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Breast Cancer Shows the Peak of Pressure in the 20-29 to 30-39

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Breast Cancer Shows the Peak of Pressure in the 20-29 to 30-39 VOL. 52, 1964 GENETICS: GIBSON AND SONNEBORN 869 Breast cancer shows the peak of pressure in the 20-29 to 30-39 periods with a decline in pressure to 40-49 and then with a very low ratio of increase (1.49, 1.40, 1.32, and 1.29) in the four remaining age groups. The degree to which the amount of pressure toward tumorigenesis follows the amount of reproductive activity is striking. Conclusion.-The relationship between the rate of increase or of decrease in cancer incidence at various sites, in successive age periods, may be used to give us suggestive information concerning the nature of the host response to tumorigenic pressures of different types and derivation. It should be emphasized that the present presentation and analysis of data is neither extensive nor exhaustive. It is intended merely to show that character- istic responses of each site exist for each sex and to call attention to differences which need further study and analysis. * The present paper is based on a statistical procedure presented before the Academy some years ago. It utilizes more recent data on incidence rather than those on mortality which were the basis of the earlier communication. 1 Cancer in Connecticut 1935-51, Conn. State Dept. of Health (1955); Cancer in New York State 1941-60, Bureau of Cancer Control, N.Y. Department of Health (1962). 2 It is recognized that other methods may be used, for example, the cohort type of tabulation and analysis. The present communication is, however, intended to raise questions rather than to answer them and must of necessity be brief. It will therefore be confined to one method of presentation of -the data. 3 It should be recognized that data which include cancer of all sites are composite. They represent a mixed population of competitive trends, the composition of which may vary from year to year. It is used here merely as an illustration of the method involved. IS THE METAGON AN m-RNA IN PARAMECIUM AND A VIRUS IN DIDINIUM?* BY IAN GIBSONt AND T. IM. SONNEBORN DEPARTMENT OF ZOOLOGY, INDIANA UNIVERSITY Communicated August 10, 1964 Certain stocks of Paramecium aurelia (syngens 1, 2, 4, and 8) contain in their cytoplasm various symbiotic organisms termed lambda, kappa, mu, etc., and the presence of these organisms results in specific "killer" phenotypes.' The mainte- nance and multiplication of some of the symbionts have been found to depend on a gene or genes in the nucleus of the host paramecium.' In stock 540 (syngen 1) either of two unlinked dominant genes (M, and M2) is necessary for the mainte- nance of mu.2 These genes act via an intermediate, the metagon.3 Active metagons can be introduced into paramecia by exposing them to cell-free extracts of metagon- bearing paramecia.4 Activity of these extracts is associated with the ribosomal fraction and with the RNA component of this fraction.4 Sonneborn5 discovered that kappa could be introduced into another ciliate, Didinium nasutum, by feeding them killer paramecia, and that kappa was thereafter maintained and multiplied even when the didinia were fed nonkillers lacking the gene essential for kappa maintenance. Downloaded by guest on September 27, 2021 870 GENETICS: GIBSON AND SONNEBORN PROC. N. A. S. The present paper describes the behavior of mu in Didinium and the nature of the metagon in Paramecium and Didinium. The evidence indicates that the metagon replicates in Didinium and that metagon RNA hybridizes specifically with DNA from paramecia bearing an M gene. Materials and Methods.-(1) Cultures: The various stocks of Paramecium aurelia used have been described elsewhere,3 as have the methods of culturing the cells in test tubes and larger mass cultures.4 Stock 540 (genotype M1 M1 M2 M2) bears mu particles and is a killer. Branches of it exist free from mu, and these are no longer killers but sensitives, containing, metagons, however. Stocks 513 and d-200 (genotype ml ml m2 m2) are sensitive and lack both metagons and mu. Stock d-200 is largely isogenic with stock 540 except for the m genes; it was derived by back-crossing the progeny of a hybrid (540 X 513) eight successive times to stock 540. Four stocks of Didinium nasutum were used: 1, from Twin Lakes, Indiana; 1*, derived from 1 by growth at 310C long enough to free it from kappa that had been introduced into it;5 2, from General Biological Supply House, Chicago; and 3, from Yellowood Lake, Indiana. Didinium nasutum feeds and thrives on live paramecia. Test-tube and larger cultures of didinia were grown by adding packed paramecia in quantities and with a frequency adjusted to yield the growth rates and population densities appropriate to the various experimental needs. Routinely, 0.3 to 3.0 gm/liter (wet weight) of packed didinia could usually be obtained. (2) Preparation of ribosomal extracts from the two ciliates: The solutions, conditions of homog- enization, and the details of differential centrifugation were essentially the same as used pre- viously4 for Paramecium, with the following modifications. The microsomal pellet obtained at 105,000 X g was treated with sodium deoxycholate and respun at the same force to yield a ribo- somal pellet. Ribosomal extracts from both Paramecium and Didinium were suspended in a solution consisting of 0.01 M Tris, 5 X 10-3 M MgClo, and bentonite 1 mg/ml. The suspension was dialyzed against this same solution overnight and was then stored at -20°C. (3) Preparation of RNA: Ribonucleic acid was extracted from the microsomes using either of two techniques: sodium lauryl sulfate and phenol (technique a)4 or guanidine hydrochloride (technique b).7 These methods gave nucleic acid preparations in which neither DNA nor protein could be detected.6 After precipitation with ethanol, the RNA was dissolved in SSC (0.015 M sodium citrate, 0.15 M sodium chloride) at a final concentration of 2 mg RNA/ml. This solution could be stored at +4°C and used over a period of up to 2 months. (4) Method of detection of metagon activity: The two methods of detecting metagon activity have been described.4 Both involve infection of metagons into cells about to lose mu because of loss of metagons. Method a, the method of 11th-fission cells, employs cells 11 fissions past the change at autogamy from genotype M1 ml m2 m2 to mI ml m2 mi. The number of metagons per cell of the latter genotype decreases until by the 11th fission about 50% of the cells have none, and therefore no mu, and the others have only one or a few metagons but a full complement of 103 or more mu. They are therefore destined to yield only one or a few descendants with a metagon, and only these can maintain mu. The method is therefore to infect with metagons at the 11th fission and examine for presence of mu the eight animals produced after three more fissions. The increased number that possess at least one metagon and therefore mu is a measure of successful infection. Most critical is the number of groups with six to eight of the eight cells possessing mu, for uninfected controls seldom, if ever yield, so many (see Table 1). Method b, the method of RNase treatment, destroys the metagons in M1 M1 M2 M2 mu-bearers by exposure to RNase (0.5 mg/ml) for about 12 hr; mu is then lost at the next fission unless reinfection with metagons occurs earlier, in which case both metagons and mu persist in the clonal progeny. (5) Nucleic acid hybridization: Paramecia were concentrated and the RNA was extracted as above. Electrophoresis on cellulose acetate paper was then carried out under ionic conditions in which a metagon-containing band, a, appeared.7 This was eluted from the paper and the eluate was concentrated to 1 ml by dialysis against polyethylene glycol. This method allowed partial purification of metagon RNA. Hybridization was accomplished using the DNA agar method of Bolton and McCarthy8 with certain modifications. Details will be published in a future paper. DNA was extracted (for method see Gibson7) from Paramecium, Didinium, Tetrahymena, Aerobacter aerogenes (the bac- terium used as food for Paramecium), and purified mu,6 incubated with agar at 1000C, treated with ribonuclease 10 wg/ml in 2 X SSC at 250C for 1 hr, and washed with bentonite solution Downloaded by guest on September 27, 2021 VOL. 52, 1964 GENETICS: GIBSON AND SONNEBORN 871 (1 mg/ml in 2 X SSC). Following incubation of the DNA with RNA containing a known amount of metagon activity and treatment of the mixture with ribonuclease solution and bentonite as above, it was placed in a column, and five 5-ml fractions (unhybridized RNA) were removed with 2 X SSC at 60'C, then a further five with 0.01 X SSC at 750C (hybridized RNA). The fractions in each group were pooled and concentrated to give 3 ml of RNA solution. This was then diluted with 2 X SSC (or in some cases-e.g., with fractions from Didinium-remained undiluted), and a 1-ml sample was used to test 20-50 cells by procedure a or b. The remainder of the solution was stored at -20'C and used if further tests were necessary. The percentage of cells infected gave a relative measure of the amount of metagon activity present. Results.-(1) Fate of mu and metagons in strains of Didinium: One didinium of each of the four strains was fed one killer paramecium (stock 540) and thereafter an excess of sensitive paramecia (stock 513) for 6 months, about 1,000 cell genera- tions.
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