Sequence analyses

The chapter is divided into four complementary sections. Sections 2 to 4 describe the process of sequence analysis, data management and reporting standards, and the design and analysis of microarray experiments. To demonstrate the described computational tools and concepts covered in these sections, a practical example is illustrated in Section 5. 

Sequence analysis is a core area of bioinformatics research. There are four basic levels of biological structure (Table 1), termed primary, secondary, tertiary, and quaternary structure. Primary structure refers to the representation of a linear, hetero-polymeric macromolecule as a string of monomeric units. For example, the primary structure of DNA is represented as a string of nucleotides (G, C, A, T). Secondary structure refers to the local three-dimensional shape in subsections of macromolecules. For example, the alpha- and beta-sheets in protein structures are examples of secondary structure. Tertiary structure refers to the overall three-dimensional shape of a macromolecule, as in the crystal structure of an entire protein. Finally, quaternary structure represents macromolecule interactions, such as the way different peptide chains dimerize into a single functional protein. 

In general, analysis increases in complexity as one attempts to predict higher levels of structure. Primary structure is highly amenable to in silico 

Secondary (2°) Local 3D structure (fold) a-helices, P-sheets RNA and protein fold prediction 

Tertiary (3°) Overall 3D structure Protein crystal Protein structure 

The classical method is the Edman degradation reaction. It involves stepwise degradation of peptides with phenylisothiocyanate (cf. 1.2.4.2.3) or suitable derivatives, e. g. dimethylaminoa-zobenzene isothiocyanate (DABITC). The resultant phenylthiohydantoin is either identified directly or the amino acid is recovered. The stepwise reactions are performed in solution or on peptide bound to a carrier, i.e. to a sohd phase. Both approaches have been automated ( sequencer ). Carriers used include resins containing amino groups (e. g. amino polystyrene) or glass beads treated with amino alkylsiloxane  

Hydrolysis of proteins with dilute acids preferentially cleaves aspartyl-X-bonds. 

The peptides are then attached to the carrier by carboxyl groups (activation with carbodiimide or carbonyl diimidazole, as in peptide synthesis) or by amino groups. For example, a peptide 

Bioinformatics is a new and exciting subject developing its own methodologies, approaches and nomenclature with an elan which has not suffered from lack of precision about defining exactly what the subject is. As might be expected of a product of the computer age, it depends heavily on internet and database access, which are the essential tools of what has become termed in silico research. A very readable introduction to the subject is provided by Attwood and Parry-Smith (1999). 

---

Protein Structure Prediction, Sequence Analysis and Protein Folding... 

Fig. 10.22 Hidden Markov model used for protein sequence analysis, are match states (corresponding in this...

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

Firefly. Firefly luciferase (EC 1.13.12.7) is a homodimeric enzyme (62 kDa subunit) that has binding sites for firefly luciferin and Mg ATP . Amino acid sequence analysis has iadicated that beetle luciferases evolved from coen2yme A synthetase (206). Firefly bioluminescence is the most efficient bioluminescent reaction known, with Qc reported to be 88% (5), and at 562 nm (56). At low pH and ia the presence of certain metal ions (eg, Pb ", ... 

Sequence analysis and mutagenesis experiments indicated that the thiolate ligand is provided by a-Cys-275 (163—165). These predictions have been... 

The frequencies of plant damage and public consequence are calculated using plant logic combined with component fragilities. Event and fault trees are constructed to identify tiic accident sequences and the damage that may result from an earthquake. In performing a plant system and accident-sequence analysis, the major differences between seismic and internal events analysis are given in Table 5.1-4... 

The first step-in plant-system and accident-sequence analysis is the identification of earthquake-induced initiating events. This is done by reviewing the internal analysis initiating events to identify initiating events relevant to seismic risk. For example. Table 5,1 -5 shows the initiating events that were used in the Seismic Safety Margins Research Program for a PWR plant (Smith et al., 1981)... 

Heilman, EJ., Wiksell, E., and Karlsson, B.-M. (1990). A new approach to micropreparative desalting exemplified by desalting a reduction/alkylation mixture, presenred ar Eighr International Conference on Methods in Protein Sequence Analysis, Kiruna, Sweden, July 1-6. 

Specificity of Representative Polypeptide Cleavage Procedures Used in Sequence Analysis... 

Amino acid sequence analysis reveals that proteins with related functions often show a high degree of sequence similarity. Such findings suggest a common ancestry for these proteins. 

Ffeijne, G. von, 1987. Sequence Analysis in Molecular Biology Treasure Trove or Trivial PursuiT Diego Academic Pre.ss. 

FIGURE 12.39 The proposed secondary structure for E. coli 16S rRNA, based on comparative sequence analysis in which the folding pattern is assumed to be conserved across different species. The molecule can be subdivided into four domains—I, II, III, and IV—on the basis of contiguous stretches of the chain that are closed by long-range base-pairing interactions. I, the 5 -domain, includes nucleotides 27 through 556. II, the central domain, runs from nucleotide 564 to 912. Two domains comprise the 3 -end of the molecule. Ill, the major one, comprises nucleotides 923 to 1391. IV, the 3 -terminal domain, covers residues 1392 to 1541. 

Fagan, T. F., et al. (1993). Cloning, expression and sequence analysis of cDNA for the Ca2+-binding photoprotein, mitrocomin. FEBS Lett. 333 301-305. 

Viviani, V. R., Bechara, E. J. H., and Ohmiya, Y. (1999). Cloning, sequence analysis, and expression of active Phrixothrix railroad-worm luciferase relationship between bioluminescence spectra and primary structures. Biochemistry 38 8271-8279. 

Three key areas are the organisation of knowledge in databases, sequence analysis, and structural bioinformatics. 

Proteases are hydrolytic enzymes with important application in industries, in particular, in detergent and in the food industry. A metagenomic study in which 100 000 plasmid clones were screened for proteolytic activity found one positive done, which was determined to be novel by sequencing analysis [84]. 

Rebmann G. Hertig C. Bull J. Mauch F. Dudler R. (1991) Complementary DNA cloning and sequence analysis of a pathogen-induced putative peroxidase from rice / / Plant Mol. Biol. V. 16. P. 329-331. 

---