SuUR protein binds to the boundary regions separating forum domains in Drosophila melanogaster

Forum domains are 50-150 kb DNA fragments that are released during spontaneous fragmentation of chromosomes. They are separated by islands of putative heterochromatin boundary regions. The SuUR protein, which is involved in the control of chromosome organization, is localized exclusively in heterochromatin and often colocalizes on chromosomes with Polycomb group proteins. To test whether the SuUR protein is associated with boundary regions, we used gel retardation assays and found that the SuUR protein binds specifically to boundary regions and that boundary regions are under-replicated. These results suggest that the regular distribution of boundary regions in chromosomes may represent the dispersion of sites designed for chromosomal silencing.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

Parallel double helical DNA. 1. Evidence for the existence of a spiral A-T-paired base

The dependence of UV and CD spectra of oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-(pApTpApTpApTpApTp ApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl on temperature and concentration was studied. It was shown that the eicosamer in concentrations below 5.10(-4) M forms a parallel stranded hairpin. From the thermal denaturation profile the thermodynamic parameters of parallel hairpin formation were determined. The values of delta H0, delta S0 and Tm were -90 +/- 8 kJ/mol, -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel helix differ from those of B-form DNA by reduction of extreme magnitude at approximately 265 nm and appearance of a negative effect at approximately 285 nm.     

Forum domain in Drosophila melanogaster cut locus possesses looped domains inside.

We have studied the relationship between chromosomal forum domains and looped domains in the cut locus of Drosophila melanogaster . Forum domains were earlier detected by separation in pulsed-field gels of 50-150 kb chromosomal DNA fragments obtained after spontaneous non-random degradation of chromosomes. We have localized the boundary region where cleavage sites are scattered between two forum domains in the regulatory region of the cut locus. We have sequenced a 13 kb region spanning few kilobases from distal domain, the boundary region and part of the proximal forum domain where several scaffold associated regions (SARs) were observed. We conclude that forum domains and looped domains are physically different types of domains and belong to different levels of organization in eukaryotic chromosomes. The boundary region between the neighboring forum domains in the cut locus possesses the Doc element insertion and a micro-satellite stretch and thus might remind a small island of heterochromatin and correspond to so-called intercalary heterochromatin that is known to be located in the 7B1-2 band where the major part of the cut locus is reside.     

Parallel double helix and tertiary structure of nucleic acids

The thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3' (parAT), 3'-d(AT)5pO(CH2)5Opd(AT)5-5' (antiAT), 3'-d(A)10pO(CH2)6Op(T)10-3' (parA-T) and 3'd(A)10pOX X (CH2)6Opd(T)10-5' (antiA-T) in 0.01 M phosphate buffer at pH 7 in presence 0.1, 0.25, 0.5 and 1.0 M NaCl have been studied. It was shown that at lower temperature (0-20 degrees C) all oligomeres exist as complexes of two (canonic duplex) or four (eight) molecules of oligonucleotides, but at higher temperature (30-70 degrees C)- as hairpins with parallel (parAT and parA-T) of antiparallel (antiAT and antiA-T) orientation of chains. Thermodinamic parameters of separated strands-hairpins and hairpins--"low temperature complexes" transition were computated from the melting curves [A260 (T)] by nonlinear regression. AntiA-T was shown by ethidium bromide binding to exist at low strength (0.01 M phosphate buffer without NaCl) as four-stranded complex from two antiparallel double stranded helices parallely oriented and bonded by satisfy hydrogen-bond of groups not involved in WC-pairing. At higher ionic strength the two of such tetramers was conjugated by hydrophobic interaction into octamers. We speculate that four-stranded complexes serves to bring together, and zipper up two antiparallel double stranded helices at replication of DNA, cross-over of gomologues chromosomes and other biochemically important processes.     

Parallel double stranded helices and the tertiary structure of nucleic acids

Thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3'(par(AT], 3'-d(AT)5pO(CH2)6Opd(AT)5-5'(anti(AT],3'-d(A)10pO(CH2) 6Op(T)10-3'(par(A-T], and 3'-d(A)10pO(CH2)6Opd(T)10-5' (anti(A-T], was studied in 0.01 M phosphate buffer, pH 7, in the presence of 0.1, 0.25, 0.5 and 1.0 M NaCl. All the oligomers were found to exist at a lower temperature (0 to 20 degrees C) as complexes composed either of two oligomer molecules (a canonical duplex) or of more oligomer molecules whereas, at a higher temperature (30 to 70 degrees C), they formed hairpins with a parallel (par(AT) and par(A-T] or antiparallel (anti(AT) and anti(A-T) orientation of the chains. Melting curves (A260(T] were used to calculate thermodynamic parameters for the formation of hairpins and "low-temperature" duplexes. Experiments on ethidium bromide binding to the oligonucleotides have shown that the oligomer anti(A-T) exists, at a low ionic strength, as a four stranded complex ("quadruplex") contains two antiparallel helices, d(A).d(T), which have a parallel orientation and are bound to one another owing to the formation of additional hydrogen bonds between nucleic acid bases. The possible biological function of quadruplexes is discussed.     

The Formation Of Parallel RNA-RNA Duplexes In Vitro

Abstract

Over the years the structural properties of nucleic acids have been of interest, providing data that may be of importance for DNA and RNA organization and function in the cell. We have attempted to look for the formation of parallel RNA-RNA duplexes in vitro. RNA molecules comprising complementary in the same polarity alternating stretches of A and U of increasing length were enzymatically synthesized and annealed in physiological conditions. The fractionation in the denaturing Polyacrylamide gels revealed the formation of two types of full-length parallel RNase A - stable duplexes established either by A-U or by A-A and U-U self pairs. These results suggest novel structural properties of versatile RNA molecules that potentially may be realized in vivo.     

Suspension array analysis of 16S rRNA from Fe- and SO(4)2- reducing bacteria in uranium-contaminated sediments undergoing bioremediation

A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of Desulfotomaculum were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.