequences in the heterogeneous nuclear and cytoplasmic messenger RNA of mammalian and insect cells

Initiation of DNA synthesis has been followed in mouse myeloma cells grown in suspension culture. In cells labeled with ³H-thymidine for short times, label first appears in short fragments of DNA which can be chased into bulk DNA (>50 S) upon further incubation in unlabeled thymidine. In a 15 min pulse, DNA fragments with a sedimentation coefficient of 30 S tend to accumulate. Our results support the contention that DNA synthesis is discontinuous in myeloma cells.However, a search for RNA associated with nascent DNA in the myeloma system was unsuccessful. Newly synthesized DNA was isolated on a benzoylated naphthoylated DEAE cellulose column. After heat denaturation, this fraction was centrifuged to equilibrium in a Cs₂SO₄ density gradient. The nascent DNA displays no shift in density greater than the density of the bulk DNA. When cells were pulse labeled with ³H-uridine and the nascent DNA fraction analyzed on Cs₂SO₄ density gradients, no ³H-labeled RNA was found associated with the DNA peak or at intermediate densities that would be indicative of a RNA-DNA molecule, covalently linked. Unless scission of the RNA primers occurs immediately after the initiation of DNA synthesis, our results indicate that DNA synthesis commences without RNA primers in myeloma cells.     

Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells.

Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.     

Balbiani ring nucleotide sequences in cytoplasmic 75 S RNA of Chironomus tentans salivary gland cells

The giant puffs, the Balbiani rings (BR) 1 and 2 of Chironomus tentans polytene chromosomes synthesize large RNA molecules sedimenting at about 75S. An RNA fraction of approximately the same size is present in nuclear sap and cytoplasm. In situ hybridization of cytoplasmic 75S RNA and other electrophoretically defined cytoplasmic RNA fractions showed BR RNA to be confined to the 75S RNA, and absent in other high molecular-weight cytoplasmic RNA fractions, which indiates that BR RNA is transferred from the nucleus into the cytoplasm without an appreciable size reduction.     

Relationship between nuclear and cytoplasmic RNA in Drosophilia cells.

Polyadenylated RNA was isolated from nuclei of cultured Drosophila cells, Schneider's line 2, and used as a template to synthesize a complementary DNA probe. Hybridization experiments were performed to study the relationship between nuclear and cytoplasmic RNA. About two-thirds of the nuclear polyadenylated RNA sequences exist in the cytoplasm. Experiments with fractionated cDNA probes demonstrated that RNA sequences that are frequent in the nucleus are also abundant in the cytoplasm. These findings are consistent with a precursor-product relationship in which some polyadenylated molecules in the nucleus are destined for the cytoplasm while other sequences are polyadenylated but not transferred.     

The structure of nuclear pre-mRNA. VII. The hybridization and renaturation properties of double-stranded sequences of nuclear pre-mRNA]

Some properties of the double-stranded regions of pre-mRNA are discribed. 1. The double-stranded regions contain approximately 80 base pairs. 2. The material contains the heterogeneous populations of sequences and some homogenous material which renatures with a COT1/2 value of (1.5-3) X 10(-4). 3. Identical sequences of fast-renaturing "hairpins" may be found in various tissues. 4. Double-stranded RNA and mRNA have some sequences complementary to each other. These results consistent with the view that the hairpin sequences may act as specific recognition sites for ribonucleases involved in processing of pre-mRNA.     

Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells

A variety of methylated oligonucleotides were derived from mouse L cell messenger RNA and heterogeneous nuclear RNA by digestion with specific ribonucleases, and the cap-containing oligonucleotides separated from those containing internal m⁶A by chromatography on diborylaminoethyl-cellulose. Cap-containing sequences of the type m⁷GpppX^mpG, m⁷GpppX^mpY^(m)pG, m⁷GpppX^mpY^(m) pNpG and m⁷GpppX^mpY^(m)p(Np)_{> 1}G have distinctive non-random compositions of the 2′-O-methylated constituent X^m; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA². For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m⁷Gppp(m⁶)A^mG, whereas less than 1% are m⁷GpppU^mpG. The high degree of similarity in cap sequences is consistent with the previously postulated precursor-product relationship between hnRNA caps and mRNA caps.The composition of the Y position in capped hnRNA molecules was determined to be (29% G, 20% A, 51% Py), which differs considerably from the composition of Y^m in the cap II forms of mRNA (8% G^m, 11% A^m, 81% Py). Given the precursor-product relationship between hnRNA caps and mRNA caps, this result provides strong evidence that only a restricted subclass of mRNA molecules receive the secondary methylation at position Y.In both hnRNA and mRNA the internal m⁶A occurs in well-defined sequences of the type: $\text{-N}{}_1\text{-(}\text{G}\text{A}\text{)-m}{}^6\text{A-C-N}{}_2\text{-}$, the 5′ nearest-neighbor of m⁶A being G in about three-quarters of the molecules and A in about one-quarter of the molecules. The nucleotide N¹ is a purine about 90% of the time and the nucleotide N² is rarely a G. These same sequences are present in large (> 50 S), as well as small (14 S to 50 S) hnRNA. These results raise the possibility that the internal m⁶A, like caps, may be conserved during the processing of large hnRNA into mRNA. Two models based on this idea are discussed.     

Cloning and study of inserted sequences of gene 28S in Drosophila melanogaster ribosomal RNA

Cloning of fragments of ribosomal genes containing insertions in the 28S RNA gene has been reported earlier. Subcloning of DNA fragments corresponding to insertion sequences and their hybridization with DNA, RNA and polytene chromosomes from different flies is described. Type 1 insertions (containing BamI sites) are highly heterogeneous in length and sequence even in homozygotes. Type 2 insertions (with EcoRI sites) are rather homogeneous. Two types of insertions are represented in the D. melanogaster genome by 50 and 30 copies, respectively. Restriction fragments with insertions significantly differ in DNA from embryos and larvae. D. simulans and D. virilis also contain the sequences of both types of insertions, though in fewer number of copies. Type 1 insertions seem to be poorly transcribed, and type 2 insertions are not transcribed at all. Among 2000 recombinant clones screened a number of DI plasmids hybridizing to isolated insertions were obtained. Six of them were mapped with restriction endonucleases and hybridized with insertion fragments. rRNA and polytene chromosomes. All of these DI plasmids hybridize with the nucleoli, one with the chromocenter and one with the 79F 3L site. In LI9, not coding for rRNA, the sequences, corresponding to two types on insertions are located only a few kilobases apart. D17a does not encode for rRNA, but hybridizes in situ only with the nucleoli.     

Oligomeric sequences in the cytoplasmic RNA of sea urchin embryos

Oligomeric stretches of adenylate and uridylate and polymeric segments of adenylate have been shown to exist in sea urchin embryo hnRNA. It is demonstrated here that at least some oligo(U)-enriched sequences are conserved in sea urchin cytoplasmic RNA, whereas apparently few, if any, oligo(A) sequences are so conserved.