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B S Vold

Publications and source records attributed to B S Vold.

At least 19 recordsLinked to original sources
PubMed1996

The catalytic core of RNase P.

A deletion mutant of the catalytic RNA component of Escherichia coli RNase P missing residues 87-241 retains the ability to interact with the protein component to form a functional catalyst. The deletion of this phylogenetically conserved region significantly increases the Km, indicating that the deleted structures may be important for binding to the precursor tRNA substrate but not for the cleavage reaction. Under some reaction conditions, this RNase P deletion mutant can become a relatively non-specific nuclease, indicating that this RNA's catalytic center may be more exposed. The catalytic core of the RNase P is formed by less than one third of the 377 residues of the RNase P RNA.

Bacillus subtilis↗
PubMed1996

DELISA: sensitive nonisotopic assay for GAD65 autoantibodies, a key risk-assessment marker for insulin-dependent diabetes mellitus.

Nonisotopic assays for the measurement of autoantibodies to 65-kDa glutamic acid decarboxylase (GAD65) have not previously achieved performance equivalent to radiobinding assays (RBA). We have developed a modified ELISA protocol, DELISA, for measuring autoantibodies to GAD65 in serum. The method overcomes the problems of poor sensitivity and specificity associated with conventional ELISAs. Serum containing GAD65 autoantibodies is incubated with biotinylated GAD65 (bGAD65). Sufficient soluble Protein A-dextran conjugate is added to bind the immunoglobulins in the sample, including GAD65 autoantibodies to which GAD65 is bound. After incubation, the mixture is transferred to a streptavidin-p4ated microtiter well, which binds free bGAD65 but not bGAD65 bound to autoantibodies. Streptavidin-bound bGAD65 is detected by means of a peroxidase-GAS65MAb conjugate. The method appears to have comparable sensitivity and specificity to those of RBAs. Reaction of the antibodies with soluble antigen to increase the binding rate and the use of high serum concentrations and very low antigen concentrations to increase sensitivity are critical elements of the method.

Autoantibodies↗
PubMed1995

High-level expression of soluble recombinant RNase P protein from Escherichia coli.

We have expressed recombinant RNase P protein from Escherichia coli in high yield. A hexahistidine sequence at the amino terminus allowed protein purification in a single step. Mass spectrometry confirmed the molecular weight of the purified protein and indicated a purity of > 95%. Protein functionality was demonstrated by reconstitution of active holoenzyme.

Bacterial Proteins↗
PubMed1993

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.

Bacillus subtilis↗
PubMed1992

A cluster of nine tRNA genes between ribosomal gene operons in Bacillus subtilis.

A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rrnJ and rrnW in Bacillus subtilis has been cloned and sequenced. This cluster contains the genes for tRNA(UACVal), tRNA(UGUThr), tRNA(UUULys), tRNA(UAGLeu). tRNA(GCCGly), tRNA(UAALeu), tRNA(ACGArg), tRNA(UGGPro), and tRNA(UGCAla). The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnI and rrnH, and of the 5'-end of trnB, a cluster of 21 tRNA genes found immediately 3' to rrnB. Neither the tRNA(UAGLeu) gene nor its product has been found previously in B. subtilis. With the discovery of this new set of tRNA genes, a total of 60 such genes have now been found in B. subtilis. These known genes account for almost all of the tRNA hybridizing restriction fragments of the B. subtilis genome. The 60 known tRNA genes of B. subtilis code for only 28 different anticodons, compared with a total of 41 different anticodons for 78 tRNA genes in Escherichia coli. This may indicate that B. subtilis does not need as many anticodons because of more flexible translation rules, similar to the situation in Mycoplasma capricolum.

Bacillus subtilis↗
PubMed1990

The discovery of new intron-containing human tRNA genes using the polymerase chain reaction.

Introns in transfer RNA genes are rare in vertebrates. Until now, the only intron-containing human tRNA genes were believed to be those coding for tRNA(Tyr). All of these introns are inserted 3' to the anticodon position in these genes. We have designed polymerase chain reaction primers that can amplify all of the tRNA(Tyr) genes for cloning and sequencing by using the conserved portions of the gene coding for the structural part of the tRNA. Our preliminary results have revealed five tRNA(Tyr) genes, each of which contains a different intron. We used the same technique to amplify, clone, and sequence the human genes for tRNA(Leu)CAA. This has resulted in the discovery that this human tRNA gene family also has introns inserted 3' to the anticodon. This polymerase chain reaction technique is useful in detecting new families of intron-containing tRNA genes as well as identifying sequence variations in the introns of individual genes.

Base Sequence↗
PubMed1990

Control of the position of RNase P-mediated transfer RNA precursor processing.

Two Bacillus subtilis tRNA(His) precursors (Green, C. J., and Vold, B. S. (1988) J. Biol. Chem. 263, 652-657) were processed by Escherichia coli RNase P in the presence of varying [Mg2+]. The wild type precursor was processed under all conditions to afford a single tRNA product containing 8 base pairs in the acceptor stem. In contrast, the position of processing of a mutant tRNA(His) precursor (containing a G27----A27 alteration) was shown to be condition-dependent. Processing occurred at A27 under conditions consistent with formation of an A27-C100 base pair in the acceptor stem but at G28 under conditions that disfavored base pair formation. The ability to control the site of RNase P-mediated tRNA precursor processing is unprecedented and permits analysis of the chemical factors that promote processing.

Bacillus subtilis↗
PubMed1988

Processing of a multimeric tRNA precursor from Bacillus subtilis by the RNA component of RNase P.

Processing of multimeric precursor tRNAs from Bacillus subtilis by the catalytic RNA component of RNase P was studied in vitro. Previous studies on processing by either Escherichia coli or B. subtilis RNase P-RNA utilized monomeric or dimeric substrates. In the experiments described here, a multimeric precursor tRNA containing six complete tRNA sequences and the partial sequence of a seventh were used. One species did not encode the 3'-terminal CCA sequence and the partial tRNA lacked 3' nucleotides and could form only a 3-base pair instead of a 7-base paired aminoacyl stem. Two species had the potential for forming extended base-paired aminoacyl stems. Processing was studied under varied ionic conditions. Chemical sequencing of the products showed that the RNase P-RNA cleavage produced the proper mature 5' termini for all of the six complete tRNA species, but no 5'-cleavage of the partial species was observed. At suboptimal ionic concentrations, the two species capable of forming extended base-paired aminoacyl stems were not observed. Thus, encoding of the 3'-CCA in a tRNA species is not critical for processing, but the formation of an aminoacyl stem with more than 3 base pairs is necessary. Particularly noteworthy was the observation that all species of the multimeric precursor could be processed at significantly lower ionic conditions than monomeric precursors used previously by ourselves and others. However, a single precursor species produced from the multimeric precursor could also be processed at the same lower ionic conditions as the multimeric precursor. This demonstrates that precursor tRNA species can differ widely in their ionic requirements for processing and that, to a large extent, the optimal conditions of MgCl2 or NH4Cl are a function of the substrate which is used.

Bacillus subtilis↗
PubMed1988

Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons. II. Unique properties of an operon containing a minor 5 S rRNA gene.

This is part of a series of two papers on gene regulation in Bacillus subtilis rRNA-tRNA operons that contain large clusters of tRNA genes. The preceding paper (Vold, B.S., Okamoto, K., Murphy, B.J., and Green, C.J. (1988) J. Biol. Chem. 263, 14480-14484) investigates the rrnB operon containing 21 tRNA genes, and this paper investigates a B. subtilis rRNA-tRNA operon containing 16 tRNA genes and a minor 5 S rRNA. Hybridization studies suggest this minor 5 S rRNA occurs as a single copy in the B. subtilis 168 genome. S1 nuclease mapping indicates that this minor 5 S rRNA gene has its own promoter. No promoters have been found immediately 5' to any of the major 5 S rRNA species in B. subtilis rRNA operons. S1 mapping of the spacer region between the 23 S and minor 5 S rRNA revealed that the maturation of the 23 S rRNA in this operon may arise from an unusual processing mechanism. S1 nuclease mapping experiments suggest the existence of a promoter element immediately upstream of the last gene, for tRNA(Leu CAA), in the operon. A precursor leucine tRNA resulting from transcription of this last tRNA gene was observed in Northern hybridizations, and the amounts of this precursor increased during sporulation. A single terminator-like element is located just upstream of this last tRNA gene; however, S1 nuclease mapping experiments suggest that some read-through transcription occurs. Thus, all 16 tRNA genes are under control of the upstream 16 S rRNA promoters and the minor 5 S rRNA promoter. However, the last tRNA gene is primarily under the control of its own unique promoter.

Bacillus subtilis↗
PubMed1988

Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons. I. The tRNA gene cluster of rrnB has an internal promoter.

Although the sequence and organization of many Bacillus subtilis tRNA genes are known, primary transcripts from these regions have not been previously analyzed. In this paper, S1 nuclease mapping, S1-type mapping, and Northern analyses were applied to the end of the 23 S rRNA, the 5 S rRNA, and the 21 tRNA genes of B. subtilis operon rrnB. Primary transcripts from the 5 S rRNA and tRNA genes up to approximately 600-800 nucleotides long were observed with S1-type mapping. The presence of discrete bands of processing intermediates indicated preferred processing points within the initial transcript. S1 nuclease mapping delineated a start point for transcription between the second and third tRNA genes. The -10 sequence was within the 37-base pair spacer region between tRNA genes, and the -35 sequence was within the structural gene for the upstream tRNA. Precursors from this region were evident during midexponential growth and two sporulation stages. Thus, in addition to promotion from the rRNA promoters, 19 of the 21 downstream tRNA genes are also under the control of an internal tRNA gene promoter. The accompanying paper (Vold, B. S., Green, C. J., Narasimhan, N., Strem, M., and Hansen, J. N. (1988) J. Biol. Chem. 263, 14485-14490) investigates the minor 5 S rRNA and 16 tRNA genes of another rRNA-tRNA gene set and emphasizes unique promoter elements in that system as well as a potentially unique rRNA processing scheme.

Base Sequence↗
PubMed1988

Ionic conditions for the cleavage of the tRNA-like structure of turnip yellow mosaic virus by the catalytic RNA of RNase P.

The 3'-end of the RNA genome of turnip yellow mosaic virus can form a pseudoknotted tRNA-like structure that can be recognized by several tRNA-specific enzymes. We have found that the catalytic RNA component of Bacillus subtilis RNase P can cleave this structure in unusually low ionic strength buffers at a site analogous to the 5'-end of an aminoacyl stem of a tRNA. Most other precursors can only be processed under low ionic strength conditions if the RNase P holoenzyme is used; processing by the catalytic RNA component alone requires a higher ionic strength buffer. The cleavage of the turnip yellow mosaic virus tRNA-like structure demonstrates the importance of the substrate in determining the optimal buffer conditions for this reaction and also shows that high ionic strength buffers are not always necessary for cleavage by the catalytic RNA.

Ammonium Chloride↗
PubMed1988

Structural requirements for processing of synthetic tRNAHis precursors by the catalytic RNA component of RNase P.

Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.

Bacillus subtilis↗
PubMed1986

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.

Bacillus subtilis↗
PubMed1986

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.

Bacillus subtilis↗
PubMed1986

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.

Adenosine↗
PubMed1985

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.

Bacillus subtilis↗
PubMed1983

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.

Bacillus subtilis↗