Determination of pyrimidine nucleotide sequences of DNA]

A modification of the Burton method for determination of pyrimidine nucleotide blocks (isopliths) of DNA, providing a higher yield of large-sized nucleotide isopliths, is described. The amount of side products (interisopliths) does not exceed their amount upon DNA hydrolysis according to the Burton method. Another advantage of the technique recommended is a considerable shortening of hudrolysis time (20 min instead of 18 hours). The modification described has been successfully used to determine the pyrimidine nucleotide blocks of some warm-blooded animals DNAs. It has been found that the DNA of animals with higher sensitivity to ionised irradiation contains more oligothymidylic sequences as compared to the DNA of animals, less sensitive to irradiation.     

DNA polymerases from non stimulated and phytohemagglutinin stimulated normal human lymphocytes

The presence and some properties of DNA polymerases isolated from normal human lymphocytes, non stimulated and stimulated by phytohemagglutinin, are described. In the non stimulated lymphocytes two cytoplasmic DNA polymerases are found, one eluting from DEAE cellulose at 0.07 M NaCl (CI_n) and the other at 0.13 M NaCl (CII_n). In the nuclear soluble fraction only one enzyme activity is found (NI_n) which does not adsorb to DEAE cellulose. In the cytoplasm of stimulated lymphocytes only one enzyme activity is detected (CI_s) which elutes from DEAE cellulose at 0.12 M NaCl. The nuclear soluble fraction contains two activities, NI_s, which does not adsorb to DEAE cellulose, and NII_s, which elutes from DEAE cellulose at 0.07 M NaCl. Some properties of the different enzymes are described which indicate that NI_n and NI_s enzymes are clearly different from the others.     

Interspersion and transcription of repeated sequences of rat DNA.

Repetitive rat DNA reassociated to Cot=0.1 and deprived of "foldback" sequences showed close interspersion with unique sequences. As measured by electron microscopy, the average length of repetitive segments was about 600 +/- 400, and of unique segments 1800-3600 base pairs. Pyrimidine tracts over 80 nucleotides in length were found mainly in foldback and repetitive fractions. Oligo(dT) tracts, 20-30 bases in length prevailed in the DNA fraction reassociated to Cot=0.1. Repetitive and unique DNA fractions were annealed to Millipore filters and hybridized with hnRNA. Up to 1.6% of repetitive DNA reassociated to Cot=0.05 showed base complementarity with hnRNA, whereas the comparative figures for DNA reassociated to Cot=10 and for the unique fraction were 0.8% and 0.3% respectively. When hybridization of hnRNA was carried out in solution in vast DNA excess, no hybrid formation with repetitive sequences reassociated to Cot=0.1 was observed, although hybridization with DNA reassociated to Cot=10 was noticeable.     

Analysis of highly repeated DNA sequences of rat with EcoR1 endonuclease

Cleavage of rat liver nuclear DNA with EcolR1 restriction endonuclease yields 14 discrete fragments ranging from 2300 to 93 base pairs in length, representing approx. 10.5% of the rat genome. Fragments of 1500, 180, and 93 base pairs are reiterated over 100 000 times; fragments of 2300, 880, 290, and 200 base pairs are reiterated over 20 000 times; the remaining fragments are present in over 1000 copies per genome. When compared to whole rate DNA, 11 were 1-5% richer in A . T base pairs and five were 1.5-2.5 times more methylated. From the criteria of the banding patterns in complete and incomplete digests, base composition and extent of methylation, none of these fragments appeared to be generated as oligomers of a basic shorter repeat. The reassociation of EcoR1 fragments was monitored on hydroxyapatite and by S1 nuclease treatment in order to assess band reiteration frequency and the possibility of interpersion or short internal repeats. The renaturation of the four smallest EcoR1 fragments gave no indication of short internal repeats from hyperpolymer formation nor interpersion with lower frequency sequences by size reduction after S1 nuclease treatment. Anomalous renaturation of several large fragments was observed, possibly due to internal repeats.     

Conservation of repeated DNA sequences in aneuploid human tumor cells

A series of human neuroectodermal tumors, all containing more than the normal diploid DNA, and each with its own distinct chromosome mode, were studied using restriction enzyme cleavage and specific DNA sequence hybridization. Methods described were quite sensitive and quantitative and as few as 40 molecules with a given restriction site were reproducibly detected in total nuclear DNA. Analysis of several fluorescent gel bands associated with different chromosomal domains revealed no changes between any of the tumor and normal cells. Specific probe hybridization, using purified complex repeating sequences, indicated fidelity of base sequence, as well as preservation of the relative amounts of each of a number of minor related multimers in both the tumor and normal cells. Centromeric regions containing arrays of such sequences may be maintained in these tumor cells and furthermore it is possible that some of these cells are polyploid with respect to DNA sequences, rather than aneuploid as their chromosome profiles suggest.This paper is dedicated to the late H.S.N. Greene, our inspired teacher     

Effects of arsenic on DNA synthesis in human lymphocytes stimulated by phytohemagglutinin

Effects of arsenic on DNA synthesis in human lymphocytes were biphasic: either trivalent (arsenic trioxide and sodium arsenite) or pentavalent (sodium arsenate) arsenic compounds at very low concentrations enhanced DNA synthesis in human lymphocytes stimulated by phytohemagglutinin (PHA), whereas higher concentrations inhibited DNA synthesis. There were differences among individual susceptibities to arsenic-induced DNA synthesis. Either stimulating or inhibiting effects of trivalent arsenic on DNA synthesis in PHA-stimulated lymphocytes were always stronger than those of pentavalent arsenic. It was also shown that both trivalent and pentavalent arsenic could be rapidly taken up into the human lymphocytes, and immediately stimulated or inhibited DNA synthesis. A possible dual effect of arsenic at very low concentrations as both comutagen and inhibitor of mutagenesis is discussed.     

Organization of nucleotide sequences in maize DNA

Nucleotide sequence organization in the genome of maize has been studied using renaturation kinetics of DNA and S-1 nuclease digestion of the renatured products. Approximately 40% of the genome consists of single copy sequences, and 15% of these sequences are interspersed between repeated sequences and are approximately 1100 nucleotide pairs long. About 54% of the genome consists of repeated sequences. Six per cent of the genome consists of foldback sequences. These sequences are distributed through at least 44% of the genome. It was found using renaturation kinetics that the sum of foldback and highly repeated DNA fractions of Dobrudzhanko maize and inbred lines differ in the amount of DNA composing the fractions. Comparison of the DNA of the Dobrudzhanko maize and inbred lines by the method of DNA-DNA hybridization indicates strong differences in the amount of polynucleotide homologies between the Dobrudzhanko maize and the D1 inbred line on one hand and the A619 inbred line on the other hand.