The EST Strategy |
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The EST Strategy: Taking a Stab at the DNA Blueprint DNA is often called the blueprint of life. Genomics researchers seek a vision of that blueprint: the grand scheme that allows an organism to build something from nothing. But the construction project itself often obscures the big picture from view. Just as a glance at a plumber's work under the kitchen sink tells nothing about the roofer's plan for the eaves, the genes building a root Trying to deduce a genome's potential by examining gene activity in one cell, one tissue type, or one stage of development would be like trying to figure out the final shape of a house by looking at one corner of a room. Likewise, one cannot gage how the rooms were built by looking at the façade. To grasp how a genome produces each stage and each tissue of a plant, researchers must study each stage and each tissue. Only then can the entire genomic scheme come into focus. The Maize Gene Discovery Project's EST Strategy is a first stab at achieving that goal. Finding Genes By Looking at Gene Activity As maize plants develop, they produce roots, stems, leaves and finally fruit. To grow each of these tissues at the appropriate stage of development, different genes are turned on and off in the plants' cells. When a gene is "on," it is transcribed into messenger RNA (mRNA), which is in turn translated into protein. An mRNA sequence matches the DNA sequence that produced it, minus certain sections that have been spliced out (introns). So, by knowing an mRNA sequence or even part of one, a researcher can find part of a gene.  ESTs Represent Active Genes Determining an mRNA sequence takes several steps. After researchers extract mRNA from a cell, they transcribe it back into stable pieces of complementary DNA (cDNA), insert the cDNA into plasmid libraries, and sequence each end of the cDNAs. These short stretches of DNA code are called Expressed Sequence Tags, or ESTs. Because they reflect mRNA sequences, ESTs represent active genes. To determine whether an EST represents a newly discovered gene, the MGDP created a computer tool (ZmDBAssembler) that compares ESTs to one another and assembles them into tentative unique contiguous sequences (TUCs). The MGDP team also compares the ESTs to known genomic sequences to identify possible introns, the segments of mRNA that are spliced out (learn more).  Looking for ESTs in a Variety of Tissues and Developmental Stages When looking for ESTs, many researchers extract mRNA from homogenized cells of the adult organism. But MGDP sequenced ESTs from separate tissues and developmental stages in order to find a more comprehensive set of ESTs while also gaining considerable information about patterns of gene expression. As shown in the table below, MGDP looked for ESTs in 13 tissue types and at 6 stages of development. The team found very little overlap in ESTs from these different sources, demonstrating the wisdom and efficiency of this approach.
MGDP has experienced a remarkable return on its EST investment. The first 73,000 ESTs corresponded to 22,000 genes – one gene for every 3.3 ESTs. That rate is gradually declining: now, it takes 4.7 ESTs to find a new gene. The team foresaw such diminishing returns because some genes produce only a few mRNAs in a single tissue during a brief stage of development. Nevertheless, MGDP expects to find more than 200,000 ESTs by project's end, and these should define about 35,000 genes or about three-quarters of the expected total number of genes in maize. For more information about ESTs, check out: |
system reveal nothing about how to make a flower.|
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