How the Zebra Gets Its Stripes: A Simple Genetic Circuit

 of University of California - San Diego)

Science Daily — Many living things have stripes, but the developmental processes that create these and other patterns are complex and difficult to untangle.

"The essential components can be buried in a complex physiological context," said Terence Hwa, a professor of physics at the University of California, San Diego, and one of the leaders of the study published October 14 in Science. "Natural systems make all kinds of wonderful patterns, but the problem is you never know what's really controlling it."Now a team of scientists has designed a simple genetic circuit that creates a striped pattern that they can control by tweaking a single gene.With genes taken from one species of bacterium and inserted into another, Hwa and colleagues from the University of Hong Kong assembled a genetic loop from two linked modules that senses how crowded a group of cells has become and responds by controlling their movements.

One of the modules secretes a chemical signal called acyl-homoserine lactone (AHL). As the bacterial colony grows, AHL floods the accumulating cells, causing them to tumble in place rather than swim. Stuck in the agar of their dish, they pile up.

Because AHL doesn't diffuse very far, a few cells escape and swim away to begin the process again.

Left to grow overnight, the cells create a target-like pattern of concentric rings of crowded and dispersed bacterial cells. By tweaking just one gene that limits how fast and far cells can swim, the researchers were able to control the number of rings the bacteria made. They can also manipulate the pattern by modifying how long AHL lasts before it degrades.

Although individual bacteria are single cells, as colonies they can act like a multicellular organism, sending and receiving signals to coordinate the growth and other functions of the colony. That means fundamental rules that govern the development of these patterns could well apply to critical steps in the development of other organisms.

To uncover these fundamental rules, Hwa and colleagues characterized the performance of their synthetic genetic circuit in two ways.

First, they precisely measured both the activity of individual genes in the circuit throughout the tumble-and-swim cycle. Then they derived a mathematical equation that describes the probability of cells flipping between swim and tumble motions.

Additional equations describe other aspects of the system, such as the dynamics of the synthesis, diffusion and deactivation of one of the cell-to-cell chemical signal AHL.

This three-pronged approach of "wet-lab" experiments, precise measurements of the results, and mathematical modeling of the system, characterize the emerging discipline of quantitative biology, Hwa said. "This is a prototype, a model of the kind of biology we want to do."

Co-authors include Jian-Dong Huang, associate professor of biochemistry at the University of Hong Kong additional researchers at Hong Kong Baptist University, the University of Marburg, and the University of Hong Kong including members of the 2008 iGEM team, which Hwa co-advised as a Distinguished Visiting Professor at UHK.

Hwa is a senior scientist with UC San Diego's Center for Theoretical Biological Physics.

Uncharted Territory: Scientists Sequence the First Carbohydrate Biopolymer

Structure of the bikunin: The portion on the left corresponds to the sugar part of the molecule, the sequence of which was determined in the current study. The portion on the right corresponds to the protein part of bikunin. (Credit: Rensselaer Polytechnic Institute)

ScienceDaily  — DNA and protein sequencing have forever transformed science, medicine, and society. Understanding the structure of these complex biomolecules has revolutionized drug development, medical diagnostics, forensic science, and our understanding of evolution and development. But, one major molecule in the biological triumvirate has remained largely uncharted: carbohydrate biopolymers.

The paper is titled "The proteoglycan bikunin has a defined sequence."

Today, for the first time ever, a team of researchers led by Robert Linhardt of Rensselaer Polytechnic Institute has announced in the October 9 Advanced Online Publication edition of the journal Nature Chemical Biology the sequence of a complete complex carbohydrate biopolymer. The surprising discovery provides the scientific and medical communities with an important and fundamental new view of these vital biomolecules, which play a role in everything from cell structure and development to disease pathology and blood clotting.

"Carbohydrate biopolymers, known as glycosaminoglycans, appear to be really important in how cells interact in higher organisms and could explain evolutionary differences and how development is driven. We also know that carbohydrate chains respond to disease, injury, and changes in the environment," said Linhardt, who is the Ann and John H. Broadbent Jr. '59 Senior Constellation Professor of Biocatalysis and Metabolic Engineering at Rensselaer. "In order to understand how and why this all happens, we first need to know their structure. And today, at least for the simplest glycosaminoglycan structure, we can now do this."

The first glycosaminoglycan sequenced was obtained from bikunin. Bikunin is a proteoglycan, a protein to which a single glycosaminoglycan chain is attached. Unlike less sophisticated carbohydrate biopolymers, such as starch and cellulose, the proteoglycans are decorated with structurally complex carbohydrates that enable them to perform more sophisticated and defined roles in the body. Bikunin, for example, is a natural anti-inflammatory that is used as a drug for the treatment of acute pancreatitis in Japan. It has the simplest chemical structure of any proteoglycan. Linhardt views the discovery of the structure of bikuin as the first step on the ladder to the discovery of the structure of more complex proteoglycans.

"The first genome sequences of DNA were on the simplest organisms such as bacteria. Once the technology was developed it ultimately led to the sequencing of the human genome," he said. "In our efforts to sequence carbohydrate biopolymers we don't yet know if the defined structure we observe for this simple protoglycan will hold for much more complex proteoglycans."

But, looking for structure in more complex proteoglycans will be among the next steps in the research for Linhardt and his team. The search for structure could help put to rest a long-running debate in the scientific community as to whether complex carbohydrate biopolymers require a defined structure to function.

"Despite all that is known about glycan formation, our understanding has not yet been deep enough to infer sequence or even determine if sequence occurs," Linhardt said. "These findings represent a new way of looking at these complex biomolecules as ordered structures."

Linhardt's research into carbohydrate sequencing began 30 years ago. In his previous work, he determined that some order existed in at least a portion of some carbohydrate biopolymers, but it did not represent the entire finished puzzle.

"Previously, we could see a pattern, but we could not see if all the chains were playing the same music. The tools did not yet exist. Now we can recognize it as a symphony."

To uncover the entire structure, Linhardt and his team, which was led by his doctoral student Mellisa Ly, borrowed a technique from the field of protein research called the proteomics top-down approach. As opposed to the bottom-up approach that first breaks apart a complex biopolymer into pieces and then rebuilds it piece by piece like a jigsaw puzzle, the top-down approach used by Linhardt and colleagues allows the researcher to picture the whole intact puzzle. This can only be accomplished with some of the most sophisticated technology available to the scientific community today, including very high-powered mass spectrometers.

Linhardt used a mass spectrometer located in the Rensselaer Center for Biotechnology and Interdisciplinary Studies (CBIS) to make his initial discoveries, and had these results independently confirmed on a separate and higher-level spectrometer at the University of Georgia. Mass spectrometers break down a molecule into separate charged particles or ions. These ions can then be categorized and analyzed based on their mass-to-charge ratio. These ratios then allow for sequencing of the entire molecule.

"This was truly the convergence of really sophisticated spectroscopy and its application to biology," Linhardt said. "We were fortunate to have a lot of time to play with the instrument at CBIS to understand its capabilities."

Beyond the technology it also took faith and determination. According to Linhardt, "It takes a student that is willing to try something even when the odds are pretty low. If it doesn't work, you make incremental progress. If it does work, you can make a great discovery. But, from the beginning you need to be a believer that it is worth taking the chance because it takes a lot of hard work in the lab."

And the odds weren't in Linhardt's favor. Despite being the most simple of proteoglycans, there were still 290 billion different possible sequences for the molecule.

"The first sample we looked at, we got the structure," Linhardt said. "In the end we did 15 chains and they all came back playing the same exact symphony."

The research is funded by the National Institutes of Health.

Linhardt and Ly were joined in the research by Tatiana Laremore of Rensselaer; Franklin Leach and Jonathan Amster of the University of Georgia; and Toshihiko Toida of Chiba University in Japan.

I’m Ready For More

“My dear Baladeva, best of our family, please come immediately with Your younger brother, Krishna. You both ate in the morning, and now You ought to eat something more.” (Mother Yashoda, Shrimad Bhagavatam, 10.11.16) No amount of affection previously offered can stop Mother Yashoda from continuing on with her motherly duties. It is not that once she performs a certain amount of work, she just sits back and relaxes the rest of the time. Rather, with each offering she makes to her children, her maternal affection only grows stronger. Though her beloved child, Lord Krishna, and His elder brother Balarama were properly fed in the morning, her worrying does not stop. To ensure that they continue to enjoy their youth properly, she agonizes over their well-being, thinking that they have not eaten enough. Therefore she calls them home, to return from the playground where the sacred pastimes of the Supreme Personality of Godhead take place. The young children ate their breakfast in the morning. From following the advice of Mother Yashoda, the queen of the farm community of Vraja, they had the strength to go out and play. Young children should be let free into the fields to enjoy their sportive tendencies. Rather than remain locked up, better the young bundles of energy be allowed to release their potential for activity. The fuel for their play comes from the mother’s love, which arrives in the form of her cooking. Mother Yashoda’s cooking is so tasty that even Krishna’s friends enjoy it. A young brahmana boy by the name of Madhumangala is known for coming over and eating more food than anyone else. Mother Yashoda loves this, as her hard work in the kitchen does not go to waste. She is ready to offer an endless amount of sumptuous delights to Krishna and His friends, and as a perfect match, Shri Krishna is willing to accept whatever His dear mother offers. Isn’t Lord Krishna considered the Supreme Personality of Godhead? If so, how can He have a mother and father? The more relevant question would be why shouldn’t He have parents if He so chooses? Why would that enjoyment be denied the Supreme Enjoyer? The Vedic tenets stipulate that God can be known by three primary characteristics. He is the original proprietor, the supreme enjoyer, and the best friend of the living entities. Enjoyment comes through association with His property and friends. In this area there can be different moods of association. Krishna is not picky; He will play the role perfectly to fit the particular devotional mood of His adherent. This even holds true with His enemies. How does this last part work? The enemies deny the existence of God. They come up with many justifications for their belief system. “Krishna is just a sectarian figure; evolution explains the creation; God is dead; religion is for those who can’t cope with death; God is made by man, not the other way around”, etc. Many excuses are made over the course of the many years of the earth’s existence. Krishna kindly reciprocates by remaining hidden from the vision of such people. If someone were to hate us and not want to see us, we would gladly oblige their request and stay away from their presence, especially if avoiding them would give us pleasure as well. Krishna is complete in Himself; therefore if someone is insistent on turning their back to Him, there is no loss on the Lord’s part. Krishna will find new ways to keep the miscreant further away from Him. “I am never manifest to the foolish and unintelligent. For them I am covered by My eternal creative potency [yoga-maya]; and so the deluded world knows Me not, who am unborn and infallible.” (Lord Krishna, Bhagavad-gita, 7.25) Why is such a person labeled a miscreant? Well, if God is the original proprietor responsible for providing the gifts we take for granted on a daily basis, wouldn’t anyone who tried to deny His existence be considered a miser? If I walk up to someone’s property and start pointing to different things and saying that they are mine, is that sane behavior? Yet this is precisely the tact followed by the staunch atheists, who continually observe material existence using their blunt sense perception and try to prove that God does not exist. They will always have the fuel necessary to continue in this endeavor, as Krishna will reveal bits and pieces of different aspects of His creation, with each successive discovery considered new and groundbreaking. Since the clock will always tick towards eventual death, the atheist is guaranteed to never acquire complete knowledge. Indeed, in the next life the same person gets to renew their search, with the slate wiped clean as far as knowledge goes. Just as Krishna engages with the atheist by remaining hidden, He fully appreciates the devotional efforts made by those willing to acknowledge the Lord’s existence. The more sincere the effort, the more information about God is revealed. As one ascends the chain of spiritual knowledge, they get to see more and more of Krishna. The most exalted devotees are those who get to interact with Krishna personally through a particular rasa, or transcendental mellow. In the beginning stages there is shanta-rasa, or venerable appreciation for the Supreme Lord. The fact that God is a person with spiritual attributes may not even be known in this stage, but at least He is respected. The concept of a “god-fearing” person correlates well with shanta-rasa. In shanta-rasa, the Supreme Lord’s blissful features are not well known. Not that these features can ever be absent, for Krishna is always inananda. He is always seen smiling and playing on His flute. As further devotional practice is followed, more of Krishna’s features are revealed, similar to how our vision clears up when more dust is removed from the eyes. The vision is what gets clearer, not the person being viewed. The steady engagement in bhakti-yoga, or devotional service, facilitated through chanting mantras like, “Hare Krishna Hare Krishna, Krishna Krishna, Hare Hare, Hare Rama Hare Rama, Rama Rama, Hare Hare”, helps one ascend to the higher platforms of spiritual interaction. Mother Yashoda and the residents of the farm community of Vrindavana are so elevated that they get to see Krishna directly. What’s even more remarkable is that Krishna uses His yoga-maya potency to keep His divinity hidden. Think of this as Krishna wanting to be treated just like one of the guys. He’s like everyone else after all, except His sparkling transcendental features cannot remain concealed. Therefore even when He appears on earth in a human form, His uniqueness is duly noted. In Vrindavana some five thousand years ago, Krishna crawled around on the floor like an ordinary infant. This behavior especially caught the interest of learned sages, who marveled at God’s ability to give the appearance of being an ordinary living entity. The purpose behind Krishna’s behavior was to grant the desire for interaction of the residents. Mother Yashoda particularly wanted interaction in vatsalya-rasa, or parental love. How amazing is Krishna? He is the Supreme Father, the fountainhead of all energies, the person from whom everything emanates, and yet He is kind enough to take on the role of a child to give pleasure to Nanda Maharaja and Mother Yashoda. For the interactions to be fully relished, Krishna behaved just like an ordinary boy, going out to the fields every day with His friends. Mother Yashoda was thus given the opportunity to feed her son, to smother Him in motherly affection. The accounts of Krishna’s pastimes are most thoroughly presented in the Shrimad Bhagavatam, which is also known as the Bhagavata Purana. Purana means “old”, so the Puranas are Vedic texts which expound upon the highest truths of life through stories relating to incidents from ancient times. Not that the events are fabricated, just the exact details provided are sequenced in such a way as to teach many lessons. The interactions between Mother Yashoda and her son show that God is always ready to accept more love from the devotees. Our responsibilities are not finished after muttering a few mantras and attending periodic religious functions. If we forget Krishna, we are automatically in a negative position. We don’t need the looming threat of punishment in hell to know that absence of God consciousness is detrimental. On the reverse side, remaining in Krishna’s company is always beneficial. Since God is absolute, His personal presence is not required. Just hearing about Mother Yashoda calling Krishna and Balarama to come home to eat is as good as witnessing the events firsthand. Prasadam cooking and distribution is modeled after this concept. The devotee follows the example of Mother Yashoda, knowing that the food they are offering to thedeity will be accepted by the Lord Himself, who never tires of eating anything offered with love and devotion. Mother Yashoda’s offerings fueled Krishna’s activities and they further bound her in a knot of loving affection to her son. In the same fashion, with every devotional act we take up, our attachment to Krishna tightens. Say Krishna’s name all the time and pretty soon you’ll see His beautiful, smiling face wherever you go. That image of the Lord holding His flute with His lotus-like hands and not showing the least stress on His face reminds the conditioned soul of what their constitutional position is. The Supreme Enjoyer is the resting place for those seeking enjoyment. The original proprietor kindly bestows gifts that can be used in furthering one’s God consciousness. The best friend of the living entities is waiting for His fragmental sparks to decide to come and play with Him. In the sacred land of Vrindavana, the food is provided by the cows and the grains, the preparations by Mother Yashoda, and the entertainment by Krishna and His sportive exploits. The ears are satisfied with the sounds emanating from the Lord’s flute and the eyes by His precious, adorable vision. With the complete package available to those who follow bhakti, why would anyone take to any other type of activity? The potential for becoming fully God conscious is the gift granted to the human being. Though the gift is there, one can’t enjoy it unless it is unwrapped. The young child doesn’t know how valuable the expensive vase in the house is; hence the parents warm them not to touch it. If not for this warning, the child may inadvertently throw the vase to the ground and break it. In a similar manner, the precious gift of the potential for tasting the fruit of one’s existence given to the human beings must be carefully unwrapped through the instructions of the spiritual master, or guru. The bona fide guru is a devotee of Krishna who always enjoys the Lord’s association in some way or another. Without the instruction of the guru, the precious gift will either remain unwrapped or get tossed aside as being unimportant. The guru will tell the student to regularly chant the holy names and hear the nectar of Krishna’s pastimes. Simply meditating on the one scene of Mother Yashoda calling Krishna and Balarama to come home can provide so much insight. From one tiny incident, so many transcendental thoughts can be awakened, leading the inquisitive mind towards the proper destination. The Shrimad Bhagavatam and its verses cannot be accurately priced; the information within is invaluable. Krishna is always ready to accept more love, so why shouldn’t we be the ones to offer it to Him? In Closing: “Krishna, I understand You want to go on playing, For I gave You plenty to eat this morning. But now it is time for You to come back home, You can’t survive on just the breakfast alone. Baladeva, it is time to come back with your brother, Do not you two hear the piteous cries of your mother?” In this way Mother Yashoda always looks to protect, Her son Krishna, who endless amount of love can accept. In these verses much wisdom and delight does abound, In the sacred Shrimad Bhagavatam of Vyasa they are found. Stay in Krishna consciousness, let not the mind drift, Tasting fruit of existence is mankind’s unique gift. To learn of this opportunity’s true value, Hear from guru, who speaks words of God that are true. Neglecting worship of Krishna do not make the mistake, In this life let the divine vision of Him the eyes take.