Pairwise sequence alignment below the twilight zone.

Improved sequence alignment at low pairwise identity is important for identifying potential remote homologues in database searches and for obtaining accurate alignments as a prelude to modeling structures by homology. Our work is motivated by two observations: structural data provide superior training examples for developing techniques to improve the alignment of remote homologues; and general substitution patterns for remote homologues differ from those of closely related proteins. We introduce a new set of amino acid residue interchange matrices built from structural superposition data. These matrices exploit known structural homology as a means of characterizing the effect evolution has on residue-substitution profiles. Given their origin, it is not surprising that the individual residue-residue interchange frequencies are chemically sensible. The structural interchange matrices show a significant increase both in pairwise alignment accuracy and in functional annotation/fold recognition accuracy across distantly related sequences. We demonstrate improved pairwise alignment by using superpositions of homologous domains extracted from a structural database as a gold standard and go on to show an increase in fold recognition accuracy using a database of homologous fold families. This was applied to the unassigned open reading frames from the genome of Helicobacter pylori to identify five matches, two of which are not represented by new annotations in the sequence databases. In addition, we describe a new cyclic permutation strategy to identify distant homologues that experienced gene duplication and subsequent deletions. Using this method, we have identified a potential homologue to one additional previously unassigned open reading frame from the H. pylori genome.

Statistical mechanical simulation of polymeric DNA melting with MELTSIM.

MOTIVATION: MELTSIM is a windows-based statistical mechanical program for simulating melting curves of DNAs of known sequence and genomic dimensions under different conditions of ionic strength with great accuracy. The program is useful for mapping variations of base compositions of sequences, conducting studies of denaturation, establishing appropriate conditions for hybridization and renaturation, determinations of sequence complexity, and sequence divergence. RESULTS: Good agreement is achieved between experimental and calculated melting curves of plasmid, bacterial, yeast and human DNAs. Denaturation maps that accompany the calculated curves indicate non-coding regions have a significantly lower (G+C) composition than coding regions in all species examined. Curves of partially sequenced human DNA suggest the current database may be heavily biased with coding regions, and excluding large (A+T)-rich elements. AVAILABILITY: MELTSIM 1.0 is available at: //www.uml.edu/Dept/Chem/UMLBIC/Apps/MEL TSIM/MELTSIM-1.0-Win/meltsim. zip. Melting curve plots in this paper were made with GNUPLOT 3.5, available at: http://www.cs.dartmouth.edu/gnuplot_inf o.html Contact : blake@maine.maine.edu;

Wide variations in neighbor-dependent substitution rates.

The pattern of 20,200 point substitutions in the 16 unique neighbor-pair environments has been determined from aligned gene/pseudogene sequences in the current database of human DNA sequences. Substitution rates, representing averages over those for different regions of the genome, are distributed over a 60-fold range with strong biases in particular neighbor-pair environments. The rates for substitutions involving the CG doublet are the most rapid overall, where changes of the C.G pair vary over a tenfold range depending on the type of substitution and the 5' neighbor-pair. In general, the rates are fastest in alternating purine-pyrimidine sequences and slowest in purine.pyrimidine tracts, suggesting that the frequencies of one or both key molecular misadventures that can occur during replication, dNTP misinsertion and transient misalignment, may be associated with structural alternations and flexibility of the backbone. By contrast, purine.pyrimidine tracts are less flexible, less prone to substitution, and therefore their proportions accumulate in sequences over time. Characteristic biases of the content and arrangement of oligonucleotide strings or tuples in all sequence elements, but particularly in non-coding regions, appear to be due to the pattern of different neighbor-dependent substitution rates. Computer simulations of numerous replicative cycles have been carried out with substitutions occurring on the same schedule found in this study for pseudogenes. Statistical analyses of tuple frequencies at periodic intervals during the simulation experiment indicate that sequences slowly change in lexical complexity toward a quasi-equilibrium state that corresponds to that for introns.

Determination of organic acids in sugar cane process juice by high-performance liquid chromatography: improved resolution using dual Aminex HPX-87H cation-exchange columns equilibrated to different temperatures.

The application of high-performance liquid chromatography to the quantitative analysis of organic acids in sugar cane process juice is described. Separation of these acids is achieved on Aminex HPX-87H cation-exchange columns. Resolution is improved by connecting two columns in series and equilibrating them at different temperatures. The acids in the sample are first isolated on DEAE-Sephadex A-25 anion-exchange resin equilibrated with 0.5 M isobutyric acid. They are then eluted with 0.5 M sulphuric acid and injected directly onto the HPLC column after filtration through a 0.45-micron membrane. This method allows the simultaneous determination of oxalic, cis-aconitic, citric, phosphoric, malic, trans-aconitic, succinic, glycolic, lactic, formic and acetic acids. Isobutyric acid is also separated on this system and provides a useful guide to the loading capacity of the anion-exchange resin.

Fructan from Erwinia herbicola.

Levan production by strains of Erwinia herbicola is common, and this property has some taxonomic significance for species differentiation within the "herbicola" group. The extracellular polysaccharide elaborated by strain 403 was characterized by nuclear magnetic resonance spectroscopy and methylation analysis. Results showed it to be a typical bacterial levan.