In E. influenzae the highly specific desoxyribonucleic acids (DNA's) which play the role of heredity determinants of type specificity and SM resistance, have induced these traits in only a small proportion of the population exposed to their action. The evidence suggests that this small proportion, "the susceptible cells" possess a property or substance needed by the DNA in order to induce an heritable change. The size of the small proportion of susceptible cells can be influenced significantly by a number of factors; when all the factors now to be listed are operating the frequency has not exceeded 1:1000. The Type of Origin of Recipient Cells.—Type a exhibits the lowest frequency, about 1:10,000,000, and type d the highest, approximately 1 per 1000 cells exposed. This type-specific property which controls the frequency of susceptible cells is an inherited trait; repeated change to a heterologous type shows no influence on the incidence of these cells. Concentration of DNA.—Within certain limits increase in the concentration of DNA controlling streptomycin resistance can increase the size of the proportion of cells in which streptomycin resistance can be induced. However, increases in concentrations greater than 10–1 µg. per ml. have not induced streptomycin resistance in a higher proportion of cells. Phase of Growth Cycle.—Predictable fluctuations in frequency of induced heritable changes have been demonstrated in both Rb and Rd populations during growth. There is no evidence that susceptible cells reproduce their kind; they emerge in all experiments when the population reaches the end of the logarithmic period and a density of 2 to 4 x 108 cells per ml. In the early logarithmic phase it is difficult to demonstrate the presence of susceptible cells. The peak frequency of susceptible cells occurs in the early stationary phase of the growth cycle. Thereafter, the decline in frequency is a gradual one. The data suggest that in a given population the same cells are susceptible to a number of different type-specific DNA's and the DNA controlling SM resistance. Comparison of Frequency of Cells Susceptible to Different DNA's.—In a given population the frequency of cells susceptible to different type-specific DNA's and the DNA controlling streptomycin resistance is not significantly different. Competition between Type-Specific DNA's—The data suggest that DNA's of types a, b, and c compete for the same cells in Rd populations. When Rd populations are exposed simultaneously to 2 of these 3 DNA's in different concentrations the proportion which each type contributes to the total type-specific cells induced is closely correlated with the concentration of the corresponding DNA. Exclusion of DNA's.—Induction of one type specificity or streptomycin resistance can be completely prevented in a population containing susceptible cells by previous exposure for 15 minutes to a 1000-fold higher concentration of another type-specific DNA.
Two genetic traits of Hemophilus influenzae populations, type specificity (1, 2) and sensitivity to streptomycin (3), can be changed with predictable regularity by exposure to desoxyribonucleic acid-containing extracts (DNA) isolated from cells of the type desired. The R cell which has lost all signs of type specificity can be changed to the homologous or a heterologons type (1). Fully equipped type-specific populations derived from a single colony, and homogeneous therefore with respect to the type-specific trait, can be changed directly to a new type by the action of DNA extract from the type desired (2). When both substances essential for the reaction are present, the DNAcontaining extract and the substrate in cells susceptible to the change--the change in the genetic trait occurs in less than 5 minutes. The data of Zamenhof et al. (4) lend support to the premise that the substance responsible for induction of new type-specific traits in H. influenzae populations is the DNA itself. The new trait is inherited; therefore, it would seem that the DNA either modifies the gene or plays the dynamic role which has been attributed to genes. In the process of studying the nature of the cell susceptible to the genetic changes just described, genetic substances which control 2 different type-specific traits were found within the same transformed H. influenzae cell. This report will present data derived from experiments designed to study the process responsible for the phenomenon; the results are consistent with the premise that the action of DNA on susceptible cells may bring about certain changes heretofore thought to occur only after the fusion of cells in sexual reproduction. Materials and Methods Strains of H. influenzae:All strains used were derived originally from a single colony. Rb was derived from type b H. influenzae strain Sb~, by selection of a non-iridescent colony which appeared spontaneously; all signs of type specificity are absent.
In the previous communication (Zamenhof et al., 1953) the study of the inactivation of the transforming principle (deoxyribonucleic acid [DNA] having transforming activity) by several physical and chemical agents has been reported. Some of those agents, especially Fe-++, are known to be potent mutagens; others such as temperature oscillation are reported to be weak mutagens, but they have not been tested on the living cell under drastic conditions, such as heating above 76 C, available for the study of the transforming principle in vitro. Mutagenic action by other
If two microorganisms are to exchange genetic material which produces a heritable change, it is alleged that they must be closely related. This premise is supported by experimental work in bacterial viruses and in recombination in Escherichia coll. Heritable changes which have been induced in microorganisms by transformation have, prior to 1955, been reported only within a species of bacteria. In 1955, two short reports were published on interspecies induction of streptomycin (SM) resistance in Hemophilus (1 a, 1 b, 2). In each report, heterospecific transformation was of a much lower order of frequency than homospecific transformation.The designations "species" and "genus" used in this paper follow the nomenclature of the American Society of Bacteriologists (3). The precise connotations inherent in the term "species" when applied to higher organisms are not implied here. With this reservation, "species" and "genus" will be used in this paper without further qualifications.All evidence on transformation supports the premise that in pneumococci, H. influenzae, and meningococci the active component of the heredity determinant is desoxyribonucleic acid (DNA). The DNA which controls SM resistance in one serological type of H. influenzae has been shown capable of inducing resistance to SM in a heterologous type (4).These results stimulated an exploration of the specificity of the SM resistance transforming agent in an attempt to answer the question: Is the DNA which controls resistance in homologous type or species different from that which controls SM resistance in heterologous species of the genus Hemophilus? The genetic marker of resistance to high concentrations of SM was selected because it may appear in a single mutational step in many species of bacteria and can be induced in transformation experiments in a single step; also, a highly selective environment is available so that transformed cells can be not only detected but also counted accurately.
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