Mitochondrial mysteries
--Christine Chase, Horticulture Sciences Department, University of Florida; and
--Susan Gabay-Laughnan, Department of Plant Biology, University of Illinois

This one-hour demonstration has been given by the Chase lab for numerous groups of high school and junior high school students. Only small pieces of equipment are required, and the demonstration has also been taken to local classrooms. The demonstration begins with a 20-minute slide show and discussion. The following points are made:

1.Many important genetic discoveries relevant to all organisms were first made studying plants (Mendel and McClintock, for example). Plants are wonderful organisms for genetic investigations!

2. The plant cell contains, among other organelles, a nucleus, mitochondria and chloroplasts. The functions of these organelles are briefly discussed.

3. While much of the genetic information (DNA) resides in the nucleus and is inherited according to Mendel's laws, some genetic information resides in the mitochondria and chloroplasts. This information is usually inherited from the maternal parent.

4. The expression of genetic information in the nucleus, mitochondria, and chloroplasts must be coordinated somehow.

5. Cytoplasmic male sterility (CMS) systems are good tools to study coordination among different genomes. The mitochondria encode this maternally inherited failure to produce functional pollen, but nuclear restorer genes can suppress or overcome the mitochondrial genotype, resulting in a male-fertile plant.

6. An experiment that we used to identify regions of the mitochondrial genome involved in CMS is described. The experiment involves the following concepts:
    a. CMS plants sometimes undergo a mitochondrial mutation to male fertility. We call these mutants fertile revertants. We can use this genetic change to help us identify parts of the mitochondrial DNA that may be responsible for the CMS trait. We do this by comparing the mitochondrial DNA from CMS plants and the fertile revertants.
    b. Mitochondria can be purified from other cell components by differential centrifugation. DNA can then be released and purified from these mitochondria.
    c. Restriction endonucleases and agarose gel electrophoresis can be used to compare the mitochondrial DNAs from CMS plants and fertile revertants. After these techniques are explained, photos of the restriction fragment patterns are passed around to see who can identify fragment differences. Fragments present in the CMS plants but not in the fertile revertants are candidates for further study.

The remainder of the hour is spent in the lab, demonstrating the various components of the experiment. We do the following:

1. Grind some etiolated seedlings in mitochondrial extraction buffer in a blender, explaining that this breaks open cells to release all of the component parts.

2. Large components (nuclei, plastids, starch grains) are removed by a low speed centrifugation (1,000 x g for 10 min). The supernatant is centrifuged at 10,000 x g for 10 min to pellet smaller components. The resulting pellet is a crude preparation of mitochondria. These spins can be accomplished in a variable speed microfuge.

3. While spins are in progress, we demonstrate agarose gel electrophoresis. We usually have three gels prepared. One (still in the casting tray with slot former) is passed around so that students can see how the gel is formed and what it feels like. A second is set up in an electrophoresis unit with buffer. Student volunteers can load pre-prepared samples of bromphenol blue and xylene cyanol in 20% glycerol. The two dyes are quickly separated during a 100 volt run, so students can observe the separation of molecules according to size. The third gel has been run, stained with ethidium bromide, and set up under a plexiglass shield on a transilluminator. We usually run molecular weight standards. These provide a striking pattern that is easy for the students to observe when the illuminator is turned on.

Susan has generously provided seeds of CMS-S maize and a cytoplasmic revertant to fertility for this work. In addition, she suggests the use of the RU male-fertile cytoplasm. These three cytoplasms are distinguished by different mitochondrial episome components. The CMS-S line carries the S1 and S2 episomes, the revertant lacks episomes and the RU line carries episomes of different molecular weight. The episomes are present in high copy number and can be readily visualized on ethidium bromide-stained gels of undigested, crude mitochondrial DNA preparations. This provides a simple and striking demonstration of cytoplasmic genetic differences (and separation of DNA molecules by size). We have plans to increase seeds of these materials and to make some reciprocal crosses, which could be used to demonstrate maternal inheritance of the mitochondrial plasmid DNAs. We'd be happy to provide seeds and further assistance to anyone interested in demonstrating mitochondrial mysteries. 


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