Marianne Dieckmann

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>108 cts/min per μg) in vitro using deoxynucleoside [α-32P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.

Competition between RNA polymerase and DNA polymerase for the DNA template

Abstract RNA polymerase reacts with native and denatured DNA to give relatively poorly dissociated complexes. Such complexes are unavailable as templates for replication by DNA polymerase or as substrates for exonucleases I, II, or III, although they are readily degraded by micrococcal nuclease of E. coli endonuclease I. We interpret these findings to mean that RNA polymerase binds strongly at or near the 3′-hydroxyl terminus of helical DNA chains.

The enzymic synthesis of amino acyl derivatives of ribonucleic acid. VI. Nucleotide sequences adjacent to the ... pCpCpA end groups of isoleucine- and leucine-specific chains.

The amino acid-acceptor ribonucleic acid chains specific for leucine and isoleucine were labeled with 32P at the 3′-hydroxyl end of the chain by replacing the terminal trinucleotide, ..pCpCpA, with 2 equivalents of CM32P to yield RNA..32pC32pC. Analysis of the labeled fragments produced by T1 RNase digestion of the isoleucine-specific labeled RNA preparation revealed that the chains which bind isoleucine are terminated by the sequence ..pGpCp(UpC)pApCpCpA. A similar digestion of the leucine-specific labeled RNA showed the existence of two types of chains and these contain ..pGpCpApCpCpA and ..pGpUpApCpCpA as terminal nucleotide sequences.