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Little Shop of Horrors: A Moving Plot of an other-world's unmanned land...
"This study was not possible even a year ago," says Michael. "Nanopore sequencing, which some refer to as the 'holy grail' of DNA sequencing, has revolutionized the reading of even the most complex regions of the genome that were completely inaccessible and unknown until now."

Quote:If a plant can be a bio-electric hybrid...what else can it be hacked with?
New technologies enable better-than-ever details on genetically modified plants
January 19, 2019, Salk Institute

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This image depicts an Arabidopsis plant overlaid on individual, labeled DNA molecules of the T-DNA-transformed Arabidopsis genome. Credit: Salk Institute
Salk researchers have mapped the genomes and epigenomes of genetically modified plant lines with the highest resolution ever to reveal exactly what happens at a molecular level when a piece of foreign DNA is inserted. Their findings, published in the journal PLOS Genetics on January 15, 2019, elucidate the routine methods used to modify plants, and offer new ways to more effectively minimize potential off-target effects.

"This was really a starting point for showing that it's possible to use the latest mapping and sequencing technologies to look at the impact of inserting genes into the plant genome," says Howard Hughes Medical Institute Investigator Joseph Ecker, a professor in Salk's Plant Molecular and Cellular Biology Laboratory and head of the Genomic Analysis Laboratory.

When a scientist wants to put a new gene into a plant—for basic research purposes or to boost the health or nutrition of a food crop—they usually rely on Agrobacterium tumefaciens to get the job done. Agrobacterium is the bacteria that causes crown gall tumors, large bulges on the trunks of trees. Decades ago, scientists discovered that when the bacteria infected a tree, it transferred some of its DNA to the tree's genome. Since then, researchers have co-opted this transfer ability of Agrobacteriumfor their own purposes, using its transfer-DNA (T-DNA) to move a desired gene into a plant.

Recently, DNA sequencing technologies had started to hint that when the Agrobacterium T-DNA is used to insert new genes into a plant, it may cause additional changes to the structural and chemical properties of the native DNA.

"Biotech companies spend a lot of time and effort to characterize transgenic plants and disregard candidates with unwanted changes without understanding—from a basic biological perspective—why these changes may have occurred," says Ecker. "Our new approach offers a way to better understand these effects and may help to speed up the process."

"The biggest unknown was whether, and how many copies of, the T-DNA were inserted at the same time as the piece you wanted," says Florian Jupe, a former Salk research associate who now works at Bayer Crop Science. Jupe, Salk Staff Researcher Mark Zander and Research Assistant Angeline Rivkin are co-first authors of the new paper, along with Todd Michael of the J. Craig Venter Institute.

Since the T-DNA approach can lead to an integration of many copies of a desired gene into a plant, it can be difficult to study the final result with standard DNA sequencing, as most technologies struggle to sequence highly repetitive stretches of DNA. But Ecker and his colleagues turned to a new combination of approaches—including optical mapping and nanopore sequencing—to look at these long stretches in high resolution. They applied the technologies to four randomly selected T-DNA lines of Arabidopsis thaliana, a commonly used model plant in biology. (These plants are derived from a large population of T-DNA insertional mutants that were created using an Arabidopsis transformation method, called floral dip, to study gene function.)

Optical mapping revealed that the plants had between one and seven distinct insertions or rearrangements in their genomes, ranging in size by almost tenfold. Nanopore sequencing and reconstruction of the genomes of two lines confirmed the insertions to single-letter resolution, including whole segments of DNA that had been exchanged—or translocated—between chromosomes in one of the selected lines. Gene insertions themselves showed a variety of patterns, with the inserted DNA fragment sometimes scrambled, inverted or even silenced.

"This study was not possible even a year ago," says Michael. "Nanopore sequencing, which some refer to as the 'holy grail' of DNA sequencing, has revolutionized the reading of even the most complex regions of the genome that were completely inaccessible and unknown until now."

Finally, when the researchers studied packets of genetic material called histones they found additional changes. Histone proteins package DNA into structural units, and modifications of these histones mediate whether a gene can be accessed for use by a cell (a level of regulation called epigenetics). Depending on where T-DNA was integrated, certain nearby histone modifications appeared or disappeared potentially changing the regulation or activation of other nearby genes.

"Now we have the first high-resolution insights on how T-DNA insertions can shape the local epigenome environment," says Zander.

In an ideal world, the researchers say, T-DNA would insert one single, functional copy of a desired gene with no nearby side effects on a plant's genome. While their findings show this is rarely the case in Arabidopsis, their methods offer a path to a better understanding and surveillance of the effects.

"This technology is exciting because it gives us a much clearer look at what's going on in some of these transgenic Arabidopsis lines," says Rivkin.

"With Arabidopsis, it's relatively easy because it has such a small genome, but because of continued improvements in DNA sequencing technology, we expect this approach will also be possible for crop plants," adds Ecker, who holds the Salk International Council Chair in Genetics. "Current methods require screening of hundreds of transgenic lines to find good performing ones, such as those without extra insertions, so this technology could provide a more efficient approach."

[Image: 1x1.gif] Explore further: Extensive variation revealed in 1,001 genomes and epigenomes of Arabidopsis

More information: Florian Jupe et al, The complex architecture and epigenomic impact of plant T-DNA insertions, PLOS Genetics (2019). DOI: 10.1371/journal.pgen.1007819 

Journal reference: PLoS Genetics [Image: img-dot.gif] [Image: img-dot.gif]
Provided by: Salk Institute

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Why do Hydra end up with just a single head?

January 18, 2019, University of Geneva

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A Hydra that produces too little Sp5 spontaneously develops multiple heads. Credit: Brigitte Galliot, UNIGE
Often considered immortal, the freshwater Hydra can regenerate any part of its body, a trait discovered by the Geneva naturalist Abraham Trembley nearly 300 years ago. Any fragment of its body containing a few thousands cells can regenerate the entire animal The one-centimeter polyp has a developmental organizer center located at the head level, and another located in the foot. The head organizer performs two opposite activities: activating, which causes the head to differentiate, and inhibiting, which prevents the formation of supernumerary heads.

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Researchers at the University of Geneva (UNIGE), Switzerland, have discovered the identity of the inhibitor, a protein called Sp5, and deciphered the dialogue between these two antagonistic activities, which maintain a single-headed adult body and organize an appropriate regenerative response. Published in the journal Nature Communications, their study reports that this mechanism has been conserved throughout evolution, both in Hydra and in humans. Sp5 could therefore be an excellent candidate as an inhibitor of human tumors in which the activator pathway is the motor of proliferation.

"Regeneration of the head relies on the transformation of the stump into a tissue called the head organizing centre, which has developmental properties, and like an architect, it directs the construction of the future head," explains Brigitte Galliot, professor at the Department of Genetics and Evolution of the UNIGE Faculty of Science.

The head organizer carries out two opposite activities, one activating and the other inhibiting. The first induces the differentiation of stem cells into specialized head cells. The activator is a growth factor called Wnt3, whose action allows the initiation of a three-dimensional cell differentiation program that directs the construction of the head. Thus, in the absence of Wnt3, the head regeneration program cannot proceed. The inhibitory activity, produced under the control of the activator activity, prevents the formation of supernumerary heads. "These two antagonistic activities establish a dialogue between them, but we knew neither the identity of the inhibitor nor the nature of this dialogue," says the biologist.

Using the results of a study conducted by a German team on the planarian flatworm, the biologists developed a gene screening strategy to identify this inhibitor. "We started from 124 candidates that met specific criteria to single out a unique winner that met all of them. It is a gene that codes for a protein called Sp5," says Matthias Vogg, researcher at the Department of Genetics and Evolution of the UNIGE Faculty of Science and first author of the study. The scientists then demonstrated that Sp5 binds to the regulatory region of the gene that codes for Wnt3, blocks its expression and thus the formation of the head.

The seven heads of the freshwater Hydra

How does the dialogue between the activator pathway and the inhibitor work? "We have quantified the expression of the genes encoding Wnt3 and Sp5 in different parts of the body of intact or amputated Hydra, and discovered that a regulatory loop between the two activities is established according to the location and quantity of each gene expressed," notes Brigitte Galliot. Thus, in intact animals, the growth factor Wnt3 will be mainly present at the tip of the head, while Sp5 will be primarily active in the surrounding area, to prevent the appearance of other heads.

When researchers block the expression of Sp5, Hydra polyps, intact or regenerating, develop multiple heads, all perfectly functional. "We also replicated these results from Hydra polyps whose cells had been completely dissociated from each other, then reaggregated and left in culture—multi-headed Hydra re-formed completely in four to five days," explains Matthias Vogg.

In humans, the cell signaling pathway activated by Wnt3 is mainly active during embryonic development, as well as in different types of tumors in adults. If the inhibitory effect of Sp5 is confirmed in our species, this protein could be a candidate treatment targeting cancer cells that use the Wnt3 pathway to proliferate.

[Image: 1x1.gif] Explore further: Hydra can modify its genetic program

More information: Matthias C. Vogg et al, An evolutionarily-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity in Hydra, Nature Communications (2019). DOI: 10.1038/s41467-018-08242-2 

Journal reference: Nature Communications [Image: img-dot.gif] [Image: img-dot.gif]
Provided by: University of Geneva

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Along the vines of the Vineyard.
With a forked tongue the snake singsss...

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RE: Little Shop of Horrors - by EA - 12-07-2018, 10:35 PM
RE: Little Shop of Horrors - by EA - 12-22-2018, 10:00 PM
RE: Little Shop of Horrors: A Moving Plot of an other-world's unmanned land... - by EA - 01-20-2019, 02:42 PM

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