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RNA Quest May Unlock Cell's Street

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"

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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-

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