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 -