Specific biological activity of deoxyribonucleic acid (DNA) was first estabfished by the experiments of Avery, MacLeod, and McCarty (1). These investigators showed that the substance responsible for the transformation of pneumococcal types was DNA. Current literature contains many examples of the biological activity of DNA including that isolated from bacteriophages. Spizizen (2) and Frazer et al. (3) infected protoplasts of Escherichia coli with subviral extracts from T, cofiphage. Guthrie and Sinsheimer (4) infected protoplasts of E. coli with purified DNA from $X174 phage. Kaiser and Hogness (5) were able to transform E. coli with DNA from kdg phage, providing that nontransducing helper phages were added to the bacteria along with the DNA. More recently Romig (6) infected a transformable strain of Bacillus subtilis with DNA from a phage which he had isolated from soil and designated as SP3.The experiments reported here extend these observations to include the mycobacteriophages. It was found that DNA from mycobacteriophage D29 could infect its host. Bacteria susceptible to infection with intact phage were also susceptible to phage DNA; no alteration in their physiologic state was required. Bu.~ersl--Tri*: Sigma Chemical Company, St. Louis. A 1 m stock solution adjusted to the desired pH between 7 and 9 by adding 10 N HC1 and used in the final concentration of 0.05 or 0.007 M. Materials and Methods
i\iIycobacteriophage D28 propagated on ill. s~~regtuatis ATCC 9626 in the presence of 0.06% Tween had a latent period of 70 minutes, a rise period of 30 minutes, and a burst size of 88. The adsorption rate in nutrient broth was increased by adding any one of several divalent cations to the medi~un, whereas the addition of sodium or potassium decreased adsorption. Approsi~nately SOYo of the adsorbed phage produced abortive infection in the absence of CaC+; of all the cations tested, only calcium increased plaque-forming ability. NIycobacteriophage D29 propagated on il{. stttegrnntis ATCC 607 had a latent period of 65 minutes, a rise period of 30 minutes, and burst size of 104. Calcium and ~uagr~esium, and, to a lesser degree, sodium and potassium, increased its adsorption rate in nutrient broth. However, only calcium increased both adsorption and productive infection.Incremental additions of Tween reduced phage adsorption, diminished burst size, and finally caused abortive infection.
In 1885 Ehrlich (1) discovered that when coerulein-s, an acidic dye, was injected subcutaneously into experimental animals, various organs were stained but the brain was left entirely uncolored. In 1913 Goldman (2) performed a series of now classical experiments which established the concept of the blood-brain barrier. After injecting trypan blue intravenously, he found that vital staining occurred in all the tissues except the brain, which remained uncolored except for the choriod plexus.In all probability, most substances penetrate the central nervous system to some degree. In addition, the blood-brain barrier is not uniform throughout the brain since well defined areas such as the hypophysis, infundibulum, area postrema, and others are readily stained by trypan blue from the circulation. However, the fact remains that many substances, i.e. electrolyte, colloid, vital dye, protein, etc., penetrate the central nervous system parenchyma with great difficulty, whereas the exchange of these substances between vessels and tissues in the rest of the body occurs much more freely. The blood-brain barrier is, in effect, a rate phenomenon and not a true barrier (3). Accumulative evidence indicates that it is a composite mechanism regulating the uptake of materials by the CNS; biochemical and physiological as well as anatomical factors all contribute to its function (eft reference 4).The role of the blood-brain barrier in the humoral spread of neurotropic viruses, and in particular poliovirus, to the CNS has long been a matter for speculation. In infected individuals, only rarely does poliovirus reach the CNS where it produces the paralysis that characterizes overt disease. The portal of entry is the oropharynx and lower intestinal tract; from these initial and/or *
In the previous publication (1) it was reported that deoxyribonucleic acid (DNA) extracted from mycobacteriophage D29 could infect its host, Mycobacterium smegmatis 607. No alteration in the structural integrity of the bacteria such as the production of protoplasts was required; caltures susceptible to intact phage could also be infected with phage D N A .This investigation has been extended to include three additional mycobacteriophages. I n the experiments reported here, D N A extracted from D4 and D32 but not that from D28, was found to be infectious. A t t e m p t s to extend the host range of intact phage with phage D N A were not successful. Materials and MethodsThe source and preparation of reagents, enzymes, buffers, and chromatography procedures were the same as those described in the preceding publication (1). One additional buffer used in this study, Versonate-potassium trichioroacetate (V-KTCA), Nutrional Biochemicals Company, Cleveland, Ohio, was prepared according to Weil (2). Versonate buffer was composed of 0.2 g disodium Versonate, NaCI 8.0 g, KC1 0.2 g, Na2HPO, 1.15 g, KHdaO4 0.02 g, distilled water to 1000 ml. Potassium trichioroacetate: 0.15 ~, pH 7.6. The final V-KTCA buffer was made up of 1.5 parts Versonate and 2.5 parts potassium trichioroacetate. Compounds not previously described (1), will be noted in the text.Pkages.--D28, D29, D32, and D4 were obtained originally from Dr. Seymour Froman.They were isolated by soil-enrichment techniques and except for D4, are active against certain strains of both virulent and saprophytic acid-fast bacilli; D4 is active against Mycobacterium smegmatis 607 (3, 4). Methods for the production of high-titered lysates and for phage assay have been described (5).Baaeria.--Mycobaaerium smegmatis ATCC 607 (propagating host for D4 and for D29), Mycobacterium sp. ATCC9033 (propagating host for D32), and Mycobacterium sp. ATCC 9626 (propagating host for D28) were employed. These bacteria will be referred to in the text by their ATCC numbers. Two additional species of Mycobacterium were employed in hostrange experiments. They were obtained from Dr. Froman and, according to him, one is M.phlei (F 89) and the other M. smegmalis (F 87). The latter can be distinguished from 607 by its phage susceptibility pattern. These bacteria will be referred to in the text as F 89 and as F 87. Stock cultures were maintained, and log-phase cultures prepared as described (5).
207mor cells, it is speculated that immunization has a lethal rather than a static effect on transplanted tumors.Discussion. McKee et aZ.(3) were unable to induce immunity by treatment with Ehrlich's ascites cells that had been killed by desiccation, freezing and thawing, mechanical grinding, and supersonic waves. The possibility exists that the physical-chemical changes of tumor antigens induced by in vitro x-irradiation are extensive enough to make the antigens less homologous and more antigenic for the mouse without destroying antigenic specificity. Indirect evidence for this is manifested by recent observations made in this laboratory. The agglutinins against normal Ehrlich's tumor cells are more readily produced and demonstrated in sera of animals that have been exposed to higher irradiation doses .Summary. Pre-immunization with irradiated Ehrlich's ascites tumor cells protected mice against subsequent ip transplants of viable cells. Immunization procedures started one day after sc challenge with the tumor, significantly reduced mortality. Such post-immunization did not inhibit the rapid multiplication that follows ip transplantation of the tumor. The evidence indicated that the immunization procedures employed resulted in a lethal rather than a static effect on transplanted tumor cells.
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