Analysis for free: comparing programs for sequence analysis

Programs to import, manage and align sequences and to analyse the properties of DNA, RNA and proteins are essential for every biological laboratory. This review describes two different freeware (BioEdit and pDRAW for MS Windows) and a commercial program (Sequencher for MS Windows and Apple MacOS). Bioedit and Sequencher offer functions such as sequence alignment and editing plus reading of sequence trace files. pDRAW is a very comfortable visualisation tool with a variety of analysis functions. While Sequencher impresses with a very user-friendly interface and easy-to-use tools, BioEdit offers the largest and most customisable variety of tools. The strength of pDRAW is drawing and analysis of single sequences for priming and restriction sites and virtual cloning. It has a database function for user-specific oligonucleotides and restriction enzymes.     

Three distinct RNA sequence elements are required for efficient apolipoprotein B (apoB) RNA editing in vitro.

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine to convert a CAA glutamine codon to a UAA translational stop codon by the direct conversion of cytidine to uridine at nucleotide 6666. We have proposed the 'mooring sequence' model for apoB RNA editing, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. One sequence element (approx. nts 6671-81, the presumed 'mooring sequence') has been previously identified as necessary for editing. We have identified two additional sequence elements which are necessary for efficient editing: (1) a 5' 'Regulator' region which modulates editing efficiency and (2) a 'Spacer' region between the editing site and the 3' mooring sequence, whose distance is critical for efficient editing. Utilizing this data, we have induced editing at a cryptic site and have defined a 22 nucleotide 'cassette' of specific apoB sequence which is sufficient to support wild-type levels of editing in vitro in a background of distal apoB RNA sequence.     

DNAAlignEditor: DNA alignment editor tool

Background

With advances in DNA re-sequencing methods and Next-Generation parallel sequencing approaches, there has been a large increase in genomic efforts to define and analyze the sequence variability present among individuals within a species. For very polymorphic species such as maize, this has lead to a need for intuitive, user-friendly software that aids the biologist, often with naïve programming capability, in tracking, editing, displaying, and exporting multiple individual sequence alignments. To fill this need we have developed a novel DNA alignment editor.

Results

We have generated a nucleotide sequence alignment editor (DNAAlignEditor) that provides an intuitive, user-friendly interface for manual editing of multiple sequence alignments with functions for input, editing, and output of sequence alignments. The color-coding of nucleotide identity and the display of associated quality score aids in the manual alignment editing process. DNAAlignEditor works as a client/server tool having two main components: a relational database that collects the processed alignments and a user interface connected to database through universal data access connectivity drivers. DNAAlignEditor can be used either as a stand-alone application or as a network application with multiple users concurrently connected.

Conclusion

We anticipate that this software will be of general interest to biologists and population genetics in editing DNA sequence alignments and analyzing natural sequence variation regardless of species, and will be particularly useful for manual alignment editing of sequences in species with high levels of polymorphism.

     

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.     

Computational prediction of RNA editing sites

MOTIVATION: Some organisms edit their messenger RNA resulting in differences between the genomic sequence for a gene and the corresponding messenger RNA sequence. This difference complicates experimental and computational attempts to find and study genes in organisms with RNA editing even if the full genomic sequence is known. Nevertheless, knowledge of these editing sites is crucial for understanding the editing machinery of these organisms. RESULTS: We present a computational technique that predicts the position of editing sites in the genomic sequence. It uses a statistical approach drawing on the protein sequences of related genes and general features of editing sites of the organism. We apply the method to the mitochondrion of the slime mold Physarum polycephalum. It correctly predicts over 90% of the amino acids and over 70% of the editing sites.     

A sequence assembly and editing program for efficient management of large projects.

We describe a sequence assembly and editing program for managing large and small projects. It is being used to sequence complete cosmids and has substantially reduced the time taken to process the data. In addition to handling conventionally derived sequences it can use data obtained from Applied Biosystems,Inc. 373A and Pharmacia A.L.F. fluorescent sequencing machines. Readings are assembled automatically. All editing is performed using a mouse operated contig editor that displays aligned sequences and their traces together on the screen. The editor, which can be used on single contigs or for joining contigs, permits rapid movement along the aligned sequences. Insertions, deletions and replacements can be made in individual aligned readings and global changes can be made by editing the consensus. All changes are recorded. A click on a mouse button will display the traces covering the current cursor position, hence allowing quick resolution of problems. Another function automatically moves the cursor to the next unresolved character. The editor also provides facilities for annotating the sequences. Typical annotations include flagging the positions of primers used for walking, or for marking sites, such as compressions, that have caused problems during sequencing. Graphical displays aid the assessment of progress.