jinian: (real scientist)
[personal profile] jinian
Now that my presentation has gone well, I'm reasonably caught up on work, and I've had a little time to relax, we return to our regularly scheduled science geeking. In particular, a recent post on Nightshade Free precipitated a possibly excessive amount of research into the issue of Roundup-Ready soybeans. Namely, does Monsanto's patented glyphosate-resistant soybean strain really contain petunia DNA, and could that be a problem for people who react to nightshades?

The first thing to consider is that each cell contains many thousands of proteins, so any food we eat has a lot of different potential allergens. (Something like rice has a minimal amount, because the rice seed has a big deposit that's just starch, and to get white rice we polish off all the parts that aren't that, but anything else is going to have loads of different proteins.) Each protein has a function, like a fiber that makes a muscle cell contract or a receptor on the cell surface that senses a flavor.

Now for the Central Dogma of Molecular Biology, and yes we do call it that unironically: DNA is made of nucleotides and it codes for proteins. A gene is generally considered to be a sequence of DNA that produces one protein. Part of that sequence is the "promoter" -- in article #2 (from comments in Nightshade Free), that's the part that came from the cauliflower mosaic virus, and I work with that promoter quite often. The promoter is a DNA sequence that determines when and where the protein is produced, but doesn't get translated itself. CaMV35S causes a protein to be expressed (almost) all the time (almost) everywhere. So if only a promoter sequence comes from an organism you're allergic to, there is no protein produced and there should be no potential allergen issue.

It doesn't sound from the article like the petunia sequence is a promoter, though; it sounds like it may be a piece of protein that tells the cell where to put the main protein. Looking at scholarly articles (which I have access to through my university) tells me a bit more about making the overall construct, but not a lot about the petunia bit in particular. I learned a lot of great information about how Roundup resistance works, though.

The idea behind Roundup Ready plants is this: We know that the herbicide glyphosate (sold by some as Roundup tee em) acts by stopping one specific enzyme that plants need to live. Specifically, glyphosate kills chloroplasts, the organelles that make plants green and allow them to photosynthesize. Transforming the plants with a gene coding for an enzyme that does the same job but resists glyphosate will allow them to survive -- but where do we get that? It turns out that bacteria also have this type of enzyme, which is called EPSPS (5-enolpyruvylshikimate-3-phosphate synthase); it makes certain types of amino acids that cells need to live. Bacteria evolve really quickly so their EPSPSes are likely to have major differences but still do the same job; i.e., if we're lucky glyphosate can't grab onto them, but they'll make the amino acids just fine. So Monsanto screened lots of bacteria looking for ones that could live when exposed to glyphosate. US Patent 5633435 covers the glyphosate-resistant bacterial variant of the EPSPS enzyme that Monsanto found, and it also appears to cover transgenic plants of many kinds that they could transform using this protein coding sequence. (Oh, this is cool, they compared the sequence to that of known plant genes and tweaked it so that it might be better expressed in plants; I will refrain from boring people with the details, but considering the era I'm impressed.)

The EPSPS enzyme is produced by a plant nuclear gene, but where it works is in the chloroplast. This is an artifact of the ancient symbiosis between the plant cell and the chloroplast, which is believed to have been a free-living photosynthetic bacterium that was taken into a plant cell but not eaten many millions of years ago. Actually, the common EPSPS enzyme between them is another piece of evidence supporting that scenario -- if the chloroplast was once a bacterium, it makes sense that it could have similar genes to a bacterium's. But the tricky part about the symbiosis is that gene migration to the nucleus; that ensures that the chloroplasts can't just get out of the plant cell again, since the plant cell really wants to keep them. Some symbioses are a little one-sided, and this is one of them. It might be a bit easier for chloroplasts to live in a big cell that protects them, but in a way they're being held captive for their incredibly useful ability to make sugar from light and carbon dioxide.

So when EPSPS is made in the plant cell, it follows the normal pattern of a plant protein: DNA in the nucleus gets transcribed into messenger RNA, which goes to the ribosomes to be translated into protein. The first part of the protein (around 75 amino acids, very small for a protein) that's made is the chloroplast transit peptide (CTP), which acts like an address on a package, and the cell routes the protein to the chloroplast accordingly. Once the protein arrives at the chloroplast, it gets imported... and the CTP is cleaved off and degraded. According to the patent, the CTP sequence is the only petunia-derived DNA in the transgene. So, there's very little petunia-derived protein ever produced, and it's present in minuscule, transient amounts.

Specifically regarding the nightshade-reaction concern, I've found some statements on allergenicity in an FDA page on the Roundup-Ready soybean. They summarize: "Monsanto also stated that CP4 EPSPS does not fit the profile of the typical allergen because 1) it is not heat stable; 2) it is not a major protein in soybeans; 3) it is not resistant to digestion; and 4) it is not glycosylated." It sounds like the FDA didn't do their own investigation but did respect the claimant's methods, and as much as I dislike Monsanto's business model I have a lot of respect for the methods I see in that patent myself. They also compared the sequences to those of known allergens; obviously this goes only so far, but it's what they could do, and it came up clean. The European Food Safety Authority accepted similar assays on glyphosate-tolerant maize in 2003 (pdf).

On the basis of my research, I recommend not worrying at all about nightshade in your soy. Plants have tens of thousands of other proteins present in much higher amounts that are far more likely to be what you're sensitive to, and the tiny snippet of petunia DNA in that genetic construct is no sooner translated into amino acids than it gets disassembled again.

Even geekier PS:
One thing I wondered was just how different the petunia EPSPS CTP was compared to the native soybean EPSPS CTP. Related genes are often somewhat different, and signal regions can diverge more than functional parts of proteins. I took the amino acid CTP sequences from the patent, but that didn't include soybean sequence. The genome sequence for Arabidopsis (the plant biologist's fruit fly) is publicly available, so I found the EPSPS gene, then used NCBI BLAST to search for similar sequences. Among the many plant EPSPS results were soybean (Glycine max) and petunia (as P. hybrida, lucky the sequence was numbered starting with PET). I translated them all to amino acid sequence (one was nucleotides, and they needed to match) and used a free program called ClustalW to align these sequences; it automatically looks at the sequences you give it to determine their similarity, and lines up the bits that are alike molecularly. I was surprised to find that all of the CTPs were very different from each other, with only 6 identical positions of about 75 total amino acids in the CTP. I guess those are important ones!

Anyway, it's clear that there are multiple ways to get the message across to the cell. Having made transgenes by molecular cloning, I can tell you honestly that molecular biologists are kind of lazy, and if the petunia CTP was already available I can completely believe that they'd just use what they had. It used to be very much more difficult to clone genes and sequence them, and we adapt our strategies based on what's handy even now.
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