MIT team synthesizes second artificial gene CARBOHYDRATE—A major step has been completed toward syn thesis of an artificial gene with the potential of functioning detectably within a livkig cell. Only some 10 days before its announcement in Chicago, a team of scientists at Massachusetts Institute of Technology, led by Dr. Har Gobind Khorana, completed synthesis of a 126-nucleotide gene found in the bacterium Escherichia coli. All that remains to make a function ing gene is synthesis of the start and stop signals. That remainder, however, may be a minor hurdle or a formidable one. The team, says team member Kanhiya Lai Agarwal, has determined 24 nucleo tides of the stop signal. But the stop signal can't be tested until the start signal is synthesized. The team is working on the start sig nal now. If it is small, Dr. Agarwal says, the team may complete it before very long. But it isn't known whether the start signal is very small, as long as the gene itself, or longer. Sequencing the control segments of
the gene is a tedious procedure, Dr. Agarwal points out. In effect, it's like putting together a puzzle. Using one strand of the natural DNA, the scien tists hook onto it a strand of their arti ficial gene, leaving the unknown natural control segment extending beyond. Then nucleotides are added, one kind at a time, in the presence of the proper enzymes, until a nucleotide correspond ing to the unknown natural nucleotide is taken up by the gene. This process is repeated unit by unit. In the sense that the team still doesn't have a functioning gene, it hasn't pro gressed beyond the point in 1970 when it synthesized the first artificial gene—a 77-unit gene for alanine transfer RNA from yeast. But that event was a dead end since the substance for which the gene coded was plentiful in the yeast cell and the product of the artificial gene couldn't have been detected. Nor did the scientists know the nature of the start and stop signals. In contrast, there is a way of testing the currently synthesized E. coli gene, which codes to tyrosine transfer RNA. And it was easier to work with since Dr. Sidney Altman and Dr. John Smith in Cambridge, England, had determined
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> ORGANIC—An asymmetric hydroboration step is the key to a new stereospecific synthesis leading to two prostaglandin precursors. Dr. John J. Partridge, Dr. Naresh K. Chadha, and Dr. Milan R. Uskokovic, all of the chemical research department, Hoffmann-La Roche, Nutley, N.J., devised the new technique, which eliminates a troublesome resolution step from previously developed prostaglandin syntheses. Cyclopentadiene is the starting material for the new synthesis. It is treated first with sodium hydroxide, then with methyl bromoacetate in tetrahydrofuran at - 7 8 ° C. Stereospecificity is imparted by hydroboration with ( + )-di3-pinanylborane. Reaction with methylsulfonyl chloride in pyridine at 0° C. and then with sodium hydroxide yields 2S-hydroxy-4-cyclopentene-1ft-acetic acid lactone. The optically active lactone can then be used in one of two methods (one developed by Dr. Elias J. Corey of Harvard, the other by Dr. Josef Fried of .the University of Chicago) to produce other intermediates ultimately leading to prostaglandin F2cv. Dr. Partridge notes that the lactone, as synthesized by the new method, is already 96% optically pure. Since it is crystalline (the racemic compound, in contrast, is an oily substance), simple recrystallization gives 100% optically pure material. Overall yields (based on cyclopentadiene) are 24% for the "Corey intermediate" and 3 6 % for the "Fried intermediate," Dr. Partridge adds.
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C&ENSept. 17, 1973
the sequence of the gene and had carried out extensive work with it. When a virus—phage phi 80—infects the E. coli bacterium, the gene is easily taken up from the bacterium into the virus. The gene becomes prominent in the virus's hereditary material, making it easy to study. Dr. Agarwal explains that testing of the gene will involve the phage phi 80 virus. In E. coli, most of the genes are used for synthesis of proteins. Sometimes, however, a certain mutation in such a gene can result in a stop signal within a gene. This mutation results in a nonfunctional protein, which is shorter than normal. The mutation can be suppressed by a second mutation in another gene of E. coli. The British scientists discovered that this second mutation was within the gene of a tyrosine transfer RNA, for this reason often called tyrosine suppressor transfer RNA gene. This gene cancels out the stop signal and introduces tyrosine instead, resulting in a protein of normal length that is often fully functional. Thus, to test the artificial gene, the Khorana team can introduce it into the phage phi 80 virus. The virus can then be made to infect the mutant bacterium E. coli, carrying the artificial gene into the cell with its own genetic material. If E. coli proteins are normal, that shows the gene works. Ultimately, Dr. Agarwal says, the team hopes to be able to use the gene to study function of the nucleic acids. They would carry out such studies by changing portions of the gene and seeing what effect the changes have in the E. coli.
Recognition factors aid cancer diagnosis BIOLOGICAL—An assay procedure to evaluate the level of recognition factors in blood plasma could be a clinically useful test in both diagnosis and prognosis of malignant disease. Support for the usefulness of such a test has come from studies by Dr. Nicholas R. Di Luzio and coworkers at Tulane University School of Medicine, New Orleans. The suggestion for such a test procedure, Dr. Di Luzio says, is based on a number of recent research findings: • Indications that surveillance or recognition of foreignness by macrophage cells of the reticuloendothelial system depends, in part, upon the presence of protein components in plasma, which the Tulane group has designated "recognition factors." • Clinical studies to determine the relationship of recognition factors and cancer snowed that recognition factors are significantly depleted in patients with malignant tumors. • The studies also demonstrated that patients with advanced malignant disease show greater losses in recognition
GLYCINE
Laser technique makes glass fibers In studies of experimental laser techniques for making high-quality glass fiber, a Bell Telephone Laboratories scientist uses a laser beam to melt glass rod so that it can be drawn into a fiber. The carbon dioxide laser, Bell says, is a highly controllable and " c l e a n " source of heat, compared to conven tional heaters that put minute impurities in the glass. Bell foresees the use fulness of glass fibers as a way to transmit light beams carrying telephone messages, data, and computer information.
Aminoacetic Acid N.F. and tech grades FOOD · COSMETICS
factor activity than do those with pri mary tumors. • Observations that recovery of rec ognition factor activity followed, in cer tain cases, initiation of therapy suggest a relationship between tumor growth and development and recognition factor activity. In a symposium on phagocytosis, Dr. Di Luzio described current studies car ried out by his group at Tulane in an attempt to evaluate the influence of recognition factor administration on tumor growth. The studies were con ducted using recognition factor fraction isolated from normal human plasma along with leukemia cells transplanted into rats. Administration of recognition factors with tumor cells, the group found, pro duced about a 90% reduction in tumor growth. By comparison, other human plasma fractions, employed as controls, showed no inhibitory effect on tumor growth and development. This result, Dr. Di Luzio says, denotes the possi bility that isolated recognition factor fraction has a specific anticancer action. To isolate recognition factor fraction from normal human plasma, the re search workers added 20,000 units of heparin per 100 ml. of plasma. The plasma was then maintained at 0° C. for three to six hours for the recognition factor fraction to precipitate. The pre cipitate, collected by centrifugation, was washed with distilled water, lyophilized, and stored at -20° C. The lyophilized fraction, about 1.5 mg. of protein per ml. of plasma, was dissolved in sterile saline to a protein concen tration of 20 mg. per ml. immediately prior to injection. Immunoelectrophoresis, Dr. Di Luzio says, showed that the plasma protein
fraction was heterogeneous. After fur ther purification, it was found that the phagocytic-promoting activity resides in the ^-globulin fraction. In evaluating the ability of recognition factor fraction to modify tumor growth, rats were injected subcutaneously with tumor cells and either the recognition factor fraction, saline solution, or hu man serum albumin. After 10 days, the animals were killed and the tumors ex cised and weighed. In rats receiving tumor cells and saline solution, mean tumor weight on the 10th day was about 10 grams. For those rats receiving recognition factor fraction, there was an 86% decrease in tumor weight. Albumin didn't modify tumor development. To check on whether the inhibitory effect of the recognition factor fraction could be due to direct cytotoxicity, the Tulane group conducted in vitro studies with tumor cells and the fraction. No direct cytotoxicity was found. In an assay of phagocytic-promoting ability of normal human plasma, heparin-precipitate fraction, and super natant, the recognition factor fraction was found to enhance phagocytosis. And studies of the role of recognition factors as chemotactic agents showed that the recognition factor fraction, in addition to enhancing phagocytosis, also has chemotactic properties. All of these findings for recognition factors—ability to enhance macrophage phagocytosis, ability to exert chemo tactic activity, and capability of modify ing tumor growth—further buttress the concept, Dr. Di Luzio says, that recognition factors may be important regulatory factors in macrophagemediated mechanisms in detection or recognition of tumor cells.
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