Technique Gives Researchers New Look At Genetic Activity In Living Cells

February 06, 1998

CHAMPAIGN, Ill. -- A new, non-invasive method for tagging chromosomes is making genetic activity in living cells easier to see, and likely will lead to insights into chromosome movements, folding and unfolding during natural events such as cell division, DNA replication and transcription.

Since the method was announced by University of Illinois researchers in December 1996, it has been applied by several laboratories in live cells taken from bacteria, yeast and mammals, as well as from Drosophila (fruit flies) and C. elegans (worms).

In an upcoming issue of Trends in Cell Biology, the use of the technique will be detailed by Andrew S. Belmont, a professor of cell and structural biology at the U. of I., and Aaron F. Straight, a physiologist in the School of Medicine at the University of California at San Francisco.

"This method is opening a clearer window into the working mechanics in cells," Belmont said. "It gives us a way to look at the dynamics that hasn't been possible before. Down the road, we would like to learn what happens to the structure of a chromosome when a gene is turned on or off."

The method uses a specific protein-DNA interaction in which a protein binds to a specific target in DNA without altering chromosomal structure. The traditional DNA hybridization technique for localizing a particular chromosome region cannot be done on living cells, and it causes some damage, a problem that has seriously limited structural and mechanical research on chromosomes.

Belmont created a roughly 10,000-base pair DNA fragment containing 256 copies of the lac-operator sequence to which the lac-repressor protein binds. The interaction between the operator and repressor, found normally in bacteria, is well-described by previous work. By detecting the lac-repressor protein, the location of the DNA fragment containing the lac-operator repeats is revealed.

The fragment is put into chromosomes by genetic engineering. To sharpen visibility, the lac repressor protein is fused with a naturally occurring green fluorescent protein, recently discovered in jellyfish, allowing for viewing the area in living cells by light microscopy. With immunogold staining, yet clearer views have been seen under electron microscopes.

Combining this with gene amplification, a process in cancer cells that duplicates chromosomal regions, has allowed scientists to see entire chromosome arms. The results have provided pictures and proof of theorized chromosomal fibers 100 nanometers in diameter in live cells as folding and unfolding occurs.

Belmont's group has seen specific bending and motion of chromosomal fibers. UCSF biologists have seen motion during mitosis in living yeast cells. They hope to understand the natural functions of proteins required for normal chromosome separation in both yeast and human cells. Harvard researchers, using the method on bacterial cells, are questioning the theories on chromosome segregation. They now suggest an active, mechanical system for the separation. Such a mechanism had been thought to exist only in eukaryotic organisms such as plants and animals.
-end-


University of Illinois at Urbana-Champaign

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