(Philadelphia, PA) - In experiments with transgenic mice, University of Pennsylvania School of Medicine researchers discovered the remaining steps in the complicated process of how the largest class of jumping genes replicates and inserts themselves within the human genome. Haig H. Kazazian, Jr. MD, Chair of the Department of Genetics, and colleagues at Penn published their findings in the February issue of Genome Research. This knowledge may shed light on the origins of "junk" DNA, parts of the genome for which no function has yet been discovered.

Jumping genes-also called mobile DNA or transposons-are sequences of DNA that can move or jump to different areas of the genome within the same cell. They are a rare cause of several genetic diseases, such as hemophilia and Duchenne muscular dystrophy.

Retrotransposons are one class of jumping genes, with the L1 family being the most abundant in the human genome. Retrotransposons move by having their DNA sequence transcribed or copied to RNA, and then instead of the genetic code being translated directly into a protein sequence, the RNA is copied back to DNA by the retrotransposon’s own enzyme called reverse transcriptase. This new DNA is then inserted back into the genome. This process of copying is similar to that of retroviruses, such as HIV, leading scientists to speculate about a viral origin for retrotransposons.

“L1 retrotransposons, which are the only active mobile DNA elements in humans, have accounted for about 30 percent of the human genome by their own insertions and by driving the insertion of other kinds of elements,” says Kazazian. “In fact, humans have over 500,000 L1 retrotransposons within an individual genome.”

In order to learn about the effects of L1 retrotransposon insertions into the human genome, the researchers made a transgenic mouse in which human L1 retrotransposons could replicate. They injected several copies of a human L1 retrotransposon to create the transgenic mouse. In subsequent generations, the retrotransposons moved within the offsprings’ genomes and each new insertion could be detected by the investigators. The researchers characterized 51 new jumps of L1, finding that insertions landed in random genomic regions. Several L1 insertions included small pieces of extra DNA.

While tracing the origin of this extra DNA, Daria Babushok, an MD/PhD student in the Kazazian lab, came up with the missing steps in the mechanism of retrotransposon replication. “It was known previously that the enzyme endonuclease cleaves one of the strands of cellular DNA and then the retrotransposon inserts by binding to that cleaved DNA strand and copying itself onto that strand,” she says. “It sneaks in there.”

How the retrotransposon finally integrated and pasted itself back together was unknown, until this paper. “What we saw in our insertions hinted at the possibility that reverse transcriptase actually jumps onto the second DNA strand and continues the synthesis,” she explains. “We think that this is how the second part of the element integrates into the genome. If this mechanism proves to be correct, it will bring us much closer to knowing how more than half a million retrotransposons have accumulated in the human genome.”

Eventually, continuous jumping by retrotransposons expands the size of the human genome and may cause shuffling of genome content. For example, when retrotransposons jump, they may take portions of nearby gene sequences with them, inserting these where they land, and thereby allowing for the creation of new genes. Even otherwise unremarkable insertions of L1 may cause significant effects on nearby genes, such as lowering their expression.

Now, by knowing the final steps in retrotransposon replication and being able to follow and map new insertions in animals, the researchers will be able to more fully understand how L1 retrotransposons are able to invade the human genome.

“We were able to obtain a snapshot of a large number of new L1 jumps in a situation closely mimicking what occurs every day in the human genome,” says Babushok. “Importantly, occasional small additions of extra DNA sequences at the ends of new L1 insertions gave us tantalizing leads to the L1 retrotransposon replication mechanism. We are very excited to follow this thread to confirm our proposed mechanism and to come closer to a complete understanding of the interaction between L1 retrotransposons and our genomes.”

The research was supported by grants from the National Institutes of Health. Study co-authors are Eric M. Ostertag, Christine E. Courtney, and Janice M. Choi, all from Penn.

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