Where does all that DNA go?

In this recent essay, I discussed studies that showed a surprisingly high rate of movement of DNA from organelles to the nuclear genome.  Curious and questioning readers should have wondered about this, as one implication is that nuclear genomes should become huge mosaics of organelle DNA in a relatively short evolutionary time.  Of course, this is not the case – organelle DNA may be found in nuclear genomes, but it makes up a tiny fraction of these genomes.

If you had read my essay and wondered along these lines, you may pat yourself on the back.  For this paradox is something that has puzzled others.  A recent paper in PLoS Genetics helps to resolve things.  Briefly, Anna Sheppard and Jeremy Timmis followed up on the earlier studies , asking what happens to the organelle genes after they wander into the nucleus.  These authors found a high frequency with which the organelle DNA (at least the marker gene that was followed in these studies) is altered or deleted.  As the authors discuss (the abstract follows beneath the fold), this suggests that the overall picture is a dynamic one – DNA can move into the nucleus at a high rate, but it is also removed relatively rapidly.  The result is a sort of steady state, one that affords the creation of new nuclear genes without the burden of vast amounts of organelle DNA.

The abstract and author summary:

“Abstract

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.

Author Summary

In eukaryotes, mitochondria and plastids are the descendents of once free-living prokaryotic ancestors. Over time, these organelles have donated a great deal of genetic material to the nuclear genome. Although usually non-functional, these DNA transfer events have, over evolutionary time, resulted in a large pool of functional nuclear genes and therefore the process of DNA transfer has been an important driving force in eukaryotic evolution. Previous studies showed that DNA transfer of a specific marker gene (neo) from the plastid to the nucleus occurred in one in every 11,000 to 16,000 male gametes. Because of this high frequency of transfer and the large size of integrants, this process would be expected to result in a cumulative increase in genome size, unless there are counterbalancing deletion events. In this study, we analysed the stability of the neo gene after integration into the nuclear genome. We found that the gene is highly unstable, with deletion often occurring within a single generation. These results indicate that plastid DNA insertion into and removal from the nuclear genome are in dynamic equilibrium, thus providing a mechanism by which the chances of functional DNA insertion are maximised without compromising the nuclear genome as a whole.”

One additional snippet, to whet the appetites of readers who should be asking why organellar DNA is so unstable, while other highly repeated DNA (from transposons, for example) persists so easily.

“Why do some kr lines show a high level of instability, while others appear to be more stable? One possibility is that the chromosomal location and sequence context of the integrant determines the level of stability. For example, nuclear integration of organellar sequences may be dependent on the formation of double strand breaks (DSBs) [13],[18],[19] and if some regions of the genome are particularly prone to DSBs, as is the case for meiotic recombination hotspots in yeast [20], this could facilitate both integration and removal of nupts in these regions. Differing levels of stability could represent differing tendencies to sustain DSBs. Another possibility is that the level of stability depends on the sequence of the integrant itself, rather than the surrounding sequence. In this case nupts may be recognised as foreign DNA and subsequently removed. For example, the recognition could occur via differences in methylation status, as plant nuclear DNA is highly methylated and ptDNA is not [21],[22]. Certain plastid sequences may be more prone to elimination than others, or alternatively the level of stability may depend on the size of the integrant. Differing levels of stability also may be related to differences in transgene copy number. Kr2.3, kr2.5, kr2.7 and kr2.10 all display instability, but kr2.7 appears to be the least unstable of this group (Figures 1 and 2). Southern blotting indicates that kr2.3, kr2.5 and kr2.10 have single or low copy insertions while kr2.7 appears to have several copies of neo[11]. Therefore it may be that in the case of kr2.7, the loss of kanamycin resistance requires several deletion events or an infrequent large deletion.”.

There are more interesting things in this paper – discussion of the well-known instability of transgenes, possible implications for evolution and biotechnology, to name two.  I encourage readers to read through the whole study.

The citation:

Sheppard AE, Timmis JN 2009 Instability of Plastid DNA in the Nuclear Genome. PLoS Genetics 5(1): e1000323 doi:10.1371/journal.pgen.1000323.

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