Urine levels of N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine, N6-(delta 2-isopentenyl)adenosine, and 2'-O-methylguanosine as determined by radioimmunoassay for normal subjects and cancer patients.

Radioimmunoassays (RIA) are presented for the evaluation of the levels of the following three modified nucleosides in human urine: 2'-O-methylguanosine (Gm), N6-(delta 2-isopentenyl)adenosine (i6A), and N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine (t6A). Competitive inhibition of the RIA was provided by 2 to 10 microliters of untreated urine and the sensitivity of each RIA was in the pmol range. Partial fractionation of urine indicated that the majority of inhibitory activity was in the fraction coeluting with a nucleoside standard. The amounts of nucleosides in 24-hr urine samples from eight normal subjects were 2.2 +/- 0.9 mg (S.D.) for t6A; 0.17 +/- 0.09 mg for Gm; and 0.050 +/- 0.019 mg for i6A. The levels of t6A, i6A, and Gm were also determined by RIA of urine samples of patients with lymphomas or solid tumors. Levels of t6A were significantly elevated for patients with lung cancer (p less than 0.001), non-Hodgkin's lymphoma (p less than 0.05), and other solid tumors (p less than 0.02) but not for patients with Hodgkin's disease. The RIA data on the other two nucleosides, i6A and Gm, showed no similarly significant variations. Increased levels of t6A in the cancerous state were substantiated by isolating the t6A fraction from the urine of normal subjects of patients with lung cancer and quantitating the amount by use of UV adsorption. These preliminary results indicate that RIA for t6A might be clinically useful by providing a complementary approach to the assessment of the levels of modified nucleosides by gas-liquid or high-performance-liquid chromatography.

Use of a monoclonal antibody to detect elevated levels of a modified nucleoside, N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine, in the urine of breast cancer patients.

Monoclonal antibodies to the modified nucleoside N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine (t6A) have been produced and characterized. These antibodies were utilized in a radioimmunoassay to quantitate the levels of this modified nucleoside in the urine of patients with benign breast disease, primary breast cancer, and metastatic breast cancer. Eighty-nine % (16 of 18) of patients with metastatic breast cancer excreted higher quantities of t6A than did patients with benign breast disease or primary breast cancer. This modified nucleoside represents a new, potential marker not previously included in similar studies using high-performance liquid chromatography. The immunoassay approach for quantitating this nucleoside has the advantage over high-performance liquid chromatography in that the former is more compatible with a clinical laboratory setting, and it does not require pretreatment of the urine or sophisticated analytical equipment. The elevation of t6A levels in metastatic breast cancer patients indicates that it may be a more sensitive marker than previously studied modified nucleosides. Furthermore, t6A might be particularly useful for monitoring transition to the metastatic stage in patients already diagnosed as having breast cancer.

Preparation and specificity of antibodies directed toward the ribose methylated nucleotide, 2'-O-methylguanosine 5'-monophosphate.

Antibodies have been produced that recognize the ribosome methylated nucleotide, 2'-O-methylguanosine 5'-monophosphate, pGm. Specificity was tested in a radioimmunoassay. Other ribose methylated nucleotides, deoxyguanosine, and guanosine 5'-monophosphate exhibited negligible cross-reactivity. Elements of antibody recognition, in descending order of importance, were the parent base, the methylated ribose moiety, and the phosphate group.

Promoter used by sigma-29 RNA polymerase from Bacillus subtilis.

Gene expression during endospore formation by Bacillus subtilis is controlled in part by a sporulation-induced form of RNA polymerase, E sigma 29. The determination of the nucleotide sequences that govern utilization of promoters by E sigma 29 has been limited by the small number of available promoters that are recognized by E sigma 29. In the present report we describe a promoter that is adjacent to the rrnB region of the B. subtilis chromosome and is utilized in vitro and in vivo by E sigma 29. S1 nuclease mapping and dinucleotide priming experiments have been used to determine the start point of transcription. The nucleotide sequences near the -10 and -35 region of this promoter, bvx, are conserved, and resemble sequences at these regions for other promoters that are utilized by E sigma 29.

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.

Activity of ribosomal and tRNA promoters of Bacillus subtilis during sporulation.

The rrnB operon in Bacillus subtilis includes 21 tRNA genes downstream of the ribosomal genes. In addition to the dual promoters upstream of these ribosomal genes, a second promoter is found within the tRNA gene region. In this study, these two promoter regions were inserted before the lacZ gene and each was integrated as a single-copy into the B subtilis chromosome to examine their activity during sporulation. Both promoters exhibited similar strength and temporal downregulation from vegetative growth through t3 of sporulation. At t3, transcription from both promoters was approximately 20% of that in logarithmic growth. No obvious function of the second promoter is evident except to boost transcription of downstream genes. This function may be important because the ribosomal promoters of rrnB are weak relative to other ribosomal promoters. For instance, the rrnO promoters were much stronger than the rrnB ribosomal promoters and considerably more active at t3. Thus, all ribosomal promoters are not of the same strength nor are they transcriptionally downregulated to the same extent during sporulation. However, both the rRNA and tRNA promoters in the rrnB operon are similar in strength and downregulation.

Production and characterization of antibodies and establishment of a radioimmunoassay for ribosylzeatin.

An antibody directed towards ribosyl-trans-zeatin has been produced and characterized. The antiserum was produced in rabbits using ribosyl-zeatin-bovine serum albumin as an immunogen. A radioimmunoassay which employed this antiserum and a tritiated antigen was established. As little as 10 picomoles ribosyl-trans-zeatin could be detected. The specificity of the antiserum was measured in the radioimmunoassay by using nonradioactive nucleosides as competitive inhibitors. Changes in position N(6) were more effective in decreasing antibody recognition than changes in position 2. Of particular interest was the interaction of the isomer ribosyl-cis-zeatin. This compound was significantly less active as an inhibitor than ribosyl-trans-zeatin, demonstrating that the antibody was sensitive to minor changes in the structure of the antigen.The use of this antibody and the radioimmunoassay for ribosylzeatin provides a rapid method for the detection of ribosylzeatin, as well as offering the potential for immunoadsorbent columns which would be useful in the purification of macromolecules, such as tRNA, which contain the ribosylzeatin moiety.

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.

Analysis of isoaccepting transfer ribonucleic acid species of Bacillus subtilis: chromatographic differences between transfer ribonucleic acids from spores and cells in exponential growth.

Differences between the transfer ribonucleic acid (tRNA) of spores and exponentially growing cells of Bacillus subtilis 168 were compared by co-chromatography on reversed-phase column RPC-5. This system gave excellent resolution of isoaccepting species in 1 to 2 hr using a 200-ml gradient. Two methods were used to extract spore tRNAs, a procedure using a Braun homogenizer and a pretreatment with dithiothreitol followed by lysis with lysozyme. Where changes were observed, column elution profiles of spore tRNAs were independent of the extraction method used. Three kinds of changes between the profiles of vegetative cell tRNA and spore tRNA were observed: (i) no change; phe-, val-, ala-, asp-, ileu-, pro-, met-, fmet-, and his-tRNAs, (ii) a change in the ratio of existing peaks; gly-, tyr-, leu-, ser-, thr-, aspn-, and arg-tRNAs, and (iii) the appearance or disappearance of unique peaks; lys-, glu-, and trp-tRNAs.

Analysis of isoaccepting transfer ribonucleic acid species of Bacillus subtilis: changes in chromatography of transfer ribonucleic acids associated with stage of development.

Changes in chromatographic profiles of tyrosyl-, leucyl-, tryptophanyl-, and lysyl-transfer ribonucleic acids (tRNAs) are presented as a function of the growth stage in Bacillus subtilis. All of the tRNA groups investigated expressed different temporal patterns of change in isoaccepting species. Tyrosyl-tRNAs were the earliest to change and were followed by changes in leucyl- and then tryptophanyl-tRNAs. Lysyl-tRNAs were unique in having two times of change: one early and one very late. As an aid in understanding the temporal aspect of tRNA alterations during sporulation, the chromatographic profiles of aminoacyl tRNAs from an early blocked asporogenous mutant were studied. The asporogenous mutant used was blocked at the axial filament stage, stage 0 of sporulation. Nevertheless, those tRNAs which showed differences between the spore and cells in exponential growth exhibited similar changes in the asporogenous mutant after 24 h of growth. The data suggest that several tRNA changes occur during development in B. subtilis but that the events leading to these changes are either independent of, or occur before, stage 0 of sporulation, except in the case of lysyl-tRNA.

Comparison of lysyl-transfer ribonucleic acid species from vegetative cells and spores of Bacillus subtilis by methylated albumin-kieselguhr and reversed-phase chromatography.

Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons.

Post-transcriptional modifications of the anticodon loop region: alterations in isoaccepting species of tRNA's during development in Bacillus subtilis.

Structural similarities of tRNA's were compared using three sets of isoaccepting species that had previously been shown to undergo significant changes in chromatographic elution properties as a function of developmental stage in Bacillus subtilis. Comparisons of the structures of the tRNA's were based on the composition of their modified nucleosides, comparisons of oligonucleotide elution profiles from RPC-5 columns, and two-dimensional electrophoretic fingerprint analysis of oligonucleotides. The tRNA's studied were tRNA(Lys) (1) and tRNA(Lys) (3); tRNA(Tyr) (1) and tRNA(Tyr) (2); and tRNA(Trp) (1) and tRNA(Trp) (2). The results suggest that the difference among these pairs of isoaccepting species is a difference in the degree of post-transcriptional modifications of the anticodon loop region. The nucleosides involved were N(6)-(Delta(2)-isopentenyl)adenosine (i(6)A), 2-methylthio-N(6)-(Delta(2)-isopentenyl)adenosine (ms(2)i(6)A), and an unknown nucleoside K, which occurred in a position analogous to N-[9-(beta-d-ribofuranosyl)purin-6-ylcarbamoyl]threonine. The amounts of i(6)A and ms(2)i(6)A, determined using total tRNA from exponential-or stationary-phase cells, suggest that the thiomethylation of i(6)A is a pleiotropic phenomenon affecting several tRNA species. As opposed to the situation in Escherichia coli tRNA, where ms(2)i(6)A constitutes about 90% of the total hydrophobic nucleosides at all growth stages, B. subtilis tRNA's have i(6)A as the predominant hydrophobic nucleoside in exponential growth and ms(2)i(6)A as the predominant nucleoside in stationary phase. Thus, the enzyme system which forms i(6)A and the enzyme system which thiomethylates i(6)A are not coordinated during growth in B. subtilis as they are in E. coli. It is suggested that these changes in anticodon loop modifications in B. subtilis may be related to changes in the translational apparatus which occur during sporulation.

Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region.

A cloned DNA segment from Bacillus subtilis containing 21 tRNA genes was introduced into Escherichia coli. In the B. subtilis genome, these tRNA genes are located after an rRNA gene set and before tandem terminators. The rRNA and tRNA genes are thought to represent a single transcriptional unit. However, another putative promoter occurs after the second tRNA gene within the tRNA gene cluster and has a sequence compatible with both the major B. subtilis (sigma 43 type) promoter and the major E. coli promoter. The B. subtilis 21-tRNA-gene cluster was introduced into E. coli to see whether this promoter would be recognized in E. coli, to determine the start point of transcription in the E. coli system, and to see whether mature B. subtilis tRNAs would be transcribed and processed in E. coli. Expression was evaluated by monitoring levels of aminoacylation of mature tRNAs extracted from E. coli containing plasmids with or without the B. subtilis tRNA genes and by examining profiles of isoaccepting species on columns of RPC-5. S1 nuclease mapping was performed to define the starting point for transcription. The results indicated that a putative promoter located within the B. subtilis tRNA gene region was functional when cloned into E. coli and that it initiated at the same nucleotide as it does in B. subtilis. In addition, at least some B. subtilis tRNA genes could be transcribed and processed in E. coli to mature tRNAs capable of accepting an amino acid.

Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.

Changes in soluble RNA and ribonuclease activity during germination of wheat.

Gross changes in protein and nucleic acid were studied in germinating wheat seeds. The nucleic acid fraction was separated on columns of methylated albumin-keiselguhr. It was found that more than 50% of the transfer RNA was lost from the embryo in the first 10 to 15 hours of germination. This was followed by a period of rapid resynthesis of transfer RNA, to the normal level at about 20 hours. The decline and increase in transfer RNA was accompanied by a change in the ratios of certain amino acid acceptor species. Evidence is also presented that an embryo ribonuclease is lost during the first 10 to 15 hours, followed by the appearance of a second seedling ribonuclease between 15 and 30 hours of germination.

Sequence analysis of a cluster of twenty-one tRNA genes in Bacillus subtilis.

The DNA sequence of a cluster of twenty-one tRNA genes distal to a rRNA gene set in B. subtilis was determined. None of the tRNA genes are repeated in the sequence. The only classes of tRNAs that are not represented are those for cysteine, glutamine, tryptophan, and tyrosine. Three of the tRNA genes in this cluster do not have the 3'-CCA sequence encoded in the gene. There is no RNA polymerase terminator sequence in the region between the 5S gene and the first tRNA gene or within the tRNA gene cluster. A terminator sequence was found directly after the last tRNA gene. This rRNA and tRNA gene cluster probably represents one transcriptional unit. However, there may be an RNA polymerase promoter site within this sequence, which raises some interesting questions concerning the regulation of transcription for these tRNA genes.

A unique method utilizing antinucleotide antibodies for evaluating changes in the levels of modified nucleosides of tRNAs from crude extracts of whole cells.

The utilization of antibodies directed toward modified nucleosides in evaluating changes in the levels of certain modified nucleosides in transfer RNA is reported. Antibodies directed toward the N6-(delta 2-isopentenyl)adenosine modification were used in this model system with a mutant strain of Escherichia coli designated ipaA. The procedure is rapid, sensitive, and specific. In addition, it does not depend on the existence of an in vitro remodification system or any radiochemical labeling of the tRNA. By varying the extraction technique, the method could be applied to procaryotic or eukaryotic cell lines. The existence of antibodies specific for other nucleoside modifications makes this a system that is potentially applicable to a variety of deficiencies in the modification of both tRNA and rRNA.