Stephen Zamenhof

Studies on depurination of DNA by heat

Depurination by heat of DNA and DNA constituents, in solution and in the dry state, has been further investigated. The extent of depurination in solution varies inversely with the ionic strength and p H. Depurination at elevated temperatures in solution appears to be mainly an acid-catalysed hydrolysis. Studies of depurination in the dry state, in the same conditions of heating used to induce mutations in dry cells and spores, indicated that approximately 30 molecules of purine are liberated per molecule of DNA, probably by the pyrolytic breakage of the N-glycosidic bond and/or destruction of the sugar. In solution and in the dry state, the nature and the extent of depurination of DNA by heat differs from that of the constituent deoxymononucleotides. Depurination, which appears to occur throughout the entire molecule of DNA, results in most conditions in a slightly greater liberation of guanine compared with adenine. The energies of activation of depurination of DNA in solution and in the dry state were calculated. The extent of depurination was unaffected by incorporation of 5-bromouracil into the DNA. Correlations between depurination and changes in the viscosity and transforming activity of DNA and the nature and extent of heat-induced mutational sites are discussed.

THE DETERMINATION OF DEOXYRIBONUCLEIC ACID AND OF CELL NUMBER IN BRAIN.

IT HAS been reported that the administration of the pituitary growth hormone to young animals produces an increase in the number of cortical neurons (ZAMENHOF, 1941, 1942) or a hypertrophy of neurons (CLENDINNEN and EAYRS, 1961). A corresponding improvement in maze performance of rats treated early with growth hormone has also been studied (WARDEN, ROSS and ZAMENHOF, 1942; CLENDINNEN and EAYRS, 1961). In these and similar problems it is imperative to devise improved methods for the comparison of total numbers of cells in samples of brain tissue. Since the amount of deoxyribonucleic acid (DNA) per diploid cell of a given species is constant (VENDRELY and VENDRELY, 1949; MIRSKY and RIS, 1951), and since normal neurons and glia cells are essentially diploid (SWIFT, 1950; HELLER and ELLIOTT, 1954), the determination of DNA in brain tissue is often used as a convenient index of the number of brain cells. The examination of some of the existing methods for the determination of the DNA content of tissue (review in HUTCHISON and MUNRO, 1961) reveals that these methods are not applicable to brain tissue for reasons to be discussed subsequently. Nevertheless, such methods have been employed ; not surprisingly, results reported in the literature are often erroneous and inconsistent even when the same species of animal is under consideration. The purpose of this work has been to examine the existing methods of DNA determination and to devise improvements and new methods which will be useful for the determination of the amount of DNA in samples of brain tissue and, therefore, for comparisons of the numbers of cells in such samples.

Study of Microbial Evolution through Loss of Biosynthetic Functions: Establishment of “Defective” Mutants

On appropriate media, mutants which have lost a biosynthetic function may exhibit a selective advantage over parental strains. Is this how “parasitism” became established ?

Studies on the resistance of desoxyribonucleic acids to physical and chemical factors.

Abstract The human and ox DNA were prepared by methods one of which included the addition of DNA having transforming activity, to serve as a marker of “non-denaturation”. The DNAs so prepared were tested for their resistance to heat, ionic strength, dehydration, DNAse, HCHO, H 2 O 2 , Fe +2 , and various other mutagenic or protein denaturing agents. No significant significant species differences in resistance were found. The resistance to heat was found to be considerably higher than in the preparations in which the injurious effects of low ionic strength, dehydration or minute traces of DNAse were not excluded. This loss of stability to heat in specified conditions was found to be a sensitive criterion of injuries to the DNA molecule. The parallel findings of the loss of transforming activity of hemophilus influenzae DNA are compared.

On the desoxypentose nucleic acids from several microorganisms

The isolation of the desoxypentose nucleic acids of three microorganisms, viz. Serratia marcescens, a facultatively autotrophic hydrogen organism Bacillus Schatz, and Hemophilus influenzae, type c, is described. The composition of these substances with respect to the contents of adenine, guanine, cytosine, and thymine was determined; this led to their classification in regard to the DNA types to which they belong. A procedure for the separation of the purines adenine and guanine from the pyrimidines cytosine and thymine by adsorption chromatography on filter paper with water as the solvent has been developed.

The desoxypentose nucleic acids of three strains of Escherichia coli.

Abstract The isolation and composition of desoxypentose nucleic acid preparations from three different strains of E. coli are described. All specimens resembled each other very closely with respect to the relative amounts of adenine, guanine, cytosine, and thymine. The purine and pyrimidine composition was unusual and distinguished these substances from all other DNAs encountered so far: all nitrogenous constituents were found in nearly equimolar amounts.

Purification of the desoxypentose nucleic acid of Hemophilus influenzae having transforming activity

Abstract The purification of DNA preparations of Hemophilus influenzae , types b and c, endowed with transforming activity is described. The mild procedure makes use of electrophoretic separation of the DNA from contaminating PNA and polysaccharides. The final DNA preparations, free of detectable amounts of these contaminants and of proteins, were active in transformation in concentrations of less than 0.0004 μg. and 0.01 μg. DNA/ml. for types b and c, respectively.

Detection and rapid differentiation of glucosamine, galactosamine, glucosamine uronic acid, and galactosamine uronic acid☆

Abstract A method is presented for the rapid differentiation, on a micro scale, of glucosamine, galactosamine, glucosamine uronic acid, and galactosamine uronic acid by paper electrophoresis of their deamination products in borate buffer. A new spray reagent for the specific detection of the 2-amino-2-deoxy sugars is also described; this is based on the color given by their deamination products with indole.

FURTHER STUDIES ON THE UNSTABLE AND “UNNATURAL” DESOXYRIBONUCLEIC ACIDS*

I n the fundamental work of I. K. Lehman and A. Kornberg (see also Bessman ~1 u1.I) on the enzymatic synthesis of desoxyribonucleic acid (DNA), two problems have a connection with the studies my associates and I have been conducting. One of these is related to the utilization of heated DNA as a primer in enzymatic synthesis. We have been subjecting to heat DNA that has transforming activity, and have found that the loss of biological activity always starts together with the loss of viscosity,2 even though different genetic markers in the same preparation have different heat ~tabilities.~ The important question of whether such hcating results in a loss of genetic information could not be answered definitely by these experiments, because a collapsed molecule might conceivably become biologically inactive in the transformation phenomenon and yet retain the information. Another problem is whether the information can change upon heating. We have presented evidence4 that one change that occurs in molecules heated in vitro is their unstabilization: the DNA remains biologically active, but loses its stability to heat. On reproduction, the injury is repaired and the molecules become stable again. Thus, the change was not a “mutation in nitro.” Our attempts to produce a true mutation in nitro (a change in DNA that is retained upon reproduction) have not yet led to positive results. The treatments used are heating, ultraviolet irradiation, and mild deamination with HNOz . Recently, however, we were able to demonstrate a very high mutability and gene unstabilization in vzvo with Escherichia ~ o l i , ~ and have advanced a working hypothesis that this is due to the unstabilization of DNA. Another aspect is the replacement of thymidylic acid by its analogues in the enzymatically synthesized DNA. The problem was to study the replacement of thymine in the DNA of living cells by the thymine analogues and to investigate the biological effects of such replacement.‘j-16 In the case of 5-bromouracil in a thymine-requiring strain, about one half of the thymine can be so replaced.‘j-* A substantial part of the thymine can also be replaced by 5-chlorouracil and 5-iodouracil (but not 2-thiothymine, 5-fluorouracil, or uracil). The amount of replacement, however, is smaller than in the case of 5-bromouracil; this amount is correlated with the similarity in size of the halogen substituent and the methyl group? The replacement of thymine by 5-bromouracil is highest in strains requiring thymine for g r ~ w t h ; ~ however, the replacement can be achieved in other cases of “disturbances” in DNA synthesis. Suchdisturbancesoccur naturally in several mutants (not only thyminerequirers, but also such forms as their “back-mutants”s and uracil-requirers)