C-rich Segment Research Articles

DSIF modulates RNA polymerase II occupancy according to template G+C content.

The DSIF complex comprising the Supt4h and Supt5h transcription elongation proteins clamps RNA polymerase II (RNAPII) onto DNA templates, facilitating polymerase processivity. Lowering DSIF components can differentially decrease expression of alleles containing nucleotide repeat expansions, suggesting that RNAPII transit through repeat expansions is dependent on DSIF functions. To globally identify sequence features that affect dependence of the polymerase on DSIF in human cells, we used ultra-deep ChIP-seq analysis and RNA-seq to investigate and quantify the genome-wide effects of Supt4h loss on template occupancy and transcript production. Our results indicate that RNAPII dependence on Supt4h varies according to G + C content. Effects of DSIF knockdown were prominent during transcription of sequences high in G + C but minimal for sequences low in G + C and were particularly evident for G + C-rich segments of long genes. Reanalysis of previously published ChIP-seq data obtained from mouse cells showed similar effects of template G + C composition on Supt5h actions. Our evidence that DSIF dependency varies globally in different template regions according to template sequence composition suggests that G + C content may have a role in the selectivity of Supt4h knockdown and Supt5h knockdown during transcription of gene alleles containing expansions of G + C-rich repeats.

A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein

The fabrication of nanomaterials-based sensing platform has attracted a great deal of interest due to their unique properties. Here, we report a novel hybrid platform of g-C3N4 nanosheets/DNA-stabilized Ag nanoclusters (CNNS/AgNCs) for sensing application. In this platform, the fluorescent AgNCs was synthesized using a pair of double-functional ssDNA sequence as a template, including the aptamer segment against thrombin and C-rich segment for AgNCs. Next, the interaction between the fluorescent Apt-AgNCs and CNNS was investigated. It is verified that DNA-stabilized AgNCs could absorb on the CNNS surface via the stronger π−π interaction to form the hybrid platform, whose fluorescence is quenched by CNNS through the photoelectron transfer effect (PET). When targets are introduced into the system, target/Apt-AgNCs complex will fall off from the CNNS surface, resulting in the fluorescence recovery. This hybrid platform can achieve the detection of biomolecule with high sensitivity and selectivity. Considering the fluorescence variability of DNA scaffold AgNCs, this hybrid platform is promising to extend to other target and even multi-target detection.

Novel alleles of the VERNALIZATION1 genes in wheat are associated with modulation of DNA curvature and flexibility in the promoter region.

BackgroundIn wheat, the vernalization requirement is mainly controlled by the VRN genes. Different species of hexaploid and tetraploid wheat are widely used as genetic source for new mutant variants and alleles for fundamental investigations and practical breeding programs. In this study, VRN-A1 and VRN-B1 were analysed for 178 accessions representing six tetraploid wheat species (Triticum dicoccoides, T. dicoccum, T. turgidum, T. polonicum, T. carthlicum, T. durum) and five hexaploid species (T. compactum, T. sphaerococcum, T. spelta, T. macha, T. vavilovii).ResultsNovel allelic variants in the promoter region of VRN-A1 and VRN-B1 were identified based on the change in curvature and flexibility of the DNA molecules. The new variants of VRN-A1 (designated as Vrn-A1a.2, Vrn-A1b.2 – Vrn-A1b.6 and Vrn-A1i) were found to be widely distributed in hexaploid and tetraploid wheat, and in fact were predominant over the known VRN-A1 alleles. The greatest diversity of the new variants of VRN-B1 (designated as VRN-B1.f, VRN-B1.s and VRN-B1.m) was found in the tetraploid and some hexaploid wheat species.For the first time, minor differences within the sequence motif known as the VRN-box of VRN1 were correlated with wheat growth habit. Thus, vrn-A1b.3 and vrn-A1b.4 were revealed in winter wheat in contrast to Vrn-A1b.2, Vrn-A1b.5, Vrn-A1b.6 and Vrn-A1i. It was found that single nucleotide mutation in the VRN-box can influence the vernalization requirement and growth habit of wheat. Our data suggest that both the A-tract and C-rich segment within the VRN-box contribute to its functionality, and provide a new view of the hypothesised role of the VRN-box in regulating transcription of the VRN1 genes. Specifically, it is proposed that combination of mutations in this region can modulate vernalization sensitivity and flowering time of wheat.ConclusionsNew allelic variants of the VRN-A1 and VRN-B1 genes were identified in hexaploid and tetraploid wheat. Mutations in A-tract and C-rich segments within the VRN-box of VRN-A1 are associated with modulation of the vernalization requirement and flowering time. New allelic variants will be useful in fundamental investigations into the regulation of VRN1 expression, and provide a valuable genetic resource for practical breeding of wheat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0691-2) contains supplementary material, which is available to authorized users.

Conformational diversity of single-stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases.

Repetitive extragenic palindrome (REP)—associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5′-G and 3′-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 585–596, 2015.

The complete mitochondrial genome of the Sichuan hot-spring keel-back (Thermophis zhaoermii; Serpentes: Colubridae) and a mitogenomic phylogeny of the snakes

The complete mitochondrial genome sequence of the Sichuan hot-spring keel-back (Thermophis zhaoermii) was determined in the present study. The genome is 17,322 bp in size, containing 2 ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and 2 control regions, similar to other alethinophidian snakes. A special 40 bp non-coding region, which was highly homologous to the start of control regions I (CR I) and II (CR II), containing a 16 bp C-rich segment, was identified upstream of the pseudo-tRNAPro gene that had been observed across Colubridae and Homalopsidae. Twelve concatenated heavy-strand encoded protein-coding genes were used for phylogenetic reconstruction employing Bayesian and maximum likelihood inference. Both analyses yielded identical topologies, demonstrating that T. zhaoermii can solidly be placed within Colubridae as a sister group to Colubrinae. The paraphyly of Scolecophidia and monophyly of Henophidia and Caenophidia were also supported. A relaxed clock molecular divergence time analysis was carried out to estimate the temporal origin of each clade. Our results indicate that the Alethinophidia began to diverge from the paraphyletic Scolecophidia approximately 130 million years ago in the early Cretaceous; the divergence of living alethinophidian snakes, the radiation of the Caenophidia, and the separation between Acrochordus and the Colubroidea might have been caused by the K/T event.

The -Globin mRNA Stabilization Complex Is Assembled in the Nucleus Supporting CP Integration of Nuclear and Cytoplasmic Controls over -Globin Gene Expression. ααα.

Stability of human -globin mRNA in erythroid cells is critically dependent on the function of a 3UTR RNA-protein (RNP) complex ( complex ). This complex is assembled by the high-affinity binding of a polyC-specific RNA binding protein (CP) to a defined C-rich segment in the 3UTR. Recently we observed that certain CP protein isoforms contain nuclear localization signals, co-localize with nuclear speckles, and shuttle between nuclear and cytoplasmic compartments. These data suggested that the -complex may initially assemble on nascent nuclear transcripts and may have nuclear as well as cytoplasmic functions. To test this model, we immuno-enriched CP-RNP complexes from K562 nuclear extracts and probed these complexes for -globin transcripts. We found that these CP-containing nuclear RNP complexes were enriched for intron-containing -globin transcripts as well as mature, spliced -globin mRNAs. The association of mature -globin mRNA in the complex was entirely dependent on the presence of the 3UTR CP binding site. In contrast, the unspliced -globin transcripts remained associated with CP complexes in the absence of the 3 UTR binding site. These data suggested that one or more previously unrecognized CP binding sites are present within and specific to unspliced -globin transcripts. Visual inspection of -globin intronic sequences revealed two prominent C-rich elements. These sequences are in an open secondary structure and conform to established parameters for high-affinity CP binding sites. RNA EMSA confirmed two high-affinity CP binding sites within intron 1; the stronger site was coincident with the pyrimidine-rich tract of the splice-acceptor site. In vitro splicing assays were used to explore nuclear functions of the a complexes. Incubation of full-length -globin transcripts with HeLa cell nuclear extract resulted in the efficient removal of both introns. Elimination of the 3UTR complex slowed removal of intron 1 but accelerated intron 2 excision. The impact of the intron-1 complexes on splicing was also studied. Incubation of an -globin transcript segment extending from exon 1 through intron 1 and into exon 2 resulted in efficient intron excision. Affinity-depletion of CP from the HeLa extract enhanced intron 1 excision from this substrate. This splicing enhancement was reversed by the addition of recombinant CP to the depleted extract. In summary, we report that assembly of the complexes on -globin transcripts takes place in the nucleus, that these RNP complexes assemble at sites within intron 1 as well as at the previously described 3UTR binding site, and that the both sets of a complexes have the potential to alter the efficiency of -globin intron excision. The fully processed -globin mRNA, containing the remaining 3UTR complex, is then exported to the cytoplasm in a pre-packaged form where it is stabilized by the UTR complex and is robustly translated. We propose that CP mediates and integrates nuclear as well as cytoplasmic post-transcriptional controls over human -globin gene expression.α′'α' αα′αααααααα′ααα′αααααα′ααααααα′αα′α“”′ααα

Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions.

Two regions of mouse rDNA were sequenced. One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA. The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years). In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions. Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments. As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems. Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation. These conserved regions include some short homologous sequence patterns and closely spaced direct repeats.

Study of ribosomal deoxyribonucleic acid of sea urchin.

The structure of ribosomal DNA (rDNA) satellite of sea urchin (Lytechinus variegatus) sperm has been re-examined after purification by two widely used methods. Saturation hybridization experiments indicate that about 28--32% of the rDNA contain sequences complementary to rRNA. Results of hyperchromic spectral analysis reveal that the rDNA melts as two distinct but unequal components. The early transition presumably corresponds to the melting of most of the transcribed part and the late transition is suggestive of the melting of a G + C-rich segment of the rDNA, which may be a nontranscribed spacer.