Viable bacteria were found in permafrost core samples from the Kolyma-Indigirka lowland of northeast Siberia. The samples were obtained at different depths; the deepest was about 3 million years old. The average temperature of the permafrost is -10 degrees C. Twenty-nine bacterial isolates were characterized by 16S rDNA sequencing and phylogenetic analysis, cell morphology, Gram staining, endospore formation, and growth at 30 degrees C. The majority of the bacterial isolates were rod shaped and grew well at 30 degrees C; but two of them did not grow at or above 28 degrees C, and had optimum growth temperatures around 20 degrees C. Thirty percent of the isolates could form endospores. Phylogenetic analysis revealed that the isolates fell into four categories: high-GC Gram-positive bacteria, beta-proteobacteria, gamma-proteobacteria, and low-GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and beta-proteobacteria, and all gamma-proteobacteria, came from samples with an estimated age of 1.8-3.0 million years (Olyor suite). Most low-GC Gram-positive bacteria came from samples with an estimated age of 5,000-8,000 years (Alas suite).

No abstract

Purified preparations of the tRNA methylase deficient in supK strains of Salmonella typhimurium transfer methyl groups from S-adenosylmethionine (SAM) to at least two tRNA species, an alanine tRNA and a serine tRNA. The identity of the tRNA substrates for this enzyme was determined by a change in the elution position of the methyl-labeled tRNA from BND-cellulose columns before and after aminoacylation with a specific amino acid followed by derivatization of the free primary amino group with phenoxy- or naphthoxyacetate. The radioactive methyl group enzymatically added to these tRNAs is both acid and base labile and can be hydrolyzed to a volatile product at pHs above 7.5 and also at pH 1. The methylated 3'-nucleotide isolated from digested tRNA is a pyrimidine derivative and chromatographs like a modified uridylic acid. Its identity has not been established, but it is likely that it corresponds to the methyl ester of V, uridin-5-oxyacetic acid.

The effect of magnesium ions on the conformation of two highly purified yeast alanine tRNAs has been studied by optical methods. The results indicate that both have base

Isoleucine and alanine tRNAs are encoded tandemly within the 16S-23S intergenic spacer of some eubacterial rRNA gene clusters. Southern hybridization analysis and DNA sequence analysis demonstrated a novel gene organization for an rRNA gene cluster on the Streptococcus pneumoniae chromosome. A sequence specifying an alanine tRNA was found within the intergenic spacer, but no sequence specifying an isoleucine tRNA was found there. Southern hybridization analysis indicated that the location of the isoleucine tRNA gene was near the 5S rRNA gene in two of four rRNA gene clusters.Typically, the gene organization of eubacterial rRNA gene clusters is 5'-16S-23S-5S-3' (2 and references within; 20), although several exceptions have been observed (12,21,23 Southern hybridization analysis of rRNA and spacer tRNA genes. Total DNA was extracted (11) and purified from late-log-phase cultures of S. pneumoniae (ATCC 33400). The concentration of the DNA preparation was determined by the diphenylamine assay as previously described (6). In separate reactions, 1 ,ug of total DNA was digested with either HindlIl or PstI. Conditions for the digestions were those specified by the supplier of the restriction endonucleases (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). The restriction digests and lambda HindIll fragments were electrophoresed in a 1% agarose gel. The fractionated DNA was transferred to a solid support membrane (Nytran; Schleicher & Schuell, Inc., Keene, N.H.) as previously described (19). The membrane-bound DNA fragments were sequentially probed with 5'-end-labeled [y-32P] ATP oligodeoxyribonucleotides specific for the three rRNA genes and the tRNAIle and tRNAAla genes. Probes are described in Table 1. The 5S rDNA probe was mixed, in that the sixth nucleotide was synthesized with equimolar ratios of all four deoxyribonucleotides, because of the variability at that position in eubacterial 5S rRNAs. Conditions for prehybridizations, hybridizations, and probe removal were those specified by the supplier of the hybridization membrane (Schleicher & Schuell). Figure 1 shows the results of the hybridization experiments. Estimated sizes of the DNA fragments that hybridized with the probes are presented in Most striking of our observations was the lack of similarity between the hybridization patterns generated from the tDNAIIe and tDNAAla probes. Two signals corresponding to 6.1 and 3.5 kb in the HindIII-digested DNA were observed from the hybridization with the tDNAI'e probe. Four signals corresponding to 6.1, 3.5, 3.2, and 2.5 kb were observed when the 5S rDNA probe was hybridized against the HindIII-digested DNA. Two signals corresponding to 8.9 and 7.1 kb in the PstI-digested DNA were observed from the hybridization with the tDNAIle probe. Four signals corresponding to 20.0, 8.9, 7.8, and 7.1 kb were observed following hybridization of the 5S rDNA probe to PstI-digested DNA. Thus, the two signals from the tDNAIle hybridization correspond to two of the four signals from the 5S rDNA hybridization (HindlIl-or PstI-di...