New gene expression in dimethylsulfoxide-treated friend erythroleukemia cells

Our previous studies had shown that a small amount of single-stranded DNA (ssDNA) separated from the bulk nuclear DNA of different animal cells by an improved method of hydroxylapatite chromatography (HAC) contains two distinct molecular fractions. The major fraction consists of non self-reassociating sequences that are reassociable to the unique component of bulk DNA and in great part hybridizable to homologous RNA. The minor fraction consists of self-reassociable sequences also reassociable to moderately repetitious bulk DNA. In the present work ssDNA from Friend leukemia cells induced to differentiate (ind FLC) by DMSO was compared with ssDNA from untreated control Friend cells (cont FLC). It was shown that the relative amount of ssDNA is greater in ind FLC than in cont FLC (1.5 – 1.6% and 1.2 – 1.3% of the total cell DNA respectively after a second step of HAC purification). The ind FLC-ssDNA contained a greater proportion of self-reassociable sequences (33–35%) as compared with cont FLC-ssDNA (18–20%). Also the relative amounts of ssDNA hybridizable to cytoplasmic RNA from homologous cells was slightly but constantly higher in ind FLC-ssDNA (33–34%) than in cont FLC-ssDNA (29–30%). Cross hybridizations were carried out between highly radioactive ssDNA and cellular RNAs in great excess, whether total cytoplasmic RNAs or polyadenylated mRNAs. At saturation levels, the hybridized ssDNA fraction was separated from the non-hybridized fraction, and both fractions were rehybridized to RNA from ind FLC or cont FLC. The results indicated that about 10% of ind FLC-ssDNA appeared to be specific for DMSO-treated cells. This may correspond to the expression of 1000–2000 different cytoplasmic mRNAs mostly belonging to the low abundance class.     

Changes in the frequency and diversity of messenger RNA populations in the course of myogenic differentiation.

Complementary DNAs (cDNAs) were synthesized from polyadenylated RNAs of myoblasts and myotubes and used to analyze changes in the sequence complexity and frequency distribution of messenger RNAs during myogenesis in vitro. cDNA . polyadenylated-RNA hybridization kinetics show the presence of messenger RNA sequences specific for myotubes in fully differentiated muscle cultures. These sequences are accumulated just prior to fusion, as was shown by hybridizations of myotube cDNA and total cytoplasmic RNAs from cells at different stages of differentiation. The myotube cDNA can be enriched 10-fold in myotube-specific RNA species by a hybridization with cytoplasmic RNAs from myoblasts and subsequent removal of these hybridized sequences by hydroxyapatite.     

Active DNA transcription sites released from the genome of normal embryonic chicken cells.

Single-stranded DNA (ssDNA) isolated from (and amounting to 1.5-2% of) native nuclear DNA of cultured embryonic chicken cells labelled 1-2 days with 3H-thymidine was analyzed by self-hybridization, hydroxyapatite chromatography (HAC) partial digestion with S1 nuclease, isopycnic centrifugation. Two main fractions were rehybridized to excess amounts of bulk nuclear DNA or total cytoplasmic RNAs. The major fraction, equivalent to 75% of total ssDNA, consists of unique DNA sequences, apparently derived from multiple coding regions of the cell genome, since they are not self-reassociating but are hybridizable to the non repetitious portion of bulk nuclear DNA and 40-45% of them are complementary to cell RNAs. About half of these ssDNA sequences hybridizable to cell RNAs seem to be closely connected with molecules belonging to the minor ssDNA fraction. The latter fraction consists of self-reassociating, moderately repeated DNA sequences, mainly derived from non coding regions of the cell genome. These findings are discussed in the light of others, showing interspersion of coding and non coding DNA sequences and susceptibility of active genes to certain nucleasic attacks.     

Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.     

Translation of an mRNA in rat L6 muscle cells is regulated within the cell cycle

In muscle cells two populations of mRNA are present in the cytoplasm. The majority of mRNA is associated with ribosomes and active in protein synthesis. A small population of cytoplasmic mRNA occur as free mRNA-protein complex and is not associated with ribosomes. This apparently repressed population of mRNA from rat L6 myoblast cells was used to construct a cDNA library. Radioactively labeled cDNA preparations of polysomal and free (or repressed) mRNA populations showed that at least ten recombinant clones preferentially annealed to the cDNA from repressed mRNA. One of these clones was extensively studied. The DNA from a recombinant plasmid D12 hybridized to a 1.3-kb poly(A)-rich mRNA. In proliferating myoblast cells, the 1.3-kb mRNA was more abundant in the polysomal fraction and mostly free in the non-dividing myotubes. In contrast to this mRNA, 90% of alpha and beta actin mRNAs were translated in both myoblasts and myotubes. Further analysis of distribution of the 1.3-kb RNA in the polysomal (active) and free (repressed) fractions in fusion-arrested postmitotic myotubes suggested that fusion of myoblasts was not necessary for the control of translation of this mRNA. Withdrawal of muscle cells from the cell cycle appeared to be involved in regulating translation of this mRNA. The presence of this mRNA was not, however, limited to muscle cells. This mRNA was also present in the repressed state in rat liver and kidney cells. These results, therefore, suggest that the 1.3-kb mRNA is probably translated during a particular phase of the cell cycle and is not translated in terminally differentiated non-dividing cells. Messenger RNA homologous to the 600-base-pair insert of the recombinant plasmid D12 was isolated by hybrid selection procedure from both polysomal mRNA of myoblasts and free mRNA of myotubes. Translation of the hybrid selected mRNAs from both myoblasts and myotubes in rabbit reticulocyte lysate cell-free system synthesized a 40-kDa polypeptide. These results suggest that the repressed population of 1.3-kb mRNA can be translated in vitro. The hybridization pattern of DNA from the recombinant plasmid D12 with rat genomic DNA suggested that the 1.3-kb mRNA is derived from moderately repetitive rat DNA with a repetition frequency of approximately 100 copies per haploid genome.     

Single-strandedness of the majority of DNA sequences complementary to mRNA-coding sites isolated from rat hepatoma tissue cultured cells

Single-stranded DNA (ssDNA), separated from bulk double-stranded DNA (dsDNA) of HTC by an improved method of hydroxyapatite chromatography, exhibited the same characteristics as ssDNA previously found in various cell species. It amounted to 1.5–2% of the total nuclear DNA. Only 24–26% could be self-reassociated, but the greatest part hybridized to non-repetitious DNA fraction and about 30% hybridized to homologous mRNA.Other results tend to prove that the complementary sequences of HTC-ssDNA probably consist of non-base-paired segments attached to double helical regions of dsDNA. In effect, after hydroxyapatite chromatography, a small portion of HTC-dsDNA (2–3%) was found to be rapidly digestible by S1 nuclease and this limited digestion was sufficient to reduce markedly the hybridization rates of dsDNA with both DNA and cell-free synthesised cDNA copies of polyadenylated RNAs. Furthermore, these ³H-cDNA copies could not be annealed to ssDNA under conditions that allowed their reassociation with total nuclear DNA. These findings complete the demonstration that the greatest part of ssDNA appears to be formed via selective nicks, probably enzymatic, in the coding strand of actively transcribed DNA regions.     

Interactions between 125-I-labelled RNA from RNA tumor viruses and RNA of other sources.

Fragmented 125I-labelled RNA from RNA tumor viruses was hybridized to unlabelled RNA from cells, viruses, and homoribopolymers. The viral RNA interacted with all RNA tested, except for certain homoribopolymers. Complex formation with unlabelled RNA was verified by nuclease resistance, buoyant density measurements, and thermal stability in solutions of different ionic strength. The RNAase-resistant complex involved 20-30% of the sequences in the 125I-labelled viral RNA and formed preferentially with nuclear RNA of cells. 125I-labelled hemoglobin mRNA, 125I-labelled immunoglobulin light chain (lambda2) mRNA, or 125I-labelled viral RNA from encephalomyocarditis virus (EMC) dit not from RNAase-resistant complexes with unlabelled cellular RNA.     

The appearance of new polyadenylated nuclear RNA sequences during rat-liver regeneration

Polyadenylated RNA populations from normal and 16-hour regenerating rat-liver nuclei were compared by heterologous hybridisation reactions with cDNA and unique DNA probes. Whereas unique DNA hybridisations did not show differences between the RNA populations, comparisons by cDNA hybridisation showed that about 10--15% by weight of polyadenylated sequences present in the nuclei of 16-hour regenerating rat livers were not found in the polyadenylated nuclear RNA of normal rat livers. These regenerating-specific nuclear cDNA sequences were isolated and characterised; the experiments showed that the complexity of the new sequences was 1-2 x 10(7) nucleotides (equivalent to 5,000--10,000 RNA sequences of 2,000 nucleotides in length) and that they were probably not potential messenger RNA sequences.     

Linc-smad7 promotes myoblast differentiation and muscle regeneration via sponging miR-125b

Long noncoding RNAs (lncRNAs) are involved in the regulation of skeletal muscle development. In the present study, differentially expressed lncRNAs were identified from RNA-seq data derived from myoblasts and myotubes. We conducted studies to elucidate the function and molecular mechanism of action of Linc-smad7 during skeletal muscle development. Our findings show that Linc-smad7 is upregulated during the early phase of myoblasts differentiation. In in vitro studies, we showed that overexpression of Linc-smad7 promoted the arrest of myoblasts in G1 phase, inhibited DNA replication, and induced myoblast differentiation. Our in vivo studies suggest that Linc-smad7 stimulates skeletal muscle regeneration in cardiotoxin-induced muscle injury. Mechanistically, Linc-smad7 overexpression increased smad7 and IGF2 protein levels. On the contrary, overexpression of miR-125b reduced smad7 and IGF2 protein levels. Results of RNA immunoprecipitation analysis and biotin-labeled miR-125b capture suggest that Linc-smad7 could act as a competing endogenous RNA (ceRNA) for miRNA-125b. Taken together, our findings suggest that the novel noncoding regulator Linc-smad7 regulates skeletal muscle development.     

Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA

Polyadenylated RNA, isolated from total cellular RNA of photoautotrophically grown Euglena gracilis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with ¹²⁵I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA.     

Globin mRNA sequences in polyadenylated and nonpolyadenylated nuclear precursor-messenger RNA from avian erythroblasts

Nuclear RNA from immature duck erythrocytes was fractionated into polyadenylated and nonpolyadenylated fractions, and globin mRNA sequences were determined by hybridization to DNA complementary to globin mRNA.80–90% of labeled nuclear RNA is found to be nonpolyadenylated, and 70–80% of the globin mRNA sequences present in the nucleus are found in nonpolyadenylated molecules. These data suggest that polyadenylation does not specifically select for globin mRNA sequences.The nonpolyadenylated globin mRNA sequences present in the nucleus are found mostly in molecules of small size, close to the size of polyribosomal globin mRNA, suggesting that polyadenylation is a later event in globin mRNA formation.