Why Plant 'Clones' Aren't Identical

A new study of plants that are reproduced by 'cloning' has shown why cloned plants are not identical.Scientists have known for some time that 'clonal' (regenerant) organisms are not always identical: their observable characteristics and traits can vary, and this variation can be passed on to the next generation. This is despite the fact that they are derived from genetically identical founder cells.

Now, a team from Oxford University, UK, and King Abdullah University of Science and Technology, Saudi Arabia, believe they have found out why this is the case in plants: the genomes of regenerant plants carry relatively high frequencies of new DNA sequence mutations that were not present in the genome of the donor plant.

The team report their findings in this week's Current Biology.

'Anyone who has ever taken a cutting from a parent plant and then grown a new plant from this tiny piece is actually harnessing the ability such organisms have to regenerate themselves,' said Professor Nicholas Harberd of Oxford University's Department of Plant Sciences, lead author of the paper. 'But sometimes regenerated plants are not identical, even if they come from the same parent. Our work reveals a cause of that visible variation.'

Using DNA sequencing techniques that can decode the complete genome of an organism in one go (so-called 'whole genome sequencing') the researchers analysed 'clones' of the small flowering plant 'thalecress' (Arabidopsis). They found that observable variations in regenerant plants are substantially due to high frequencies of mutations in the DNA sequence of these regenerants, mutations which are not contained in the genome of the parent plant.

'Where these new mutations actually come from is still a mystery,' said Professor Harberd. 'They may arise during the regeneration process itself or during the cell divisions in the donor plant that gave rise to the root cells from which the regenerant plants are created. We are planning further research to find out which of these two processes is responsible for these mutations. What we can say is that Nature has safely been employing what you might call a 'cloning' process in plants for millions of years, and that there must be good evolutionary reasons why these mutations are introduced.'

The new results suggest that variation in clones of plants may have different underlying causes from that of variation in clones of animals -- where it is believed that the effect of environmental factors on how animal genes are expressed is more important and no similar high frequencies of mutations have been observed.

Professor Harberd said: 'Whilst our results highlight that cloned plants and animals are very different they may give us insights into how both bacterial and cancer cells replicate themselves, and how mutations arise during these processes which, ultimately, have an impact on human health.'

Journal Reference:

1. Caifu Jiang, Aziz Mithani, Xiangchao Gan, Eric J. Belfield, John P. Klingler, Jian-Kang Zhu, Jiannis Ragoussis, Richard Mott, Nicholas P. Harberd. Regenerant Arabidopsis Lineages Display a Distinct Genome-Wide Spectrum of Mutations Conferring Variant Phenotypes. Current Biology, 2011; DOI: 10.1016/j.cub.2011.07.002

Courtesy: ScienceDaily

Increasing Potency of HIV-Battling Proteins

If one is good, two can sometimes be better. Researchers at the California Institute of Technology (Caltech) have certainly found this to be the case when it comes to a small HIV-fighting protein.

The protein, called cyanovirin-N (CV-N), is produced by a type of blue-green algae and has gained attention for its ability to ward off several diseases caused by viruses, including HIV and influenza. Now Caltech researchers have found that a relatively simple engineering technique can boost the protein's battling prowess.

"By linking two cyanovirins, we were able to make significantly more potent HIV-fighting molecules," says Jennifer Keeffe, a staff scientist at Caltech and first author of a new paper describing the study in the Proceedings of the National Academy of Sciences (PNAS). "One of our linked molecules was 18 times more effective at preventing infection than the naturally occurring, single protein."

The team's linked pairs, or dimers, were able to neutralize all 33 subtypes of HIV that they were tested against. The researchers also found the most successful dimer to be similar or more potent than seven well-studied anti-HIV antibodies that are known to be broadly neutralizing.

CV-N binds well to certain carbohydrates, such as the kind found in high quantities connected to the proteins on the envelope that surrounds the HIV virus. Once attached, CV-N prevents a virus from infecting cells, although the mechanism by which it accomplishes this is not well understood.

What is known is that each CV-N protein has two binding sites where it can bind to a carbohydrate and that both sites are needed to neutralize HIV.

Once the Caltech researchers had linked two CV-Ns together, they wanted to know if the enhanced ability of their engineered dimers to ward off HIV was related to the availability of additional binding sites. So they engineered another version of the dimers -- this time with one or more of the binding sites knocked out -- and tested their ability to neutralize HIV.

It turns out that the dimers' infection-fighting potency increased with each additional binding site -- three sites are better than two, and four are better than three. The advantages seemed to stop at four sites, however; the researchers did not see additional improvements when they linked three or four CV-N molecules together to create molecules with six to eight binding sites.

Although CV-N has a naturally occurring dimeric form, it isn't stable at physiological temperatures, and thus mainly exists in single-copy form. To create dimers that would be stable under such conditions, the researchers covalently bound together two CV-N molecules in a head-to-tail fashion, using flexible polypeptide linkers of varying lengths.

Interestingly, by stabilizing the dimers and locking them into a particular configuration, it seems that the group created proteins with distances between binding sites that are very similar to those between the carbohydrate binding sites in a broadly neutralizing anti-HIV antibody.

"It is possible that we have created a dimer that has its carbohydrate binding sites optimally positioned to block infection," says Stephen Mayo, Bren Professor of Biology and Chemistry, chair of the Division of Biology, and corresponding author of the new paper.

Because it is active against multiple disease-causing viruses, including multiple strains of HIV, CV-N holds unique promise for development as a drug therapy. Other research groups have already started investigating its potential application in prophylactic gels and suppositories.

"Our hope is that those who are working to make prophylactic treatments using cyanovirin will see our results and will use CVN2L0 instead of naturally occurring cyanovirin," Keeffe says. "It has higher potency and may be more protective."

The work was funded by the National Security Science and Engineering Faculty Fellowship program, the Defense Advanced Research Projects Agency Protein Design Processes program, and the Bill and Melinda Gates Foundation through the Grand Challenges in Global Health Initiative.

Journal Reference:

1. J. R. Keeffe, P. N. P. Gnanapragasam, S. K. Gillespie, J. Yong, P. J. Bjorkman, S. L. Mayo. Designed oligomers of cyanovirin-N show enhanced HIV neutralization. Proceedings of the National Academy of Sciences, 2011; DOI: 10.1073/pnas.1108777108

Courtesy: ScienceDaily