How Low Can You Go? Designing a Minimal Genome

By Elizabeth Ohneck, PhD

How many genes are necessary for life? We humans have 19,000 – 20,000 genes, while the water flea Daphnia pulex has over 30,000 and the microbe Mycoplasma genitalium has only 525. But how many of these genes are absolutely required for life? Is there a minimum number of genes needed for a cell to survive independently? What are the functions of these essential genes? Researchers from the J. Craig Venter Institute and Synthetic Genomics, Inc., set out to explore these questions by designing the smallest cellular genome that can maintain an independently replicating cell. Their findings were published in the March 25th version of Science.

The researchers started with a modified version of the Mycoplasma mycoides genome, which contains over 900 genes. Mycoplasmas are simplest cells capable of autonomous growth, and their small genome size provides a good starting point for building minimal cells. To identify genes unnecessary for cell growth, the team used Tn5 transposon mutagenesis, in which a piece of mobile DNA is introduced to the cells and randomly “jumps” into the bacterial chromosome, thereby disrupting gene function. If many cells were found to have the transposon inserted into the same gene at any position in the gene sequence, and these cells were able to grow normally, the gene was considered non-essential, since its function was not required for growth; such genes were candidates for deletion in a minimal genome. In some genes, the transposon was only found to insert at the ends of the genes, and cells with these insertions grew slowly; such genes were considered quasi-essential, since they were needed for robust growth but were not necessary for cell survival. Genes which were never found to contain the transposon in any cells were considered essential, since cells that had transposon insertions in these genes did not survive; these essential genes were required in the minimal genome.

The researchers then constructed genomes with various combinations of non-essential and quasi-essential gene deletions using in vitro DNA synthesis and yeast cells. The synthetic chromosomes were transplanted into Mycoplasma capricolum, replacing its normal chromosome with the minimized genome. If the M. capricolum survived and grew in culture, the genome was considered viable. Some viable genomes, however, caused the cells to grow too slowly to be practical for further experiments. The team therefore had to find a compromise between small genome size and workable growth rate.

The final bacterial strain containing the optimized minimal genome, JCVI-syn3.0, had 473 genes, a genome smaller than any autonomously replicating cell found in nature. Its doubling time was 3 hours, which, while slower than the 1 hour doubling time of the M. mycoides parent strain, was not prohibitive of further experiments.

What genes were indispensable for an independently replicating cell? The 473 genes in the minimal genome could be categorized into 5 functional groups: cytosolic metabolism (17%), cell membrane structure and function (18%), preservation of genomic information (7%), expression of genomic information (41%), and unassigned or unknown function (17%). Because the cells were grown in rich medium, with almost all necessary nutrients provided, many metabolic genes were dispensable, aside from those necessary to effectively use the provided nutrients (cytosolic metabolism) or transport nutrients into the cell (cell membrane function). In contrast, a large proportion of genes involved in reading, expressing, replicating, and repairing DNA were maintained (after all, the presence of genes is of little use if there is no way to accurately read and maintain them). As the cell membrane is critical for a defined, intact cell, it’s unsurprising that the minimal genome also required many genes for cell membrane structure.

Of the 79 genes that could not be assigned to a functional category, 19 were essential and 36 were quasi-essential (necessary for rapid growth). Thirteen of the essential genes had completely unknown functions. Some were similar to genes of unknown function in other bacteria or even eukaryotes, suggesting these genes may encode proteins of novel but universal function. Those essential genes that were not similar to genes in any other organisms might encode novel, unique proteins or unusual sequences of genes with known function. Studying and identifying these genes could provide important insight into the core molecular functions of life.

One of the major advancements resulting from this study was the optimization of a semi-automated method for rapidly generating large, error-free DNA constructs. The technique used to generate the genome of JCVI-syn3.0 allows any small genome to be designed and built in yeast and then tested for viability under standard laboratory conditions in a process that takes about 3 weeks. This technique could be used in research to study the function of single genes or gene sets in a well-defined background. Additionally, genomes could be built to include pathways for the production of drugs or chemicals, or to enable cells to carry out industrially or environmentally important processes. The small, well-defined genome of a minimal cell that can be easily grown in laboratory culture would allow accurate modeling of the consequences of adding genes to the genome and lead to greater efficiency in the development of bacteria useful for research and industry.

On Cheap Dates and cheapdate

By Michael Burel

As a graduate student, I don’t have many chances to go on dates. My definition of a good “date” would be my fruit flies and I taking a romantic walk on the beach, sipping fine Italian wine, and getting perfect experimental results on a Saturday night. And by those standards, I’ve gone on a lot of bad dates.

You’ll have to forgive me, then, if I don’t know what constitutes a “cheap date.” So, by peeking over my shoulder to ensure my baymate couldn’t see what I was shamelessly about to Google, I searched “define: cheap date.” I was unsurprisingly led to Urban Dictionary, whose definitions of (*ahem*) colorful terms I’ve never heard of—even while walking the streets of New York City—remain suspect at best. But I did end up finding a palatable definition for what a cheap date really is: “A person who has a low tolerance for alcohol and becomes intoxicated quickly after relatively few drinks.

By that definition, scientists sure got it right this time. In honor of Valentine’s Day, I would like to open this first segment of Drosophila Diaries with the Drosophila melanogaster gene, cheapdate. Yes, scientists may receive a tremendous amount of erroneous flack for being painfully logical and uptight (I’m looking at you, Big Bang Theory). If you are in search for a humorous reprieve, however, look no further than the gems that are Drosophila genes, which are named after their mutation-induced phenotypes. It’s a chance for scientists to leave a lasting impression and mentally quip, “Thank you, thank you, I’ll be here all night!” as if at a stand-up comedy club where colleagues roar with laughter at clever genetic knee-slappers. To each their own.

So how did the cheapdate gene get its name? In 1998, Monica Moore and colleagues performed a genetic screen in Drosophila in order to identify genes that affect alcohol sensitivity. Fruit flies, with their astounding genetic tractability, offer a chance to understand how mutations in genes and their resulting dysfunctional proteins cause observable changes in animal behavior (a feat cell culture does not allow…unless you count annoying contamination as an acquired skill akin to acrobatics, a point I will beg to differ to my grave).

I know, I know, I can hear you now: “Wait…so you mean they looked for genes that made flies act like a lightweight? How do you even tell if a fly is drunk?” Enter the inebriometer: A torture-chamber sounding device that is actually a vertical glass tube filled with several meshed rings on which flies can perch. Controlled concentrations of ethanol vapor diffuse throughout the tube, and as time goes by, flies get tipsy, lose motor coordination, and fall off the mesh. Think like the traditional “walk and turn” field sobriety test for drunk drivers, but instead of shouting sloppy profanities and getting arrested, flies are simply measured for how quickly they fall down.

In their screen, Moore et al. identified a particular gene that caused “inebriated” flies to fall faster than wild-type flies. They determined that this gene was actually an allele of the already discovered gene amnesiac, which plays a role in associative memory and encodes a protein that increases levels of cAMP. Moore et al. reasoned that cAMP levels were jeopardized in cheapdate-mutant flies. Indeed, by bolstering cAMP back to normal, they were able to sober up cheapdate mutants, watching them perch with all the dignity and grace that comes with balancing delicately on a wire-like lining while knocking a few back. By the way, did I mention cAMP signaling levels are altered in cells from alcoholic patients (1, 2)? Yeah, turns out fruit flies are actually good for something.

So this Valentine’s Day, if you are wining and dining a special someone (or preparing to snuggle up solo with a big bottle of Pinot like myself), remember that if your final bill comes out mysteriously discounted, you may have cheapdate to thank.

 

Spooky Science

Sally Burn

Happy Halloween! It’s been a spooky old few months for science. Here is a selection of the creepiest papers to rise recently from the publishing crypt:

 

“Dracula’s children: Molecular evolution of vampire bat venom”…

… This is the fantastic title of a ghoulish offering from the Venom Evolution Lab at the University of Queensland. Vampire bat venom is secreted by the submaxillary gland of blood-sucking bats. It contains toxins which interfere with the prey’s normal hemostatic response to injury, resulting in prolonged bleeding from the attack wound and providing the bat with an epic meal. Researchers in Bryan Fry’s lab set out to characterize the components of vampire bat venom and understand their molecular evolutionary history. They isolated novel isoforms of known venom toxins and also detected previously unknown peptides, many of which showed homology to proteins involved in blood clotting and vasodilation. The researchers found that mutation of the venom proteins’ surface chemistry may facilitate evasion of the host’s immune response. They also uncovered new molecular information about the appropriately named Draculin, a major component of vampire bat venom. Fry’s lab isolated a large fragment of Draculin’s amino acid sequence and discovered that it produces a mutated form of the lactotransferrin scaffold. These findings shed light on the molecular evolution of vampire bat venom and may aid in the search for novel compounds to use in anti-venom drugs.

 

Prepare to be spooked

Which is the scariest family in the world? The Addams family? The Munsters? No, it’s the Halloween family of genes of course! Members of this family encode cytochrome P450 enzymes and perform roles in insect development and reproduction. The genes have eerie names like phantom, spook, spookier, disembodied, and shade. A recent paper in BMC Molecular Biology reports the cloning and functional characterization of spook in the planthopper insect Sogatella furcifera. The lab of Guo-Qing Li characterized the expression pattern of the gene and showed that loss of expression caused delayed development and lethality. They also found evidence for a role in ecdysteroid hormone synthesis in the hemiptera order of insects – a known function of spook in other insect orders.

 

Halloween II

It’s definitely that Halloween time of year. Guo-Qing Li’s group, clearly working at full speed in time for Halloween, have now uncovered a role for a second Halloween gene in planthopper ecdysteroid hormone synthesis: shade. Taking a similar approach to that in their first paper, they isolated the gene and characterized its expression pattern. Once more they found that expression loss resulted in nymphal lethality. This phenotype could be rescued by providing the insects with ecdysteroid hormone, supporting a role for shade in the hormone’s synthesis in hemiptera. So, kids, when you are out trick-or-treating tonight try to remember what you are really celebrating: the synthesis of insect sex hormones – hurrah!

Porpoise-ful Evolution

Sally Burn

[quote style=”boxed” float=”right”]The happiness of the bee and the dolphin is to exist. For man it is to know that and to wonder at it.  Jacques Yves Cousteau[/quote]   

Dolphins are an object of fascination to many of us, with their quirky “smiles” and apparently playful nature appealing to young and old alike. Human uses for these sea mammals have ranged from service in the US Navy, to controversial food item, to subject of hippy artwork and questionable tattoos. The things that intrigue us the most about these animals though are probably their intellectual skills and the fact that they are fully aquatic mammals. But how did a mammal end up adapted to and completely living in an aquatic environment? Continue reading “Porpoise-ful Evolution”