ABSTRACT
A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.
Subject(s)
Biotechnology , Chimera , DNA , DNA, Single-Stranded , Genome, Bacterial , Retroelements , Reverse Transcription , RNA , RNA-Directed DNA PolymeraseABSTRACT
Given the importance of RNA secondary structures in defining their biological role, it would be convenient for researchers seeking RNA data if both sequence and structural information pertaining to RNA molecules are made available together. Current nucleotide data repositories archive only RNA sequence data. Furthermore, storage formats which can frugally represent RNA sequence as well as structure data in a single file, are currently unavailable. This article proposes a novel storage format, ‘FASTR’, for concomitant representation of RNA sequence and structure. The storage efficiency of the proposed FASTR format has been evaluated using RNA data from various microorganisms. Results indicate that the size of FASTR formatted files (containing both RNA sequence as well as structure information) are equivalent to that of FASTA-format files, which contain only RNA sequence information. RNA secondary structure is typically represented using a combination of a string of nucleotide characters along with the corresponding dot-bracket notation indicating structural attributes. ‘FASTR’ – the novel storage format proposed in the present study enables a frugal representation of both RNA sequence and structural information in the form of a single string. In spite of having a relatively smaller storage footprint, the resultant ‘fastr’ string(s) retain all sequence as well as secondary structural information that could be stored using a dot-bracket notation. An implementation of the ‘FASTR’ methodology is available for download at http://metagenomics.atc.tcs.com/compression/fastr.
ABSTRACT
Human papillomavirus (HPV) is an etiologic agent of the uterine cervix cancer and several other neoplasias in women globally. E6 protein of HPV type 16 is highly conserved and plays the key role in an inducing cancer via suppressing activity of P53. We have used different bioinformatics tools for generation of phylogenetic tree, modeling of RNA secondary structure, gene designing and codon optimization of HPV E6 gene. The size of E6 gene sequences of nine strains HPV type 16 was estimated to be 456 to 477 bp and G+C content was ranged between 37.06 to 37.94%. We used E6 gene sequences for construction of phylogenetic relationship and these divided into five groups. RNA secondary structures of E6 gene were modeled and analyzed that folding free energy of wild genes was -093.96, -106.21,-040.48, -090.76, - 072.68, -092.86, -039.32, -044.78, -047.88 kcal/mol and after codon optimization free energy was -122.70, -107.40, - 104.80, -121.40, -127.40, -110.80, -105.20, -122.30, -110.40 kcal/mol respectively. Moreover, gene designing and codon optimization have used to improve the heterologous expression in living organisms by increasing translational efficiency. All strains of HPV16 were used for codon optimization in E. coli. Codon adaptation index (CAI) and G+C contents of E6 gene in optimized DNA were enhanced by 3.6 (72.7%) and 1.3 (25.2%) fold, respectively. The present study provides useful insights into phylogenetic and evolution in the cervical cancer causing Human papillomavirus type 16. The optimized DNA can be chemically synthesized and over expressed in E. coli as compare to its wild type counterparts. Alternatively, the secondary structure and free energy of E6 were investigated that will be helpful to predict the evolution of primitive and genetically stable HPV type 16 strains. This finding provides new insight in better understanding of cervical cancer.
ABSTRACT
Aeromonas veronii is a human pathogen that causes diarrhea, wound infections and hemorrhagic septicemia. A. veronii isolated from Gomti river water and identified on basis of 16S rRNA sequences, which showed the high degree of homology with existing sequences. Moreover, the 16S rRNA sequences were also being used for modeling of RNA secondary structures and resembles with Gibbs free energy of other available strains. The result indicates the accurate molecular identification of A. veronii and its phylogeny shows the high degree of homology with diverse source of other strains.
ABSTRACT
Three pSIREN-siRNA plasmids were constructed using a pSIREN-RetroQ vector to silence the expression of muhidrug resistance-associated protein (MRP1) gene, and subsequently characterized by restriction endonuclease digestion and DNA sequencing. A truncated MRP1 and a full-length MRP1 were cloned into pEGFP-N2 and PeDNA3.1 respectively as pEGFP-MRP1T and pcDNA-MRP1. The plasmid pEGFP-MRP1T was co-transferred with each of the three pSIREN-siRNAs into HEK293 cells for MRP1T-GFP targeted silencing, and pSIREN-siRNA1 was used as the negative control, pSIREN-siRNA2 and pSIREN-siRNA3 appeared to be more effective to silence MRP1T-GFP compared to pSIREN-siRNA1 as shown by fluorescence microscopy. For the silencing of full-length MRP1 expression, HEK293 ceils were co-transferred with pcDNA-MRP1 and either of the three pSIREN-siRNAs, then subjected for Western blot analysis and MTT assays, pSIREN-siRNA2 and pSIREN-siRNA3 were able to inhibit the expression of 190 kD MRP1, but not pSIREN-siRNA1. The MDR of MRP1-transfected HEK293 ceils was abolished with pSIREN-siRNA2 or pSIREN-siRNA3 transfections. RNA secondary structure predictions demonstrated that the mRNA local free energy (△G) of the siRNA1 targeted sequence was lower, as the GC content and Tm value of siRNA1 were higher than those of siRNA2 and siRNA3. These data suggest that the local structure siRNAs and target mRNA may influence the silencing efficiency of MRP1 expression.
ABSTRACT
Attention was gradually paid by biologists to the using of RNA secondary structure in the classification of microbiology and phylogenetic relationship analysis in recent years. The development around the research was summarized here briefly. And more emphasis was given to the part introducing the application of RNA secondary structure to the analysis of phylogenetic relationship.
ABSTRACT
Small and large subunits of Escherichia coli ribosome have three different rRNAs, the sequences of which are known. However, attempts by three groups to predict secondary structures of 16S and 23S rRNAs have certain common limitations namely, these structures are predicted assuming no interactions among various domains of the molecule and only 40% residues are involved in base pairing as against the experimental observation of 60 % residues in base paired state. Recent experimental studies have shown that there is a specific interaction between naked 16S and 23S rRNA molecules. This is significant because we have observed that the regions (oligonucleotides of length 9–10 residues), in 16S rRNA which are complementary to those in 23S rRNA do not have internal complementary sequences. Therefore, we have developed a simple graph theoretical approach to predict secondary structures of 16S and 23S rRNAs. Our method for model building not only uses complete sequence of 16S or 23S rRNA molecule along with other experimental observations but also takes into account the observation that specific recognition is possible through the complementary sequences between 16S and 23S rRNA molecules and, therefore, these parts of the molecules are not used for internal base pairing. The method used to predict secondary structures is discussed. A typical secondary structure of the complex between 16S and 23S rRNA molecules, obtained using our method, is presented and compared briefly with earlier model building studies.