Barbara S. Vold

Nucleotide sequence of the Bacillus subtilis ribosomal RNA operon, rrnB.

The primary sequence of DNA covering a complete ribosomal RNA (rRNA) operon from Bacillus subtilis, designated rrnB has been elucidated. Following a set of tandem promoters, rrnB encodes: (i) a 16S and a 23S rRNA determinant with no tRNA spacer region in between; (ii) a 5S rRNA determinant; and (iii) 21 contiguous tRNA species; before (iv) two characteristic terminator hairpins are found. More than 7 kb are included within this operon, which maps between aroG and thr5 on the B. subtilis chromosome. This represents the first report of the entire sequence of an rRNA operon from B. subtilis or from any Gram-positive organism.

Radioimmunoassays for the modified nucleosides N[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine and 2-methylthioadenosine.

Radioimmunoassays were established for the modified nucleosides N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine, t6A, and 2-methylthioadenosine, ms2A. The assays depended on the production of antisera specific for t6A and ms2A that have not been previously reported. The nitrocellulose membrane filtration and saturated ammonium sulfate RIA techniques were compared for efficiency. Various radioactive antigens were employed to establish which type of antigen would give the best binding. The tritium post-labeling procedure of Randerath and Randerath was used to obtain labeled nucleosides of high enough specific activity to be useful for RIAs when the labeled nucleoside was not available commerically. The specificity of the antibodies toward nucleosides and purified tRNAs is reported. Although the titer of the t6A antiserum was low, the specificity was very sharp. An interesting finding was that threonine, a major structural component of the side-chain modification of t6A, was completely infective as an inhibitor.

Chapter 9 Immunoassays for Modified Nucleosides of Ribonucleic Acids

Publisher Summary This chapter presents a survey of antibodies directed toward modified ribonucleosides and their basic and clinical applications. Immunoassay methodology for radioimmunoassays and enzyme linked immunosorbent assays applicable to modified nucleosides are presented.. These methods are used to characterize polyclonal and monoclonal antibodies directed toward the modified nucleosides N 6 -[9-( β -D-d)-6-ylcarbamoyl]-L-threonine, t 6 A; N6-(∆ 2 -isopentenyl)adenosine, i 6 A;2ʹ-O-methyl guanosine; and zeatin. Two illustrations are presented for quantitation of nucleoside levels using immunoassay technology. One is a method for quantitating levels of modified nucleosides in tRNAs from bacterial extracts, using as an example the Escherichia coli mutant miaA that is deficient in i 6 A derivatives. This procedure can be adapted to screening for either prokaryotic or eukaryotic cells deficient in a specific modification on transfer RNA (tRNA) or ribosomal ribonucleicacid (rRNA). Quantitation of levels of modified nucleosides in human urine is described and polyclonal antibodies directed toward three different modified nucleosides are studied using urine samples from normal subjects and patients with cancer. The nucleoside t 6 A shows promise for monitoring the transition to the metastatic stage for breast cancer patients.