Sequence-based approaches

SEQUENCE-BASED APPROACHES TO ALKALOID BIOSYNTHESIS GENE IDENTIFICATION... 

This chapter is an historical perspective with selected examples from our own laboratory, first at the University of Munich, and now at the Leibniz Institute of Plant Biochemistry in Halle, that reflect the changes in how we define and exploit sequence-based approaches to alkaloid biosynthesis gene identification. ... 

Sequence-Based Approaches to Alkaloid Biosynthesis Gene Identification... 163 Toni M. Kutchan... 

There is some error in the sequences (Ewing et al, 1998), which is no problem for the usual whole-sequence-based approach to expression analysis, but a drawback when individual sites are studied. In particular, the automatic base calling by a computer may increase the error. 

A second widely used sequence-based approach relies on matrices of residue frequencies, pioneered by Parry (1982). He showed that the residue distribution at the seven heptad positions of the putative coiled-coil segments of myosin, tropomyosin, o-keratin, and hemaglutinin are asymmetric, and proposed a method by which the residue frequencies could be used to predict whether a sequence of unknown structure would form a coiled coil. This approach was implemented with modifications in... 

It is also possible to use antibody based approaches to assay the accumulation of enzymes involved in denitrification pathways in environmental samples. Ward and Cockroft (1993) developed antisera to nitrite reductase from a marine Pseudomonas stutzeri isolate, and assayed sediment, water column and microbial mat samples. The antibody identified nitrite reductase in mat samples and was more specific than a DNA probe designed against the same species (Ward and Cockroft, 1993). JVIore recently, other strains of denitrifiers have been isolated from both sediment and water column samples. Sequence information regarding the denitrifying genes should prove invaluable for future refinement of both sequence based approaches and the development of additional antibodies to denitrifiers important in the marine environment. 

A sequence-based approach to the identification of differentially expressed genes through comparative analysis. Allows simultaneous analysis of sequences that derive from different cell populations or tissues. This is not a chip-based method. Identification of sequences relies on completeness of public sequence databases and, therefore, can only be used to analyse known genes. 

A major barrier to current sequence-based approaches to community diversity studies remains the specificity of PGR primers. Simulation analysis in our laboratory has revealed that several universal primer sets for bacteria and archaea are unable to amplify certain phyla. There are also no truly universal PGR primers that amplify phyla across all domains and so information on the overall community is lacking for many microbial habitats. One way to address this shortcoming is to construct datasets for each domain (Archaea, Bacteria, Eukarya) and then use quantitative tools such as real-time PGR to estimate relative abundance of domains. We have applied this approach to lithic microbial communities with some success (Pointing et al., 2009). 

Whereas the Dunedin (1990) and Sudhury (1995) meetings and books each had one report of genetic data, the London (2000) meeting heard seven reports, five represented by papers in this part. This reflects the worldwide growth of the DNA sequence-based approach, providing taxonomy with its first characters of certain genealogical value. Some main points of these contributions will be highlighted here. 

As indicated above, because threading uses structure, it should be superior to sequence-based approaches that are one-dimensional and that assess the evolutionary relationship between sequences and thereby, by inference, their structural relationship. In practice, however, many of the most successful... 

Genome sequence-based approaches can be a buffer against the distorting evolutionary events at a single locus the investigation of several sequences can lead to a further comprehension of the events, which occurred during the evolution of an organism (Konstantinidis and Tiedje 2005). 

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