Yo Kikuchi

Hairpin plasmid ― a novel linear DNA of perfect hairpin structure

The terminal structures of deletion derivatives of linear DNA killer plasmid from yeast were analyzed. The yeast Kluyveromyces lactis harbors two unique double‐stranded linear DNA killer plasmids, pGKL1 of 8.9 kb and pGKL2 of 13.4 kb. The killer toxin and the resistance to the killer are coded by pGKL1, while pGKL2 is required for the maintenance of pGKL1 in the cell. When the pGKL plasmids from K. lactis were transferred into Saccharomyces cerevisiae by transformation, non‐killer transformants harboring pGKL2 and new plasmids, F1 of 7.8 kb and F2 of 3.9 kb, were obtained. F2 was shown to be a linear DNA arising from a 5‐kb deletion of the right part of pGKL1. F1 was an inverted dimer of F2. Here we show that F2 has two different terminal structures: one end has a protein attached at the 5′ terminus whereas the two strands of duplex are linked together at the other end, thus forming a hairpin structure. This is a novel type of autonomously replicating DNA molecule.

Ligation of endogenous tRNA 3' half molecules to their corresponding 5' halves via 2'-phosphomonoester,3',5'-phosphodiester bonds in extracts of Chlamydomonas.

tRNA preparations from Chlamydomonas and wheat germ contain small amounts of tRNA 5′ halves and corresponding 3′ halves. Incubation of cell‐free extracts from the two sources with [γ‐32P]ATP yielded 5′‐32P‐labeled tRNA 3′ halves which were joined to their corresponding 5′ counterparts to form mature tRNA containing 2′‐phosphomonoester,3′, 5′‐phosphodiester bonds. tRNA 3′ halves labelled with T4 kinase were purified, sequenced and also joined to their 5′ counterparts. It is proposed that these tRNA halves may be intermediates of the tRNA splicing process, and that the RNA kinase and ligase activities observed here are part of the tRNA splicing complex.

An efficient synthesis of polyguanylic acid by a thermophilic polynucleotide phosphorylase

Polyguanylic acid (poly(G)) was synthesized from GDP in a yield of 60-75% by Thermus thermophilus polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) at 70 degrees C, pH 8.5 in the presence of Mg2+. The yield was dependent on the ratio of GDP to Mg2+, but was independent of the concentrations of enzyme or substrate. The maximal rate of GDP polymerization was obtained when the ratio of GDP to Mg2+ was 3:1. However, by prolonged incubation, the higher initial ratio of over 4:1 was preferred because of the rapid consumption of GDP in the reaction mixture. Poly(G) prepared by 1 h incubation was heterogeneous in size from 5 S to over 23 S, but by prolonged incubation of 19 h the size of product converged to 9 S as judged by sucrose density gradient centrifugation. Its chain length was determined by terminal nucleoside analysis to be 200 nucleotides long.