Julius Marmur

A procedure for the isolation of deoxyribonucleic acid from micro-organisms†

A method has been described for the isolation of DNA from micro-organisms which yields stable, biologically active, highly polymerized preparations relatively free from protein and RNA. Alternative methods of cell disruption and DNA isolation have been described and compared. DNA capable of transforming homologous strains has been used to test various steps in the procedure and preparations have been obtained possessing high specific activities. Representative samples have been characterized for their thermal stability and sedimentation behaviour.

Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature

The previously discovered linear relation between the base composition of DNA, expressed in terms of percentage of guanine plus cytosine bases, and the denaturation temperature, T m , has been further investigated. By means of measurements on 41 samples of known base composition the previously observed relation has been confirmed. It can be summarized thus : for a solvent containing 0·2 M -Na + , T m = 69·3 + 0·41 (G-C) where T m is in degrees Centigrade and G-C refers to the mole percentage of guanine plus cytosine. The deviations of experimental points from this relation are no more than that expected from the uncertainties of base analysis and the variations of a half degree in the reproducibility of determining the T m . Consequently it appears that the measurement of the T m is a satisfactory means of determining base composition in DNA. The T m values are most simply measured by following the absorbance at 260 m μ as a function of temperature of the DNA solution and noting the midpoint of the hyperchromic rise. Only 10 to 50 μ g of DNA are required. A number of other DNA samples of unknown base composition have been examined in this manner and their base compositions recorded.

Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl.

A comprehensive study of the buoyant density of DNA as a function of composition has been made. The linear relation previously reported has been confirmed. Based on a value of 1·710 g cm −3 for DNA from Escherichia coli the following relation was obtained from the best fit of measurements on 51 different DNA samples: ρ = 1 ⋅ 660 + 0 ⋅ 098 ( G C ) where ρ refers to buoyant density and (GC) to the mole fraction of guanine plus cytosine. On this basis the composition of DNA from 36 other sources, not previously reported, has been estimated. Several specific observations were made. Bimodal distributions in the density-gradient band patterns were found in DNA from calf thymus and salmon sperm. The DNA of the commonly studied T-even bacteriophages exhibits altered densities due to the presence of gluco-sylated hydroxymethylcytosine. The DNA of φ X174 phage is abnormally heavier suggesting less base-pairing than normal denatured, single-stranded DNA.

Thermal renaturation of deoxyribonucleic acids

The conditions that produce the optimal re-formation of the native DNA conformation from denatured DNA have been examined. The restoration of the native conformation, called thermal renaturation, has been found to depend markedly on the source of the DNA; mammalian DNA coming from cells with very large DNA content renatures only slightly, bacterial DNA with greatly reduced DNA content per cell undergoes extensive renaturation, and the very smallest bacteria together with bacteriophage, having the lowest DNA contents, show nearly complete renaturation. With a given DNA, the optimal renaturation was found to occur at about 25° below the denaturation temperature, T m . The extent of renaturation was optimal above 0·4 M -Na + and increased with molecular weight. The identity of the renatured DNA and the native material can be shown in two ways: the similarity of the absorbance-temperature curves and the similarity of the rate of thermal inactivation of biological markers at temperatures somewhat above T m . This reproduceability of the helix-coil transition and the course of thermal inactivation demonstrates that the same secondary structure has re-formed and that non-specific hydrogen bonding is not involved.

A study of the base sequence homology among the T series of bacteriophages

Abstract When a mixture of two DNA samples is denatured and then renatured, single strands from the two types of DNA will specifically recombine if the base sequences are essentially the same. Such hybrid DNA molecules can be identified in density gradient ultracentrifugation if one of the samples has been made heavy. Using 5-bromouracil substitution or N15 and D replacement, heavy DNA from T4 and T7 bacteriophages was prepared and used with normal DNA from T2, T3, and T6 bacteriophages in a test of sequence homology. A high degree of homology was found between pairs of T-even phage DNA and between the DNA from T3 and T7. No indication of homology was found between DNA from T-even and T-odd phages, nor between these and Escherichia coli DNA. These results are in general agreement with genetic evidence except that genetic exchange between T3 and T7 bacteriophages has not yet been reported.