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D G Barker

Publications and source records attributed to D G Barker.

At least 37 records · Page 2Linked to original sources
PubMed1986

Cloning and characterization of the Schizosaccharomyces pombe DNA ligase gene CDC17.

The Schizosaccharomyces pombe CDC17 gene has been cloned by complementation of the cdc17 mutant coding for temperature-sensitive DNA ligase. An allele-specific suppressor active only in the presence of a high osmotic pressure was also isolated. The cloned CDC17 gene failed to complement the analogous DNA ligase mutation, cdc9, in Saccharomyces cerevisiae, although the reverse complementation was successful [Barker and Johnston, Eur. J. Biochem. 134 (1983) 315-319]. The CDC17 gene specifies a 2.8-kb transcript.

Cloning, Molecular↗
PubMed1985

DNA ligase-AMP adducts: identification of yeast DNA ligase polypeptides.

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [alpha-32P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.

Adenosine Monophosphate↗
PubMed1985

The nucleotide sequence of the DNA ligase gene (CDC9) from Saccharomyces cerevisiae: a gene which is cell-cycle regulated and induced in response to DNA damage.

The CDC9 gene of Saccharomyces cerevisiae encodes a DNA ligase, and we have determined the nucleotide sequence of a 3.85 kb fragment of DNA which encompasses the convergently transcribed CDC9 and CDC36 genes. S1 nuclease mapping has revealed a major 5' end for the CDC9 mRNA, and one major and one minor site for 3' polyadenylation. These two sites lie within the C-terminal coding region of the CDC36 gene, implying that these two genes are transcribed from overlapping sequences. An interesting structural feature of the CDC9 gene is a series of 6 hexanucleotide repeats (ATGATT) which occur within the 650 bp immediately upstream from the site of transcription initiation. These repeat elements may be implicated in the cell division cycle regulated expression of CDC9. Comparison of the predicted amino acid sequence of the yeast DNA ligase (Mr 84,806) with the sequences of the T4 and T7 bacteriophage DNA ligases reveals little similarity except for a stretch of approximately 45 amino acids, comprising 3 short homologous segments. This region may represent an ATP-binding domain common to polynucleotide ligases.

Amino Acid Sequence↗
PubMed1985

An improved assay for DNA ligase reveals temperature-sensitive activity in cdc9 mutants of Saccharomyces cerevisiae.

A sensitive and quantitative assay for DNA ligase has been developed which is suitable for the analysis of crude cell extracts of yeast. The assay is sufficiently sensitive to detect the low levels of DNA ligase activity remaining in cdc9 mutants of Saccharomyces cerevisiae. Indeed, we have been able to show that this residual activity is temperature-sensitive, thus establishing finally that CDC9 is the structural gene for DNA ligase.

Base Sequence↗
PubMed1985

Electron microscopic studies of condensed mitotic chromosomes in the fission yeast Schizosaccharomyces pombe.

By blocking cells in mitosis with the anti-fungal drug thiabendazole, it has been possible to carry out ultrastructural studies on the condensed chromosomes of the fission yeast, Schizosaccharomyces pombe. It is estimated that the DNA in these chromosomes is compacted approximately 1000-fold, and that the nucleoprotein density is similar to that of higher eukaryotic metaphase chromosomes. A basic structural component of the condensed chromosomes appears to be a 50-60 nm fibre, which is often visible in a loop configuration on the periphery of the chromatids. This is reminiscent of the 50-60 nm fibre loops which are frequently seen in preparations of metaphase chromosomes, and suggests that mechanisms of nucleoprotein folding may be similar in both lower and higher eukaryotes.

Chromosomes↗
PubMed1983

The amino acid sequence of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus.

The primary structure of the tyrosyl-tRNA synthetase (TyrTS) of Bacillus stearothermophilus has been deduced from the nucleotide sequence of the cloned gene and from the amino acid sequence of peptides isolated from the purified enzyme. TyrTS (B. stearothermophilus) has a molecular weight of 47316 and the sequence is 56% homologous with that of TyrTS (Escherichia coli). The binding domain for the substrate intermediate tyrosyl adenylate is located in the N-terminal portion of the polypeptide and is highly conserved in both enzymes. Several lysine residues, which are shielded from acetylation in the TyrTS-tRNATyr complex, are also located in a stretch of highly conserved sequence.

Amino Acid Sequence↗
PubMed1982

The tyrosyl-tRNA synthetase from Escherichia coli. Complete nucleotide sequence of the structural gene.

The structural component of the tyrS gene of Escherichia coli, comprising 1269 base pairs, has been fully sequenced by the combined M13/dideoxychain termination approach. The gene has a codon usage pattern which is typical of highly expressed proteins and similar to other Escherichia coli aminoacyl-tRNA synthetase genes. Peptide purification and sequencing has been used to locate the N-terminus and to provide confirmation of 95% of the translated protein sequence. This latter yields on Mr of 47,403 for the Escherichia coli tyrosyl-tRNA synthetase, and reveals considerable homology with the primary structure of the analogous enzyme isolated from Bacillus staerothermophilus.

Amino Acid Sequence↗
PubMed1982

Cloning and amplified expression of the tyrosyl-tRNA synthetase genes of Bacillus stearothermophilus and Escherichia coli.

Two fragments of DNA which carry the genes coding for the tyrosyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus have been cloned into the plasmid pBR322 and were selected by complementation of an E. coli temperature-sensitive mutant. Transformation of this strain with either of the recombinant plasmids results in a 100-fold increase in tyrosyl-tRNA synthetase activity measured in vitro and the protein products co-migrate with the corresponding purified enzymes on polyacrylamide gels.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Methionyl-tRNA synthetase from Escherichia coli. Primary structure of the active crystallised tryptic fragment.

A 3300-base segment of Escherichia coli chromosomal DNA, cloned into pBR322, will complement a methionine auxotroph in which the lesion is a defective methionyl-tRNA synthetase with a much reduced affinity for methionine. Crude extracts of these transformants contain elevated levels of a protein which has a subunit molecular weight of 66 000, methionyl-tRNA synthetase aminoacylation activity in vitro and which cross-reacts with anti-(methionyl-tRNA synthetase) antibodies. This polypeptide is very slightly larger than the well-characterised and crystallised tryptic fragment of methionyl-tRNA synthetase. A DNA sequence of 1750 residues at one end of the cloned insert codes for a non-terminated open reading frame in which we can locate a large number of methionyl-tRNA synthetase tryptic and chymotryptic peptides. We have also sequenced 300 nucleotides upstream of this coding segment where we find a large invert repeat in the putative methionyl-tRNA synthetase promoter region.

Amino Acid Sequence↗
PubMed1976

Factor analysis of body hair distributions.

Using observations on the presence or absence of terminal hair in 13 locations on 700 white adult males, descriptive statistics were tabulated, phi coefficients of intercorrelation were computed, principal axis factor loadings were extracted, and varimax rotation of factors to simple structure was performed. The resulting rotated factors were interpreted as five independent components of body hair distribution: dorsal (less sacral), pubic extension, thoracic, sacral, and abdominal.

Factor Analysis, Statistical↗