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M J Bibb

Publications and source records attributed to M J Bibb.

86 records · Page 5Linked to original sources
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

Nucleotide sequences encoding and promoting expression of three antibiotic resistance genes indigenous to Streptomyces.

Promoter-probe plasmid vectors were used to isolate putative promoter-containing DNA fragments of three Streptomyces antibiotic resistance genes, the rRNA methylase (tsr) gene of S. azureus, the aminoglycoside phosphotransferase (aph) gene of S. fradiae, and the viomycin phosphotransferase (vph) gene of S. vinaceus. DNA sequence analysis was carried out for all three of the fragments and for the protein-coding regions of the tsr and vph genes. No sequences resembling typical E. coli promoters or Bacillus vegetatively-expressed promoters were identified. Furthermore, none of the three DNA fragments found to be transcriptionally active in Streptomyces could initiate transcription when introduced into E. coli. An extremely biased codon usage pattern that reflects the high G + C composition of Streptomyces DNA was observed for the protein-coding regions of the tsr and vph genes, and of the previously sequenced aph gene. This pattern enabled delineation of the protein-coding region and identification of the coding strand of the genes.

Base Sequence↗
PubMed1985

Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus.

A DNA fragment containing the coding and regulatory sequences of the erythromycin (Er) resistance (ermE) gene of the Er produces Streptomyces erythraeus was cloned in Streptomyces lividans using the plasmid vector pIJ61. The approximate location and orientation of ermE were deduced from studies of its expression after subcloning in Escherichia coli. Sequences responsible for transcription of ermE in Streptomyces were studied by nucleotide (nt) sequencing, high resolution S1 and exonuclease VII mapping, in vitro transcription and in vivo promoter-probing. Tandemly arranged promoters of typical prokaryotic appearance initiate transcription of the coding region of ermE; a promoter of similar sequence was identified that initiates transcription of a likely coding region running in the opposite direction to ermE. It is suggested that these sites represent a class of vegetatively expressed Streptomyces promoter that is utilised by a form of RNA polymerase holoenzyme that also recognizes typical promoters of other bacterial genera.

Base Sequence↗
PubMed1984

The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences.

Bacterial genes that code for proteins appear to possess a codon usage characteristic of their overall base composition. This results in different but predictable non-random distributions of nucleotides within codons, permitting the recognition of protein-coding sequences in a wide range of bacterial species. The nature of this distribution depends on the base composition of the coding sequence. The position-specific differences are especially conspicuous in genes of extreme G + C content, allowing the particularly reliable prediction of the reading frame and coding strand of experimentally determined DNA sequences. This finding has been exploited to identify the coding sequence of the viomycin phosphotransferase (vph) gene of Streptomyces vinaceus. An easily applied computer program ("Frame") has been written to carry out and display such analyses.

Bacterial Proteins↗
PubMed1983

Plasmids, recombination and chromosome mapping in Streptomyces lividans 66.

Streptomyces lividans 66 was shown to harbour two self-transmissible plasmids: SLP2, which acts as a sex factor, and SLP3. Derivatives of this strain which had lost both plasmids were used as host strains to study a range of Streptomyces plasmids for their ability to promote their own transfer and to mobilize chromosomal markers. A linkage map of the S. lividans chromosome containing ten markers was derived from the results of matings using several different sex plasmids, and protoplast fusions. SLP2 was transferred interspecifically to S. parvulus ATCC 12434 and to S. coelicolor A3(2); in the latter it acted as a fertility factor. Interspecific crosses also led to the discovery of a further plasmid, SLP4, from S. coelicolor, SLP2, SLP3 and SLP4 could not be visualized on agarose gels using standard plasmid isolation procedures, but their presence was detected by transformation into S. lividans.

Chromosome Mapping↗
PubMed1982

Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans.

Promoter-probe plasmid vectors were constructed for Streptomyces lividans using expression of the Escherichia coli chloramphenicol acetyltransferase gene as an indicator of promoter activity. These vectors have been used to isolate and to study the activity of DNA sequences that contain transcriptional control signals from Streptomyces, Bacillus licheniformis, E. coli, and Serratia marcescens. Studies of these promoter regions in heterospecific hosts indicate that genus or species-specific factors may present barriers to the expression of bacterial genetic material in certain heterologous cellular environments. While promoter regions isolated from E. coli, S. marcescens and B. licheniformis all appear to be recognized by the RNA polymerase of S. lividans, the Streptomyces transcriptional control signals isolated do not appear to function normally in E. coli.

Bacteriophages↗
PubMed1981

Excision of chromosomal DNA sequences from Streptomyces coelicolor forms a novel family of plasmids detectable in Streptomyces lividans.

When strains of Streptomyces coelicolor A3(2) lacking the previously identified autonomous plasmids SCP1 and SCP2 are crossed with Streptomyces lividans 66, some of the S. lividans progeny are able to elicit zones of growth inhibition (lethal zygosis), previously associated with the transfer of conjugative Streptomyces plasmids, when grown in contact with S. lividans 66. Some such progeny yield covalently closed circular (CCC) plasmid DNA, the size and restriction endonuclease cleavage pattern of which is constant for a particular isolate, but varies among isolates. These plasmids, which have been named SLP1.1, SLP1.2, etc., all confer resistance to lethal zygosis elicited by the others. Genetic and molecular characterization of the plasmids reveals that they are derived from the strA region of the chromosome of S. coelicolor. It is proposed that, before or during mating with S. lividans, the SLP1 sequences are excised from the chromosome, bringing varying regions of the surrounding chromosome with them, and can circularize to yield the SLP1 family of plasmids. Autonomous SLP1 plasmids can also be generate by cleaving total DNA of S. coelicolor with certain restriction enzymes, ligating it, and transforming the DNA into S. lividans. The autonomous SLP1 plasmids exist within S. lividans in a few copies per chromosome, and act as fertility factors. They provide suitable vectors for DNA cloning since the segments of chromosomal DNA carried by the larger members of the family are dispensable.

Conjugation, Genetic↗
PubMed1981

Sequence and gene organization of mouse mitochondrial DNA.

The complete sequence of the 16,295 bp mouse L cell mitochondrial DNA genome has been determined. Genes for the 12S and 16S ribosomal RNAs; 22 tRNAs; cytochrome c oxidase subunits I, II and III; ATPase subunit 6; cytochrome b; and eight unidentified proteins have been located. The genome displays exceptional economy of organization, with tRNA genes interspersed between rRNA and protein-coding genes with zero or few noncoding nucleotides between coding sequences. Only two significant portions of the genome, the 879 nucleotide displacement-loop region containing the origin of heavy-strand replication and the 32 nucleotide origin of light-strand replication, do not encode a functional RNA species. All of the remaining nucleotide sequence serves as a defined coding function, with the exception of 32 nucleotides, of which 18 occur at the 5' ends of open reading frames. Mouse mitochondrial DNA is unique in that the translational start codon is AUN, with any of the four nucleotides in the third position, whereas the only translational stop codon is the orthodox UAA. The mouse mitochondrial DNA genome is highly homologous in overall sequence and in gene organization to human mitochondrial DNA, with the descending order of conserved regions being tRNA genes; origin of light-strand replication; rRNA genes; known protein-coding genes; unidentified protein-coding genes; displacement-loop region.

Animals↗
PubMed1981

Mitochondrial DNA of chloramphenicol-resistant mouse cells contains a single nucleotide change in the region encoding the 3' end of the large ribosomal RNA.

The complete DNA sequence of the rRNA genes of mouse L cell mtDNA provides a basis for the examination of the nucleotide sequence of this region in a mutant mouse cell line that is resistant to chloramphenicol, a known inhibitor of mitochondrial protein synthesis. Resistance to chloramphenicol (CAPr) is conferred by a cytoplasmic determinant that is linked to mtDNA restriction endonuclease site polymorphisms. We have determined the sequence of a 212-nucleotide region of mtDNA from a CAPr mouse cell line that encodes a portion of the 1582-nucleotide large rRNA. This sequence is located 107-318 nucleotides from the 5' end of the heavy strand coding sequence, which corresponds to the 3' end of the rRNA. There is a single nucleotide difference in the large rRNA gene from CAPr cells, an A-to-G transition 243 nucleotides from the 5' end of the coding sequence. This single transition is located within a region of 10 nucleotides tht is otherwise completely homologous to human and yeast mitochondrial large rRNAs and Escherichia coli 23S rRNA and is positioned immediately adjacent to a single nucleotide transversion known to occur in a yeast CAPr mutant. This characterization of a mammalian mitochondrial mutant at the nucleotide level directly demonstrates that a mutant phenotype may result from a single mtDNA nucleotide change in an animal cell.

Animals↗
PubMed1981

Cloning and expression in streptomyces lividans of antibiotic resistance genes derived from Escherichia coli.

Hybrid plasmids that replicate in both Escherichia coli and Streptomyces lividans were constructed in vitro by joining the E. coli-derived plasmid pACYC184 or pACYC177, at their BamHI or PstI restriction site, respectively, to S. lividans plasmid pSLP111. After introduction of the composite replicons into S. lividans by transformation, chloramphenicol (Cm) resistance encoded by pACYC184 and kanamycin resistance encoded by pACYC177 were phenotypically expressed in the S. lividans host. A Sau3A restriction endonuclease-generated deoxyribonucleic acid fragment from pACYC184 containing the entire structural gene for the Cm acetyltransferase enzyme, but lacking the nucleotide sequence ordinarily serving as the Cm resistance gene promoter, also specified resistance to Cm when introduced in either orientation into the BamHI or BclI endonuclease cleavage site of pSLP111 or into corresponding sites of the analogous plasmid pSLP101. These findings make it unlikely that the biologically active CM acetyltransferase was being made in S. lividans as part of a fused protein, but instead indicate that the ATG start codon used for initiation of translation of the Cm resistance gene in E. coli was also utilized in S. lividans. In contrast, the synthesis of messenger ribonucleic acid that encodes the Cm acetyltransferase in S. lividans was, in at least one instance, apparently initiated at nucleotide sequences within the S. lividans plasmid vector, with resulting transcriptional read-through into the E. coli-derived deoxyribonucleic acid segment.

Acetyltransferases↗
PubMed1978

Tryptophan genes in Rhizobium--their organization and their transfer to other bacterial genera.

R. leguminosarum trp alleles mapped by R68.45-mediated recombination were located in three distinct chromosomal regions. We isolated three derivatives of R68.45 that carried different trp genes of R. meliloti. Each of the plasmids suppressed all of the R. leguminosarum trp alleles in a particular region. The R-primes were transferred to strains of P. aeruginosa carrying mutations in different trp genes. The plasmid pAJ24JI suppressed trpA, B and F mutants, pAJ73JI suppressed trpC and D and pAJ88JI suppressed a trpE mutant. When the R-primes were transferred to E. coli trp strains they failed to suppress any trp mutants. A derivative of pAJ24JI was isolated which was able to suppress trpA and F mutants of E. coli.

Escherichia coli↗
PubMed1977

Chloramphenicol acetylransferase-independent chloramphenicol resistance in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) and S. lividans 66, which lack chloramphenicol acetyltraferase, gave rise to vhloramphenicol-sensitive (CmlS) variants spontaneously at frequencies of 0-5 to 2%. The fertility type of S. coelicolor in respect of the SCPI plasmid (SCPI+, SCPI- or NF) had no effect on chloramphenicol sensitivity or on the frequency at which CmlS variants arose. CmlS isolates spontaneously reverted to CmlR at frequencies one to three orders of magnitude lower than the frequency with which CmlS strains arose from CmlR. CmlR revertants obtained spontaneously from CmlS clones again produced CmlS isolates at the normal frequency of several per cent. Therefore, CmlS and CmlR are reversible phenotypes. In crosses between marked CmlR and CmlS S. coelicolor strains, transfer of chloramphenicol resistance into the sensitive strain apparently occurred independently of chromosomal recombination. Mapping experiments excluded the possibility that segregation of a chromosomal locus determines CmlR versus CmlS phenotype. In crosses between SCPI- strains, fertility was not significantly different in CmlR x CmlS, CmlR x CmlR and CmlS x CmlS combinations. Covalently closed circular DNA from CmlS and CmlR strains of S. coelicolor was indistinguishable in molecular weight and restriction endonuclease cleavage pattern. It is suggested that chloramphenicol resistance in S. coelicolor A3(2) is affected by some kind of transposable genetic element that may be capable of extra-chromosomal existence.

Acetyltransferases↗