Thursday, December 7, 2017

Many Paths To The Top Of The Mountain

Biology concepts – hydrogenosome, FeS cluster protein, loricifera, erythrocyte

More than one way to skin a cat seems to
be a newer version of the old British saying,
“there are more ways to kill a cat than by
choking it with cream.” Mark Twain was one
of the first to use the cat skinning version, in his
classic A Connecticut Yankee in King Arthur’s
The old Chinese proverb says, “There are many paths to the top of the mountain, but the view is always the same.” Put somewhat less delicately, “There’s more than one way to skin a cat.” Who wants to skin a cat? I think there is something to be said for the wisdom gained in 4000 years of culture, to say nothing of the ability to say it better.

In biology, this is particularly relevant; organisms have found different ways to do the same things, and different ways to do different things, but the end goal is always the same – live long enough to reproduce and the more offspring the better.

Last week we talked about how some organisms have degraded their mitochondria into mitosomes, and how they get along fine just using glycolysis and fermentation for energy (and maybe some arginine dihydrolase action). But there is another mitochondrial remnant in some other species of anaerobic eukaryotes called the hydrogenosome, and it works more like a mitochondrion than does the mitosome.

Here is the T. vaginalis protist. The blue probe
binds to DNA (just one nucleus for this guy) and
the yellow probe binds to a hydrogenosome
protein. The strands at the top are the flagella it
uses to move, not its hair.

Trichonomas vaginalis is a eukaryotic amitochondriate, and therefore is an anaerobic (without oxygen) protozoan. Unlike many protozoans, T. vaginalis does not have an environmentally resistant form (something that can live outside the host for a prolonged time – often called a cyst). It is transmitted directly from host to host, in this case sexually. Trichomoniasis is the most common curable sexually transmitted disease, but 70% of cases have no symptoms (asymptomatic). This is unfortunate because T. vaginalis infection can predispose to HIV infection and even cervical cancer. Having symptoms initially might prevent some of the later tragedies.

Unlike the mitosome containing protists, T. vaginalis does use its mitochondrial remnant (hydrogenosome) to make ATP. The hydrogenosome was discovered much earlier than the mitosome, although they have the same origin and general morphology. Because of this difference in timing, amitochondrial organisms with hydrogenosomes are called type II amitochondriates. Type I’s were the organisms that presumably didn’t have any mitochondrial-like organelle (and were seen first), like the Giardia and E. histolytica that we now know have mitosomes.

Pyruvate generated by glycolysis enters the hydrogenosomes just like it does in mitochondria. The Krebs cycle would be next for aerobic organisms, but in the hydrogenosome, iron-containing enzymes convert the pyruvate into an intermediate that has CoA (coenzyme A) bound to it. When this CoA is removed, energy is released, and this energy is used to convert ADP to ATP.

Because ATP production occurs at the level of substrate (a molecule being chemically changed, in this case by an enzyme), it is called substrate level phosphorylation. This is in contrast to the use of oxygen and the electron transport chain of proteins to produce ATP through the proton gradient (oxidative phosphorylation). One of the byproducts of the pathway is hydrogen, hence the name of the organelle.

In terms of energy production, the pyruvate:ferredoxin oxido-reductase (the iron/sulfate-containing enzyme in hydrogenosomes, often abbreviated as FeS cluster enzymes) pathway is about as efficient as the arginine dihydrolase pathway (ADH) in some mitosome-containing organisms. However, T. vaginalis also contains the ADH pathway, so it comes out ahead of Giardia in terms of energy production.

While the hydrogenosome has some activity in energy production via the FeS-protein mediated metabolism of pyruvate with production of ATP, the mitosome seems to be limited to the assembly of the FeS clusters only. A study of the proteins of the mitosome show the parts are there to make the FeS clusters, but that there are not the enzymes needed to break down pyruvate and produce ATP.

A study trying to quantify the amount of methane
gas produced by cows was carried out recently
in Argentina. The method involved a big backpack
and a delicately placed rubber hose. At some point,
scientist A approached scientist B and said, I’ve
got a great idea….”
Other hydrogenosome-containing organisms include the anaerobic unicellular fungus, Neocallimastix frontalis (it lives in the guts of rumen animals like cows). N. frontalis byproducts are used by gut methanogens (methane-producing bacteria) and therefore contributes to the generous amount of gas produced by cows. Many estimates name dairy and beef cattle flatulence as a bigger source of greenhouse gases than automobiles!

Another hydrogenosome-containing protozoan is Nyctotherus ovalis. It lives in the GI tract of cockroaches, and efficiently works with an archaeal bacterium that uses the hydrogen that the hydrogenosomes release. Just one more reason that cockroaches will outlast us all. The fact that some fungi and some protozoans have hydrogenosomes indicates that this organelle has evolved independently from mitochondria at least three different times in history – they must be a good idea.

Even with the exception of anaerobic protists and fungi, it was believed until just recently that at least all multicellular eukaryotic (metazoan) organisms depended aerobic respiration for energy production. However, there are even metazoan exceptions. A 2010 study of the bottom of the Mediterranean Sea found three different animals that survive without using oxygen and therefore don’t have mitochondria.

The deepest basin of the Med, near Greece, is nearly anoxic (an environment without oxygen).  In the muds of this basin were found three loriciferan (lorici = corsette and fera = bearing, so organisms with a sort of girdle) species that live in this area all the time. Other animals can survive in an anoxic environment for a while, but they don’t call it home.

Loriciferans weren’t even discovered until 1983.
Now we have some that live as anaerobes. Most
species of this phylum live in the deep waters,
but only a few are obligate anaerobes, meaning
they can only perform anaerobic respiration.
Oxygen can be damaging, it likes to scavenge
electrons, I wonder if it is toxic to the loriciferans.
These new loriciferans have hydrogenosomes instead of mitochondria, and produce ATP in the same ways as T. vaginalis and the other anaerobic eukaryotes. This is a completely new door being opened in biology, because the multicellular animals evolved after Earth turned from an anoxic environment to a place where oxygen was plentiful. It seems that even some of the more advanced organisms don’t have a problem reverting to more ancient systems if they find themselves in a place where they need it.

Would you believe that some of your cells might not have mitochondria? Well, about 26 trillion of your cells (if you’re an adult male) are amitochondrial – your red blood cells. That’s right; the erythrocytes that deliver oxygen to your cells in order so they can make ATP in their mitochondria don’t have any mitochondria of their own! In an attempt to carry as much oxygen as possible (bound to a big molecule called hemoglobin) your red blood cells have evicted their mitochondria.

This is probably a good idea, since making energy in the erythrocytes would use up the oxygen they are supposed to deliver to other cells. Instead, they act more like prokaryotes, and carry out glycolysis and lactic acid fermentation in their cytoplasm for the energy they need. To gain more room for hemoglobin, the RBCs have also done away with their nucleus.  They have no way to produce more proteins or repair themselves, so they work as long as they can and then they are replaced.

Old erythrocytes are phagocytosed (eaten) by macrophages in the spleen and liver and are destroyed. New RBCs (about 2 million per second) are produced in your bone marrow. The spleen also acts as a reservoir for blood cells, a ready supply for when you need them, but you can get along without it, you are just more susceptible to infections, since the spleen houses many white blood cells just waiting to recognize a pathogen that needs to be taught a lesson.

Human red blood cells (left) are round and biconcave,
but the camel RBCs are oval. You can see why so many
people believe they have a nucleus, but what you are seeing
is their biconcave side staining darker. The large cell in the
middle is an immune cell.
Anucleate (a = without, and nucleate = pertaining to a nucleus) erythrocytes are the norm for mammals. Many people think that camels are the exception, that they have nucleated RBCs, but this is not so. But they do have ovoid RBCs. When they run low on water, camels can remove water from their blood and use it in their cells. This leaves their blood thicker and harder to push through the small capillaries. Round RBCs would be impossible to squeeze through when the blood is viscous, so the camel has evolved RBCs that are longer in one direction and smaller in the other, to help blood flow in times of dehydration.

On the other hand, almost all non-mammalian vertebrates do have erythrocytes that do have nuclei. The only exceptions are a few salamander species that have some anucleate erythrocytes. For example, 95% of the Batrachoseps attenuatus salamander’s RBCs are anucleate. There is also the pearlside fish which is known to have non-nucleated red blood cells.

However, the crocodile icefish is even a bigger exception; it is the only vertebrate animal that has gotten rid of its RBCs altogether. This species lives in cold, highly oxygenated waters. The oxygen it needs just travels in the blood as a dissolved gas and is carried to every cell. These fish have even lost the DNA for making hemoglobin – now that is efficiency!

Given our apparent complexity, it is amazing
just how few genes humans have; the grape
has almost 30% more. The chicken doesn’t have
many fewer than us, and we don’t have to worry
about laying eggs. What is more amazing is that nine
years after the completion of the human genome
project, we still aren't exactly sure how many
genes we have.
Or is it? We have recently discovered that the majority of proteins have more than one function. Scientists gave this idea more thought when the results of the human genome project started to role in and we discovered far fewer genes than we expected. It is now accepted that humans have about 22,000 genes, not even as many as the grape, which has 31,000. Even the lowly fruit fly has 15,000 genes! How do we get so many functions out of so few gene products? Multitasking!

Take hemoglobin for example, it doesn’t just carry oxygen in the blood. It also acts as an antioxidant in several types of immune cells, and in certain neurons. It is a regulator of iron uptake and metabolism, since it carries iron at its core. It destroys nitric oxide, which is one reason why the little blue pill doesn’t work forever. You have to wonder what else the crocodile icefish has lost by giving up its hemoglobin and how it has made up for these losses. One change probably requires many more to be made as well.

We have seen how some organisms get along without mitochondria. What about the other end of the energy equation? Plants can make their own carbohydrate in the chloroplast – but is that what makes it a plant? Let’s look at this next time.

Roberto Danovaro, Antonio Dell'Anno1, Antonio Pusceddu, Cristina Gambi1, Iben Heiner and Reinhardt Møbjerg, & Kristensen (2010). The first metazoa living in permanently anoxic conditions. BMC Biology DOI: 10.1186/1741-7007-8-30

For more information or classroom activities on hydrogenosome, FeS cluster protein, loricifera, erythrocyte, see:

Hydrogenosome –

FeS cluster protein –

Loricifera –

Erythrocytes –

Thursday, November 30, 2017

A Biological Energy Crisis

Biology concepts –  mitochondria, aerobic respiration, anaerobic respiration, glycolysis, fermentation, mitosome

The bee hummingbird is the smallest bird in the world. 
Living on the 2 largest islands of Cuba, this little 
guy is only 5 cm (1.9 in) long and weighs just a bit more 
than a paperclip. The males and females live in separate
nests and never see each other again after mating.
Birds in flight use an astounding amount of energy, and the smallest birds use the most energy. Hovering hummingbirds must flap their wings 50-80 times a second, which requires a lot of energy. To meet this demand, they use 10x the amount of oxygen that a person uses (per gram of body weight)! To move this much oxygen in their blood when flying, their hearts must beat over 1200 times per minute. At that rate, a red blood cell can traverse the bird’s entire circulatory system in less than one second!

It is a vicious circle; the hummingbird must eat constantly in order to have the energy to hover, and it must hover in order to eat constantly. Hummingbirds convert their carbohydrate intake into cellular energy (ATP) on the fly, using the sugars ingested only a few minutes earlier to support up to 90% of their need. Contrast that to humans; elite athletes can draw only about 15% of their needed energy from the sugars they ate recently. 

So how is all this energy made? Since we have been talking about the mitochondria on and off for several weeks, you would be right to guess that this organelle is involved, but it doesn’t start there.

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This is an extremely simple cartoon of glycolysis.
If you want more detail, like which step calls for
glyceraldehyde phosphate dehydrogenase, then
look here.
Dietary glucose ends up in the cytoplasm after it is eaten and transported through the blood to each and every cell in the body. In the cytoplasm, the sugar is broken down in a process called glycolysis (gly = glucose and lysis = splitting). This process takes the carbohydrate from a six-carbon sugar down to two three-carbon sugars (pyruvate). In the process, there is a net gain of two ATP molecules (four are actually made per glucose but you have to invest 2 ATPs to get the process rolling). That isn’t much of a payoff. There must be something more, and this is where the mitochondria figure in the process.

The pyruvates are taken into the mitochondria and a second process begins to consume them. First there is a carbon and two oxygens removed from each pyruvate to form acetyl-CoA in what is called a linking reaction, since it links glycolysis to the next step - the citric acid cycle (Kreb’s cycle).   In this cycle, a series of reactions takes place to sequentially remove carbons from the sugar, leaving a four-carbon molecule (oxaloacetate) that then joins to the acetyl-CoA produced from another pyruvate. The series of reactions results in 2 ATPs and 6 NADH’s formed. This latter molecule (long name = nicotinomide adenine dinucleotide + hydrogen) will become important in the final step.

Remember that the mitochondrion has two membranes, and the inner membrane is folded into many cristae, in order to increase its surface area. The NADH’s produced during the Krebs cycle work with a series of proteins embedded in the inner mitochondrial membrane (called the electron transport chain) to create a proton gradient.

This is a Goldilocks version of the electron transport chain; the level 
of detail is juust right. Keep an eye out for the NADH, the water, and 
the protons moving in and out. They are important, as is the flow of 
the electron, hence the name; the electron transport chain.
When the NADH is broken down, a hydrogen ion (the same thing as a proton) is pushed into the inner membrane space. This is against its gradient and creates a high-energy situation, since it wants to move back into the matrix (the space inside the inner membrane). The ATP synthase allows the proton to move back in, but uses the energy of the gradient to convert ADP into ATP. One ATP is made for every proton that is pushed out and then allowed back into the matrix by oxidative phosphorylation.

The driving force behind NADH’s release of an electron and a proton (hydrogen atom) is that some atom must be waiting to scoop the extra electron, and this something is oxygen (this is why it is called oxidative phophorylation). This is why we have to breathe, the oxygen is a big magnet (metaphorically speaking) for the electron. The oxygen plus the electron plus two hydrogens bind together to form water. This is the metabolic water that is so important to many animals that don’t drink water

All told, the electron transport chain produces 36 ATP molecules per glucose, much more than the paltry 2 resulting from glycolysis (called substrate level phosphorylation as opposed to oxidative phosphorylation). It is a good thing that hummingbirds have mitochondria to wring so much energy out of their food (not so bad for us either).

And herein lies the exception, some eukaryotes have decided to try to live without mitochondria. It isn’t as though they just never underwent endosymbiosis; recent evidence is showing us that all eukaryotes had mitochondria at some point in their evolution. These exceptional organisms just worked out another way to produce energy, and allowed their mitochondria to disappear or change over time.

The human gut pathogen Giardia intestinalis (or lamblia) is a good example. Look as long as you like, but you won’t find a mitochondrion in this protozoan. Until 2003, scientists hypothesized that the lack of mitochondria in G. lamblia meant that it was a very early eukaryote, diverging from other eukaryotes before the endosymbiotic event that created mitochondria. But, then we discovered it was an even bigger exception.

Meet Giardia intestinalis; he looks happy to see you.
The blue probe binds to DNA, those are the two nuclei.
The green probe binds to the mitosomes. Just like the
duck in A Christmas Story – “it’s smiling at us!”
Instead of mitochondria, Giardia has 2-50 cryptons, also called mitosomes. These are mitochondrial remnant organelles (crypton = cryptic mitochondrion), with no genome of their own. They are completely reduced; all of their DNA has been transferred to the nucleus or lost, so mitosomes do not replicate on their own.

In Giardia, the mitosomes line up and down the sides of the organism’s two nuclei, with some between the nuclei. Yes, you're right -  Giardia doesn’t have any mitcohondria, but it has two nuclei – go figure. This specific and repeated arrangement suggests a specific function for these organellar remants. We aren’t sure what the functions might be, but it is not energy production. G. intestinalis produces its energy by glycolysis and by fermentation – the same process that yeast use to produce alcohol.

In alcohol fermentation of yeast, the 3-carbon pyruvates from glycolysis are converted to 2-carbon ethanol and some NADH is converted back to NAD+. This prevents a critical shortage of NAD+ in the cell. The amount of NAD+ in the cell is limited, so if glycolysis is to continue there must be NAD+ must be recycled from NADH.  The conversion of NADH back to NAD+ is the main purpose behind fermentation; it doesn’t produce any more energy than glycolysis alone.

Notice how fermentation doesn’t make more
ATP than glycolyis alone. In both lactic acid
fermentation and alcohol fermentation change
NADH to NAD+. This is the purpose behind
fermentation. Lots of energy is left on the
table -you can power a car engine on ethanol.
By the way - you ferment too. Yes, you. When oxygen is scarce, mammals will resort to fermentation, we just don’t produce alcohol. Instead, our waste product is lactic acid. In 1929, Nobel laureate Archibald Hill stated that it was the buildup of lactic acid in the muscles that caused muscle soreness after exercise, but his experiment was flawed. It wasn’t until just a few years ago that we discovered that lactic acid is crucial in keeping the muscles working (and brain) working when they are taxed. Lactic acid isn’t the problem, it is part of the solution.

But back to Giardia. Unlike yeast, G. lamblia doesn’t have a choice, it undergoes alcohol fermentation all the time. Make that almost all the time. Without oxygen (even though it doesn’t use it to make ATP) most of the pyruvate is converted to alanine, an amino acid, during fermentation. With even a little bit of oxygen, this switches over to alcohol production.  But there is another way Giardia can make some energy.

A mechanism called the arginine dihydrolase pathway has been seen only in prokaryotes and two eukaryotic anaerobes (Giardia and Trichomonas vaginalis). This speaks to the primitive nature of Giardia; no wonder scientists thought that it didn’t ever have mitochondria, like prokaryotes. In the arginine dihydrolase pathway, a whole bunch of steps lead to a little bit of ATP formation. It must make a difference for the organism’s survival, otherwise they wouldn't invest the energy in maintaining the pathway.

Giardia isn’t the only eukaryote to choose mitosomes over mitochondria. Entamoeba histolytica also causes diarrhea when it takes up residence in your gastrointestinal tract. I think this suggests that we are providing them with all the carbohydrates they need so that glycolysis and fermentation pay off. Was there less diarrhea before twinkies and french fires? Could be – there is probably grant money available for that study.

Entamoeba histolytica and Giardia intestinalis
are not closely related, they are very different
types of protozoa. For instance, Giardia is a
flagellete (moves by flagella), but E. histolytica
is an amoeboid (moves by body movement).
But they both cause diarrhea, and Giardia has
two nuclei and E. histolytica has four!
E. histolytica was also thought to be an ancient eukaryote that never had a mitochondrion, but mitosomes were discovered in this pathogen way back in 1999; the good old days. Another pathogen, Cryptosporidium parvum is also a mitosome-containing amitochondriate. Again, this is an intestinal parasite that causes diarrhea. I think that living in the gut must have turned these organisms into mutants, like the 1950’s animals exposed to radiation in great old movies like Them! and Godzilla.

C. parvum is closely related to the organism that causes malaria (Plasmodium falciparum), but they make ATP in different ways. P. falciparum  has mitochondria and can carry out oxidative phosphorylation via the electron transport chain. So how can they be related?

Here’s how: P. falciparum might have mitochondria, but they look like they are on their way out. They only have a few genes, and at least one principal enzyme is completely missing. In one stage of the infection, Plasmodium survives only by fermentation (although it goes to lactate, not alcohol), so maybe these two parasites are not so different after all. They have another similarity, but we will talk about that in a couple of weeks when we discuss plants without chloroplasts.

Fermentation is one way eukaryotic organisms get along without mitochondria, but there are many paths to the top of the mountain. Next time we will look at organisms that found another path.

Makiuchi T, & Nozaki T (2014). Highly divergent mitochondrion-related organelles in anaerobic parasitic protozoa. Biochimie, 100, 3-17 PMID: 24316280

Raj D, Ghosh E, Mukherjee AK, Nozaki T, & Ganguly S (2014). Differential gene expression in Giardia lamblia under oxidative stress: significance in eukaryotic evolution. Gene, 535 (2), 131-9 PMID: 24321693

For more information or classroom activities on glycolysis, oxidative phosphorylation, or fermentation, see:

Glycolysis –

oxidative phosphorylation –

fermentation –

Thursday, November 23, 2017

Life Outside The Chromosome

Biology concepts – plasmid, linear organelle genomes, extrachromosomal circular DNAs, conjugation,

Planet of the Apes (1968) – a good movie, but not a great movie.
Every ape was a ventriloquist; you never saw their lips move.
But it did have the first reciprocal interspecies kiss. The pan and
scan version loses the, see no evil, hear no evil, speak no evil joke;
you only see what is in the red box.
I love older movies, but only if shown in full aspect (wide screen or letterbox format). So much of old cinema had interesting things going on outside the field of focus.  Take Charlton Heston testifying before the panel of apes in Planet of the Apes. In the pan and scan version, you see one ape covering his ears when he doesn’t like what Heston is saying, but you miss the other two apes – one is covering his eyes and one is covering his mouth! You only get the joke in wide screen.

Biology can be the same. So much emphasis is placed on chromosomal DNA that we sometimes miss interesting things going on elsewhere, or we start to investigate years later than we might have if we would just look at the whole picture.

Last week we focused on the big DNA in prokaryotes, the chromosome(s). But this doesn’t mean prokaryotes don’t have other DNA. Most prokaryotes have extrachromosomal DNA in the form of plasmids (plasma = shape, and id = belonging to). These are smaller loops of DNA that have fewer genes than a chromosome, and the genes are not essential for survival.

However, "smaller than chromosomes" doesn't mean they have to be small. The "megaplasmids" are over 100,000 nucleotides, and can be more than 2 million nucleotides in length, but even these are smaller than the chromosome. The exception might be in bacteria that have multiple chromosomes. Often one chromosome is much smaller; a megaplasmid could be larger than the secondary chromosome.

Plasmids replicate on their own, so sometimes they are called autonomously replicating elements. As such, they do not depend on the chromosome for their existence. Plasmids have internal control features that keep the number of a certain plasmid within limits in any one bacterium. Some plasmids have other controls that keep certain plasmid types from surviving in cells that have other types of plasmids. But this doesn’t mean that a cell may have only one type of plasmid. Our lyme disease-causing example of last week, B. burgdorferi, has 21 different plasmids. What is more, some are linear and some are circular. It just can’t help but be an exception in all things molecular.

The plasmid is different from the chromosome. It is
smaller and is not tethered to the cell membrane.
New data is showing that eukaryotes also possess
plasmids, especially yeast. They are being used to
produce complicated proteins in a system more
like our own cell
Even though plasmids do not carry genes essential for survival, they can still have an influence on the life of the cell. For instance, most antibacterial resistance genes are carried on plasmids. These extrachromosomal elements can be transferred from bacterium to bacterium, and can be passed on to the daughter cells, producing populations of bacteria that can laugh at our puny efforts to kill them.

Plasmids may also transfer metabolic genes, allowing the recipient cell to degrade other sources of food, or virulence genes, allowing them to colonize different portions of the body. This is sometimes what happens with E. coli.  Species that live in the large bowel pick up a plasmid that codes for a system that lets them cling to the wall of the small intestine, higher in the gastrointestinal tract. Having them live here can cause diarrhea in several different ways, but it all depends on the presence or absence of  that plasmid.

One type of plasmid, called the F plasmid, has a role in bacterial sex determination. O.K., it isn’t like the sexes we think usually think of; bacteria with the F plasmid are considered F+ or “male” and those without are considered F- or “female.” The F plasmid codes for proteins that will create a tube (pilus) that can link one bacterium to another and permit the replicated F plasmid to be transferred to the F- cell, thereby turning a female in to a male. Tada – sex change the easy way.

The F plasmid contains tra genes that build the pilus
and control the integration of the DNA into the
chromosome. Helicase, the enzyme that unwinds
DNA for replication or insertion, was first identified
in the F plasmid.
Most of the time this is not such a big deal, but sometimes the F plasmid sequences can integrate into the chromosome of the bacterium, and when it cuts itself back out and becomes circular again, it may bring piece of the chromosome as well. This is now a F’ plasmid. When the F’ gets transferred to a F- cell, it takes those chromosomal sequences with it. This is one important source of genetic diversity in bacteria, called conjugation.

Plasmids are an integral part of the prokaryotic genome, so I have never considered them exceptions. What is more, you and I both know that there are circular DNAs in eukaryotic cells. Remember that the mitochondrion and chloroplast have their own chromosomes, although significantly reduced from what they had when captured by our ancestor cells underwent endosymbiosis.

Since the organelles were derived from prokaryotes, it would follow that their DNA is kept in a single, circular chromosome. In most cases this is true, but there are those organisms that demonstrate linear organelle DNA or multiple chromosomes in their organelles.

For example, the human blood sucking louse Pediculus humanus doesn’t have a single mitochondrial chromosome. Its 34 remaining mitochondrial genes are housed on 18 separate minichromosomes. Why ? – IDK (with a nod to my texting children). Even stranger, the fungus Candida parapsilosis has a linear mitochondrial genome, while its very close relative, the human pathogen C. albicans, has a conventional mitochondrial genome geometry.

The moon jellyfish is a cnidarian. Cnidarins are named
for cnidocytes, the stingers that allow them to defend
themselves or catch food. However, the sea turtle is
immune to the toxin of the moon jelly, so they are
happy with jellyfish sandwiches, like on SpongeBob.
Many other examples of linear organelle chromosomes exist, especially in the cnidarians (animals like corals and jellyfish). The relationships between these groups, phylogenetically speaking, have been hard to work out. The evidence that the hydrozoans (like the fire coral and the Portugese man-o-war) and scyphozoans (like moon jellyfish) have linear mitochondrial genomes indicate that they are probably closely related to each other and are younger than the other groups of cnidarians, like anthozoans (most corals and sea anemones).

Finally, corn (maize, species name Zea mays) cells have been show to have linear, complex, and circular forms of the chloroplast genome. In seedlings, the areas of high cellular division seem to be more active in the linear copies of the chloroplast chromosome. This may indicate that while the circular form is still present, it is the linear form that is functional in the Z. mays cells. Maybe we are catching a peak at evolution in action.

Most prokaryotes have circular chromosomes, and most eukaryotic species have organelles with circular chromosomes. It would follow that the instances of linearization of mitochondrial or chloroplasts sequences occurred after endosymbiosis was established, but why? What is their advantage? What would the text abbreviation be for “nobody knows?”

The above examples indicate that extrachromosomal DNA in eukaryotes can be more dynamic than previously surmised. But we haven’t touched on the interesting part. Eukaryotic linear chromosomes can sometimes give rise to circular pieces of DNA that then replicate on their own and stick around for varying lengths of time, just like plasmids.

Probably for reasons of "species prejudice" we don’t use the term plasmid for circular DNA in higher organisms; it makes us sound too similar to our prokaryotic ancestors. Circular DNA in plants and animals is called extrachromosomal circular DNA (eccDNA) or small poly-dispersed circular DNA (spcDNA) – and the scientists are right, these sound much more advanced: a plasmid that a eukaryote can be proud of.

The sources of these eccDNA sequences are several. They can be formed from non-coding DNA (sequences that don’t lead to the production of a particular RNA or protein), or they can be derived from tandem repeat (two copies of the same gene) DNA that are plentiful in the eukaryotic genome. A June, 2012 study identified a new type of eccDNA in mice and humans that actually has coding sequences that are non-repetitive.

eccDNA has been found in every species in which it has been looked for, so its presence is not unusual. What is unusual is that eccDNA can come and go, and can be formed from normal intrachromosomal recombination (the crossing over of sequences within one chromosome) or by the looping out of sequences from a chromosome and then being cut out. As of now, we don’t know what controls their occurrence or why they form.

Importantly, they do seem to have a function. Small numbers are seen in normal cells, but the number is increased in cancer cells or normal cells that have been exposed to cancer-causing or DNA-damaging agents. This was first demonstrated using a cancer cell line called HeLa, named for the mother from whom they were isolated, Henrietta Lacks. I highly recommend the biography of her tumor cells called, The Immortal Life of Henrietta Lacks, authored by Rebecca Skloot.

Xenopus laevis is a good model organism for
Studying development. Notice how the tadpole
Only takes 3 days to develop into a tadpole, and
every stage can be visualized. Plus, they can lay
up to 2500 eggs at a time.
The function of eccDNA in normal tissues is suggested by a study in Xenopus laevis, the African clawed frog. This animal is a much used model for studies of development because the eggs and embryos are big, the frogs can be induced to mate year round, and the embryos develop outside the body.

During development of the embryo, different levels of eccDNA are seen. Some sequences are seen early, while different sequences are seen later, and most of the eccDNA is gone by the time the embryos mature to tadpoles. This suggests specific functions for eccDNA in normal development. We wish we knew what the specific functions are – again, your opportunity for a Nobel Prize. 

The type of eccDNA in X. laevis is called a t-loop circle. The “t” stands for telomeres, like we mentioned last week. Telomeres have many units of a repeated sequence and are used to help replicate the ends of linear chromosomes. We have talked about how each replication of the chromosome leads to a slightly shorter telomere and how some scientists hypothesize that telomere shortening has something to do with aging defects.

Early in development, embryonic cells are dividing rapidly; in the 4-week human embryo, new cells are produced at a rate of 1 million/second! All this cell division requires replication, and replication shortens the telomeres. Could it be that the t-loop circle eccDNA has a function in preserving telomere length?

The telomere has many copies of a repeat sequence. Each repeat 
is recognized by an enzyme that helps to replicate that end of 
the chromosome. The enzyme called telomerase contains 
an RNA primer that can’t be converted to DNA, so the last
repeat is always lost. The telomere gets shorter with every 
replication. Sooner or later, this is going to cause a problem.

A study in 2002 suggested just that, these eccDNA telomere sequences might serve as a reserve of long telomeric sequences. These repeats could later be added back on to the telomeres through recombination events, thus preserving telomere length despite high levels of chromosome replication.

One the other hand, eccDNA is more plentiful in ageing cells and damaged cells. This might be an attempt to save the cell from the defects induced by telomere shortening or by damaging agents, or it may have a completely different function, perhaps even to induce cell suicide (apoptosis), so as to prevent damage to other cells. Once again, the small DNAs that are so easy to ignore may very well be the ones that allow us to live.

We have talked directly and indirectly about the mitochondria for the past few weeks; a crucial structure for energy production. Next time lets talk about the organisms that think they can do without this organelle.

Shibata, Y., Kumar, P., Layer, R., Willcox, S., Gagan, J., Griffith, J., & Dutta, A. (2012). Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues Science, 336 (6077), 82-86 DOI: 10.1126/science.1213307

For additional information or classroom activities about plasmids, extrachromosomal DNA, or telomeres, see:

Plasmids –

Extrachromosomal DNA –

Telomeres -

Thursday, November 16, 2017

On Geometry And Genomes

Biology concepts – linear chromosomes, circular chromosomes, taxonomy, replication, telomere

Organization is helpful in learning and work,
and apparently in crafts. But there is a fine
line between organization and obsessive
compulsive disorder.
Everyone (teenagers excepted) knows that getting organized helps you to learn and work. When you group tasks, items, or facts, it helps in remembering or working with them. In biology, grouping organisms has a history as old as language.

In the older grouping systems, the name of an organism was a phrase that described some characteristic of the organism. When a new relative was identified, the name phrase had to be lengthened to separate this new organism from those similar to it. As you can imagine, the names got very long very fast.

In the 1750’s, Carolus Linnaeus developed a much easier system of naming. In his “trivial system,” each organism had two descriptors in its name; a binary naming system. Linnaeus’ system (and others) of taxonomy (taxis is Greek for “arrangement”) is based on shared characteristics.

Carolus Linnaeus (he let me call him Carl) had many
names. His knighthood name was Carl von Linne, his
born name was Carl Nilsson Linnaeus. In his naming
system Linne came up with the name mammal, so I guess
he named himself again.
At first, it was the characteristics people could see that were used to group organisms. Then it was the characteristics on the macroscopic and the microscopic levels. Now it is based on molecular characteristics, forming both a taxonomic classification and an evolutionary tree; this is now called the science of phylogenetics.

Molecular characteristics usually mean DNA. Differences in DNA sequence and in the number of mutations that have occurred provide a relationship between organisms. Using these factors, a time line for their divergence can be estimated. We changed the ways we determine similarity, and that changed the rules. With new rules come new exceptions.

Many of the DNA rules start with chromosomes (chromo = color and soma = body, this comes from the dark and light banding pattern of stained DNA). Cellular DNA is very long and very thin, perhaps only 12-22 nanometers wide (about 1/5000 the width of a human hair). In this form, it can only be seen with an electron microscope.

In eukaryotes, this DNA becomes complexed with many proteins during cell division so that all the DNA can be packed up and moved more easily to the daughter cells.  Called chromosomal packaging, the DNA is wound around proteins called histones, then folded many times over, so that the finished chromosome is packed 10,000 times more compact than the original DNA helix. This is the packed DNA that we see as dark and light bands and gives it its name.

DNA packaging with proteins is a eukaryotic characteristic, unless 
I find an exception! The DNA wraps around the histones, then the 
histones line up into a coil, then the coils fold up into the
chromatid. Total packing – about 10,000 fold; it takes a piece of 
DNA 1.5 cm long and makes it 0.0000002 cm long!
By definition, a chromosome is a piece of DNA that contains genes that are essential for the survival and function of the organism. This implies that there may be other pieces of DNA that contain genes that are not necessary for survival.

The molecular rules of biology state that prokaryotes have one chromosome, a single piece of double stranded DNA that contains all the genes that the prokaryote (archaea or bacteria) needs. This is efficient for the organism; it is one stop shopping for replication of all its instructions and only two chromosomes (after replication) need to be segregated to the two daughter cells that are being made.

And here begins our exceptions. There are several prokaryotic organisms that have more than one chromosome. That is to say, their essential genes are located on more than one piece of DNA.

The first identified example of multiple chromosomes in a prokaryote was Rhodobacter sphaeroides, a photosynthetic species of true bacteria that can also break down carbohydrates it takes up. This bacterium was found to have two chromosomes, although one was more than three times the size of the other.

Genes encoding essential products for making proteins and carrying out day-to-day functions are located on each of the two R. sphaeroides circular chromosomes. There are other genes that exist on both of the chromosomes, but appear to be turned on and off via different signals. This implies that the same gene may serve its function at different times in the organism's life, or under different environmental conditions.

R. sphaeroides is by no means the only prokaryote that possesses multiple chromosomes. More than a dozen different groups of bacteria have at least some members with more than one chromosome. This includes Vibrio cholerae, the causative organism of the disease cholera. V. cholerae is responsible for a diarrheal infection that affects more than 3-5 million people per year and causes 130,000 deaths each year.

This is a crown gall in a birch tree caused by R. radiobacter.
Like in cancer tumor in animal tissues, a gallis unregulated 
growth. In grape vines, it has been responsible for the ruin 
of entire Kentucky vineyards. Kentucky makes wine?
In addition to these organisms there is Agrobacterium tumefaciens, whose name was recently changed to Rhizobium radiobacter. This is a very interesting two chromosome bacterium. It usually is a pathogen of plants, forming galls (tumors) on several cash crops, such as nut trees and grape vines. This is an important tool in the molecular biologist’s toolbox, since it has been found that R. radiobacter easily transfers DNA between itself and the plants it infects, via lateral gene transfer (a subject we have discussed in depth, When Amazing Isn’t Enough and Evolution of Cooperation). But R. radiobacter goes further, it can also cause disease in humans who have poorly functioning immune systems. For folks battling cancers, HIV, or other diseases that wreak havoc with their ability to fight off infections, R. radiobacter can cause bacteremia (bacteria colonizing the blood) or endopthalmitis (infection of the two hollow cavities of the eye).

The second molecular rule of biology is that prokaryotic chromosomes take the shape of a circle; the DNA forms a single loop. This shape is helpful in terms of replicating the prokaryotic chromosome prior to cell division. Start anywhere, and you can keep going to replicate the entire thing.  In point of fact, they don’t start just anywhere, but one start point (called an origin of replication) leads to complete replication.

There are advantages to having a circular chromosome. Prokaryotic chromosomes do not complex with proteins to become more densely packed, so it remains as a thin, long molecule. This means that fewer proteins are needed to maintain a circular, prokaryotic chromosome. In addition, since replication requires the doubling of just one piece of DNA from one origin of replication, this takes less time and fewer proteins to accomplish. Together, these features of a circular chromosome result in a more efficient and simpler process, with fewer chances for mistakes to be made.

Borrelia burgdorferi, a spirochete (spiral) bacterium was
Named for the researcher who discovered, it in 1982, Willy
Burgdorfer. It is one of the few pathogens that can function
without iron; it uses manganese instead. The ways this bug
gets around the rules is astounding.
However, there are exceptions in which prokaryotes have linear chromosomes. The Borrelia burgdorferi bacterium has a single chromosome, but it has the geometry of eukaryotic chromosomes, a line segment with two ends. This was the first prokaryote found to have a linear genome, way back in 1989. This lyme disease pathogen has one major linear chromosome and other pieces of smaller DNA that are circular or linear (which we will discuss in the next post); you just can’t trust a pathogen to follow the rules. Other prokaryotes that have linear chromosomes include our friend R. radiobacter. Even more interesting, while this pathogen has two chromosomes; one is circular and one is linear. How does that happen?

The previous discussions do not mean that all prokaryotes with multiple chromosomes or linear chromosomes are disease-causing agents, just the interesting ones. Since they cause pathology in animals or crops, they hit us in the wallet. It makes sense that we have studied them in more detail and have discovered their hidden exceptions. There are probably thousands of innocuous prokaryotes that have more than one chromosome or have linear chromosomes, we just don’t have a reason to look at them in that much detail.

There may be more than one way that prokaryotes end up with linear chromosomes. In some cases, the linear chromosomes still have bacterial origins of replication, indicating that they may have evolved from circular chromosomes. There is also evidence that some linear chromosomes might have developed from other linear DNAs in the cell, something we will talk about next time.

The rules of defining prokaryotes and eukaryotes also state that eukaryotes have linear chromosomes. The essential genes are stored on more than one piece of DNA, and these pieces have two ends apiece, like a line segment in geometry.

Linear chromosomes are a disadvantage because it is hard to replicate the ends. Because of the way that DNA replicates, the ends of the chromosomes, called telomeres, end up being shortened every time the DNA is replicated. Over time, this leads to shorter chromosomes that might lose DNA sequences that the cell needs in order to function.

Some lines of evidence suggest that telomere shortening is a direct cause of ageing. The loss of important sequences at the ends of chromosomes cases cells to perform at less than optimal levels, and mistakes and toxic products then build up and lead to larger dysfunctions of cells, organs, and systems, ie. getting old.

This is a very simple cartoon depicting recombination. When
sequences are exchanged, it isn’t necessarily a 1:1 exchange.
Sometimes parts of genes are sent one way but not the other,
So new genetic sequences can result. Some help, some hurt, and
some have no effect until the environmental conditions show
them for what they are. Most exchanges do not increase diversity
to any great degree, but the fact that some do has helped move
evolution along.
On the other hand, linear chromosomes may promote genetic diversity. In eukaryotes, the division of the cell requires each chromosome to be replicated, then the matching chromosomes of a pair (one from mom and one from dad) line up together. This is a prime opportunity for the chromosome to exchange some sequences in a process called homologous recombination; a mixing of genes beyond just getting one from each parent.

However, a study published in 2010 indicates that the geometry of the chromosome doesn’t matter when it comes to recombination rates. Scientists took a circular chromosome organism and linearized its genome (they cut it so it had ends). They also did the reverse experiment, taking a linear chromosome organism and circularizing its DNA.

In both cases, there was no change in the rate that its DNA recombined and produced slightly different offspring (the two circular chromosomes after replication can swap some pieces). So geometry does not appear to affect genetic diversity – so why did each type evolve? Good question – that can be your Nobel Prize project.

Next week we will continue the discussion of exceptions in DNA structures, including DNA that isn’t part of a chromosome, and mitochondrial and chloroplast genomes that don’t look like they should.

Casjens SR, Mongodin EF, Qiu WG, Luft BJ, Schutzer SE, Gilcrease EB, Huang WM, Vujadinovic M, Aron JK, Vargas LC, Freeman S, Radune D, Weidman JF, Dimitrov GI, Khouri HM, Sosa JE, Halpin RA, Dunn JJ, & Fraser CM (2012). Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids. PloS one, 7 (3) PMID: 22432010

Ramírez-Bahena MH, Vial L, Lassalle F, Diel B, Chapulliot D, Daubin V, Nesme X, & Muller D (2014). Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid. Molecular phylogenetics and evolution, 73, 202-7 PMID: 24440816

Suwanto A, & Kaplan S (1989). Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. Journal of bacteriology, 171 (11), 5850-9 PMID: 2808300

For more information or classroom activities on prokaryotic chromosomes or eukaryotic chromosomes, see:

Prokaryotic chromosomes –

Eukaryotic chromosomes –