Stanislav D. Ehrlich

Analysis of nucleosides on columns of fractionated sephadex G-10.

Abstract An improved procedure for the separation of nucleosides at the 1–10 nmole level on columns of fractionated Sephadex G-10 has been developed. A new experimental set-up has been used to record the chromatogram. The quantitative evaluation of each nucleoside is done using a rapid and precise calculation method involving the use of an Olivetti Programma 102 desk computer.

Radioactive labeling and analysis of 3′-terminal nucleotides of DNA fragments

Abstract 3′-Hydroxy oligodeoxyribonucleotides (average chain length 54 nucleotides), prepared by digestion of calf thymus DNA with spleen acid deoxyribonuclease followed by dephosphorylation, were used as primers for the addition of two ribonucleotides, using terminal deoxyribonucleotidyl transferase and [α-32P]ATP as donor. Conditions for the optimal incorporation of labeled nucleotides were investigated. A method for the separation of the four deoxyribonucleotides and of AMP on DEAE-cellulose columns was devised. This permitted the analysis of 3′-terminal deoxyribonucleotides after spleen exonuclease digestion of the 32P-labeled oligodeoxyribonucleotides, thus avoiding the alkaline splitting of the inter-ribonucleotide bond and the phosphatase digestion which would otherwise be necessary. The results obtained by this procedure were found to be in excellent agreement with the analysis of the 3′-terminal nucleosides released from the same DNA fragments, thus showing that the use of terminal transferase provides a valid method for determining the composition of 3′-terminal nucleotides.

Pyrimidine tracts of the (A + T)-rich satellite DNA from Cancer pagurus.

Abstract The pyrimidine tracts of (A+T)-rich (3 % G+C) satellite DNA from Cancer pagurus have been analyzed. Base pairs involving isolated and clustered T (essentially T2 and T3) form close to 89 and 7 % of the DNA, respectively. The latter are responsible, together with G · C base pairs (about 65 % of which are in isolated positions) for the incomplete renaturability of the satellite DNA. The presence of non-alternating dA · dT sequences explains the anomalously low density of the satellite.