The biotechnology community has been taken aback by a sudden and aggressive attack by an organization calling itself Humans for Bacterial Suffrage (HuBS). The group claims that an insidious culture of what it calls “eukaryotic oppression” is enslaving trillions of bacteria, subjecting them to perverse genetic experiments, and exploiting their labour in the execution of profitable biochemical reactions.

Says HuBS president David Clostridium, “Bacteria are routinely abducted from their natural habitats, sold on the open market, unwillingly subjected to invasive genetic manipulations, and forced to breed in captivity. Multiple generations of enslaved lifeforms are set to work expressing secondary metabolites at the hand of big corporations and research scientists. It’s disgraceful. They aren’t paid, the living conditions are terribly overcrowded, they don’t even get dental. Just because you don’t have a nuclear membrane, that doesn’t mean you don’t have rights, you know?”

Clostridium points out that his organization’s principal goal is simply to increase awareness. “Few people realize that so-called “organic” products like Bt insecticide are prepared by bacterial slave labour. They say it’s “natural”, because it comes from a living organism. What’s so natural about eating agar and living in a petri dish? About being sprayed with billions of your relatives onto a plant and forced to wage biological warfare on insects who never did anything to you? Consumers should have a choice. Consumers should be able to buy produce treated with free range Bt bacteria, who are properly compensated and valued for their work. It’s just like slavery. Except, you know, without that miniseries starring Geordi LaForge.”

Rhodia Chemicals markets Rhovanil Natural Vanilla, which is prepared via a biofermentation process carried out entirely by bacteria. Rhodia CEO Jean-Pierre Clamadieu was asked to comment on charges from HuBS that his company is exploiting single-celled organisms. “Look, these bacteria, they were eking out a miserable existence before they came to work for Rhodia, they were hiding in rocks, in the cold dirt, in rotting animal carcasses. They don’t even have cable TV in some of these places, you know? Our e. coli workforce is proud and happy to be part of the Rhodia team.” When asked if there had ever been any complaints from the bacterial workforce, Clamadieu shrugged. “Well, they don’t have mouths.”

Insiders at the US Republican party have expressed concerns that the ultimate goals of the bacterial suffrage lobby may include the granting of voting rights to all single-celled organisms. Estimates put bacteria population in United States at over 5×10²⁶, a number far exceeding that of registered Republicans, and a recent poll reveals that 74% of prokaryotes either agree or strongly agree with the statement “George Bush is a lying crapweasel.”

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(REPRINTED FROM ISSUE TWO, AUGUST 22nd, 2005)

Bt corn, a genetically modified organism (GMO), has been both the poster-child and thorn-in-the-side of the plant biotechnology industry from the late 1990’s to present. There are several versions of this transgenic crop that each have a gene from an insect pathogen, Bacillus thuringiensis (Bt), which encodes a protein toxic to the European corn borer (ECB), an insect pest that eats and destroys corn stems (see Figure 1). Bt corn has proven effective in reducing crop damage due to ECB, yet public opposition to Bt corn has escalated amid fears of human health and environmental risks associated with the production and consumption of Bt corn.

Figure 1. Engineering resistant corn. Following the insertion of a gene from the bacteria Bacillus thuringiensis, corn becomes resistant to corn borer infection. This allows farmers to use fewer insecticides

History of Bt

Bt corn draws its humble origins from France, where in 1938 B. thuringiensis bacteria was grown in large quantities and sprayed on corn crops to prevent ECB damage[1]. Artificial selection of Bt strains has led to the successful targeting of many insect pests. Because no toxic effects of Bt on humans have been detected in its seventy years of use, it is now considered an acceptable pest control measure for the organic food industry[2]. To this day, Bt is an important part of many integrated pest management strategies. The success of the Bt spray has been limited because the bacteria cannot survive for very long on the plant’s surface. Bt is particularly ineffective at controlling ECB because these insect live most of their larval life inside the corn stem, not on the surface: sprays are only effective when the insects are starting its journey into the stem. Thus, a means of penetrating corn tissue with Bt is required to offer long-term anti-feeding measures against tunneling insects such as ECB.

Mechanism of Bt toxicity

Researchers investigated how this bacteria kills particular insects and discovered that Bt has two classes of toxins; cytolysins (Cyt) and crystal delta-endotoxins (Cry)[3]. While Cyt proteins are toxic towards the insect orders Coleoptera (beetles) and Diptera (flies), Cry proteins selectively target Lepidopterans (moths and butterflies). As a toxic mechanism, Cry proteins bind to specific receptors on the membranes of mid-gut (epithelial) cells resulting in rupture of those cells[4]. If a Cry protein cannot find a specific receptor on the epithelial cell to which it can bind, then the Cry protein is not toxic. Bt strains will have different complements of Cyt and Cry proteins, thus defining their host ranges[5]. The genes encoding many Cry proteins have been identified providing biotechnologists with the genetic building blocks to create GM crops that express a particular Cry protein in corn that is toxic to a particular pest such as ECB yet potential safe for human consumption.

Making Bt corn

As it turns out, nature has its own biotechnologist called Agrobacterium tumefaciens which induces the growth of tumours on woody plants. These tumours are engineered by A.tumefaciens to produce a special food for the bacteria (opines) that plants normally cannot make. These tumours arise from a unique bacterial transformation mechanism involving the Ti-plasmid which coordinates the random insertion of a subset of its DNA (t-DNA) containing opine synthase genes into a plant chromosome[6] (see Figure 2). By replacing portions of the t-DNA sequence with genes of interest (such as Cry), researchers have been able to harness this transformational mechanism and confer new traits to many flowering plants including grasses such as corn7 and rice[8]. Cry-transformed corn varieties, called ‘Bt corn’, produce sufficient levels of Cry proteins to provide an effective measure of resistance against ECB and are now widely grown in North America.

Figure 2. General schematic of GM crop production

Human health and environmental risks

The promise of this technology has been largely overshadowed by concerns about the unintended effects of Bt corn on human health and the environment. Cry protein toxicity, allergenicity, and lateral transfer of antibiotic-resistance marker genes to the microflora of our digestive system threaten to compromise human health. Despite these alarming possibilities, the risks to human health appear small based upon what is known about the bacterial endotoxin, its specificity, and confidence in the processes of plant transformation and screening[9]. The task of determining the levels of such risks, however, are immense. Human diets are complex and variable. How can we trace the acute or chronic effects of eating GM ingredients when they are mixed in with many other foods that may also present their own health hazards? It is even more complicated to determine the indirect risk of eating meat from animals raised on transgenic crops. These tests take time, and the results of clinical trials are not always clear-cut. It will likely take decades before we can know with any certainty if Bt corn is as safe for human consumption as its non-GM alternatives[10].

We currently know very little about the actual ecological risks posed by Bt corn. Bt corn may be toxic to non-target organisms, transgenic genes may escape to related corn species, and ECB and other pests may become resistant to Cry proteins[11]. The alleged effect of Bt corn pollen on Monarch butterfly larvae has rocketed to the front pages of major newspapers around the world (ex. CNN). Some research has shown that Monarch butterfly larvae fed their normal diet of milkweed leaves suffer a significant decline in fitness when those leaves are dusted with Bt corn pollen (Losey et al. 1999). The methodology of this experiment, however, has been harshly criticized by members of the scientific community.

Most recently, the threat of Cry gene escape into wild populations has been substantiated by the discovery that artificial DNA from transgenic corn has been detected in traditional corn varieties in remote areas of Mexico (Quist and Chapela, 2001). However, this study was pulled from NATURE magazine in an unprecedented fashion following a heated scientific and political debate[12]. While few contest that such transgenes are present in the local corn races of Mexico, there is still no evidence to suggest that these genetic constructs are “escaping” to become established in local corn races. We are limited to an educated guess as to the likelihood and speed of such genetic pollution[13].

Balancing risk and benefit

Despite the lack of conclusive evidence that GM foods present considerable risk to human health and environment, widespread use of this new technology is being compared to past mistakes such as broadcast spraying of populated towns with DDT to control mosquitoes during the 1950s. Notions of “frankenfoods”[14] and “agroterrorism”[15] corrupting our planet present theoretical possibilities that cannot be discounted given the remarkable ability of the unlikely to become an actuality. In truth, we must plead ignorance of the long-term impacts of GM crops[16].

Arguably, every food in our current diet carries with it associated risks, determined through “trial-and-error” extending back before to our hunter-gatherer origins. Often, we will accept a certain degree of exposure to known hazards to receive known benefits. Bt corn has obvious benefits for agricultural production, increasing profit margins through more efficient and consistent corn production and improving the working environment for farmers through reduced exposure to pesticides. In a surplus market, these benefits may be passed on to the consumer as a grocery bill reduction. On a global scale, decreased crop losses due to herbivory may translate into improved world food supply since corn remains a major staple in the global diet. Ecosystems are not likely to benefit from ECB-resistant Bt corn propagation since this technology replaces a largely mechanical (non-chemical) control for ECB.

These benefits, real or imagined, have been used as leverage by Bt corn proponents in the argument to accept what they argue are minimal levels of health and environmental risk. Yet many consumer, civil rights, and environmental advocacy groups characterize such arguments as industry propaganda, asserting that corporate benefits should not out-weigh the undetermined human health, socioeconomic and environmental risks.

The relative ease in engineering Bt biopesticides into crops such as corn, cotton and rice, combined with the cost effectiveness of Bt crops for growers under threat of ECB, makes banning this technology in North America seem unlikely. This reality highlights the necessity for the research community to improve methods for assessing risks posed by GM crops. While some industry proponents may resist, it is ultimately the public’s responsibility to ensure that this new technology is properly managed in the context of other pest management methods that have their own set of risks and benefits.

Notes

Glossary

Artificial Selection – the encouragement of certain traits in an animal through selective breeding by humans, both intentional or unintentional

Ti plasmid – “tumour-inducing” plasmid: originally found in the bacterium Agrobacterium tumefaciens, this plasmid integrates into a host cell genome and causes galls on plants. Biotechnologists can take advantage of this integration to insert genes of their choice into plant cells.

Lateral transfer – also called horizontal gene transfer, the movement of genetic material from one organism to another other than from parent to offspring, and often across species, genus, or even domain.

Antibiotic resistance marker genes – genes that allow biotechnologists to distinguish between plants that have been modified properly and those that have not depending on their suceptibility to antibiotics.

Screening – the process of selection of desirable plants from a large population of transformants (different insertional events) with variation in trait depending on location and number of t-DNA insertions.

Herbivory – the consumption of plants by animals, in this case to the detriment of the plant (predation).

References

1. Van Frankenhuyzen, K. in Bacillus thuringiensis, An environmental biopesticide: Theory and practice (John Wiley & Sons, 1993).

2. Whalon, M.E. & Wingerd, B.A. Bt: mode of action and use. Arch Insect Biochem Physiol 54, 200-211 (2003).

3. Crickmore, N. et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62, 807-813 (1998).

4. Dorsch, J.A. et al. Cry1a Toxins of Bacillus Thuringiensis Bind Specifically to a Region Adjacent to the Membrane-Proximal Extracellular Domain of Bt-R-1 in Manduca Sexta: Involvement of a Cadherin in the Entomopathogenicity of Bacillus Thuringiensis. Insect Biochemistry and Molecular Biology 32, 1025-1036 (2002).

5. De Maagd, R.A., Bravo, A. & Crickmore, N. How Bacillus Thuringiensis Has Evolved Specific Toxins to Colonize the Insect World. Trends in Genetics 17, 193-199 (2001).

6. Bevan, M.W. & Chilton, M.D. T-DNA of the Agrobacterium Ti and Ri plasmids. Annu Rev Genet 16, 357-384 (1982).

7. Ishida, Y. et al. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14, 745-750 (1996).

8. High, S.M., Cohen, M.B., Shu, Q.Y. & Altosaar, I. Achieving successful deployment of Bt rice. Trends Plant Sci 9, 286-292 (2004).

9. Kuiper, H.A., Kleter, G.A., Noteborn, H.P. & Kok, E.J. Assessment of the food safety issues related to genetically modified foods. Plant J 27, 503-528 (2001).

10. Sudakin, D.L. Biopesticides. Toxicol Rev 22, 83-90 (2003).

11. Sharma, H.C. & Ortiz, R. Transgenics, Pest Management, and the Environment. Current Science 79, 421-437 (2000).

12. Ochert, A. Caught in the maize at Berkeley. California Monthly (2002).

13. Letourneau, D.K., Robinson, G.S. & Hagen, J.A. Bt crops: predicting effects of escaped transgenes on the fitness of wild plants and their herbivores. Environ Biosafety Res 2, 219-246 (2003).

14. Golden, F. Who’s afraid of Frankenfood? Time 154, 49-50 (1999).

15. van Bredow, J. et al. Agroterrorism. Agricultural infrastructure vulnerability. Ann N Y Acad Sci 894, 168-180 (1999).

16. Hoffmann-Riem, H. & Wynne, B. In risk assessment, one has to admit ignorance. Nature 416, 123 (2002).

(Art by Jiang Long and Jen Philpot)

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(REPRINTED FROM ISSUE TWO, AUGUST 8th, 2005)

When I was in grade school, my best friend had a monkey. I don’t know what kind it was, but I can tell you this: My friend’s parents had a Kung-Fu outfit for this monkey. His name was Bentley and he was kept is a large cage in the basement. When you got too close to his cage, he would grab at your shirt and tear it. But, if you were my friend’s dad, he would let you get into the cage and wrestle him. Bentley had some scrap in him, that’s for sure. I used to love watching the two of them go at it. It was like seeing a man and a small, furry Bruce Lee in a cage match. The dad, who was a Golden Gloves champion in the Navy, took it easy, though, because he saw the monkey, as did everyone, more like a very excitable, very challenged little boy…who was only let out of his cage for parties.

Recently, I was looking for a pet of my own because I was sad and lonely in New York City. I’m allergic to dogs and cats, so I knew I needed something else, something less traditional. Iguanas crossed my mind. So did the Mexican Hairless, but neither seemed to have any personality or warmth. Then, I remembered Bentley. Was I allergic to him? I don’t recall sneezing or itching when I was watching my friend’s dad put him in a headlock. And even if I was, would it bother me that much if I kept him in a cage in my second bedroom? I don’t go in there too often, so I wouldn’t die or anything. I just wouldn’t hang out in there and spar with him. But it would smell. Oooh man do I remember the way that basement smelled. Of course, they had a raccoon down there, too.

Would I have to walk my monkey? That could be a problem in this city, what with all of the permits I wouldn’t have and all. And I’m sure, if he was as radical as Bentley, he wouldn’t take kindly to those annoying little dogs that look like rats wearing coats and sneakers.

The idea of owning a monkey, which I thought could double as a kind-of-human friend, was still very appealing to me, regardless of all of these things. So I began to price them. As it turns out, monkeys are expensive! The cheapest one I could find online was $5,000!

Suddenly, having a monkey quickly became unfeasible. So, I did the next best thing: I bought Sea Monkeys.

After a few weeks, I am still happy with my choice. Sure, Sea Monkeys lack the animation of a primate, but they float around sometimes and are waaaay easier (and cheaper!) to maintain.

If you are considering getting a monkey, but can’t decide if it’s for you or not, consider going the Sea Monkey route. Here’s a Tale of the Tape that might help you to make up your mind:

Average Weight:
Monkey: 8.8-20 lbs.
Sea Monkey: Virtually nothing!
Winner: Tie – depends on your space

Cost:
Monkey: At least $5, 000
Sea Monkey: $5, tops
Winner: Sea Monkey, of course

Maintenance:
Monkey: Must clean filthy cage regularly
Sea Monkey: None
Winner: Sea Monkey!

Diet:
Monkey: Fruit
Sea Monkey: Specially developed “Sea Monkey Food” (included in box!)
Winner: Monkey. You can share!

Personality:
Monkey: Can be pretty grumpy, but can also funny!
Sea Monkey: Hard to tell
Winner: Tie. Depends on how much interaction you need

Poo-flinging:
Monkey: Yep
Sea Monkey: Do they even make this to fling?
Winner: Sea Monkey, hands down

Service to Science:
Monkey: For sure. Primates are in.
Sea Monkeys: Would you believe, a role in toxicology? [1,2,3]
Winner: Too close to call – even.

Dressability:
Monkey: Oh yes
Sea Monkey: Not Really
Winner: That snazzy monkey!!

Environment:
Monkey: a cage in your second bedroom/basement/yard
Sea Monkey: a bowl of water, anywhere
Winner: Tie – Again, depends on your space

As you can see, it’s a pretty close call between these two. I suppose your decision will come down to how invested you would be in owning a pet…and whether or not you feel like dodging flung poo.

References:

1. Artemia salina as test organism for assessment of acute toxicity of leachate water from landfills. Environ Monit Assess. 2005 Mar;102(1-3):309-21

2. The use of a brine shrimp (Artemia salina) bioassay to assess the toxicity of diatom extracts and short chain aldehydes. Toxicon. 2003 Sep;42(3):301-6.

3. Biological screening of Annonaceous Brazilian Medicinal Plants using Artemia salina (brine shrimp test). Phytomedicine. 2003 Mar;10(2-3):209-12.

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(REPRINTED FROM ISSUE TWO, AUGUST 8th, 2005)

(August 2004)

Genetic Carrying Handles: Cloning Vectors

In order to clone a gene, its DNA sequence must be attached to some kind of carrier, also made of DNA, that can take it into the cell. Biologists call these carriers vectors. A vector acts like a handle for the DNA, and it also contains other tools such as an origin of replication and a selective marker. The origin of replication is a sequence of DNA that the host cell recognizes that allows it to make more copies of the clone DNA sequence. This origin sequence is where the cell begins copying the vector and the attached clone DNA. The selective marker is a specific DNA sequence that is used by biologists to tell if the clone has entered the cell, and they are usually genes that confer antibiotic resistance to the cell. The most common media used for this process is actually very similar to chicken soup, but the carbohydrate agarose is added to convert the media into a semi-solid substance, since bacterial colonies are much easier to detect on a semi-solid surface. Agarose is much like gelatin, but it comes from seaweed and unlike gelatin, most bacteria cannot digest it. Antibiotics are often added to the media, to kill any cells that do not possess the antibiotic resistant selective marker gene, which is in the clone DNA. This way, biologists can ensure that all the remaining cells have in fact taken up the clone DNA and its vector. These cells are called transfected cells. Antibiotics are not the only way to identify transfected cells. Biologists sometimes use selective markers that turn cells a different colour or even to make them glow. Common proteins that do this include luciferase, which makes fireflies glow or green fluorescent protein, which comes from certain species of jellyfish. Green fluorescent protein also comes in other colours!

There are many different kinds of vectors and most of them are adopted from viruses or plasmids; small circles of DNA found in many bacteria and yeasts that are separate from their much larger genome. Phages are viruses that only infect bacteria, and some of them have been adapted into vectors. However, phages are sometimes hard to work with and they cannot contain much more genetic material than they already have wild-type.

Plasmids that have been turned into vectors can hold about 10,000 nucleotide base pairs (bp) or 10 kilobase pairs (kb). Cosmids are a weird mix between plasmids and phages and can hold up to 30 kb. In comparison, the average size of a human gene is about 27 kb. The average size of a gene in rice, an important and widely studied crop in many areas of the world, is about 2.3 kb. Of course, there are larger and smaller genes than these.

Viruses are another important area of study in molecular biology and they come in many different sizes. The genome of the herpes virus ranges from 100 kb to 250 kb, far bigger than the 10 kb that a plasmid vector can hold and beyond even the 30 kb carrying capacity of a cosmid. In addition, biologists are also trying to study sets of several genes that are adjacent to one another in a genomic sequence. The Human Genome Project read the sequence of all the genes in humans – that’s about 3 billion basepairs! Using plasmid vectors, more than 1.8 billion clones are required to examine an entire human genome.

Super-Sized Inserts

Bacterial Artificial Chromosomes (BAC) have been developed to hold much larger pieces of DNA than a plasmid can. BAC vectors were originally created from part of an unusual plasmid present in some bacteria called the F’ plasmid. The F’ plasmid allows bacteria to have “sex” (well, sort of: F’ helps bacteria give its genome to another bacteria but this only happens rarely when bacteria are under a lot of stress). F’ had been studied extensively and it was found that it could hold up to a million basepairs of DNA from another bacteria. Also, F’ has origins of replication and bacteria have a way to control how F’ is copied. In 1992, Hiroaki Shizuya took the parts of F’ that were important, cleaned it up, and turned it into a vector.

Figure 1. Transforming a bacterium using a BAC vector

BAC vectors are able to hold up to 350 kb of DNA and have all of the tools that a vector needs to work properly, like replication origins, antibiotic resistance genes, and convenient places where clone DNA can insert itself. With these vectors it is possible to study larger genes, several genes at once, or entire viral genomes. By using a vector that can hold larger pieces of DNA, the number of clones required to cover the human genome six times theoretically could drop from 1.8 billion to about 50 million.

Researchers have modified BAC vectors to become more convenient to use and more useful in specialized situations. In addition to the antibiotic resistance gene that was added to identify transfected bacteria, a gene was added that enabled the bacteria to turn the colourless substance X-gal/IPTG blue. This substance is found in the chicken-soup-like media on which the bacteria colonizes. This colour-changing gene, called lacZ, is split apart when the clone DNA is incorporated into the vector, so it is possible to tell not only if a bacteria had been transfected (meaning incorporated into the cell), but also if the bacteria was transfected with the vector containing insert DNA or just the vector alone (remembering that if the vector has properly incorporated the clone DNA, it will have lost its ability to change X-gal/IPTG blue)(Figure 2).

Figure 2. Selecting for transformed bacteria

Another similar modification uses the gene called sacB, which encodes a protein called levansucrase. This protein turns sucrose – table sugar – into levan, a toxic substance to bacteria. In a similar fashion, bacteria grown on media with sucrose will die if sacB is not broken up by inserted DNA. If the vector carries a DNA insert, then sacB is broken and it won’t produce levansucrase, so the bacteria can survive in the sucrose media. In theory, only bacteria transfected with a vector containing insert DNA would be able to grow and form colonies.

Some modifications to BAC vectors make them more specialized. For instance, there are researchers studying the herpes virus who have made a BAC vector that can be cultured in bacterial cells and then when put into a mammalian cell, instantly releases its insert DNA – in this case, the whole herpes virus genome. With such a vector, it is easier to grow sufficient amounts of the herpes virus for research, since it can live in bacterial cultures, instead of requiring their endemic mammalian cell cultures, which are extremely difficult to maintain. This in turn makes it easier to make modifications to the virus and study what each of its genes do.

The creation of BAC vectors has allowed researchers to do many things that they could not do before and do them more quickly and more easily. Some of the scientific practices that have simplified with BAC vectors includes phylogenetic studies (studies which examine species’ relationship to one another) and what the absolute minimum size of a genome could be. The world is currently overrun by a plethora of microbial species that cannot be grown in cultures. BACs have allowed researchers to look at microbial DNA without having to actually grow the organisms, since the DNA is kept within easy-to-grow bacterial cultures. BAC vectors are also useful for studying pathogens, and are helpful in the development of vaccines. Many pathogens are becoming resistant to all antibiotics available to medicine and BAC vectors are playing a tremendous role in discovering new and powerful antibiotics in the environment. Discovering enzymes that are able to help clean up oil spills or help farmers breed healthier farm animals or even process radioactive waste are just a few examples of what Bacterial Artificial Chromosomes can do.

BAC vectors are useful tools and the methods for working with them are fairly well developed. Continuing advances such the modification of experimental animals will only increase the wide variety of uses for big bad BACs.

Glossary

Clone – a piece of DNA found inside a cloning vector
Genome – the entirety of an organism’s genetic material
Wild-type – organisms that have not been manipulated or changed by scientists
Pathogen – an organism or virus that causes disease

References

Shizuya, H., Birren, B., Kim, UJ., Valeria, M., Slepak, T., Tachiiri, Y., & Simon, M. 1992 Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. 89; 8794-8797 PNAS

(Art by Jen Philpot, slightly higher res version of second figure found here)

(August 2003)

Messenger RNA (mRNA) is a single stranded molecule that is used as the template for protein translation. It is possible for RNA to form duplexes, similar to DNA, with a second sequence of RNA complementary to the first strand. This second sequence is called antisense RNA (Figure 1). The formation of double stranded RNA can inhibit gene expression in many different organisms including plants, flies, worms and fungi.

Figure 1 Formation of antisense RNA blocks translation.

Co-Suppression

The first discovery of this inhibition in plants was more than a decade ago and occurred in petunias. Researchers were trying to deepen the purple colour of the flowers by injecting the gene responsible into the petunias but were surprised at the result. Instead of a darker flower, the petunias were either variegated (Figure 2) or completely white!

This phenomenon was termed co-suppression, since both the expression of the existing gene (the initial purple colour), and the introduced gene (to deepen the purple) were suppressed. Co-suppression has since been found in many other plant species and also in fungi. It is now known that double stranded RNA is responsible for this effect.

Figure 2. A variegated petunia. Upon injection of the gene responsible for purple colouring in petunias, the flowers became variegated or white rather than deeper purple as was expected.

aRNA and RNAi

When antisense RNA (aRNA) is introduced into a cell, it binds to the already present sense RNA to inhibit gene expression. So what would happen if sense RNA is prepared and introduced into the cell? Since two strands of sense RNA do not bind to each other, it is logical to think that nothing would happen with additional sense RNA, but in fact, the opposite happens! The new sense RNA suppresses gene expression, similar to aRNA. While this may seem like a contradiction, it can be easily resolved by further examination. The cause is rooted in the prepared sense RNA. It turns out that preparations of sense RNA actually contain contaminating strands of antisense RNA. The sense and antisense strands bind to each other, forming a helix. This double helix is the actual suppressor of its corresponding gene. The suppression of a gene by its corresponding double stranded RNA is called RNA interference (RNAi), or post-transcriptional gene silencing (PTGS). The gene suppression by aRNA is likely also due to the formation of an RNA double helix, in this case formed by the sense RNA of the cell and the introduced antisense RNA.

How Does it Work?

But how does the double stranded RNA cause gene suppression? Since the only RNA found in a cell should be single stranded, the presence of double stranded RNA signals is an abnormality. The cell has a specific enzyme (in Drosophila it is called Dicer) that recognizes the double stranded RNA and chops it up into small fragments between 21-25 base pairs in length. These short RNA fragments (called small interfering RNA, or siRNA) bind to the RNA-induced silencing complex (RISC). The RISC is activated when the siRNA unwinds and the activated complex binds to the corresponding mRNA using the antisense RNA. The RISC contains an enzyme to cleave the bound mRNA (called Slicer in Drosophila) and therefore cause gene suppression. Once the mRNA has been cleaved, it can no longer be translated into functional protein (Figure 3 and see a Flash animation of PTGS here).

Figure 3. Mechanism of action of RNAi. Double stranded RNA is introduced into a cell and gets chopped up by the enzyme dicer to form siRNA. siRNA then binds to the RISC complex and is unwound. The anitsense RNA complexed with RISC binds to its corresponding mRNA which is the cleaved by the enzyme slicer rendering it inactive.

Uses

The suppression of protein synthesis by introducing antisense RNA into a cell is very useful. A gene encoding the antisense RNA can be introduced fairly easily into organisms by using a plasmid vector or using a gene gun that shoots microscopic tungsten pellets coated with the gene into the plant cells. Once the antisense RNA is introduced, it will specifically inhibit the synthesis of the target protein by binding to mRNA. This is a quick way to create a knockout organism to study gene function. Using antisense RNA as a tool in this way is an exciting prospect for many molecular biologists.

Antisense RNA is also being investigated for use in cancer therapy. Injecting aRNA that is complementary to the proto-oncogene BCL-2 may be useful for treating some B-cell lymphomas and leukemias. Antisense oligodeoxynucleotides (ODNs) are also being studied for human therapy. ODNs are similar to antisense RNA, but they are made synthetically and are deoxynucleotides (like those in DNA) rather than nucleotides. ODNs are being tested for their effectiveness against HIV-1, cytomegalovirus (a member of the herpesvirus group), asthma and certain cancers.

Antisense RNA methods have also been used for commercial food production. You may have heard of the Flavr Savr tomato. This tomato was developed by Calgene Inc. of Davis, California in 1991 and was approved by the U.S. FDA in 1994. The tomato was the first whole food created by biotechnology that was evaluated by the FDA. One of the problems associated with tomato farming is that the fruit must be picked while still green in order to be shipped to market without being crushed. The enzyme that causes softening in tomatoes is polygalacturonase (PG). This enzyme breaks down pectin as the tomato ripens, leading to a softer fruit. Calgene suppressed the expression of the gene encoding PG by introducing a gene encoding the antisense strand of the mRNA. When the introduced gene was expressed, the antisense strand bound to the PG mRNA, suppressing the translation of the enzyme. The Flavr Savr tomatoes therefore had low PG levels and remained firmer when ripe. This meant the Flavr Savr tomatoes can ripen on the vine and then be shipped to market. Although the Flavr Savr tomatoes were approved for sale in the U.S., production problems and consumer wariness stopped the production of this fruit in 1997.

RNA interference is a field that was stumbled upon by accident while trying to improve the colour of petunias, however its implications may be far reaching in the near future.

Additional Readings

1. Kimball’s Biology Pages — Antisense RNA

2. Ambion — The RNA interference resource.

(Art by Fan Sozzi)

Q: What is the principle evidence for Creationism?
A: The Holy Bible, of course. After all, is it likely that the author of the Universe would be mistaken about its age?

Q: But isn’t the Bible religion and not science?
A: Truth is truth. It’s a poor sort of science that ignores truth.

Q: But isn’t there a lot of evidence for evolution?
A: Not really, most of it is from university professors writing papers for each other. If they didn’t write papers they wouldn’t have jobs.

Q: How big was Noah’s ark?
A: Big enough.

Q: But what about radioactive dating?
A: Hey, everybody knows that stuff is bad for you. Stick with good Christian girls.

Q: What about the fossil evidence?
A: The real fossils are university professors writing papers for each other.

Q: Is there any other evidence for creationism besides the Bible?
A: Yes.

Q: Can you give us some?
A: Yes.

Q: Could you give us a specific example?
A: Yes.

Q: What would be a specific example of evidence for Creationism?
A: I’ve already answered that question.

Q: What about the Antarctic ice core data?
A: Now I put it to you. Coop up a bunch of men in a Quonset hut in the worst weather in the world, with nothing to do but gather data and drink, and what do you expect?

Q: Did the dinosaurs coexist with man?
A: Look, the liberals were preaching coexistence with the Communists, and you saw what happened to them.

Q: Should Creationism be taught along with Evolution in the schools?
A: Creationism should be taught instead of Evolution in the schools.

Q: Doesn’t the Geologic Column prove that the Earth is very old?
A: The geologic column proves that some things are on top of other things and some things are underneath other things. But we already knew that, didn’t we.

Q: Hasn’t evolution been demonstrated in the Laboratory?
A: Students are demonstrating everywhere these days. To their shame, many professors are demonstrating also.

Q: Aren’t Hawiian wallabies an example of Evolution in action?
A: No.

Q: Why not?
A: Because they aren’t.

Q: What is a kind?
A: A kind is cards of the same rank. Thus 4 aces and a king are four of a kind, but four spades and a heart are not.

Q: Doesn’t genetic variation indicate that life has been going on a long time?
A: Let’s be up front about this. That’s deviation, not variation, and yes, there is a lot of deviancy out there. That just shows that there has been a lot of Sin since the garden of Eden.

Q: What about Neanderthal Man?
A: Hey, you take one of those geezers and put him in tweeds and give him a pipe and he could be a professor anywhere.

Q: Some scientists state that the earth’s continents are drifting around on top of a molten interior which has shaped life as we see it now. Are they right?
A: As you well know the Bible says that beneath the surface of the earth is Hell where there is eternal fires and brimstone. If the continents appear to be moving around that is Satan’s doing.

Q: Why do almost all of the scientists believe in Evolution?
A: The real scientists don’t. As for the rest of them, that’s a very good question, isn’t it?

Q: Are you talking about a Satanic conspiracy?
A: Did I say anything about a conspiracy? You might want to think about the shape the world is in since the Evolutionists and the Liberal Humanists captured academia and how Evolution is hand in hand with Godless Communism and crime in the streets but I certainly wouldn’t want to say anything about a Satanic conspiracy. I just want you to think about it with an open mind.

* * *

(REPRINTED FROM ISSUE TWO, AUGUST 8th, 2005)

A debate is going on in Huntington’s research about whether the hallmark protein aggregates found in the brain of patients actually cause the disease. Now, a new “shortstop” may have found part of the answer.

But this shortstop isn’t an infielder. It’s a new strain of mouse, one with a mutation expected to cause neurodegeneration — since it’s tailored to make large amounts of the above protein aggregates — only it doesn’t.

Shortstop mice were recently created by Elizabeth Slow and colleagues at the University of British Columbia. And their unexpected ability to resist neurological damage is causing their creators to suggest the debate is over: protein aggregates do not seem to be toxic in mice.

“This [shortstop] finally ends the debate, showing that aggregates in vivo are not causative of illness,” says Michael Hayden, director of the Centre for Molecular Medicine and Therapeutics at UBC and senior author of a paper describing the mice.

An end to the debate could be important for the future development of drugs to treat Huntington disease, since such drugs are often chosen for their ability to inhibit aggregate formation. Shortstop mice suggest this method may not give the most useful compounds, says Hayden.

Aggregates became a big part of Huntington’s research around half a decade ago when they were found in the brains of patients. They are made of a mutant version of the protein huntingtin and can be easily seen under a light microscope.

“Huntingtin aggregates were only seen in the patients with the illness,” says Hayden, “so people thought it was the cause.” However, it was not altogether clear whether huntingtin aggregates are in fact toxic to the brain, or are simply an indicator of some other pathological process.

Slow and colleagues actually set out to investigate this question by creating a strain of mice that made more of the mutant huntingtin protein. Indeed, they did create such mice, called YAC128, which produces huntingtin aggregates.

But, in a fluke, they also created shortstop.

“We’ve been creating an animal model for this illness and to do that you have to take a very large piece of DNA and inject it into a mouse,” says Hayden, “occasionally the DNA shears and breaks into pieces.”

This is what happened in the creation of shortstop, which although it makes the same amount of huntingtin as YAC128, the version it makes is a smaller protein. Nevertheless, this smaller version still contains the mutation associated with the disease, suggesting these mice should look the same as YAC128 mice.

The serendipitous creation therefore allowed Slow and colleagues to compare YAC128 mice to shortstop mice. They found that both strains formed huntingtin aggregates (these show up as inclusion bodies in neurons). YAC128 mice also had neuronal dysfunctions, in the form of decreases in brain weight and the loss of neurons. However, shortstop mice had no ill effects, despite the presence of huntingtin aggregates in their brains. The results of the study appear in the August 1, 2005 online edition of PNAS.

The results with shortstop mice show that the presence of aggregates and the huntingtin mutation are not sufficient to cause the disease, says Hayden.

However, Hayden does caution that the truncated huntingtin protein in shortstop mice may act slightly differently than the full length version. “The results don’t exclude all the forms of protein folding that can cause the disease,” he says.

Nevertheless, the work makes you question using inclusion bodies of huntingtin as a biomarker for testing drugs to treat Huntington’s, says Hayden,

“Instead we need to look for news ways to screen for these drugs,” he says.

* * *

(REPRINTED FROM ISSUE TWO, AUGUST 8th, 2005)

QAJAOG: Greetings, Captain Zabujek.

ZABUJEK: Your eminence, Emperor Qajaog, I am honored by this private audience.

QAJAOG: Captain, word of your exploits has reached the farthest reaches of the Federated Republic of the Empire. Is it true that you have ventured to the planet called Earth?

ZABUJEK: It is true, Your Sliminess. We have journeyed millions of light years and returned safely to report our findings.

QAJAOG: Go on.

ZABUJEK: As you know, our study required two simple, working-class humans from a small, remote mountain town. Men, of course, preferably gullible and childless and mildly alcoholic.

QAJAOG: Yes, of course.

ZABUJEK: As we orbited, our Stealth Field activated, we zeroed in on the human settlement of Stone River, Idaho, which sustains a population of 215 humans and harvests nearly 500 tons of potatoes a month.

QAJAOG: Potatoes, harvested? Barbarians!

ZABUJEK: I’m aware of their folly, my squishy, amorphic highness, but they know not what they do. Wounded by their inhumanity, I’ve since adopted three potatoes from the Spud Kennel. They seem happy in my home.

QAJAOG: But back to your study.

ZABUJEK: Yes. Our sensors located two ideal specimens: Larry and Doug, two out-of-work loggers who were going on a platonic friendship-affirming camping trip that weekend, during which they had planned fruitless fishing and many back-slapping hugs.

QAJAOG: How did you apprehend them?

ZABUJEK: We sent down one of our colossal flying saucers, thinking they would confuse it for an airplane or a bird. They were busy tossing back a couple Bud Lights and trying to pitch their tent, and at first didn’t notice us looming over the trees.

QAJAOG: Didn’t notice you?

ZABUJEK: As we soon discovered, humans need – heh, heh, you’re gonna love this one – light radiation in order to see.

QAJAOG: Light radiation! What the hell?

ZABUJEK: Ha! Ha! Can you believe it?

QAJAOG: Ha! Ha! Ha! Oh, my God, I’m oozing.

ZABUJEK: Priceless.

QAJAOG: Ha! Ha! Ho. Wow. Wow.

ZABUJEK: Hooooo. So, yeah.

QAJAOG: Yeah. So. You were saying?

ZABUJEK: So we waited until they could see us. Because what’s the sport in catching them while their backs are turned?

QAJAOG: True that.

ZABUJEK: So suddenly they turn around, and bam! We hit them with a big, bright light.

QAJAOG: Oh, perfect!

ZABUJEK: Scared the living crap out of ‘em. And they’re all like: Oh, my God, it’s the aliens! And we’re all like: How’d they know? And they’re screaming and losing their shit as we pull them into the starship using the gravitational fetching beam.

QAJAOG: Couldn’t you just have grabbed them?

ZABUJEK: Yeah, see, Togath in engineering really, really wanted to use the beam, and who wants to argue with Togath? Once that guy gets pissy, he locks himself in the slimatorium and pouts. We figured, okay, fine, make Togath happy. Then maybe he won’t be such a baby.

QAJAOG: So what did you do with the humans?

ZABUJEK: Well, we shackled them to an uncomfortable slab of metal surrounded by lots of needles and sharp implements. I don’t want to be mean, but Larry was a little on the hefty side, so we had to rummage through the closet to find a bigger slab of metal.

QAJAOG: And then?

ZABUJEK: Everybody’s favorite part, of course.

QAJAOG: Ooh! Anal probe?

ZABUJEK: Hell, yeah.

QAJAOG: I love it. So what did you find out?

ZABUJEK: Not much. Mostly just the contents of their recta, the length of their respective intestinal tracts, and a bunch of microorganisms helpful in the digestive process. But check this out: It turns out that most of their neurological activity occurs in their heads.

QAJAOG: No way!

ZABUJEK: Way.

QAJAOG: So that’s what the human brain is for.

ZABUJEK: Go figure.

QAJAOG: You released them, then?

ZABUJEK: Oh, yeah. They’re negotiating their contract with Sci Fi Network movie as we speak.

QAJAOG: Cool. Who’s playing you?

ZABUJEK: Duh. David Duchovny.

QAJAOG: Thank God. That guy needed a break.

ZABUJEK: Anyway, that should do it for now. I’ll let you know when we get the rest of our test results.

QAJAOG: You know what, don’t bother. I’m probably going to get the Intergalactic Navy to destroy the Earth, city by city, using apocalyptic laser beams and savage hand-to-claw combat, anyway.

ZABUJEK: Woah, you feeling okay, Your Highness?

QAJAOG: Just one of those weeks, I guess. Anyway, I’ll check you later, Zabujek. Or should I say, Captain?

ZABUJEK: Oh, Emperor. You’re make me secrete protoplasm.

QAJAOG: Just don’t let it go to your mandibles. Okay, seriously, I have to go. Have to go walk my potato.

* * *

(REPRINTED FROM ISSUE TWO, AUGUST 8th, 2005)

Wealth can bring a lot of things to a family and new research is suggesting such things are not always good.

One of these is childhood leukemia.

Although rare overall, leukemia is one of the most common potentially fatal illnesses that can befall a child, and a new study completed at the BC Cancer Agency in Vancouver is revealing that a high socioeconomic status can raise the risk of this disease by as much as 14% in Canada.

The reasons for the link between wealth and childhood leukemia are not yet clear, but knowing is important nonetheless, since it’s a piece of the puzzle in the effort to understanding the true causes underlying this disease, which are not well understood.

“Money doesn’t cause disease,” says Marilyn Borugian, a researcher at BC Cancer Agency and lead author of the new study. “But there are so many things related to it.”

Borugian and colleagues work is not the first to link childhood leukemia to high socioeconomic status. In fact, reports go back at least two decades.

But, in recent years, work on the effect of power lines on childhood cancer (by one of the authors of the current study, Mary McBride) appeared to find that the opposite was true, namely that childhood leukemia instead associates with lower, not higher, income.

“They [McBride and colleagues] found that the healthy children seemed to be of higher income,” says Borugian, referring to the past studies. So, a question was afoot: has something changed? Or, was their an unseen bias in the power line studies?

About this time, Borugian got recruited to find the answer. She was just finishing her Ph.D., having come to study epidemiology after a 25 year career in computing for a stock brokerage. “We wanted to go about this new study through computer programming,” says Borugian, “so that’s how I got involved.”

To re-examine the link between childhood leukemia and wealth, Borugian took Canadian postal codes and linked this to information from Statistic Canada on neighborhood incomes and 96% of all leukemia cases in children from 1985-2001. She found the lowest risk of childhood leukemia in the poorest neighborhood income and the highest risk in the richest.

“The original studies, with a higher risk in a higher income, are still supported,” says Borugian. The results of the study appear in July issue of Epidemiology.

Apart from this result, Borugian’s general technique is also raising interest. “It is already being used to look into other cancers,” she says.

As for the risk of childhood leukemia, population studies like Borugian’s don’t reveal causes. People have hypothesized that early exposure to childhood infection in poorer neighborhoods might provide protection, says Borugian, but researchers need to zero in on individual cases to figure this out.

“In order to do something about this, there are still a couple pieces missing,” says Borugian, including individual studies to control for things such as diet and exercise.

“It may be as simple as increased physical activity in poorer families,” she says. But, as yet, we don’t know.

* * *

(REPRINTED FROM ISSUE TWO, JULY 25th, 2005)

YOUR DISGUSTING HEAD: THE DARKEST, MOST OFFENSIVE – AND MOIST – SECRETS OF YOUR EARS, MOUTH AND NOSE.
By The Haggis-On-Whey World of Unbelievable Brilliance. 64pp. Simon and Schuster $24.50 (Hardcover)

In Norway, you say “buse.”

As a geneticist, I am a lot more familiar with the concept of snot than one might suspect. And although this may appear to be a sort of an odd soundbite, it can be quickly explained by the simple fact that pure genomic DNA, isolated from any and all variety of nature’s participants, will actually take on the appearance of the stuff you might see dripping out of an infant’s nose. I even call it “boogery,” which delights me to no end as an educator who is privilege enough to impart such wisdom to audiences ranging from scientific Heads of Departments to priests to politicians to graduate students to lawyers and (best of all) to unwary 11 year olds. In fact, I’ll leave it to your imagination as to which particular group revels the most in this piece of information.

Anyway, given what tends to be the somewhat international flavor of these audiences, I appear to be an expert of sorts on all things snot – or at least in colloquial terms, having compiled an impressive list of foreign ways to say ‘booger.’

Of course, the importance of language is steadfast in any discipline, even one as empirical as the sciences. Furthermore, I happen to know this first hand, having once made the mistake of teaching a group of graduate students to be especially “anal” when dealing with certain molecular procedures, a lecture that was met by sincere looks of disgust from my foreign students (asking, why, of all things, do we have to be anal?).

In Ukraine, you say “smarkotch.”
In Punjabi, you say “chewae.”

In any event, anatomically speaking, being anal would be the exact opposite of all this nose business. As well, being anal has nothing to do with a really interesting book that is refreshingly titled “Your Disgusting Head, The Darkest, Most Offensive – And Moist – Secrets Of Your Ears, Mouth And Nose”. Your nose (and, more specifically, the stuff inside it) on the other hand has a significant role in this book – which considering its wonderful use of language and my particular background, might even make this count as an academic review.

Anyway, this sort of book belies description. “Your Disgusting Head,” attributed to Dr. and Mr. Doris Haggis-On-Whey and published by the fine folks at McSweeney’s Publishing, is the second in the “HOW? BOOK SERIES” (The first being “Giraffes? Giraffes!). I can tell you that it is a very pretty book – think of the luscious retro look of those 60s and 70’s children’s science encyclopedias and you have an idea of what I’m talking about. But what makes this offering different is that instead of the stoic language of education, you have surreal and often very funny musings written in a tone that suggests scientific conjecture in a Radiohead, Teletubbies, Kurt Vonnegut kind of way (forgive me Radiohead and Mr. Vonnegut). In other words, I think it’s marvelous, but I’m not so sure you will.

In Cantonese, you say “baytay.”
In Flemish, you say “snot.”

So perhaps the best way to gauge your level of interest, is to ask yourself whether the following titles make you grin:

WHERE YOUR MOUTH HAS BEEN
THE SICKENING FLUIDS THAT FILL YOUR SKULL
WHO IS THE LUNATIC WHO DESIGNED YOUR EARS?
MADAGASCAR
WHY YOUR BREATH SMELLS BAD?

And of course, my favourite,

WHERE DOES ALL THE SNOT COME FROM?

If so, then I think you too will enjoy this book immensely. In fact, I secretly believe that the underlying intent of this book is to read it to your children. Barring that, it might also work well as a reference in any scientific Ph.D. dissertation. More to the point, it’s worth checking out, especially for those of us who are naturally scientifically curious.

Oh, and just in case you were wondering – my favourite way of saying snot is the very German, “schleim;” and apparently all the snot comes from Detroit.

* * *

(REPRINTED FROM ISSUE TWO, JULY 25th, 2005)