Journal of Nucleic Acids最新文献

The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific.

Damaged DNA, generated by the abstraction of one of five hydrogen atoms from the 2'-deoxyribose ring of the nucleic acid, can contain a variety of lesions, some of which compromise physiological processes. Recently, DNA damage, resulting from the formation of a C3'-thymidinyl radical in DNA oligomers, was found to be dependent on nucleic acid structure. Architectures relevant to DNA replication were observed to generate larger amounts of strand-break and 1-(2'-deoxy- β -D-threo-pentofuranosyl)thymidine formation than that observed for duplex DNA. To understand how this damage can affect the integrity of DNA, the impact of C3'-thymidinyl radical derived lesions on DNA stability and structure was characterized using biophysical methods. DNA architectures evaluated include duplex DNA (dsDNA), single 3' or 5'-overhangs (OvHgs), and forks. Thermal melting analysis and differential scanning calorimetry measurements indicate that an individual 3'-OvHg is more destabilizing than a 5'-OvHg. The presence of a terminal 3' or 5' phosphate decreases the ΔG 25 to the same extent, while the effect of the phosphate at the ss-dsDNA junction of OvHgs is dependent on sequence. Additionally, the effect of 1-(2'-deoxy- β -D-threo-pentofuranosyl)thymidine is found to depend on DNA architecture and proximity to the 3' end of the damaged strand.

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.

Formation of RNA-DNA hybrid, or R-loop, was studied in vitro by transcribing an AGGAG repeat with T7 RNA polymerase. When ribonuclease T1 was present, R-loop formation in cis was diminished, indicating that the transcript was separated from the template and reassociated with it. The transcript was found to form an R-loop in trans with DNA comprising the AGGAG repeat, when the DNA was supercoiled. Results of chemical modification indicated that the duplex opened at the AGGAG repeat under negative supercoiling.

Aptamers are single-stranded synthetic DNA- or RNA-based oligonucleotides that fold into various shapes to bind to a specific target, which includes proteins, metals, and molecules. Aptamers have high affinity and high specificity that are comparable to that of antibodies. They are obtained using iterative method, called (Systematic Evolution of Ligands by Exponential Enrichment) SELEX and cell-based SELEX (cell-SELEX). Aptamers can be paired with recent advances in nanotechnology, microarray, microfluidics, and other technologies for applications in clinical medicine. One particular area that aptamers can shed a light on is biomarker discovery. Biomarkers are important in diagnosis and treatment of cancer. In this paper, we will describe ways in which aptamers can be used to discover biomarkers for cancer diagnosis and therapeutics.

MicroRNAs (miRNAs) are short RNA sequences that repress protein synthesis by either inhibiting the translation of messenger RNA (mRNA) or increasing mRNA degradation. Endogenous miRNAs have been found in various organisms, including animals, plants, and viruses. Mammalian miRNAs are evolutionarily conserved, are scattered throughout chromosomes, and play an important role in the immune response and the onset of cancer. For this study, the author explored the base composition characteristics of miRNA genes from the six mammalian species that contain the largest number of known miRNAs. It was found that mammalian miRNAs are evolutionarily conserved and GU-rich. Interestingly, in the miRNA sequences investigated, A residues are clearly the most frequent occupants of positions 2 and 3 of the 5' end of miRNAs. Unlike G and U residues that may pair with C/U and A/G, respectively, A residues can only pair with U residues of target mRNAs, which may augment the recognition specificity of the 5' seed region.

Thermus thermophilus MutS, a thermostable mismatch-recognizing protein, is utilized in PCR to suppress nonspecific amplification by preventing synthesis from mismatched primers. T. thermophilus RecA also decreases nonspecific amplification by promoting proper hybridization between the primer and template. We observed that MutS and RecA function under the same reaction conditions and that MutS and RecA do not preclude each other. Furthermore, there were some DNA sequences for which only one of the 2 proteins effectively suppressed nonspecific amplification. The simultaneous use of MutS and RecA is a more attractive error-suppressing technique than the use of either of the 2 proteins alone.

An expanded CTG-repeat in the 3' UTR of the DMPK gene is responsible for myotonic dystrophy type I (DM1). Somatic and intergenerational instability cause the disease to become more severe during life and in subsequent generations. Evidence is accumulating that trinucleotide repeat instability and disease progression involve aberrant chromatin dynamics. We explored the chromatin environment in relation to expanded CTG-repeat tracts in hearts from transgenic mice carrying the DM1 locus with different repeat lengths. Using bisulfite sequencing we detected abundant CpG methylation in the regions flanking the expanded CTG-repeat. CpG methylation was postulated to affect CTCF binding but we found that CTCF binding is not affected by CTG-repeat length in our transgenic mice. We detected significantly decreased DMPK sense and SIX5 transcript expression levels in mice with expanded CTG-repeats. Expression of the DM1 antisense transcript was barely affected by CTG-repeat expansion. In line with altered gene expression, ChIP studies revealed a locally less active chromatin conformation around the expanded CTG-repeat, namely, decreased enrichment of active histone mark H3K9/14Ac and increased H3K9Me3 enrichment (repressive chromatin mark). We also observed binding of PCNA around the repeats, a candidate that could launch chromatin remodelling cascades at expanded repeats, ultimately affecting gene transcription and repeat instability.

In the study of cellular RNA chemistry, a major thrust of research focused upon sequence determinations for decades. Structures of snRNAs (4.5S RNA I (Alu), U1, U2, U3, U4, U5, and U6) were determined at Baylor College of Medicine, Houston, Tex, in an earlier time of pregenomic era. They show novel modifications including base methylation, sugar methylation, 5'-cap structures (types 0-III) and sequence heterogeneity. This work offered an exciting problem of posttranscriptional modification and underwent numerous significant advances through technological revolutions during pregenomic, genomic, and postgenomic eras. Presently, snRNA research is making progresses involved in enzymology of snRNA modifications, molecular evolution, mechanism of spliceosome assembly, chemical mechanism of intron removal, high-order structure of snRNA in spliceosome, and pathology of splicing. These works are destined to reach final pathway of work "Function and Structure of Spliceosome" in addition to exciting new exploitation of other noncoding RNAs in all aspects of regulatory functions.