Deletion of Noncoding DNA Suggests Some Sequences Are Disposable

Conservation Limited

At first blush, it may seem no surprise that mammals can get along very well, thank you, without the regions of gene-free DNA sequence labeled "junk DNA". But the phrase has a checkered past. Recent studies have shown good reason to consider the gene-free portions of genomes indispensable. Various stretches of noncoding DNA have been shown to function as enhancers of gene expression, causing more of this protein or that to be produced. More to the point, these and other gene-free segments were also found to occur almost identically (that is, they are conserved) in organisms as distantly related as humans and fish. The standard belief in genomics is that evolution would not preserve sequences that were not performing some function worth maintaining. Yet, here we have examples of sequences, occurring in evolutionarily distant creatures, that seem to violate that compelling logic. Researchers must now rethink the supposition that conserved sequence is necessarily useful sequence.

In yet another twist in the story of understanding "junk DNA," researchers from JGI and Berkeley Lab's Genomics Division have found that deleting large stretches of noncoding DNA can yield surprisingly healthy mice. Mice with the noncoding sequences removed were essentially indistinguishable from wild-type mice, even though the deleted sequences contained many sections that are conserved between mice and humans. This result suggests that some noncoding sequences, even those that persist through the pressures of evolution, may indeed be disposable.

Very similar organisms, like these two rat species, can have radically different genome sizes, suggesting that a large portion of a given genome may be "junk."

The deleted sequences were a 1,511-kb length on mouse chromosome 3 and an 845-kb stretch of chromosome 19. In order to obtain mice with these two sequences deleted, the researchers used a combination of genomic targeting and Cre/lox excision (a technique that makes use of the Cre protein's ability to delete bases between inserted loxP sequences) in embryonic stem cells. They eventually obtained mice that were heterozygous for the deletions, which were in turn bred to create a generation including mice that were homozygous for the deletions.

Regulatory sequences in the deserts are identified by an in-vivo assay that stains transgenic mouse embryos in blue.

The first surprise was the percentage of offspring from heterozygous mice that were homozygous for the deletions, which was equal to the percentage of homozygous wild-type offspring, indicating no decrease in viability for those without the targeted sequences. The next step was evaluation of the overall health of the mice with the smaller genomes. They showed no differences in morphology, reproductive fitness, growth, longevity, or general homeostasis. Quantitative PCR analysis showed that the expression levels of genes flanking the deleted sequences were almost completely unaffected. Of 108 tissue expression assays in nearby genes, only two showed altered expression levels. Finally, the scientists used reporter vectors to identify potential regulatory sequences in the deleted segments. The 15 elements tested were those with the highest degree and longest stretches of sequence conservation between humans and mice. The selection also favored sequences conserved over the longest evolutionary time scales. Only one of the 15 elements--one conserved in vertebrates as varied as humans, mice, frogs, and chickens--gave a positive result.

The researchers acknowledge the impossibility of testing all potential phenotypic differences between the wild-type mice and those with sequences deleted. They also point out that the missing DNA could be compensated by redundant sequence elsewhere in the mouse genome. Still, the tantalizingly small impact of removing the sequences suggests that the mammalian genome is not densely encoded. Representing the largest reported homozygous sequence deletions in viable mice, the results give renewed relevance to the oft-maligned moniker "junk DNA."

Authors

M.A. Nóbrega, Y.Zhu, I. Plajzer-Frick, V. Afzal, and E.M. Rubin (JGI and Lawrence Berkeley National Laboratory).

Publication

"Megabase Deletions of Gene Deserts Result in Viable Mice," Nature 431, 988-993 (2004), doi: 10.1038/nature03022.

Funding

This research was funded by the National Heart, Lung, and Blood Institute and the U.S. Department of Energy.