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

• Using Transposons to Tag Genes
• Designing the RescueMu Transposon
• Breeding RescueMu Plants
• Converting Grids of RescueMu Plants into Library Plates
• Finding the Mutated Genes
• Tracing a RescueMu Tagged Gene to a Specific Plant

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.

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.

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.

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.

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).

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.