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Project Documentation & Protocols: Maize Gene Discovery Project: Education:
Finding Maize Genes

Contents: Maize Gene Discovery | The Challenge of Maize Genetics | Why Discover Maize Genes? | Finding Genes
Linking Genes to Function | Creating Databases | Building a Storehouse | Accomplishments | What's Next? | Glossary

The Maize Gene Discovery project looks for genes in two complementary ways. One method searches for expressed sequence tags or ESTs. These represent genes that are turned "on" in a specific maize tissue at a specific point in time. (Learn more about MGD's EST strategy) http://www.maizegdb.org/documentation/mgdp/est/index.php

The second method takes advantage of special transposons -- stretches of DNA that insert copies of themselves inside maize genes. The researchers grew grids of maize plants that all contained a specific, engineered transposon tag called RescueMu. The transposon, whose sequence is known and easily traceable, inserts itself in new chromosome locations, but always within a gene. The researchers then find genes by sequencing the DNA on both sides of RescueMu wherever it's found in those many plants. (Learn more about RescueMu gene tagging) http://www.maizegdb.org/documentation/mgdp/library-plate/index.php

Both approaches to gene discovery have been enormously successful. Thus far, the project has found more than 155,000 ESTs. After determining the overlap among these sequences, the ESTs appear to identify more than 31,000 unique genes (learn more about MGD Accomplishments).

The RescueMu strategy has discovered about 5000 genes. At least 3300 of these match known ESTs, indicating that these are indeed genes with active functions in the maize plant. During the final year of the project, the rate of gene discovery using RescueMu will accelerate. (Learn more about MGD Accomplishments).

 

The EST Strategy

 

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 system reveal nothing about how to make a flower.

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 fašade. 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.

Library 486 - Immature Leaf Organ - Shoot
ESTs found - 5867 Development stage - P5/6 - P10/11
Library 487 - Apical meristem Organ - Shoot
ESTs found - 684 Development stage - Immature
Library 496 - Stressed shoot Organ - Shoot
ESTs found - 1306 Development stage - Salt stress
Library 603 - Stressed root Organ - Root
ESTs found - 2023 Development stage - Salt stress
Library 605 - Endosperm Organ - Kernel
ESTs found - 6565 Development stage - 10-14 days post pollination
Library 606 - Ear tissue Organ - Immature ear
ESTs found - 5516 Development stage - Ear length from 0.5cm - 2.0cm
Library 614 - Root Organ - Root
ESTs found - 10611 Development stage - 3-4 days old
Library 618 - Tassel primordia Organ - Tassel
ESTs found - 3407 Development stage - Tassel length from 0.1cm - 2.5cm
Library 660 - Mixed stages of anther and pollen Organ - Anther, pollen
ESTs found - 6444 Development stage - Premieotic anthers to pollen shed
Library 683 - 14 day immature embryo Organ - Embryo
ESTs found - 1138 Development stage - 14 days after pollination
Library 687 - mixed stages of embryo development Organ - Ear
ESTs found - 4763 Development stage - 14, 21, 28, and 35 days after pollination
Library 707 - Mixed adult tissues Organ - Tassel, kernel, silk, husk, root, and leaf
ESTs found - 4443 Development stage - Adult
Library 829 - Silk infected with Fusarium Organ - Silk
ESTs found - 269 Development stage - Adult
Library 945 - Mixed adult tissues Organ - Tassel, kernel, silk, husk, root, and leaf
ESTs found - 4346 Development stage - Adult
Library 946 - tassel primordium prepared by Schmidt lab Organ - Tassels
ESTs found - 10547 Development stage - just after the transition from vegetative to inflorescence
Library 947 - 2 week shoot from Barkan lab Organ - Shoot
ESTs found - 8878 Development stage - 2 week old seedling (3 leaves)
Library 949 - Juvenile leaf and shoot cDNA from Steve Moose Organ - Juvenile vegetative shoots
ESTs found - 10708 Development stage - 4 stages from 3-13 days after imbibing
Library 950 and 9524 - Mature pollen from Sheila McCormick's lab Organ - Pollen
ESTs found - 427 Development stage - Mature
Library 951 - BMS tissue from Walbot Lab (GR) Organ - N/A
ESTs found - 600 Development stage - Mixed logarithmic and stationary growth phases
Library 952 - BMS tissue from Walbot Lab (GR) Organ - N/A
ESTs found - 13483 Development stage - Mixed logarithmic and stationary growth phases
Library 953 - Immature ear with common ESTs screened by Schmidt lab Organ - Immature ear
ESTs found - 248 Development stage - 0.5cm - 2.0cm
Library 1091 - Immature ear tissue with common inserts from library 606 screened by Schmidt lab Organ - Immature ear
ESTs found - 7645 Development stage - Before pollen shed
Library 3524 - Mature pollen from Sheila McCormick's lab Organ - N/A
ESTs found - 3466 Development stage - Mature

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:
ESTs Fact Sheet: http://www.ncbi.nih.gov/About/primer/est.html

 

Finding Genes Using RescueMu Tagging

By tagging genes with transposons, researchers can find and sequence them more easily. This section explains MGDP's RescueMu tagging strategy from start to finish.

 

Using Transposons to Tag Genes

Looking for a gene among billions of nucleotides is like looking for a needle in a haystack unless the gene has a tag on it that says "Here I am." Transposons, which have distinct nucleotide sequences at either end, can function as such tags in the maize genome.

Also known as "jumping genes," transposons are short stretches of DNA that can insert themselves into new locations in a cell's DNA. For a transposon to "jump," the cell must contain transposase, an enzyme that is produced by a gene located either in the transposon itself or somewhere else in the cell's DNA. When the transposase recognizes the distinct sequence of nucleotides at each end of the transposon, it will either move or copy the transposon into a new chromosomal address.

Lines of corn that contain both a transposon and the transposase gene can produce new transposon tags each time the transposon jumps.

 

Designing the RescueMu Transposon

Mu elements are maize transposons that are commonly used for gene tagging. They preferentially insert into genes, rather than into noncoding regions of maize DNA. And they're also relatively stable: they jump by being copied rather than moving; and for the most part, they're only jumping in the eggs and pollen. So a maize line containing a Mu element will have transposon tags in a new set of genes in every generation.

For the Maize Gene Discovery Project, researchers specially designed and engineered RescueMu, a Mu element that researchers can easily find and save for future study. RescueMu contains DNA for a plasmid that can later carry many copies of the cloned transposon and its neighboring maize DNA into E. coli bacteria. These bacteria can then be saved in refrigerated plates that serve as libraries containing the RescueMu plasmids and their flanking maize genes.

 

Breeding RescueMu Plants

Before tagging maize genes with RescueMu, the MGDP team had to create lines of corn containing the transposon. They began by inserting the engineered transposon into maize lines that did not contain a gene for transposase. They then grew these plants and checked that the transposon was present. Next, they crossed these lines with a line hat included the transposase gene, allowing RescueMu to jump into new locations. As a result, the seeds from these plants should each contain newly tagged genes. These seeds were then grown into grids, as described below.

 

Converting Grids of RescueMu Plants into Library Plates

During each year of the project the team sews several grids of 2304 seeds -- 48 rows and 48 columns . of their special breeds of maize.

At adulthood, the team takes punches from the plants in each row and each column, pooling them to create 96 DNA samples. This research design allows scientists to convert the DNA samples into a plasmid library on a standard 96-well plate. Thus, a single grid of corn can be transformed into a storable plate of E. coli about the size of a tape cassette. And a set of plates from the entire MGD project can be stored in a box the size of a large dictionary.

 

Finding the Mutated Gene

The MGDP team looks for genes on either side of the RescueMu tag for every pooled row in the plasmid libraries.

They expect to find approximately one tag in each of the 48 plants in a row. To be 95 percent sure they find each of the 48 tags at least once, they grow 288 E. Coli colonies containing the DNA from each row. They then sequence the DNA on each side of the easily identifiable RescueMu tag. On average, the team finds each of the 48 RescueMu tags (and its flanking DNA) three times. By this method, the MGDP has sequenced more than 73,000 stretches of genomic DNA (otherwise known as a genomic survey sequences (GSS)). These sequences constitute more than 70% of the total maize GSS data in GenBank, the federal depository for genetic data.

The GSSs, which are not unique, amount to about 5000 genes found in just three grids: G, H and I. Many more will be found in the coming year as the RescueMu program accelerates, with two grids to be sequenced in the summer of 2002 (K and M) and four others to be completed by the project's end.

After finding the genes, researchers match the genomic sequences to maize ESTs. This confirms that the genomic sequence is in fact a gene that gets turned on in maize. It also identifies the stretches of genomic DNA that are spliced out when the gene is expressed (learn more).

 

Tracing a RescueMu Mutation to a Specific Plant

Though the sequencing of grid rows accomplishes the MGDP's goal of finding genes using RescueMu tagging, the stored plasmid libraries allow further refinement by future researchers. Those interested in studying a particular gene from a pooled row might want to identify the specific grid plant it came from. To do that, they can use a procedure called PCR to search the column libraries looking for the known row sequence. A row and column match identifies a specific plant. For example, if the desired row 1 sequence is found in column 47, then a researcher can order self-fertilized seed from the row 1 column 47 plant (learn more).

The MGDP phenotyping project connects mutant phenotypes to specific grid plants as well, allowing researchers to postulate and test whether a specific RescueMu insertion caused an observed trait (learn more). But because the grid plants contain other Mu elements in addition to RescueMu, a mutant trait could well be caused by a different transposon. The MGDP determines sequences only for the RescueMu insertion sites. If the mutant phenotype turns out to be associated with a different Mu element, techniques are available to clone and sequence that site as well.


Katherine Miller, a freelance science writer, contributed the text for this page to the Maize Gene Discovery Project. You can reach her at .

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