Whether told in different languages, Sleeping Beauty is a fairytale most little girls around the world are familiar with. The story of a beautiful princess who slept under the curse of a dejected fairy for hundreds of years only to be awakened by her true love’s kiss became so popular that it inspired a number of literary and movie adaptations many centuries after Charles Perrault wrote the piece in 1697.
In 1997, exactly three hundred years later, genetics (that field of science that deals with the study of genes, heredity and variations in living organisms) has a quite similar story to tell—the awakening of a gene found in salmon which had been dormant for 15 million years not through a kiss from a prince but by the meticulous work of a team of scientists led by Dr. Peter B. Hackett of the University of Minnesota. The amazing story of the gene’s awakening from millennia of evolutionary slumber has earned it the name Sleeping Beauty (SB). Yet, contrary to the fairytale character from which it got its name and all the other fairytale characters we knew who stand for the archetypes of meekness and subtlety in our society, the Sleeping Beauty gene is far from being docile.
Sleeping Beauty is identified as a transposon, a highly mobile gene which is capable of jumping and inserting itself in one string of genes to another that triggers a mutation. It could also jump out again from such genetic string causing the mutation to revert. But why did it just turn itself off and lay in silent suspension for millions of years being active as it was? And if Sleeping Beauty’s pre-occupations are only to transpose and disrupt the activities of working genes, what function does it hold for life? To answer these questions we need to look at Sleeping Beauty from a larger picture—the composition of the genome, a picture in which genes combine and recombine to determine the characteristics of a living organism.
Decoding Sleeping Beauty’s Identity: Transposons and the Genome
In his book GENOME, The Autobiography of a Species in 23 Chapters, Matt Ridley provided a very simple illustration of understanding the genome (the human genome specifically in this case). He explained that the human body is made up of approximately 100 trillions of cells, each cell containing two complete sets of human genome protected inside that dark blob called nucleus. One genome is derived from the female parent and the other from the male parent. The genome, according to him, can be likened to that of a book: it consists of chapters called chromosomes; each chapter is composed of several thousand stories called genes; each story is structured by paragraphs called exons and are “interrupted by advertisements called introns”; each of these paragraphs are built from words called codons where each word is represented by letters called bases.
But unlike a true book which we read from left to write and are made up of words with different lengths, as Ridley explained further, the genome, our metaphorical book, could be read from left to right and right to left but not both at the same time. Also, each word in the book is composed only of four letters following the sequence ACGT. In the process of replicating itself, ACGT is now reproduced as TGCA which in turn is replicated as ACGT the copy of which is sequenced as TGCA. And this alternation continues for thousands and thousands of sequences. However, at this point lays an all too human story: replication is not always perfect. Sometimes a letter is being missed out or inserted in the wrong place—a process called mutation—and the sequence is altered to a previously unrecognized form.
There is another way of illustrating this phenomenon. Suppose that we have strings of three words which are BREAK, SPIN, and STEP (although no genetic sequence ever looked this way). By random movement, E jumps out of BREAK and attaches itself at the end of SPIN; consequently the word SPIN now turns into SPINE which carries an entirely different meaning from its previous form. After sometime, E jumps out again and inserts itself in the middle of STEP to make it STEEP which changes once more the meaning of the word. After losing the E SPINE now reverts into its original form which is SPIN. The insertion of E in different locations changes not only the meaning of the words but also the expressions that can be derived from them. By the same account, genetic sequencing and the disruption of a sequence changes the activities of genes in the vicinity that results to phenotypic variations or differences in physical appearances. These unstable letters that delete or insert themselves in another location are what geneticists called transposons (as mentioned earlier in the article), a genetic classification from which SB belongs.
The American geneticist and Nobel Prize Awardee Barbara McClintock was the first to observe that the genome is not a stationary but a dynamic entity subject to alteration and rearrangement when she studied the variegated color pattern of maize kernels in the 1930s. She bred homozygous female maize for C and bz alleles (absent were the Ds alleles) with homozygous males for C,Bz and Ds alleles to yield heterozygous offspring with an aleurone layer—that outermost cell layer of the endosperm or the tissue produced inside the seed of most flowering plants, which had the genotype C’CCbzbzbz—Ds. Since the dominant inhibitor allele C was present, it was expected that the offspring kernels would be colorless no matter their genetic makeup within the Bz/bz locus or area. Indeed, many of these kernels turned out colorless. However, varied amounts of dark brown spots and streaks were also observed in kernels where individual cells have lost their C and Bz alleles because of a chromosomal break at the Ds locus that followed transposition. Without the C allele that controlled color expression or the Bz allele responsible for the purple coloring of kernels, the cells that experienced disruption at the Ds locus turned out with a brown coloring.
Because of this mobile behavior geneticists considered transposons as parasites that propagate themselves using the resources of the host cell. The model of their life-cycle provides a picture of invading new species, passing copies of itself from generation to another, thriving within the genome until they reach an ultimate phase where they exist as fossils, a model consistent with the selfish DNA theory. The host organism is its survival machine; the transposon is the shielded warrior. However, transposons’ activities, even as they replicate themselves, are confined within the cell. Due to this restrictive mechanism, transposons have to learn to live in harmony with the host cells lest they cannot survive. As such, transposons evolved different strategies to avoid causing detrimental effects to the host. The host has, likewise, developed ways to silence the activity of transposons, fighting back and suppressing the jumping habits of some genes until they settle down. This was Sleeping Beauty’s fate 15 million years ago. The same mobile ability that gives it power to replicate itself and modify the expression patterns of genes became the trigger for both its inactivity and the activation of the host organism’s silencing weapon. Ultimately, Sleeping Beauty learned to be an agreeable cellular inhabitant and sat still.
Resurrecting the Mobile Gene
Advances in genetic research allowed scientists to study the behavior of genes and this paved the way for rediscovering the dormant Sleeping Beauty in salmonid fish. Dr. Hackett’s team at the University of Minnesota in the United States was able to identify numerous copies of salmon transposase gene which became inactive through years of mutations. What they did was took one copy of the genetic expression and aligned the mutated transposase. After which, they used the resulting consensus—the commonly expressed characteristics of the gene, as a template for a fully functional transposon.
On the other side of the Atlantic Ocean, scientists holed in one of the labs at the Max Delbrück Institute of Molecular Medicine in Berlin, Germany led by Zoltan Ivics, a guy endowed with a pair of soft but searching blue eyes and a Freudian mustache, generated the first active form of Sleeping Beauty transposase which they called SB10. They cloned a TC1/mariner element which they successfully isolated from white cloud mountain fish (Tanichthys albonubes). Like Hacket’s team, they speculated that the sequence of the element could be deduced from the consensus sequence of the alignment of many independent Tc1-like transposons from a number of fish species, each of which also encountered inactivating mutations. By fixing these mutations, they were able to revive Sleeping Beauty from dormancy and restore its ability to function as a transposon. SB10 was found to have limited applications though and other independent researchers modified it to increase its efficiency. Many subsequent studies found Sleeping Beauty to be active in numerous cells of zebrafish, medaka, senopus, rat and even humans. Once more, Sleeping Beauty is back into motion!
With these ground-breaking studies, Sleeping Beauty is now known as a type of transposon that inserts itself in one location to another through a cut-and-paste mechanism. It consists of two functional parts: the transposase enzyme and the transposon. The transposase enzyme is basically a type of protein that mediates the jumping of transposon. For illustrative purposes, let us focus on the mechanism of a transposon’s movement as determined by the Tn5 transposase. Imagine the transposon as a small horizontal bar flanked by other horizontal bars called ‘donor’ DNA. At the start of the transposition or gene jumping activity, individual molecules of transposase attaches themselves to both ends of the bar. After that the transposon forms a loop that brings the two ends of the transposable elements close together. Once this loop, called synaptic complex, is formed, the Tn5 transposase cuts the transposon DNA away from the genes flanking it. After cleavage, the process in which the transposon has completely excised itself from its original location, the Tn5 transposase/DNA complex duo can now move freely until it encounters and binds to a ‘target’ DNA. In a process called strand transfer, the transposase catalyzes the insertion of the transposon into the target DNA completing the transposition process. And thus, going back to our simple illustration earlier, in the strand BREAK E broke out from the flanks of R and A, moved about until it spotted the strand SPIN and bound itself with N.
Owing also to the increasing number of studies that described the capacity of transposons as an important force in the evolution of gene regulation and the creation of genetic novelty the dominant perception of the selfish nature of transposons has also evolved considerably in the past two decades. Presently, various efforts are directed at exploring the potential of transposons not only to explain genetic variation but also to find explanations to the development of biological conditions and diseases. A number of such explorations focused on the clue that Sleeping Beauty could provide regarding the development of cancer.
Sleeping Beauty, Cancer and all the Juanitas in the World
Five years ago, I met a 48-year old woman named Juanita in a rural area in the Philippines while on a school-related field immersion. With her small build, Juanita was no different from all the other village women of her generation. If you miss the slight stoop in her shoulders, you would not be able to guess that she carried the whole world on her back. No one new in the village would know of the dark force spreading itself inside her body at an ever increasing speed. Sooner it will engulf every normal cell that remained in her system. Sooner breast cancer will defeat her.
Having just witnessed one of my aunts’ grueling fight with and ultimate surrender to ovarian cancer, I was drawn to Juanita. Almost every morning, throughout the three months that I stayed in her village I would visit her in the small nipa and bamboo hut where she lived alone. In her firewood stove I would boil turmeric tubers for her to drink while we talked. In one of those talks I asked her how breast cancer felt like physically.
With a cloud of tears gathering behind those black pair of eyes that seen much suffering, she gathered herself and looked at me saying, “It felt like being stabbed by one-thousand knives…especially during the night.”
“And to think I didn’t feel anything nine months ago except for a growing lump in my breast,” she added.
Her words left a stab in my heart too especially when, two months after, in the comfort of my own home someone from the village came to deliver the news I already anticipated to hear but was not prepared to accept. Juanita, my closest friend in the village, died two weeks ago.
The thing is people may differ in skin color, economic status, and level of education but there are so many Juanitas all over the world. The World Health Organization (WHO) reported cancer as a leading cause of death worldwide. But cancer does not only affect women, it affects men and children as well. In 2004 alone, cancer accounted for 7.4 million deaths throughout the world.
The increasing incidence and mortality rates of cancer presents a big challenge for cancer research in which Sleeping Beauty might just play a very important role. Five years ago, while Juanita’s cries of pain mingled with the shrill of the night wind and the chirping of the crickets under the grasses, in developed countries like the US scientists begun to turn their lens on Sleeping Beauty to understand which genes contribute to cancer.
“There is a need to know for each and every kind of human cancer which genes could become mutated. Once we have identified such genes, researchers can start working on drugs or forms of treatment tailored to fight that type of cancer,” declared Dr. David Largaespada, an associate professor of genetics, cell biology and development who headed the team of researchers working under a Masonic Cancer Center and National Cancer Institute (NCI) cancer research collaboration.
To identify genes that mutate and stimulate cancer development, the team modified the Sleeping Beauty transposon to contain parts of DNA that can turn on and off the process by which the cell derives information or signal from genes to make proteins. The modified transposons were placed in the genomes of mice that were bred with mice who are predisposed to cancer. The genes of the cancer cells taken from the offspring of this union that developed cancer were studied. By tracking the insertions made by Sleeping Beauty, researchers could then identify the genes that mutated and resulted in the development of cancer. At the outset, when transposons like Sleeping Beauty caused a gene to mutate, it leaves a ‘tag,’ a sort of marker that pinpoints their location in the genomic expanse. In this manner, Sleeping Beauty plays two important functions. First it serves as a vessel that ferries potentially cancer-causing genes from one genetic string to another within the cell. Then, it provides information where the mutation occurs and what are the genes that facilitated this action.
Researching cancer with the use of Sleeping Beauty still has a long way to go. While it is true that cancer prevention and/or cure rests on a myriad of factors beyond Sleeping Beauty’s ability to handle, still, finding cancer solutions within the frontiers of science will find an increasing need for the helping ability of this jumping gene. And while a clear solution to the problem posed by cancer is not yet in place Sleeping Beauty does not need to go back to sleep.
