RNA Quest May Unlock Cell's Street

NOW that J. D. Watson and company have outlined their construction manual for DNA (deoxribonucleic acid), the carrier of genetic information for all cells, many scientists agree that the best target for further research is RNA (ribonucleic acid). The cell copies specific genetic instructions from the DNA into RNA and then transports the RNA from the nucleus to the cytolpasm. It has not yet been determined how the cell chooses which information to copy and how the RNA is transmitted.

Fotis C. Kafatos, assistant professor of Biology and popular lecturer in Biology 15b, has spent the last two years investigating the cellular and molecular aspects of cell differentiation (how the cell decides what role it will play). Kafatos, a 28-year-old Greek citizen, has already published a dozen scientific communications which have received international attention. The editors of Nature cited his scientific promise and the crucial nature of his work in a rare burst of praise in the May, 1967, issue. Born in Crete, he came to America immediately after high school and enrolled at Cornell University. He finished the four year program in three years, graduating first in a class of 100 with high honors in Zoology.

He took his Ph.D. degree at Harvard with Carroll Williams, professor of Biology. When asked about Kafatos, Williams said "Fotis is in a group by himself. I've never seen anyone with such a green thumb for identifying the critical phenomenon and devising the necessary experiment." Williams relates how one of his colleagues, after watching Kafatos present his findings, said "God may be dead but Zeus isn't." Williams used to be one of the heads of the developmental biology course Kafatos now teaches with John G. Torrey, professor of Botany. But "Kafatos is such an enchanting and lovable teacher that I soon realized he could do the job as well as I could, so I resigned," Williams said.

Escaping the Cocoon

KAFATOS' FIRST major research project at Harvard, performed while a graduate student, was an investigation of how a moth escapes from its cocoon. The Elementary Science Study program has published an account of the way he found "How a Moth Escapes from its Cocoon." It will be used in elementary schools in September. In the pamphlet's preface, Kafatos states that science courses should not teach only the well-ordered results of research but also the daily progress of research, but also the daily progress of research, including the disappointments as well as the illuminations. He claims this would encourages frustrated beginners and would present a realistic picture of scientific work.

The answers Kafatos found in this project eventually presented him with a tailor-made system in which to pursue his old interest in cell differentiation. Through other scientists' research he found that different species of moths had various means of escaping from their cocoons. He found that, for example, one Australian species has a hard, pointed structure at the front of its head that it uses as a saw and that the caterpillar of one kind of silk moth leaves an exit hole when it builds the cocoon. The species Kafatos chose to work with, the Chinese Oak Silk Moth, however, had no such obvious method.

He soon found that the moth opens its cocoon by wetting it with some kind of liquid that softens the glue holding the silk threads together. Kafatos discovered that a certain drug made the liquid appear on the moths' face, even when they were not trying to escape from a cocoon, and collected the liquid efficiently by applying the drug. After realizing that the liquid must contain some material in addition to salt and water, Kafatos guessed that an enzyme, a chemical agent that helps break down or "digest" other chemicals, was the liquid's active element.

At first he believed the liquid was produced in the moth's digestive system. Analysis of the moth's digestive apparatus showed that there was a large amount of protein-digesting enzyme present but it was not certain this was the same enzyme as in the liquid. To confirm that the enzymes were the same, Kafatos became a bug surgeon, removing the midgut of twenty moths. As expected, none of the moths produced any liquid. Yet he was not satisfied with the result: it seemed too easy. He later realized that the pupae he used had been refrigerated for some time to prevent them from developing into moths at the usual time. Months later, as a safeguard, he repeated his surgery on twenty young, healthy moths. They were able to produce the liquid. At that point Kafatos still had no idea where the active element was synthesized.

The First Drop

WHILE dissecting pupae in search of the liquid's production site, he noticed a pair of long, very thin tubes in the front part of the pupa. These were the remains of the silk-producing tubes of the caterpillar. They led to a single opening on the moth's face just underneath the mouth. It was impossible to tell exactly where the liquid comes from since the first drop appears suddenly and covers the face. Yet the mouth and the old silk tubes were the only two openings and the mouth could be discounted because Kafatos had already shown that the enzyme came from nowhere in the digestive system.

He plugged up the silk tubes with melted wax, leaving the mouth open. When the liquid-producing drug was administered, no liquid appeared until the wax was removed. Kafatos was certain that the liquid did come from the tubes. Yet when he mashed the tubes, expecting to find the enzyme, no enzyme was present. One of Kafatos' typical brainstorms saved the day. He realized that the liquid he had been collecting was produced by two glands. The old silk tubes produced the inert part of the liquid while a special gland on the face secreted the enzyme itself. Kafatos substantiated these findings by proving that the moths could produce the enzyme even if the silk tubes were removed at an early stage of the pupa's development.

He soon noticed that there was some white, crystal-like powder on the face of a moth that was ready to emerge from its cocoon. Most of the enzyme crystals were on two cone-shaped structures on the face, called maxillae, which scientists had hither-to believed useless to the silk moth. Kafatos also found concentrated enzyme solution in the maxillae's cells, which squeeze the solution out through fine tubes leading to the surface of the maxillae. The enzyme, mixed with the liquid of the old silk tubes, gets painted over the cocoon's tip, thus dissolving the cocoon's glue and enabling the moth to push the strands apart and escape.

The Cellular Level

Puzzled by the changing function of the moth's old silk tubes and fascinated by the process which commits cells to their various fates, Kafatos turned to developmental biology on the cellular level. He presents his own findings in this field in two lectures in his course. He often involves his undergraduates, as well as his graduate students, in his projects. Kafatos is investigating both how cells become specialized and how they sometimes change from one specific function to another. These questions are crucial for man's understanding of the cell's nature. Furthermore, since cancerous cells are previously normal ones which, for some reason, begin to change and spread wildly, his research may eventually prove to be valuable in the fight against cancer.

THE Cell Theory, formulated in 1839, states that all living matter is composed of cells, which are made up of nuclei and cytoplasm. In the last two decades scientists have found that DNA, a long double--chained molecule found in the nucleus, carries the cell's genetic information. This information is the blueprint that describes how the working parts of the cell, the proteins, should be assembled from 20 building blocks, the amino acids. Thus DNA ultimately determines the cell's function. The DNA's information is transferred to RNA (the cell's working manual), which is chemically like DNA except that it is usually single-stranded. Specific RNA messages ("messenger RNA") are shipped to the cytoplasm and there direct synthesis of the specific proteins.

All cells apparently have the same genetic information. That is, red blood cells, which produce hemoglobin, have the same information as muscle cells, although obviously have widely different functions. Cell specialization thus may depend on the process by which the cell chooses which genetic information to copy into RNA and, perhaps more importantly, to transport and use in the cytoplasm.

The Stacks of Widener

Kafatos is investigating precisely this information-retreival which goes on during differentiation. He likens the information selection to the ordeal of obtaining a book from the stacks of Widener. Just as an industrious Cliffie chooses one book from the stacks by some apparently mystical method, so the cell somehow selects which information to copy. Next, the Cliffie has to decide whether or not the book is worth reading after all. She may carry it with her to the reading room or she may immediately discard it in the stacks. Similarly, not all the information which is copied into RNA is transported to the cytoplasm. Finally, as the girl may postpone reading the tome once in the reading room, so sometimes the RNA message is not immediately utilized in the cytoplasm. On the other hand, the girl may fall in love with the book and decide to carry it with her forever. Likewise, the RNA messages may persist in the cytoplasm and be read over and over again. Kafatos adds that the critical choice may lie at any of these levels. If we discover where and when the choice occurs concerning what the cell will do, we may be able to understand how a cell specializes and ultimately perhaps to control this specialization.

Kafatos points out that even if the nucleus of a fertilized egg is removed, the egg can still give rise to a very young embyro. This demonstrates that stable messenger RNA for making an embryo had been stored in the egg's cytoplasm. Storage may well be the level where the critical choice might reside in the cells of higher organisms. This is not the case for very simple cells like bacteria, where the genetic information is transcribed into RNA and immediately translated into protein. Kafatos explained that, due to the great instability of the bacterial world, bacteria do not think ahead because they must be able to adjust continually to constantly changing conditions. The DNA sends a steady stream of messenger RNA which adjusts the cell's protein-synthesizing orders. Any one message remains in the cytoplasm for about three minutes. There is thus immediate control. As soon as an environmental change makes a change in the cell desirable, the DNA can erase its old orders and send new messenger RNA.

"Cocoonaise"

IN CONTRAST, the highly-differentiated cells of more developed organisms are much more stable. Once a cell is programmed to fulfill a specific role, it will continue to do so. In this case continual messages to the cytoplasm are superfluous. Kafatos has been able to correlate stability and differentiation. He made this important discovery in a research project which began with an undergraduate, Julianne Reich '67, a former Bio 15 student now at the Medical School. The gland on the moth's face which produces large amounts of a single enzyme is an example of a highly differentiated organ. About 70 per cent of the protein made by this gland is one enzyme, "cocoonaise." The rest is proteins needed for cell maintenance and growth. The message for making the differentiation - specific protein is extra stable. That is, each molecule of cocoonaise - messenger RNA remains active in the cytoplasm for at least two days. By contrast, the rest of the cell's messengers only survive for a few hours. Presumably, their decay introduces flexibility in the non-specialized functions of the cell.

These conclusions were obtained through an unorthodox combination of techniques. In addition to classical biochemical methods, he used a microscopist's approach. He took advantage of a structural peculiarity of the cells. As it is produced, cocoonaise gets stored in a separate part of the cell, away from the rest of the cell's proteins. Kafatos thus could measure how much of each kind of protein the cell was synthesizing at any one time, simply by looking at how much new protein was added to each region.

He stopped the synthesis of all RNA by treating the cells with the specific antibiotic Actinomycin D, which is, incidentally, used to stop the growth of cancer cells. From then on, all protein synthesis depended on pre-existing messengers. He detected new protein molecules by exposing the cells to radioactive amino acids, which are incorporated into any protein the cell synthesizes. Kafatos has made thin sections of the cell and covered them with a thin photographic film. The radioactivity behaves like light and activated the film. His process is called autoradiography. He could then develop the film, count the activated silver grains over the two regions of the cell, and know how much of each protein had been synthesized. He found that a few hours after actinomycin treatment the cell stopped making its non-specialized proteins, but it continued to make cocoonaise for at least two days.

Do Informosomes Exist?

After investigating the stability of RNA, Kafatos turned to possible mechanisms for transporting the RNA to the cytoplasm. Extending his Widener metaphor, he said the first possibility is similar to a Cliffie going into the stacks to obtain the book herself while the second is like her using the library's call system, having the book delivered up to the reading room. In the first possibility, the actual users of the genetic information, the ribosomes, or protein-synthesizing particles, may carry messenger RNA from the nucleus to the cytoplasm, the cite of protein synthesis. Second, there may exist a distinct kind of particle which binds the RNA messenger, protects it, and possibly even stores it in the cytoplasm till it is needed. The existence of these hypothetical particles, which are called "informosomes," or information-carriers, was first postulated by Russian scientists working with fish embryos four years ago. Using essentially their method, Kafatos last year identified particles that may be informosomes in insects. His report was published in the latest issue of the Proceedings of the National Academy of Science. He regards both his and the Russians' results as somewhat inconclusive, however, because of the technical difficulties in identifying the informosomes.

Kafatos' first step in testing the reality of informosomes was to get an idea of the composition of his messenger-carrying particles in silk worms. Ribosomes are about 50 per cent protein and informosomes, which may be similar to ribosomes, have been postulated by the Russians to contain a higher amount of protein. Chemical analysis of the particles suspected to be informosomes was impossible because Kafatos was dealing with such minute amounts of particles. Finally he turned to a method for testing a particle's density devised ten years ago by Harvard's Matthew Meselson, professor of Biology. Particles are placed in a centrifugal tube containing a salt gradient, a solution with various density levels. Kafatos used a centrifuge capable of creating a gravitational field 400,000 times greater than that of the earth. The particles soon settle to the level of the solution which has the same density.

Salt Breakthrough

CESIUM CHLORIDE, a salt somewhat similar to table salt, is commonly used in density studies, but animal ribosomes were found to be unstable in this solution. The Russians circumvented this difficulty by "fixing" the ribosomes by tanning them with formaldehyde. Yet, according to Kafatos, this did not end the problem because tanning may alter the particles chemically so that the results may not be definitive. After a years's work, Kafatos, in collaboration with a colleague, Ned Feder, now at the National Institute of Health, synthesized a different salt to use in the centrifuge in which ribosomes would be stable. It took some detective work to decide what that salt should be, but finally they succeeded. Their salt, made from cesium and, in substance, similar to vinegar, preserves ribosome structure, at least. This by itself is a major success because it should allow studies on animal ribosome structure, which up to now were only possible with the more stable ribosomes from bacteria.

Kafatos is right now engaged in further experiments with the new salt, to test that informosomes actually do exist. It has been suggested that cancer is a special case of a cell's function being transformed by a virus. A cancer cell has broken out of the normal limits on its growth and function. If scientists can discover what makes cells less stable in their commitment to a limited, differentiated career, and thus more liable to become cancerous, he might be able to find ways to treat this process. Yet Kafatos repeatedly emphasizes the fact that he is still distant from tangible results in his research. He said, in addition, that not only are scientists working on cell differentiation far from answers, but they are still groping for the right questions. "It's a great field but we are only now beginning," he said.

He has participated in many other research projects, including a survey of heart disease in Crete in the sum-