And Now There Are Six!!

Scientists eh! What a drag they can be! Forever coming up with new things that the rest of us have to wrap our minds around (or at least feel we should try).

Readers of these pages will know I’m periodically apt to wax rhapsodic about ‘the secret of life’ – the fact that all living things arise from just four different chemical units, A, C, G and T. Well, from now on it seems I’ll need to watch my words – or at least my letters – though maybe for a while I can leave it on the back burner in the “things that have been but not yet” category, to use the melodic prose of Christopher Fry.

Who dunnit?

The problem is down to Floyd Romesberg and his team at the Scripps Research Institute in California.

Building on a lot of earlier work, they’ve made synthetic units that stick together to form pairs – just like A-T and C-G do in double-stranded DNA. But, as these novel chemicals (X & Y) are made in the lab, the bond they form is an unnatural base pair.

Left: Two intertwined strands of DNA are held together in part by hydrogen bonds. Right top: Two such bonds (dotted lines) link adenine (A) to thymine (T); three form between guanine (G) and cytosine (C). These bases attach to sugar units (ribose) and phosphate groups (P) to form DNA chains. Right bottom: Synthetic X and Y units can also stick together and, via ribose and phosphate, become part of DNA.

After much fiddling Romesberg’s group derived E. coli microbes that would take up X and Y when they were fed to the cells as part of their normal growth medium. The cells treat X and Y like the units they make themselves (A, C, G & T) and insert them in new DNA – so a stretch of genetic code may then read: A-C-G-T-X-T-A-C-Y-A-T-… And, once part of DNA, the novel units are passed on to the next generation.

Science fiction?
If this has you thinking creation and exploitation of entirely new life forms?!!’ you’re not alone. Seemingly Romesberg is frequently asked if he’s setting up Jurassic Park but, as he points out, the modified bugs he’s created survive only as long as they’re fed X and Y so if they ‘escape’ (being bugs this would probably be down the drain rather than over a fence), they die. Cunning eh?!!

Is this coming to a gene near you?
No. It is, however, clear that more synthetic bases will be made, expanding the power of the genetic code yet further. What isn’t yet known is what the cells will make of all this. In other words, the whole point of tinkering with DNA is to modify the code to make novel proteins. In the first instance the hope is that these might be useful in disease treatment. Rather longer-term is the notion that new organisms might emerge with specific functions – e.g., bugs that break down plastic waste materials.

At the moment all this is speculation. But what is now fact is amazing enough. After 4,000 million years since the first life-forms emerged, more than five billion different species have appeared (and mostly disappeared) on earth – all based on a genetic code of just four letters.

Now, in a small lab in southern California, Mother Nature has been given an upgrade. It’s going to be fascinating to see what she does with it!

Reference

Zhang, Y. et al. (2017). Proceedings of the National Academy of Sciences 114, 1317-1322.

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Our Inner Self

Richard Gettner is the anti-hero of Christopher Fry’s wonderful play The Dark is Light Enough, set in the Austro-Hungarian war of 1848. Viewing himself as a failed author, failed husband and all-round disaster, he’s just absented himself from the Austrian Army on the basis of not being too nifty at soldiering either. Their minions are hot on his heels, intent on meting out the retribution that the military traditionally reserve for deserters, and he’s taken refuge in the family home of his former wife. In a tête á tête with her she rebukes him for his knack of self-destruction and points out that his book was actually quite well received and wasn’t really a failure. All Gettner’s frustration then bursts forth in a tirade of brutal philosphising:

‘Is there another

Word in the language so unnecessary

As ‘fail’ or ‘failure’?

No one has ever failed to fail in the end;

And for the very evident reason

That we’re made in no fit proportion

To the universal occasion; which, as all

Children, poets and myth-makers know,

Was made to be inhabited

By giants, fiends, and angels of such size

The whole volume of human generations

Could be cupped in their hands;

And very ludicrous it is to see us,

With no more than enough spirit to pray with,

If as much, swarming under gigantic

Stars and spaces.’

Fry deserves to be remembered as one of the great poetic wordsmiths of the English language, if only for The Dark is Light Enough but, had he known that nine out of ten cells in our bodies are bugs, he might have added a final blast to his demolition of the human condition:

Our failings should not surprise as we are but a sinister symbiosis,

More bacterial than human,

Helpfully poised such that when our hour is done

The microbial hordes surge forth to reduce us to our component parts.

bacteria and virus cartoon

The range of the hordes

Our rising preoccupation with the bug army (see it’s a small world & The Best Laid Plans In Mice and Men …) has been promoted by several recent studies that have propelled our ‘inner organism’ from the bowels of biology into the limelight. The story is somewhat fragmented but it’s a good time to see if we can make sense of the current threads.

We’ve known for many years that a motley collection of microorganisms are happy residents in most of our nooks and crannies, ranging from tummy buttons and through the skin, to saliva and our guts. They include bacteria and fungi, they’ve become known as the human microbiome (or microbiota), are said to outnumber human cells 10 to 1 and, all-told, can be viewed as a co-evolved ‘super-organism’ that has many benefits, including making our metabolism more efficient and hence improving nutrition. However, as with everything else in biology, this close relationship is a balancing act, the disturbance of which carries risks for disease development.

It’s critical to note that this vast microbial army, toiling away on our behalf in the dungeon of our innards, mostly dwelling in our gut, is a really mixed lot. It’s estimated to include about 700 different species of bacteria, of which perhaps thirty or forty species make up the bulk. It’s a bit like a mini Great Barrier Reef, well known as the world’s largest coral reef system and extraordinary in that, although it’s made up of billions of tiny organisms, the thing can behave in an integrated way, most dramatically illustrated by mass spawning.

Within the gut there are two major sub-families of microorganisms (Bacteroidetes (Bs) and Firmicutes (Fs)). Although more close-knit genetically speaking, each of these still includes many different classes of microbe. So, they’re a bit of a rabble but, by and large, not only are they harmless, they actually play a vital part in keeping us healthy.

Bacterial army manoeuvres

The power of DNA sequencing means that we can now interrogate our inner armies as to their make up under different conditions, because each type of microbe has a distinctive genome. The first thing to emerge is a dramatic shift in the balance between the major sub-families in obese individuals, be they mice or humans. That is, obese animals have about half the number of Bs and double that of Fs, compared to normal. And the link here is that the bug switch alters the pool of genes available, the upshot being increased energy harvest from nutrients consumed. In other words the switch helps animals get fatter.

It’s possible to breed mice that do not have any gut bugs and ask what happens when you transfer a colony from another animal. Bacteria-free mice on receipt of a normal gut army promptly double body fat: microbiota transferred from obese mice makes ’em twice as fat and, remarkably, human gut microbes from someone who’s obese also makes mice obese, if fed a high-fat rather than a normal diet.

Chemical warfare

Because we use antibiotics on a massive scale to control infections, we might ask whether they cause the good guys to suffer what the military call collateral damage – the point being that antibiotics don’t target bacteria on the basis of whether they’re good for us or potentially fatal. Inevitably, it turns out that ‘good guys’ do get hit by some antibiotics, and when this happens mice gain weight and build up fat. Unsurprisingly, a high-fat diet makes things worse. The sequence is that the drug changes the balance in microbiota before mice become obese and – a real shock – one course of antibiotic treatment imprints these effects on the animal permanently: it acts for life.

To clever for our own good

In our panic to avoid obesity and still pander to our sweet tooth, mankind has taken to using artificial sweeteners on a massive scale in the mistaken belief that these low-calorie agents do no harm. Only recently has this come to light as yet another example of the old adage about there being no such thing as a free lunch. It’s remarkable: saccharin, the most commonly used artificial sweetener, causes big shifts in the proportions of different types of gut bacteria – some increasing whilst others go down – the overall effect again being much more efficient energy harvesting from food. This is a direct effect of saccharin on the bugs, blocked by commonly used antibiotics.

The story so far

The regiments from which our foot soldiers are drawn (i.e. the species that form the microbiota) affect our metabolism and in particular can influence obesity – and that’s inextricably linked with type 2 diabetes and heart disease. With that in mind, it seems obvious that upsetting them with drugs is a risky business. What’s more, seemingly harmless food supplements can also be fraught with danger.

Marching to a beat

Yet another amazing feature of our inner army is that it keeps time. That is, the abundance of different sub-types fluctuates in synchrony with the day/night cycle. Put another way, it marches to a circadian rhythm along with many other physical, mental and behavioral changes that respond mainly to light – and hence roughly follow a 24-hour cycle. These can be big changes in composition: a particular type of bug can double in amount in 6 hours and return to its initial level by 6 hours later. One of the most familiar examples of the importance of biological rhythms comes from upsetting them by flying long distances on an east–west axis. Sure enough, mice have the same problem and, just like us, their clock is disturbed by jet lag (rather than shuttling them business class across the Atlantic you can simulate the effect simply by shifting the light-dark cycle under which they live forwards or backwards by 8 hours every three days). This largely blocks microbiota rhythmicity, the overall effect being to reduce the total number of bacteria. This in turn raises blood sugar level and the mice become obese. These events are absolutely dependent on what has happened to the microbiota because they are replicated in germ-free mice after transfer of jet-lagged faeces.

That’s more astonishing than might appear at first glance because it places the daily variation in gut bug populations alongside the basic circadian rhythms of the sleep-wake cycle, body temperature and other important functions. Circadian rhythms are driven by a ‘master clock’ in the brain that coordinates all the body clocks so that they are in synch. Four proteins are at the heart of the clock (CLOCK and BMAL1, highly expressed during the light phase, and cryptochromes (CRYs) and period proteins (PERs) expressed in the dark phase). These regulate the expression of many genes, thereby controlling the overall response (see Twenty More Winks). The implication is, therefore, that far from being a kind of add-on that occasionally gets upset, our microbiota play central role in a healthy body.

A recent example of it doing just that comes from another mouse model showing our ‘inner organism’ acting to protect against bacteria from the outside world. In response to infection, cells that line the small intestine switch on the production of a particular sugar (fucose): that is then released from the cells and consumed by members of the microbiota – this novel energy source seemingly helping the host to survive the onslaught of infectious microorganisms.

And finally …

All this stuff about germs being our best friends is riveting but what about the important question? Well, there appears to be a complex interaction between diet, microbial metabolism and colorectal cancer, with bacteria able to make some agents that protect against cancer and some others that drive carcinogenesis. There’s evidence that a wide range of tumours can be promoted by transferring microbiota to germ-free mice and, on the other hand, that depleting intestinal bacteria reduces the development of liver and colon cancers.

Space invaders

Personal space is, apparently, a big thing for many of us these days. So big that ‘scientists’ have had a go at measuring it – they never miss an opportunity do they? Actually, boffins being boffins, they measured something called the defensive peripersonal space (DPPS) – a ‘vital safety margin surrounding the body’ – by sticking a pair of electrodes to the wrists of volunteers who held their hands different distances from their faces whilst receiving bursts of current through the electrodes. That made them blink (!) and the nearer the hand to the face the more they blinked, as the shock was perceived to be a greater threat to their face. There is, seemingly, a sharp boundary: up to somewhere between 20 cm and 40 cm is a high-risk area where we get very aerated: beyond that we don’t much care – with large personal variations depending on how twitchy you are. Debrett’s, which styles itself as the arbiter of society etiquette, has a simpler test, its distilled wisdom revealing that if you can feel the warmth of someone’s anxious breath upon your face, then you’re standing too close.

With all this neurosis it’s probably a good job no one mentioned our inner army: a ten-to-one cellular takeover (albeit that bugs are much smaller) is not so much a bit of heavy breathing as a blitzkrieg. Even so, it’s a delicately poised occupation upon which we depend for survival – and it’s one that we disturb at our peril.

References

Sambo, C.F. and Iannetti, G.D. (2013). Better Safe Than Sorry? The Safety Margin Surrounding the Body Is Increased by Anxiety. The Journal of Neuroscience 33, 14225-14230; doi: 10.1523/JNEUROSCI.0706-13.2013.

Twenty more winks

In Episode One we alerted ourselves to the large amount of evidence saying that a good night’s sleep really is essential if you wish to reduce your chances of a wide variety of medical misfortunes. But what do we know about how molecules respond to sleep disruption to produce such nasty effects?

Molecular Clocks

Life on earth depends on energy sent forth by the sun and, in synchrony with the rotation of our planet, many of the inner workings of mammals fluctuate over each period of roughly 24 hours. This pattern is called the circadian clock, its most obvious manifestation being the sleep-wake cycle. Over the years considerable evidence has accumulated that the link between shift-work and cancer is probably due to circadian rhythm disruption and suppression of nocturnal production of a hormone called melatonin. All living things make melatonin (in mammals in the pineal gland of the brain) and it signals through a variety of protein receptors on cells to regulate the sleep-wake cycle but it also plays a role in protecting DNA from damage.

Melatonin production is regulated by the circadian oscillator, itself controlled by two sets of proteins that control each other’s expression in a feedback loop. Thus one pair, CLOCK and BMAL1, activates Cryptochrome and Period. They in turn repress CLOCK and BMAL1 – the upshot being that the activities of both pairs oscillate over a day-night cycle: as one goes up the other comes down. These central regulators are encoded by evolutionarily ancient genes (two for Cryptochromes and three for Period proteins). In plants and insects CRY1 responds to light but in mammals CRY1 and CRY2 work independently of light to inhibit BMAL1-CLOCK.

Two interlocked feedback loops control clock protein expression

CRY-CLOCK

OUTCOME: ≈ 24 hour cycle expression of PER & CRY

BMAL1 & CLOCK 12 hours out of phase

Alarming the Clock

So having sounded the alarm that just one night’s sleep shortage has obvious effects, what do the genes make of it? Well, the short answer is they get upset. A recent study took blood samples from a group of normal people and found that more than 700 genes (about 3% of our total number) significantly changed their level of expression over 1 week of insufficient sleep (5.7 h) by comparison with 1 week of sufficient sleep (8.5 h). About two-thirds were reduced whilst one-third was up-regulated (made more of their protein product). Unsurprisingly, among those that went down were the major clock regulators. It’s worth noting that the sleep perturbation in this experiment was relatively mild – intended to be similar to that experienced by many individuals. The genes most strongly affected play roles in a wide range of biological processes – DNA structure (hence gene expression), metabolism, stress responses and inflammation. The responses of genes to changes in sleep patterns are not the result of mutation (i.e. changes in the sequence of DNA)  but, at least in part, they’re caused by small changes in the structure of DNA. {These are epigenetic modifications – any modification of DNA, other than in the sequence of bases, that affects how an organism develops or functions. They’re brought about by tacking small chemical groups either on to some of the bases in DNA itself or on to the proteins (histones) that act like cotton reels around which DNA wraps itself}. Thus there is evidence for gene silencing by hyper-methylation of CRY2 (adding methyl groups (CH3) to its DNA) and the converse effect of hypo-methylation (removing methyl groups) of CLOCK occurs in women engaged in long-term shift work and is associated with an increased risk of breast cancer.

Inflaming the Problem

The cells that mediate inflammation and immune responses also have circadian clocks – meaning that normally these processes are rhythmically controlled and clock disruption (for example by sleep loss) affects this pattern. Disabling the clock in mice (by knocking out CRY altogether) switches on the release of pro-inflammatory messengers and knocking out one of the Period genes (PER2) makes mice cancer-prone – reflecting the fact that MYC (the key proliferation driver) is directly controlled by circadian regulators and is consistently elevated in the absence of PER2.

Clock Faces

The mass that comprises a tumour is a mixture of cells – cancer cells and normal cells attracted to the locale – so it’s a quite abnormal environment and in particular there may be regions where the supply of oxygen and nutrients is limited. This is sensed as a stress by the cells, one response being to lower protein production until normal conditions are restored. If this doesn’t happen within a given time the response switches to one leading to cell suicide. One way in which overall protein output can be reduced is by activating an enzyme (IRE1α) that breaks down code-carrying messenger RNAs that direct assembly of new proteins. Remarkably, it has emerged that one of the mRNAs targetted by IRE1α is the core circadian clock gene, PER1. The degradation of PER1 mRNA means that less PER1 protein is made, which in turn disrupts the clock. However, it seems that PER1 has other roles that include helping the cell suicide response – a major anti-cancer defence. All of which suggests that disruption of the IRE1α/ PER1 balance might have serious consequences. Indeed IRE1α mutations have been found in a variety of cancers including brain tumours in which low levels of PER1 are an indicator of poor prognosis. The IRE1α mechanism coincidentally activates the transcription factor XBP1 (as well as PER1 mRNA decay) and one target of XBP1 is the gene encoding a messenger (CXCL3) that makes blood vessels sprout offshoots. Thus this master regulator suppresses cell death, activates proliferation (lowering PER1 deregulates MYC) and promotes new blood vessel formation.

A Tip for Snoozing

If you’re still wide awake it just goes to prove the utter fascination of biology – but today’s story says that you have to find ways of, if not falling asleep, at least courting insensibility (as Christopher Fry put it). If it’s a real problem for you may I make a really radical suggestion? Turn to our physicist friends and select from their recent literary avalanche. A ‘brief history of …’ something or other will do fine. It’s a knock-out! Sweet dreams!!

References

Möller-Levet, C.S., Archer, S.N., Bucca, G., Laing, E.E., Slak, A., Kabiljo, R., Lo, J.C.Y., Santhi, N., von Schantz, M., Smith, C.P. and Dijk, D.-J. (2013). Effects of insufficient sleep on circadian rhythmicity and expression amplitude of the human blood transcriptome. PNAS 110, E1132-E1141.

Fu, L.N. et al. (2002). The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111, 41-50.

Zhu, Y. et al. (2011). Epigenetic impact of long-term shiftwork: pilot evidence from circadian genes and whole-genome methylation analysis. Chronobiol Int, 28, 852–861.

Pluquet, O. et al. (2013). Posttranscriptional Regulation of PER1 Underlies the Oncogenic Function of IREα. Cancer Res., 73, 4732-4743.