Edna Seaman

CHARACTERIZATION OF A DEOXYRIBONUCLEASE OF MUSTELUS CANIS LIVER.

Summary The presence of an enzyme which hydrolyzes thermally-denatured T4 DNA has been demonstrated in extracts obtained from Mustelus canis livers. The enzyme was detected by its ability to decrease the serologic activity of thermally-denatured T4 DNA when tested with experimentally produced antibodies to denatured T4 DNA. This unique assay was used during purification and characterization of the enzyme. Thermally-denatured DNA was shown to be the preferred substrate for the enzyme. Selective destruction of denatured DNA by the enzyme was demonstrated when mixtures of native and thermally-denatured T4 DNA were exposed to the nuclease and then subjected to CsCl 2 centrifugation. The activity towards thermally-denatured DNA was optimal in 0.1 M NaCl. It showed a requirement for divalent metals which could be satisfied by Mg 2+ , Mn 2+ , and Co 2+ . The nuclease has a pH optimum of 8.0. The rate of hydrolysis of thermally-denatured DNA suggested that the digestion proceeded endonucleolytically.

Immunological Evidence for the Existence of Thymine Dimers in Ultraviolet Irradiated DNA

Rabbits immunized with complexes of methylated bovine serum albumin (MBSA) and denatured DNA produce antibodies directed toward DNA (Plescia et al., 1964). These antibodies, like those described for the glucosylated DNA of the T-even phages (Levine et al., 1960) react with the denatured antigen; a conformation in which the bases are not involved in hydrogen bonding and are therefore available for interaction with the antibody. The antibodies to the T-even phage DNA’s are highly specific for the glucosylated cytosine moieties (Murakami et al., 1961; Townsend et al., 1965) and can be used to determine the extent of phage DNA synthesis in T4-infected E. coli cells even though host cell DNA is present in large excess (Levine et al., 1960).

Immunological Evidence for the Identity of a Photoproduct Formed during Photooxidation of DNA with Methylene Blue, Rose Bengal, Thionin, and Acridine Orange

Irradiation of DNA with visible light, in the presence of oxygen and a photosensitizing dye such as methylene blue, is known to destroy specifically the guanine residues in nucleic acids (Simon and Van Vunakis, 1962). The chemistry of the photooxidation reaction is being studied actively in several laboratories (cf. Bellin and Grossman, 1965) but it is not yet understood. It is known that the photoproducts may vary depending upon the composition of the reaction mixture. Immunological experiments provided the first evidence that, during the photooxidation of DNA, a reactive photointermediate condensed with the available amino groups of the buffer, Tris(hydroxymethyl)amino methane (Tris) to yield an adduct (Van Vunakis et al., 1966). The evidence that established the existence of this adduct will be reviewed only briefly. DNA was photooxidized in Tris buffer at pH 8.5 using methylene blue as the photosensitizing dye. DNA thus treated (DNATris-MB) was complexed with methylated bovine serum albumin (Plescia et al., 1964) and injected into rabbits. The serologic activities of native and denatured DNAs with the anti-DNATris-MB became apparent as the reaction mixture containing DNA, Tris buffer and methylene blue was irradiated with visible light.

Antibodies to the Terminal Moieties of Guanine Mononucleotides and Guanine-Containing Dinucleotides

Antibodies specific for nucleic acids have been obtained by immunizing rabbits with conjugates composed of bases, nucleosides or nucleotides covalently linked to proteins or polyamino acids. The chemical methods used to prepare such immunogens have been reviewed (Levine and Stollar, 1967) and the characteristics of some of these antibodies are considered in this volume.