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P F Sims

Publications and source records attributed to P F Sims.

51 records · Page 3Linked to original sources
PubMed1989

Isolation of alpha-tubulin genes from the human malaria parasite, Plasmodium falciparum: sequence analysis of alpha-tubulin.

As a step towards identifying exploitable differences between host and parasite at the molecular level, we have isolated and sequenced genomic clones encompassing an entire alpha-tubulin gene (designated alpha-tubulin I) from the human malaria parasite, Plasmodium falciparum. The gene, which contains two introns, encodes a product with a predicted length of 453 amino acid residues (50.3 kD). The protein sequence shows a high degree of homology to other alpha-tubulins, particularly that of the coccidian parasite, Toxoplasma gondii (94%), whose gene carries introns in identical positions. Only one copy of the alpha-tubulin I gene itself was found, although a second gene designated alpha-II was also identified which is closely related but which differs at both the nucleotide and amino acid sequence levels. The alpha-I and beta-tubulin genes were found to reside on different chromosomes.

Amino Acid Sequence↗
PubMed1988

Multiple ligninase-related genes from Phanerochaete chrysosporium.

We have investigated four distinct sequences in genomic DNA of Phanerochaete chrysosporium that show strong homology to a lignin peroxidase cDNA sequence [Tien and Tu: Nature 236 (1987) 520-523, and 328 (1987) 742]. The complete nucleotide sequence of one of these genes is presented here. Comparison of the sequenced gene, LIG1, with the published cDNA sequence reveals the presence of eight short intervening sequences. The open reading frame encodes a polypeptide of 372 amino acids (aa), which includes a 28-aa residue hydrophobic leader sequence. The predicted Mr of the mature protein product is 37,000. Partial sequencing of the other three genes reveals substantial nucleotide and amino acid homology.

Agaricales↗
PubMed1988

Functional expression of the yeast Mn-superoxide dismutase gene in Escherichia coli requires deletion of the signal peptide sequence.

Oligodeoxyribonucleotide-directed mutagenesis has been used to delete the leader sequence-coding region from the yeast gene specifying Mn-superoxide dismutase and thus enable its expression in Escherichia coli. The deletion of this leader was demonstrated to be an absolute requirement for the synthesis of an active yeast enzyme in the bacterium. Complementation analysis in E. coli has confirmed that the product of the truncated yeast gene is active in vivo as well as in vitro.

Amino Acid Sequence↗
PubMed1987

Anomalous dinucleotide frequencies in both coding and non-coding regions from the genome of the human malaria parasite Plasmodium falciparum.

We have statistically analysed the distribution of nucleotides and dinucleotides in 21 genes of the 81% A + T-rich human malaria parasite Plasmodium falciparum. The mRNA-synonymous strands of this protozoan show in general a marked excess of purines over pyrimidines, correlated with abnormally high levels of Lys and Glu. We have used the large differences in base composition between coding and non-coding regions to estimate that the parasite possesses in the range of 2700-5400 genes. The dinucleotide preference patterns are compared with consensus patterns derived from other organisms [Nussinov, Nucl. Acids Res. 12 (1984) 1749-1763]. Patterns in the coding regions surprisingly resemble those of higher, rather than lower eukaryotes, particularly with respect to TG elevation and CG suppression. The latter is correlated with an abnormally low level of Arg in these parasites. In the non-coding regions, the four dinucleotides made up of C and/or G are found with significantly higher frequencies than expected (approx. 50-150%), specifically to the 5' side of the coding regions. The possible role of these dinucleotides in control sequences is discussed.

Amino Acids↗
PubMed1985

An E. coli-yeast shuttle cosmid with positive selection for inserted fragments.

We describe the construction of a cosmid cloning vector, pMT555, which allows positive selection for the presence of an inserted DNA fragment. The vector contains sequences which enable its replication and selection in either E. coli or Saccharomyces cerevisiae. We demonstrate that pMT555 may be used for the efficient construction of total genomic banks from small quantities of donor DNA. The positive selection permits the stable maintenance of the cosmid in E. coli and the faithful replication of inserted sequences.

Cosmids↗
PubMed1985

Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei.

The nucleotide sequence of the dihydrofolate reductase (DHFR) gene of a methotrexate-resistant strain of Lactobacillus casei, which is the source of DHFR for nuclear magnetic resonance (NMR) studies, has been determined. The derived amino acid sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at position 8 and proline instead of leucine at position 90. The nucleotide sequences of 320-bp 5' and 335-bp 3' flanking regions of this gene have also been determined.

Base Sequence↗
PubMed1982

Effects of iodoamphenicol on ribosome assembly in two strains of Escherichia coli.

When the growth of Escherichia coli strain 15TP was inhibited by iodoamphenicol, three 'iodoamphenicol particles' accumulated with sedimentation coefficients of 25S, 33S and 45S. The 25S and 33S particles differ in sedimentation properties from equivalent ribosome precursor particles detected during pulse-labelling of exponentially growing cells. Inhibition of a mutant, strain 15-28 (defective in ribosome assembly), by iodoamphenicol resulted in the accumulation of 38S iodoamphenicol particles that are different from the particles made by the parent. The results support the contention that assembly of 50S ribosomal subunits by the mutant is altered at an early stage.

Bacterial Proteins↗
PubMed1980

Intermediates in the assembly of ribosomes by a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant that accumulates ribonucleoprotein ('47 S') particles during exponential growth. These particles contain mature 23 S rRNA, but lack three of the proteins of the larger ribosomal subunit, to which they are a precursor. In organisms growing at 20 degrees C, assembly of 47 S particles involves three intermediates that contain precursor 23 S rRNA, one of which has the same sedimentation properties as 47 S particles. Assembly of 50 S ribosomal subunits in the parent strain is 'normal'. There are three intermediates; each contains precursor 23 S rRNA, and one cannot be distinguished from completed subunits by sedimentation. Synthesis of 30 S ribosomal subunits in parent and mutant strains is qualitatively similar, but quantitatively different. When growth is at 37 degrees C, assembly in the mutant alters. There are now two sequential precursors to 47 S particles. Both contain precursor 23 S rRNA; one has the same sedimentation coefficient as 47 S particles. In some respects, synthesis in the mutant proceeds as though 47 S particles, rather than 50 S ribosomal subunits, are the end-product of assembly.

Centrifugation, Isopycnic↗
PubMed1979

Abnormal ribosome assembly in a mutant of Escherichia coli.

The mutant strain, 15--28, of Escherichia coli accumulates ribonucleoprotein ('47S') particles that were previously shown [Markey, Sims & Wild (1976) Biochem. J. 158, 451--456] to be an unusual intermediate in the assembly of 50S ribosomal subunits...

Centrifugation, Density Gradient↗
PubMed1978

Binding of chloramphenicol and a fragment of aminoacyl-transfer ribonucleic acid to ribosomes and a ribosome precursor from a mutant of Escherichia coli.

During exponential growth, the mutatn strain Escherichia coli 15-28 accumulates 47S particles, which are unusual precursors to 50S ribosomal subunits. The 47S particles have little ability to bind chloramphenicol, but binding of a fragment of aminoacyl-tRNA is about half that by completed subunits. The 70S (and 50S) ribosomes of strain 15-28 and its parent (strain 15TP) do not differ in chloramphenicol binding. Although ribosomes from the mutant are less able than those from the parent to bind the fragment, this difference is not as marked as was found previously [Sims & Wild (1976) Biochem. J. 160, 721-726] for the binding of an analogue of peptidyl-tRNA and for peptidyltransferase activity. The altered activities may arise because strain 15-28 misassembles 50S subunits of altered conformation and because the few proteins that 47S patricles lack have vital functions in some of the partial reactions of protein synthesis.

Binding Sites↗
PubMed1976

Peptidyltransferase activity of ribosomes and a ribosome precursor from a mutant of Escherichia coli.

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that caused the accumulation of ribonucleoprotein ('47S') particles during exponential growth. These particles are precursors to 50S ribosomes that lack three ribosomal proteins. Peptidyltransferase activity and binding at the peptidyl site of the peptidyltransferase centre are greatly decreased in 47S particles. Both these activities are lower in the 50S and 70S ribosomes of strain 15-28 than in its parent. Unusual assembly of the larger ribosomal subunit in strain 15-28 may produce completed ribosomes with diminished biological activity.

Acyltransferases↗
PubMed1976

The composition of an unusual precursor of 50 S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes. The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.

Bacterial Proteins↗
PubMedDate not supplied

A modified two primer approach to oligonucleotide-directed in vitro mutagenesis.

Using oligonucleotide-directed mutagenesis, we are trying to define the features of the protein structure that are important for the DNA and c-AMP binding by CAP from E. coli, the enzymic activity and putative DNA binding of dihydrofolate reductase of L. casei, and the functionally important regions of the self-splicing RNA of the r-RNA intron of Tetrahymena thermophila. We have used a modification of the method described by Norris et al. [1]. A mutagenic primer and an M13 universal sequencing primer are annealed simultaneously to a template from an M13 clone containing the DNA to be mutagenised and, after DNA strand extension, the fragment is cut out and recloned into either M13 or plasmid vectors. We have analysed the effect on the frequency of mutation of: the temperature used for strand extension; the class of base change attempted; the host mismatch repair system. A recently developed system for phenotypic detection of mutations in the Tetrahymena intron aided in determining mutation frequencies.

Animals↗