Mar. 8, 1999 In the century and more since Charles Darwin first advanced his theory of evolution, the proposal has come to dominate biological understanding. Current thinking has it that new traits arise spontaneously through genetic mutations, and, if they promote the survival of the individual, the genes may be selected for in subsequent generations. Over time, significant changes in a species can occur in this way -- indeed, entirely new species may eventually appear.
In this age of molecular biology, highly specific new questions about the process of evolution are being framed. Scientists would like to know whether and how evolution operates at the molecular level in an organism's DNA.
Now, experiments performed at the University of Pennsylvania Medical Center have revealed a molecular mechanism that may be a significant driver of evolution in humans and other mammals. Certain retrotransposons -- bits of DNA able to copy themselves from one region of the genome to another -- are able to pick up flanking genetic sequences and then insert themselves and the tag-along DNA at new locations. A report on the new study will appear in the March 5, 1999, issue of Science. (Advance copies of the paper are available to reporters through the journal's news office at 202-326-6421.)
"These findings suggest a new mechanism for shuffling important genetic sequences -- exons, promoters, enhancers -- that may lie downstream from these active mobile DNA elements," says Haig H. Kazazian, MD, chairman of the department of genetics and senior author on the paper. "From an evolutionary perspective, here is a way to create novel genetic combinations. While many such changes might prove lethal, some could improve function in individuals, leading to a selective advantage for those individuals."
"Previously proposed explanations for how such rearrangements of DNA might occur have been rather murky," says lead author John V. Moran, PhD, now an assistant professor of human genetics and internal medicine at the University of Michigan Medical School. "This mechanism, however, provides a relatively straightforward and powerful means for generating genomic diversity."
Working with a family of retrotransposons called long interspersed nuclear elements, also known as LINE-1s or L1s, the scientists performed two groups of experiments in cultured human cells. Between 30 and 60 active L1s are estimated to exist within the human genome. Other species, such as the mouse, are thought to have as many as 3,000 such elements in their DNA.
The aim of the first experiments was to discover whether L1s retrotranspose efficiently into genes and, if so, how often. To do this, the investigators engineered an L1 to include a marker sequence that would only be activated when the mobile L1 inserted itself into a working gene, a gene subject to transcription. The results showed that a minimum of 6 percent of all retrotransposition events effectively targeted genes. Because the total fraction of the human genome estimated to be functioning genes is only about 15 percent, this finding suggests little or no bias against genes as sites for retrotransposition.
The second set of experiments was designed to ascertain whether L1s are capable of copying and moving DNA sequences adjacent to themselves during retrotransposition. Earlier results had suggested this possibility, beginning with the surprising observation that a flanking sequence had accompanied an instance of retrotransposon insertion into the gene responsible for Duchenne's muscular dystrophy. Additional examples of such stowaway DNA with the L1s were subsequently found.
"Several years ago, we found a retrotransposon insertion into a dystrophin gene that carried about 600 base pairs of flanking sequence," says Kazazian. "When we looked elsewhere in the genome for the precursor of that insertion, we found that same 600 base pairs with the precursor."
The scientists already knew that at one end of an L1 is a sequence that initiates transcription and at the other is a sequence that terminates transcription. Closer analysis, however, revealed that the L1s use a terminating sequence that is both unusual and somewhat weak, leading to so-called readthrough, meaning that transcription at times continues past what would ordinarily have been the stop point to the next downstream termination sequence.
To better understand this readthrough phenomenon, the scientists engineered an L1 with its marker sequence outside the L1, beyond the naturally occurring termination signal. And then beyond the marker itself, they placed a well-understood and highly efficient stop signal sequence, so that the gene products of any readthrough events could be easily identified because they would end with this marker. With the stronger downstream signal in place, the engineered L1s readily picked up neighboring DNA for retrotransposition.
"Overall, we found that, not only can these retrotransposons jump into genes, but readthrough events that pick up flanking DNA are not uncommon," Moran says.
In addition to Kazazian and Moran, the third author on the paper is graduate student Ralph J. DeBerardinis. While at Penn, Moran was supported by a Damon Runyon postdoctoral fellowship. Primary funding for the study was provided by the National Institutes of Health.
The University of Pennsylvania Medical Center's sponsored research and training ranks third in the United States based on grant support from the National Institutes of Health, the primary funder of biomedical research and training in the nation -- $175 million in federal fiscal year 1997. In addition, for the third consecutive year, the institution posted the highest annual growth in these areas -- 17.6 percent -- of the top ten U.S. academic medical centers.
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