| | Introduction | Genomic analysis | ESTs and microarrays | Proteomic analysis | Perspectives | Acknowledgements | References
Introduction
The "Genomic Era" was ushered in by rapid advances in nucleic acid sequencing, making it possible to sequence whole genomes (all the genetic material in the chromosomes of a particular organism) relatively quickly (Lee and Lee, 2000). The Human Genome Project accelerated development of the various techniques and technologies involved in genomics, and opened up new "-omics" fields, notably Proteomics (the study of the complement of proteins expressed in a given cell, tissue or organism under particular conditions at a particular time). The huge amount of data generated by the systematic analysis and documentation of genomes and proteomes has in turn given rise to the field of Bioinformatics, comprising the informatics capacity and skills needed to organize and annotate the data and to further predict structure, function and inter-relationships among biomolecules of interest. The post-genomics era is characterised by an holistic approach to the study of biological systems, involving many new analytical tools. This short paper will focus on selected technological developments in the areas of genomics and proteomics, and their application in malting barley development.
Genomic Analysis
The introduction of molecular markers (segments of DNA whose pattern of inheritance can be determined), the polymerase chain reaction (PCR) to amplify genomic DNA fragments, and high throughput DNA sequencers which determine nucleotide sequences of amplified DNA by gel or capillary electrophoresis methods, are all examples of advances in genomic analysis. These advances have led to the sequencing of the genomes of several "model organisms" including human, bacteria (Escherichia coli), yeast (Saccharomyces cerevisiae), and plants such as Aradopsis and rice (Oryza sativa).
Barley researchers and breeders were quick to make use of the molecular markers for barley fingerprinting based on restriction fragment length polymorphisms (RFLP’s) and PCR-generated random amplified polymorphic DNAs (RAPDs), sequenced tagged sites (STSs), microsatellites and amplified fragment length polymorphisms (AFLPs). The North American Barley Genome Mapping Project (NABGMP) and other initiatives made great strides in locating quantitative trait loci (QTL) - specific genomic regions that affect quantitative traits (Han et al., 1997; Mather et al., 1997) - and in exploiting molecular markers in breeding for specific traits (Swanston et al., 1999) or for barley malt fingerprinting (Faccioli et al., 1999).
ESTs and Microarrays
While DNA sequence information defines the genome, it does not of itself provide information on the relative expression levels of genes. This information is more readily obtained by examining cellular RNA content. Reverse transcription of mRNA produces complemcntary DNA (cDNA), a single-stranded form of DNA that can be amplified by PCR for further study. Recent sequencing efforts have been directed towards compiling short sequences of cDNA, known as expressed sequence tags (ESTs), which can be used as genetic probes to relate sequence information to differences in mRNA expression between cell populations. ESTs, or cDNAs of known sequence, can be deposited onto glass slides in known locations to create microarrays, or gene chips. The same reverse transcription process that produces cDNAs for microarrays is used to prepare cDNA fragments from cell populations. These cDNAs can be labelled with different fluorescent tags and allowed to hybridize with the cDNA on the chip so that differences in mRNA expression between the cell populations can be examined. Genome-wide analyses of gene expression patterns are thus enabled, and can be used to study, for example, abiotic stress responses in cereal crops, including wheat and barley. Jones (personal communication) has used the commercially available Rice Chip (Affymetrix Inc., Santa Clara, CA, USA) to investigate signalling pathways and metabolic regulation in barley aleurone layers, and noted an 80% hybridization of barley cDNA to the rice oligochip.
Proteomic Analysis
The need to go beyond nucleic acid analysis, and reach an understanding of total protein expression and regulation in biological systems is motivating the field of ‘Proteomics’ (Dutt and Lee, 2000). The field of proteome analysis has largely been driven by technological developments which have improved upon the basic strategy of separating proteins by two-dimensional gel electrophoresis (O’Farrell, 1975). As with genomic information or data derived from microarray analysis, an informatic framework is required to organize proteomic data, as well as to generate structural and predictive models of the molecules and interactions being studied.
The use of 2-D gel elctrophoresis coupled with mass spectrometry (MS) is the most familiar proteomics approach, with advances in the MS ionization techniques and mass analyzers enabling the generation of different types of structural information about proteins of interest. The techniques of nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography rank alongside mass spectrometry as the major tools of proteomics. The development of protein chips (analogous to microarrays) and protein-protein interaction maps will offer new ways to rapidly characterize expression levels and relationships among proteins in normal or “perturbed” cell populations, helping researchers to bridge the ‘genotype-phenotype’ gap.
Perspectives
A combination of functional genomics and proteomics approaches allows researchers to cross interdisciplinary boundaries in "trawling" the genome, transcriptome, proteome and metabolome for new insights into structure and function that can be applied to improving the agronomic and end use traits of malting barley. As Fincher (2001) has pointed out, functional genomics and related technologies are now being used to re-visit the difficult questions of cell wall polysaccharide biosynthesis, involving synthase enzymes that had proved impossible to purify and characterize by classical biochemical methods. In addition to the resolution of their structure, the genes encoding for the more extensively studied hydrolase enzymes, responsible for cell wall breakdown and starch degradation, are being identified, generating knowledge of how characteristics such as thermostability might be enhanced to improve malting and brewing performance (Kristensen et al., 1999; Ziegler, 1999) Structural and functional studies can thus be linked back to protein and mRNA expression patterns, and ultimately to the families of genes that we might wish to conserve or alter in breeding programs.
Ultimately, the extent to which advances in functional genomics and proteomics will be embraced and adopted by the malting and brewing industry depends heavily on the industry’s ability to "keep up" with the rapidly moving field, and on the economics (perhaps the most important of the "-omics") of doing so. Brewing Science has a long and distinguished history, closely associated with the need of the industry to profitably apply science in pursuit of product quality and consistency. To this end, generations of chemists, biochemists, microbiologists, botanists and plant breeders have applied their skills to elucidating and manipulating the structural and functional characteristics of barley, yeast and hops. In the case of barley, one of the results of the close linkage between science and industry has been a relatively strong base of research and breeding activities - certainly more than would be expected from barley’s position among the world’s major crops. It is perhaps ironic that today, when the emerging technologies of genomics and proteomics are allowing us to revisit and build upon the knowledge generated in the past, industry’s engagement in the research process has become less active. The danger in moving from a position of active engagement in the generation of new knowledge lies in the potential loss of "absorptive capacity" (Cohen & Levinthal, 1990). According to these authors, once an organization ceases investing in its absorptive capacity in a quickly moving field, it may never assimilate and exploit new information in that field, regardless of the value of that information.
Acknowledgements
The advice of the following researchers, several of whom provided graphics to assist in the presentation of this paper, is gratefully acknowledged:
M. Arcellana-Panlilio, E. Armstrong, M.P. Cochrane, J. Coorssen, G.B. Fincher, R.L. Jones, A.W. MacGregor, M. Moran, B.G. Rossnagel, L.Shugar.
References
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P.L. Freeman
Alberta Network for Proteomics Innovation
2275-3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1
Presented at the 3rd Canadian Barley Symposium, June 19-20, 2003 |
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