Biomedical subjects

M J Buttner

Publications and source records attributed to M J Buttner.

44 records · Page 3Linked to original sources

PubMed1989

RNA polymerase heterogeneity in Streptomyces coelicolor A3(2).

Recent genetic and biochemical experiments have revealed the existence of at least seven different sigma factors in Streptomyces coelicolor and demonstrated a role for alternative sigma factors in the control of differentiation and also in the transcription of primary metabolic genes. S. coelicolor has four genes predicted to encode sigma factors that are very closely related to the sigma 70 polypeptide of Escherichia coli.

Amino Acid Sequence↗

PubMed1989

Transcription from the P1 promoters of Micromonospora echinospora in the absence of native upstream DNA sequences.

We demonstrated previously that the 0.4-kilobase DNA fragment from Micromonospora echinospora contains multiple tandem promoters, P1a, P1b, P1c, and P2, which are also functional when cloned into Streptomyces lividans. We now show by in vitro transcription with Streptomyces RNA polymerase that each of these promoters is an authentic initiation site, rather than a processing site for transcripts which initiate further upstream. The DNA sequence requirements for the closely spaced promoters P1a, P1b, and P1c, which are coordinately induced during stationary phase in M. echinospora, were examined by deletional analysis in S. lividans. The P1a and P1b promoters were functional despite deletion of native sequences 5 and 17 base pairs upstream of each initiation site, respectively. Thus, P1a and P1b had greatly reduced upstream DNA sequence requirements compared with typical procaryotic promoters. In contrast, transcription from promoter P1c was significantly decreased when native sequences 34 base pairs upstream were replaced.

Base Sequence↗

PubMed1988

At least three different RNA polymerase holoenzymes direct transcription of the agarase gene (dagA) of Streptomyces coelicolor A3(2).

Using a combination of gel filtration and anion exchange FPLC, three different RNA polymerase holoenzymes from Streptomyces coelicolor A3(2) have been separated. Each holoenzyme transcribes from only one of the four promoters of the S. coelicolor A3(2) dagA gene. Holoenzyme reconstitution experiments identified the sigma factors responsible for recognition of two of the promoters. The previously identified E sigma 49 transcribes from the dagA p3 promoter, whereas a novel species, E sigma 28, recognizes the dagA p2 promoter. Circumstantial evidence suggests that the third holoenzyme, which transcribes from the dagA p4 promoter, is the previously identified E sigma 35. This level of transcriptional complexity supports the idea that RNA polymerase heterogeneity may play a central role in the regulation and coordination of gene expression in this biochemically and morphologically complex bacterium.

Base Sequence↗

PubMed1987

The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis.

The DNA sequence of a 1.77 kb region of the Streptomyces coelicolor chromosome containing the coding and regulatory regions of the extracellular agarase (dagA) gene was determined. The sequence predicts a primary translation product of 309 amino acids and Mr 35132. Comparison of the N-terminal sequence determined for the mature extracellular protein with that of the primary translation product deduced from the DNA sequence predicts the presence of a 30 amino acid signal peptide. Analysis of the transcription of the dagA gene using high resolution S1 mapping, in vitro transcription, dinucleotide-primed in vitro transcription and in vivo promoter probing identified four promoters, initiating transcription approximately 32, 77, 125 and 200 nucleotides upstream of the coding sequence.

Amino Acid Sequence↗

PubMed1987

Two promoters from the Streptomyces plasmid pIJ101 and their expression in Escherichia coli.

An RNA polymerase-binding restriction fragment from the small, high-copy-number Streptomyces plasmid pIJ101 has been shown to have promoter activity in vivo using a promoter-probe vector. The nucleotide sequence of the promoter (the pIJ101B promoter) and the approximate position of the transcription start point as identified by in vitro run-off transcription are presented. Both the pIJ101B promoter and the previously characterised pIJ101A promoter were found to promote transcription in Escherichia coli. The transcription start point in E. coli for the pIJ101A promoter has been determined using high-resolution S1 mapping. Initiation occurs at the same point or within 1 or 2 nucleotides of the transcription start point previously identified in Streptomyces lividans, indicating that the same transcriptional signals are recognised in both genera. The data support the idea that one type of RNA polymerase holoenzyme in Streptomyces recognises a class of promoters similar to the major consensus promoters of E. coli, and that the manner of promoter recognition is similar in both genera.

Base Sequence↗

PubMed1987

The agarase gene (dag A) of Streptomyces coelicolor A3(2): affinity purification and characterization of the cloned gene product.

The coding and regulatory sequences of the agarase gene of Streptomyces coelicolor A3(2) were cloned in Streptomyces lividans 66 on the plasmid vector pIJ61, resulting in a several hundred-fold increase in the production of the secreted protein. Subcloning experiments localized the sequences required for agarase production and for the mediation of carbon catabolite repression to a segment of about 1.2 kb. A simple protein purification procedure that uses affinity binding of agarase to agarose beads was developed. Preliminary characterization of the enzyme, together with the results of in vitro transcription-translation studies, suggest that the intracellular form of agarase (about 34 kDa) possesses a signal sequence that is cleaved upon secretion across the cell membrane to produce an extracellular protein of about 29 kDa.

Chromatography, Affinity↗

PubMed1986

Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator.

Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a polylinker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.

Cloning, Molecular↗

PubMed1985

RNA polymerase-DNA interactions in Streptomyces. In vitro studies of a S. lividans plasmid promoter with S. coelicolor RNA polymerase.

DNA fragments of the Streptomyces lividans plasmid pIJ101 have been tested for their ability to bind Streptomyces coelicolor RNA polymerase in vitro or to promote transcription in Streptomyces in vivo. One DNA fragment which does both was shown to encode a transcript which was expressed at low cell-density in cultures of pIJ101-containing cells. The transcript start was located on the DNA sequence of the fragment by nucleotide-primed RNA polymerase binding experiments and by S1 nuclease mapping. The pattern of DNase I protection, the sites of enhanced DNase I cleavage and the DNA sequence of the fragment suggest that the RNA polymerase holoenzyme form, which recognizes this promoter, is similar in its interaction with DNA to the major RNA polymerase of Escherichia coli. Regions showing 3/6 nucleotide homology with each of the -35 and -10 regions of the consensus sequence of E. coli promoters are present in the same positions relative to the transcript start. Symmetrical sequences which may be involved in the regulation of expression of the promoter and a potential polypeptide coding sequence can be identified.

Autoradiography↗