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.     

Relationship between single-stranded DNA isolated from cultured muscular cells during differentiation and the transcription of messenger RNA.

Single-stranded DNA (ssDNA), equivalent to about 2% of the total nuclear DNA, was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA of rat myoblast cells and myotubes of the L6 line. Small quantities of 125I-labelled ssDNA were annealed with a large excess of unlabelled DNA, cytoplasmic RNA and mRNA from myoblasts or myotubes. The results indicated that ssDNA belongs to the non-repetitious portion of the cell genome and is formed of two distinct molecular fractions. The major ssDNA fractions (75%) consist of non-self-reassociating DNA sequences and the minor fraction (25%) consists of self-reassociating DNA sequences. About 30--32% and 25--26% of ssDNA from myoblast represent DNA sequences complementary to total cytplasmic RNAs and polyadenylated RNAs respectively. Hybridizations of ssDNA with an excess of RNA from myoblasts and/or myotubes show differences in the abundance and the diversity of mRNA during mascular differentiation. These differences were confirmed by DNA-driven reactions between 125I-labelled polyadenylated RNA and ssDNA in great excess.     

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.     

Characterization of Allomyces genome

Allomyces arbuscula DNA isolated from whole cells (bulk DNA) is composed of a major (alpha) and two minor components (beta & gamma) with buoyant densities in neutral CsCl corresponding to 1.721, 1.710 and 1.702 g/cm3, respectively. The DNA obtained from purified nuclei contains alpha component only. The beta component corresponds to mitochondrial DNA. The gamma component is also extra-nuclear but has not been characterized. The reassociation kinetics of sheared, bulk and nuclear DNA show that (i) 25 % bulk and 10% of nuclear DNA reanneal very rapidly and contain highly repeated sequences; (ii) moderately repeated sequences, accounting for 15% of both bulk and nuclear DNA, have a sequence complexity of approximately 7.2-10(6) daltons and are repeated about 320 times; (iii) the slow reannealing fraction accounts for about 60% of the genome and has kinetic properties similar to single copy sequences. The sequence complexity of this fraction was determined in relation to that of Escherichia coli. After a correction for the size of the repeated sequences the genome size of A. arbuscula was calculated to be 1.7-10(10) daltons.     

Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2-infected and transformed cells.

The concentrations, in copies per cell, of viral RNA sequences complementary to different regions of the genome were determined at 8, 18 and 32 hours after infection of human cells with adenovirus type 2: separated strands of fragments of ³²P-labelled adenovirus 2 DNA, generated by cleavage with restriction endonucleases EcoR1, Hpa1 and BamH1, were added to reaction mixtures at sufficient concentrations to drive hybridizations with infected or transformed cell RNA. Under these conditions, the fraction of ³²P-labelled DNA entering hybrid is directly proportional to the absolute amount of complementary RNA in the reaction.At 8 hours after infection in the presence of cytosine arabinoside, “early” viral messenger RNA sequences are present at a frequency of 300 to 1000 copies per cell. The abundance of early mRNA sequences in different lines of adenovirus 2-transformed rat cells is markedly lower than their concentration in lytically infected cells. Moreover, the abundance of early mRNA in a given transformed rat cell line reflects the number of copies of its template DNA sequences per diploid quantity of cell DNA. After the onset of the late phase of the lytic cycle, the abundance of one early mRNA species, that coding for a single-stranded DNA binding protein required for viral DNA replication, is amplified. Viral RNA sequences complementary to regions of the genome coding for other early mRNA sequences remain at the level observed at 8 hours after infection.Exclusively “late” viral mRNA sequences are present over a range of concentrations, 500 to 10,000 copies per cell, depending on the region of the genome. By 18 hours after infection, the nucleus contains approximately three times as much total, viral RNA as the cytoplasm. The abundant nuclear, viral RNA sequences at 18 hours are transcribed from a contiguous region, 65% of the genome in length. In some cases, viral RNA sequences complementary to mRNA sequences are very abundant in the nucleus. When cytoplasmic and nuclear fractions are mixed and incubated under annealing conditions, some mRNA sequences will anneal with more abundant, anti-messenger nuclear RNA sequences to form double-stranded RNA. Such annealing of nuclear, viral RNA to early, cytoplasmic mRNA sequences probably accounts for the inability to detect, by filter hybridization, certain classes of early mRNA sequences during the late stage of infection.     

Viral-encoded small RNAs in herpes virus saimiri induced tumors.

DNA sequences from the left terminus of herpes virus saimiri L-DNA are essential for the oncogenic and transforming potential of the virus, but these sequences are not required for replication. RNA derived from 0.0 to 6.7 map units (7.4 kbp) on the herpes virus saimiri genome was studied by Northern blot hybridization and by nuclease protection analyses. Although several poly(A)-containing RNAs were detected from this region in permissively-infected monolayer cells in vitro, these RNAs could not be detected in cells taken directly from viral-induced lymphomas nor in the lymphoblastoid tumor cell line 1670. Instead, these transformed T-cells expressed four small RNAs of approximately 73, 105, 110 and 135 nt derived from this region. These small RNAs were not detected at all during the course of lytic infection of monolayer cells. Thus, synthesis of these RNAs is stringently regulated in a cell-type specific manner. Genomic coding sequences for each of these small RNAs were mapped to 0.5-1.2 kbp DNA fragments stretched over 4.3 kbp of viral genetic information. These findings together with the biological properties of mutants with deletions in this region have led us to speculate that one or more of these small RNAs play an essential role in cell growth transformation by herpes virus saimiri.     

Characterization of small RNAs in Xenopus tropicalis gastrulae

Here, we report and characterize deep sequencing data and bioinformatics analysis of small RNAs from Xenopus tropicalis gastrula. A total of 17,553,124 reads with perfect match to the genome derived from 2,616,053 unique sequences were identified. Seventy-seven percent of theses sequences were not found in previous reports from X. tropicalis oocytes and somatic tissues. Bioinformatics analyses indicate that a large fraction of the small RNAs are PIWI-interacting RNAs. Up to 23.9% of small RNAs mapped to transposable elements and 27% to genic regions. Most of abundant transposable derived small RNAs are found in oocyte and gastrula libraries, suggesting that transposon needs to be silenced also during early development. Additionally, miRNAs were identified and many of them are not present in oocytes, suggesting that miRNA expression is stage specific. To the best of our knowledge, this is the first high throughput data release and bioinformatics characterization of small RNAs during Xenopus development.     

Few mitochondrial DNA sequences are inserted into the turkey (Meleagris gallopavo) nuclear genome: evolutionary analyses and informativity in the domestic lineage

Mitochondrial DNA (mtDNA) insertions have been detected in the nuclear genome of many eukaryotes. These sequences are pseudogenes originated by horizontal transfer of mtDNA fragments into the nuclear genome, producing nuclear DNA sequences of mitochondrial origin (numt). In this study we determined the frequency and distribution of mtDNA‐originated pseudogenes in the turkey (Meleagris gallopavo) nuclear genome. The turkey reference genome (Turkey_2.01) was aligned with the reference linearized mtDNA sequence using last . A total of 32 numt sequences (corresponding to 18 numt regions derived by unique insertional events) were identified in the turkey nuclear genome (size ranging from 66 to 1415 bp; identity against the modern turkey mtDNA corresponding region ranging from 62% to 100%). Numts were distributed in nine chromosomes and in one scaffold. They derived from parts of 10 mtDNA protein‐coding genes, ribosomal genes, the control region and 10 tRNA genes. Seven numt regions reported in the turkey genome were identified in orthologues positions in the Gallus gallus genome and therefore were present in the ancestral genome that in the Cretaceous originated the lineages of the modern crown Galliformes. Five recently integrated turkey numts were validated by PCR in 168 turkeys of six different domestic populations. None of the analysed numts were polymorphic (i.e. absence of the inserted sequence, as reported in numts of recent integration in other species), suggesting that the reticulate speciation model is not useful for explaining the origin of the domesticated turkey lineage.     

Attempted separation of transcriptively active and inactive chromatin by hydroxylapatite thermal chromatography

Chromatin and purified DNA were fractionated by hydroxylapatite thermal chromatography. Fractions of varying thermal stability were tested for the proportions of transcribed sequences and repetitive sequences relative to the unfractionated genome. The first 80--85% of either total chromatin or purified DNA eluted from hydroxylapatite contained the same proportion of hybridizable sequences as total DNA. The remaining 15--20% of chromatin eluting at the highest temperatures was depleted of transcribed sequences. Analysis of the 20% highest melting fraction of purified DNA showed that, while the first two-thirds of this fraction contained the same proportion of transcribed sequences as unfractionated DNA, the last third, comprising about 6% of total DNA, was depleted of active sequences. Although no major differences were detected in nonrepetitive sequence complexity of chromatin fractions, there was a correlation between relative thermal stability and repetitive sequence content in fractions of both chromatin and DNA separated by thermal chromatography. Fragments eluting at higher temperatures contained a greater proportion of repetitive sequences, as indicated by a rapidly renaturing component. Most likely, the latest eluting fractions from both chromatin and purified DNA were enriched for a nontranscribed, highly reiterated, G+C rich satellite component of the chicken genome.     

Congruent evolution of different classes of non-coding DNA in prokaryotic genomes

Prokaryotic genomes are considered to be 'wall-to-wall' genomes, which consist largely of genes for proteins and structural RNAs, with only a small fraction of the genomic DNA allotted to intergenic regions, which are thought to typically contain regulatory signals. The majority of bacterial and archaeal genomes contain 6-14% non-coding DNA. Significant positive correlations were detected between the fraction of non-coding DNA and inter- and intra-operonic distances, suggesting that different classes of non-coding DNA evolve congruently. In contrast, no correlation was found between any of these characteristics of non-coding sequences and the number of genes or genome size. Thus, the non-coding regions and the gene sets in prokaryotes seem to evolve in different regimes. The evolution of non-coding regions appears to be determined primarily by the selective pressure to minimize the amount of non-functional DNA, while maintaining essential regulatory signals, because of which the content of non-coding DNA in different genomes is relatively uniform and intra- and inter-operonic non-coding regions evolve congruently. In contrast, the gene set is optimized for the particular environmental niche of the given microbe, which results in the lack of correlation between the gene number and the characteristics of non-coding regions.