Biomedical subjects

C J Daniels

Publications and source records attributed to C J Daniels.

At least 37 records · Page 2Linked to original sources

PubMed1996

Comparative analysis of ribonuclease P RNA structure in Archaea.

Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.

Archaea↗

PubMed1995

In vivo definition of an archaeal promoter.

We have used a plasmid-based transcriptional reporter system to examine the transcriptional effects of 33 single point mutations in the box A region (TATA-like sequence) of the Haloferax volcanii tRNA(Lys) promoter. The most pronounced effects on transcriptional efficiency were found when the nucleotides corresponding to the TATA-like region were altered. Promoters with wild-type or higher levels of transcriptional activity conformed to the general archaeal box A consensus, 5'-T/CTTAT/AA-3'. The preference for a pyrimidine residue in the 5' position of this region and the exclusion of guanine and cytosine in the next four positions in the 3' direction are defining characteristics shared by all efficient archaeal promoters. We have also observed that replacement of a 10-nucleotide purine-rich sequence, located 5' of the H. volcanii tRNA(Lys) box A element, completely abolished transcription from this promoter. These data show that the H. volcanii tRNA(Lys) promoter is dependent on two separate, and essential, sequence elements. The possible functions of these sequences, in view of the recent descriptions of eucaryal-like transcription factors for Archaea, are discussed.

Archaea↗

PubMed1994

A transcriptional reporter for in vivo promoter analysis in the archaeon Haloferax volcanii.

We have used a modified intron-containing tRNA(Pro(UGG) gene (tRNA(ProM), derived from the Saccharomyces cerevisiae tRNA(Pro(UGG) gene, as a reporter to measure in vivo transcription from a halophilic archaeon promoter. Coupling of the yeast tRNA(ProM) gene to the Haloferax volcanii tRNA(Lys) promoter on the H. volcanii plasmid pWL201 led to the production of a single stable transcript that was readily quantitated by Northern (RNA) blot analysis. Comparison of tRNA(ProM) RNA production from constructs containing the wild-type tRNA(Lys) promoter and those containing mutant tRNA(Lys) promoters demonstrated that this assay system can be used to measure expression from strong and weak promoters.

Base Sequence↗

PubMed1994

Expression of a yeast intron-containing tRNA in the archaeon Haloferax volcanii.

Expression of the yeast tRNAPro(UGG) gene in Haloferax volcanii resulted in the production of a single stable transcript that had not undergone intron processing or processing of 5' and 3' flanking sequences. Mutation of the exon-intron boundary region of this RNA to produce a precursor RNA with the preferred halobacterial consensus exon-intron boundary structure did not restore intron processing. Processing of 5' and 3' flanking sequences was restored when the acceptor stem U6-U67 pair was changed to A6-U67. The significance of these results in defining the recognition requirements of tRNA maturation enzymes in the halophilic domain Archaea is discussed.

Base Sequence↗

PubMed1993

In vivo processing of an intron-containing archael tRNA.

In vitro studies on the processing of halobacterial tRNA introns have led to the proposal that archaeal and eukaryotic tRNA intron endonucleases have distinctly different requirements for the recognition of pre-tRNAs. Using a Haloferax volcanii in vivo expression vector we have examined the in vivo processing of modified forms of the halobacterial intron-containing tRNA(Trp) gene. As observed in vitro, changes in the exon-intron boundary structure of this pre-tRNA block processing. Intron sequences, other than those at the exon-intron boundaries, are not essential for processing in vivo. We also show that conversion of the tryptophan anticodon to an opal suppressor anticodon is tolerated when the exon-intron boundary structure is maintained.

Archaea↗

PubMed1992

Transfer RNA genes from the hyperthermophilic Archaeon, Methanopyrus kandleri.

Genes encoding the Leu (GAG), Ser (UGA), Gln (UUG) and Lys (UUU) tRNAs have been cloned and sequenced from the deep sea hyperthermophilic Archaeon, Methanopyrus kandleri. Sequences conforming to the TATA box element established for methanogen promoters are located upstream of the tRNA(Gln) and tRNA(Lys) genes. All four of the tRNA genes appear to encode the 3' terminal CCA residues of the mature tRNA. These methanogen tRNAs are predicted to contain most, but not all, invariant residues and are characterized by a high level of G + C base pairing, consistent with the 98 degrees C optimum growth temperature of M. kandleri.

Base Sequence↗

PubMed1991

The RNA component of RNase P from the archaebacterium Haloferax volcanii.

RNase P, an endoribonuclease responsible for generating the mature 5' termini of tRNA precursors, is composed of both RNA and protein. It has been demonstrated that the eubacterial RNase P RNA will, under the appropriate reaction conditions, exhibit catalytic activity in vitro. Evidence has not been obtained for catalytic activity by the RNAs of eukaryotic RNase P enzymes. Using a cDNA probe prepared from RNA copurifying with RNase P activity from the archaebacterium Haloferax volcanii, we have characterized the gene encoding the RNase P RNA. The proposed transcript from this gene can assume a structure resembling the eubacterial RNase P RNA and includes many of the highly conserved sequences of these RNAs. This RNA was incapable of cleaving pre-tRNA substrates in the absence of protein under a variety of in vitro conditions. Catalytic activity was observed when this RNA was combined with the protein subunit of the Bacillus subtilis RNase P complex. Similarities among the archaebacterial, eubacterial, and eukaryotic RNase P RNA sequences and structures are discussed.

Archaea↗

PubMed1990

Recognition of exon-intron boundaries by the Halobacterium volcanii tRNA intron endonuclease.

The intron-containing tRNA(Trp) precursor from Halobacterium volcanii, like many intron-containing archaebacterial precursor tRNAs, can assume a structure in which the two intron endonuclease cleavage sites are localized in two three-nucleotide loops separated by four base pairs. To investigate the role of this structure in cleavage by the halophilic endonuclease, a series of mutant tRNA(Trp) RNAs were prepared and evaluated as substrates. We find that alterations in this structure result in the loss of cleavage at both 5' and 3' sites. Cleavage of a 35-nucleotide model RNA substrate, containing only these features, demonstrates that sequences and structures present at the exon-intron boundaries are sufficient for recognition and cleavage. We have also examined the mechanism used by the halophilic endonuclease to identify the cleavage sites. Addition of a single base, or a base pair in the anticodon stem above the cleavage sites, does not affect the cleavage site selection. The addition of nucleotides between the two cleavage sites significantly decreases cleavage efficiency and has an effect on the cleavage site selection. These results demonstrate that the halophilic endonuclease requires a defined structure at the exon-intron boundaries and does not identify its cleavage sites by a measurement mechanism like that employed by eukaryotic tRNA intron endonucleases.

Bacterial Proteins↗

PubMed1990

Genes encoding the 7S RNA and tRNA(Ser) are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermus fervidus.

Analysis of gene structure in the extremely thermophilic archaebacterium, Methanothermus fervidus, has revealed the presence of a cluster of stable RNA-encoding genes arranged 5'-7S RNA-tRNA(Ser)-16S rRNA-tRNA(Ala)-23S rRNA-5S rRNA. The genome of M. fervidus contains two rRNA operons but only one operon has the closely linked 7S RNA-encoding gene. The sequences upstream from the two rRNA operons are identical for 206 bp but diverge at the 3' base of the tRNA(Ser) gene. The secondary structures predicted for the M. fervidus 7S, 16S rRNA, tRNA(Ala) and tRNA(Ser) have been compared with those of functionally homologous molecules from moderately thermophilic and mesophilic archaebacteria. A consensus secondary structure for archaebacterial 7S RNAs has been developed which incorporates bases and structural features also conserved in eukaryotic signal-recognition-particle RNAs and eubacterial 4.5S RNAs.

Archaea↗

PubMed1990

An expression vector for the archaebacterium Haloferax volcanii.

The recent development of an efficient transformation method and shuttle vectors for Haloferax volcanii has set the stage for rapid progress in archaebacterial molecular biology. We describe a shuttle-expression vector that can be selected for and maintained in either H. volcanii or Escherichia coli and permits the expression of cloned genes in H. volcanii. The vector, pWL204, was constructed by incorporating an H. volcanii tRNA(Lys) gene promoter into a derivative of the H. volcanii-E. coli shuttle vector pWL102. The vector has been used to express a modified, intron-containing, H. mediterranei tRNA(Trp) gene (tRNA(Trp)-O167). Transcription from the tRNA(Lys) gene promoter in vivo was detected by Northern (RNA) analysis with an oligonucleotide probe complementary to the unique intron sequence of tRNA(Trp)-O167. Dependence of transcription on the tRNA(Lys) promoter was demonstrated by the absence of transcription when the promoter sequence was deleted from the vector and by mapping the transcription initiation site by primer extension.

Archaea↗

PubMed1989

Genes encoding 5S rRNA and tRNAs in the extremely thermophilic archaebacterium Methanothermus fervidus.

Methanothermus fervidus was shown to have two 5S rRNA-encoding genes linked in rRNA operons to 16S and 23S rRNA-encoding genes. Sequencing of a cloned 5S rRNA gene confirmed that M. fervidus is a member of the Methanobacteriales, although its 5S rRNA is also similar in both primary sequence and predicted secondary structure to the 5S rRNA of the non-methanogenic, but also extremely thermophilic archaebacterium, Thermococcus celer. Two clusters of tRNA genes have also been cloned and sequenced form M. fervidus. The smaller cluster, cloned in pET5401, is composed of 5'-tRNA(UGUThr)-tRNA(UGGPro)-tRNA(GUCAsp)-tRNA(UUUL ys)-3' and the larger cluster, cloned in pET5475, is composed of 5'-tRNA(GUUAsn)-tRNA(CAUMet)-tRNA(UUCGlu)-tRNA(UAGL eu)-tRNA(GUGHis)-3'. The encoded tRNAs, with the exception of the tRNA(Leu), translate abundant codons in M. fervidus. The tRNA genes do not contain introns or encode 3'-terminal CCA residues. Homologous clusters of tRNA genes have been sequenced from Methanococcus vannielii and Methanococcus voltae, so that comparisons of transcription signals, gene organizations and primary sequences can be made and features possibly related to thermostability identified. During evolution, a 5S rRNA gene appears to have been incorporated into the cluster of tRNA genes in the methanococci but not in M. fervidus.

Amino Acid Sequence↗

PubMed1989

Transfer RNA intron processing in the halophilic archaebacteria.

An in vitro assay system has been developed for the Halobacterium volcanii tRNA intron endonuclease using in vitro generated precursor RNAs. A partially purified enzyme preparation is capable of precise and accurate excision of the intron from the halobacterial tRNA(Trp) precursor. The cleavage reaction produces products having 5' hydroxyl and 2',3' cyclic phosphate termini. Processing of precursor molecules containing deletions within the exon regions indicates that the halobacterial endonuclease does not require intact mature tRNA structure in the substrate; this is in contrast to the eukaryotic endonuclease enzyme that has an absolute requirement for these structures. The large halobacterial tRNA(Trp) intron does not appear to be a primary site for recognition by the endonuclease, however, its removal affects cleavage efficiency. Through a comparison of the structural and sequence features of the halobacterial substrates and the precursors of other archaebacterial intron-containing precursors, a common element is proposed for the recognition of substrates by intron endonuclease.

Base Sequence↗

PubMed1989

Gene structure, organization, and expression in archaebacteria.

Major advances have recently been made in understanding the molecular biology of the archaebacteria. In this review, we compare the structure of protein and stable RNA-encoding genes cloned and sequenced from each of the major classes of archaebacteria: the methanogens, extreme halophiles, and acid thermophiles. Protein-encoding genes, including some encoding proteins directly involved in methanogenesis and photoautotrophy, are analyzed on the basis of gene organization and structure, transcriptional control signals, codon usage, and evolutionary conservation. Stable RNA-encoding genes are compared for gene organization and structure, transcriptional signals, and processing events involved in RNA maturation, including intron removal. Comparisons of archaebacterial structures and regulatory systems are made with their eubacterial and eukaryotic homologs.

Archaea↗

PubMed1988

A tRNA(Trp) intron endonuclease from Halobacterium volcanii. Unique substrate recognition properties.

We report on the properties of a partially purified tRNA intron endonuclease from the archaebacterium Halobacterium volcanii. This enzyme is capable of precise excision of the 104-nucleotide intron from halo-bacterial pre-tRNA(Trp) substrates generated in vitro by T7 RNA polymerase transcription. The reaction requires divalent cations (Mg2+ or Ca2+) or spermidine, is inhibited by monovalent cations, and produces 5'-hydroxyl and 2',3'-cyclic phosphate termini. Unlike the universal substrate recognition properties characteristic of the eukaryotic tRNA intron endonucleases, this enzyme is specific for halophilic tRNA(Trp) substrates. The partially purified enzyme is not capable of removing the intron from a yeast pre-tRNA(Phe) substrate. Analysis of the enzyme's ability to cleave tRNA(Trp) substrates lacking exon sequences demonstrated that the mature tRNA-like structure is not required in the substrate. A substrate retaining the intact intron and only the anticodon stem and loop exon regions was efficiently cleaved. Deletions within the intron indicated that the intron was not a primary site for recognition by the endonuclease; however, its presence affects the efficiency of the cleavage reaction. The possible relationship of this enzyme to other RNA endonucleases is discussed.

Base Sequence↗

PubMed1987

Nucleotide sequence and characterization of toxR: a gene involved in exotoxin A regulation in Pseudomonas aeruginosa.

We have previously reported the discovery and subsequent cloning of a regulatory gene, designated toxR, which appears to regulate the expression of the exotoxin A (ETA) structural gene toxA. Subsequent work by this laboratory has resulted in the subcloning of the toxR gene and its transfer to a high copy number plasmid (pGW28). Functional analysis of the toxR gene using a Tn5 insertion along with toxR deletions indicates that inactivation of toxR results in a dramatic reduction of ETA production. Nucleotide sequence analysis of pGW28 has revealed a 675 bp major open reading frame (225 codons) which could encode for a protein of 24,626 daltons. Using S1 nuclease mapping, the toxR RNA transcript has been shown to originate 20 bp upstream of the presumptive translation initiation codon. Experiments using a toxA specific probe have revealed the the toxR gene product appears to regulate the expression of ETA at the transcriptional level.

ADP Ribose Transferases↗

PubMed1985

Transcription and excision of a large intron in the tRNATrp gene of an archaebacterium, Halobacterium volcanii.

The archaebacterium Halobacterium volcanii contains a single expressed gene for tRNATrp. This gene is interrupted, at a position corresponding to two nucleotides 3' to the anticodon, by an 105-base pair intervening sequence. Northern hybridization experiments show that the gene is transcribed in its entirety in vivo into a precursor of at least 180 nucleotides, and that one of the products of post-transcriptional processing is a 105-nucleotide species containing intron-specific sequences.

Base Sequence↗

PubMed1985

Sequence of 5S ribosomal RNA gene regions and their products in the archaebacterium Halobacterium volcanii.

We show that the archaebacterium Halobacterium volcanii contains two ribosomal RNA gene clusters, in which genes for individual rRNAs lie in the order 16S-23S-5S. We have cloned the 5S rRNA genes of both clusters and present sequences of the two 5S rRNA genes and their 5' and 3' flanking regions, as well as the sequence of H. volcanii 5S rRNA. We show that a gene for a tRNACys lies downstream from one, but not the other, 5S rRNA gene, and have obtained evidence that this tRNA gene is transcribed in vivo. We discuss regions of potential secondary structure which may be involved in transcription termination. We note regions of unexpected flanking sequence conversation both within H. volcanii 5S rRNA gene regions, and between them and the corresponding 5S rRNA gene region of H. cutirubrum (Hui and Dennis 1984).

Base Sequence↗

PubMed1984

Archaebacterial heat-shock proteins.

The response to heat shock was examined in seven archaebacterial strains from the genus Halobacterium. Upon heat shock each strain preferentially synthesized a limited number of proteins which fell into three narrow mol. wt. ranges. Further examination of the heat-shock response in H. volcanii revealed that heat-shock protein (hsp) synthesis was greatest at 60 degrees C. Synthesis of hsps at this induction temperature was both rapid and transient. Cells recovered their normal protein synthesis patterns rapidly upon returning to their normal growth temperature following heat shock. H. volcanii cells also responded with a ;heat shock-like' response to salt dilution, a natural environmental stress for these organisms. These results indicate that the heat shock or stress response which is charactertistic of eukaryotic and eubacterial cells is also present among members of the archaebacterial genus Halobacterium.

Journal Article↗