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N Hardman

Publications and source records attributed to N Hardman.

At least 37 records · Page 2Linked to original sources
PubMed1988

Phylogeny of Onchocerca volvulus and related species deduced from rRNA sequence comparisons.

A rapid, direct method for determining the partial nucleotide sequence of large subunit ribosomal RNA was adapted and applied to a group of helminth parasites. Small samples of total, unfractionated cellular RNA isolated from each organism were analysed and the nucleotide sequences of equivalent portions of the large subunit ribosomal RNA compared. The data obtained were used to construct a phylogenetic tree showing the evolutionary relationships within this group of organisms.

Animals↗
PubMed1987

Yeast phosphoglycerate kinase: investigation of catalytic function by site-directed mutagenesis.

A salt link buried in the domain interface of phosphoglycerate kinase has been implicated as being important in controlling the conformational transition from the open, or substrate-binding, to the closed, or catalytically competent, form of the enzyme. The residues contributing to the salt link are remote from the active site, but are connected to the substrate-binding sites through strands of beta-sheet. It has been suggested that these residues may also mediate sulphate and anion activation. These assumptions have been tested by examining the properties of a site-directed mutant (histidine-388----glutamine-388). The expression and overall structural integrity of the mutant, produced in yeast from a multicopy plasmid, remains essentially unaltered from the wild-type enzyme. However, the mutant enzyme has a kcat. reduced by 5-fold. The Km for ATP is lowered by 3-fold, and the Km for 3-phosphoglycerate is unaffected. The effects of sulphate on activity over a wide range of substrate concentrations appear to be the same for both the mutant and wild-type enzymes. These results lead to a reappraisal of the mechanistic role of the inter-domain histidine-glutamate interaction, as well as a refinement of the kinetic model of the enzyme.

Amino Acid Sequence↗
PubMed1986

Phylogeny of helminths determined by rRNA sequence comparison.

The nucleotide sequence of the 5' ends of the 28S-like rRNA molecules of five species of helminths was determined directly, using a variation on the dideoxynucleotide chain-termination method which requires only 10 micrograms of total cellular RNA for analysis. Nucleotide sequence comparisons over 208 bases allowed the phylogeny of these organisms to be determined. The data show that the rRNA sequence of Nematospiroides dubius, a nematode, is as divergent from that of two platyhelminths, Hymenolepis diminuta and Schistosoma mansoni, as it is from the rRNA sequence of the two nematodes Onchocerca gibsoni and Brugia pahangi. The latter two appear to be very closely related, whereas the two platyhelminths are more distant from each other. The study demonstrates the usefulness and generality of rRNA sequencing for the systematic phylogenetic classification of parasitic organisms whose tissues are only available in relatively small amounts.

Animals↗
PubMed1986

Antigenic variation between human respiratory syncytial virus isolates.

Three hybridoma antibodies, prepared against the RSN-2 strain of human respiratory syncytial (RS) virus, have been used to identify antigenic variation between 41 isolates of RS virus collected from widely separated geographical regions over a period of 29 years. One antibody was directed against an antigenic site on the virus fusion protein, VP70. This site was shared by 21 virus isolates tested and its recognition by the antibody was sensitive to the presence of 2-mercaptoethanol. The remaining two antibodies used react against the virus phosphoprotein, VPP32. Two independent sites were recognized on VPP32 by these antibodies. One antibody reacted with all of the virus isolates screened while the second reacted with only 21 out of the 41 virus isolates. On the basis of the variable epitope, two antigenic types of human RS virus were identified. The distribution of each antigenic group among 28 RS virus isolates from the Grampian Region, north-east Scotland, collected between 1982 and 1984 was determined. The reactivity of these antibodies was examined using immunofluorescence staining and by immunoblotting; the latter technique also revealed that the electrophoretic mobility of VPP32 varied in parallel with the variable antigenic site.

Animals↗
PubMed1985

Transposon-like properties of the major, long repetitive sequence family in the genome of Physarum polycephalum.

A family of long, highly-repetitive sequences, referred to previously as ;HpaII-repeats', dominates the genome of the eukaryotic slime mould Physarum polycephalum. These sequences are found exclusively in scrambled clusters. They account for about one-half of the total complement of repetitive DNA in Physarum, and represent the major sequence component found in hypermethylated, 20-50 kb segments of Physarum genomic DNA that fail to be cleaved using the restriction endonuclease HpaII. The structure of this abundant repetitive element was investigated by analysing cloned segments derived from the hypermethylated genomic DNA compartment. We show that the ;HpaII-repeat' forms part of a larger repetitive DNA structure, approximately 8.6 kb in length, with several structural features in common with recognised eukaryotic transposable genetic elements. Scrambled clusters of the sequence probably arise as a result of transposition-like events, during which the element preferentially recombines in either orientation with target sites located in other copies of the same repeated sequence. The target sites for transposition/recombination are not related in sequence but in all cases studied they are potentially capable of promoting the formation of small ;cruciforms' or ;Z-DNA' structures which might be recognised during the recombination process.

Journal Article↗
PubMed1985

Isolation and characterisation of a variant allele of the gene for human apolipoprotein E.

Size-selected human DNA fragments enriched in the Apolipoprotein E (ApoE) gene sequence were cloned from an individual of known ApoE phenotype, E2/E2. The clone bank was screened using a human cDNA clone for the ApoE locus (1), and a single genomic clone was isolated. Sequence data obtained from appropriate subcloned fragments confirmed that the codon for Arg-158 (CGC) in the E3 allele is altered to the codon for Cys (TGC) in the E2 allele. Hybridisation data indicated the presence of at least one intron in the ApoE gene, consistent with the structure of an independently isolated human ApoE4 allele (2).

Alleles↗
PubMed1985

Structural organization of a hypermethylated nuclear DNA component in Physarum polycephalum.

Digestion of Physarum polycephalum nuclear DNA using the restriction endonuclease HpaII generates two components, distinguishable on the basis of their molecular size. The high-molecular-weight, HpaII-resistant component, which accounts for 20% of the DNA, contains a fivefold greater concentration of 5-methylcytosine residues than the low-molecular-weight HpaII-digested fraction. Segments of hypermethylated (M+) DNA are largely composed of a single, long, highly repeated sequence, and this major element is sometimes associated with other less highly repetitive sequences in the M+ DNA fraction. Restriction mapping of cloned Physarum M+ DNA segments, and Southern blot analysis of genomic DNA using subcloned segments of M+ DNA as a probe, provide evidence for sequence variation within different copies of the dominant highly repeated element, and possibly the other associated repeats in M+ DNA, and additionally that almost complete tandemly repeated copies of the major repeat are found in some M+ DNA segments.

5-Methylcytosine↗
PubMed1983

An abundant family of methylated repetitive sequences dominates the genome of Physarum polycephalum.

A significant portion (20%) of the Physarum genome can be isolated as a HpaII-resistant, methylated fraction. Cloned DNA probes containing highly-repeated sequences derived from this fraction were used to define the pattern of structural organisation of homologous repeats in Physarum genomic DNA. It is shown that the probes detect an abundant, methylated family of sequences with an estimated genomic repetition frequency greater than 2100, derived from a large repeated element whose length exceeds 5.8kb. Sequences comprising the long repetitive element dominate the HpaII-resistant compartment and account for between 4-20% of the Physarum genome. Detailed restriction/hybridisation analysis of cloned DNA segments derived from this compartment shows that HpaII/MspI restriction sites within some copies of the long repeated sequence are probably deleted by mutation. Additionally, segments of the repeat are often found in different organisational patterns that represent scrambled versions of its basic structure, and which are presumed to have arisen as a result of recombinational rearrangement in situ in the Physarum genome. Preliminary experiments indicate that the sequences are transcribed and that the structural properties of the repeat bear some resemblance to those of transposable genetic elements defined in other eukaryotic species.

Cloning, Molecular↗
PubMed1983

Sequence organisation in nuclear DNA from Physarum polycephalum. Genomic organisation of DNA segments containing foldback sequences.

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hybridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.

Base Sequence↗
PubMed1983

Sequence organisation in nuclear DNA from Physarum polycephalum. Arrangement of highly-repeated sequences.

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.

DNA, Fungal↗
PubMed1983

Tubulin genes of Trypanosoma brucei: a tightly clustered family of alternating genes.

African trypanosomes are the causative agents of many medically and economically important diseases of man and domestic animals. The cell body of these blood-dwelling protozoa is enveloped with a dense layer of pellicular microtubules, which confer both motility and mechanical stability on these cells; microtubules are also important components of the flagellum. The major structural components of the microtubuli are two related proteins, alpha- and beta-tubulin. We have analyzed the genomic organization of alpha- and beta-tubulin genes in Trypanosoma brucei. In this organism, the majority of these genes are arranged in a single, tightly packed cluster of alternating alpha- and beta-tubulin genes with a basic repeat length together of 3.6 kilobase pairs. A genomic library of T. brucei was constructed by using the phage vector A 1059, and recombinant phages carrying tubulin genes were isolated by screening the library with heterologous chicken tubulin cDNA probes. The results of restriction endonuclease and hybridization analysis of DNA isolated from recombinant phages, and subcloned fragments thereof, were compatible with the restriction maps derived from digestion and Southern blot hybridization of genomic DNA.

Animals↗
PubMed1982

Analysis of foldback sequences and repetitive sequences in cloned segments of nuclear DNA from Physarum polycephalum.

The properties of DNA segments containing foldback elements were studied after their selection from a "random" recombinant library of Physarum polycephalum nuclear DNA sequences, cloned using the plasmid vector pBR322. Hybridisation of in vitro labelled recombinant plasmids to Southern blots of genomic restriction fragments demonstrated that each cloned segment contained repetitive elements located at several hundred sites in the genome. Two of the DNA clones generated hybridisation patterns which suggested that they contain repetitive elements with internal cleavage sites for the restriction endonuclease HaeIII. Cross-hybridisation of all combinations of the cloned sequences showed that most contain different arrangements of repetitive elements derived from different sequence families. The results are consistent with a model proposed previously on the basis of studies on total nuclear DNA, for the organisation of sequences closely associated with foldback elements in the Physarum genome.

Base Sequence↗
PubMed1981

Sequence organisation in nuclear DNA from Physarum polycephalum: methylation of repetitive sequences.

Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.

Base Sequence↗
PubMed1981

The effect of age on the properties of poly(A)-containing messenger RNA in Physarum polycephalum.

The effect of ageing on the properties of polysomal poly(A)-containing messenger RNA [poly(A)+ mRNA] of Physarum polycephalum has been investigated. Using poly(U)--Sepharose affinity chromatography it was shown that shortening of the poly(A) tract occurred as the age of the mRNA population increased. Analysis of the poly(A) segments by use of polyacrylamide gel electrophoresis, after digestion of polysomal poly(A)+ mRNA molecules with RNAase A and RNAase T1, revealed that their lengths ranged from 140 to 220 nucleotide residues. A reduction in the efficiency of utilization of mRNA for translation as the age of the mRNA population increased was demonstrated by measuring the proportion of poly(A)+ mRNA present in the polysomal fraction as compared with post-polysomal material.

Electrophoresis, Polyacrylamide Gel↗
PubMed1980

Methylation of nuclear DNA in Physarum polycephalum.

The restriction endonucleases HpaII and HhaI, whose action is inhibited by the presence of methylated base analogues at the recognition sequences in the DNA substrate, were used to investigate the distribution of 5-methylcytosine in nuclear DNA from Physarum polycephalum. Physarum DNA is digested into two fractions by these enzymes: a low-molecular-weight (M--) compartment comprising 80% of the DNA, and a high-molecular-weight (M+) compartment containing 20% of the DNA. The DNA fraction showing resistance to digestion by restriction endonuclease HpaII is cleaved by its isoschizomer MspI, indicating that methylated endonuclease-HpaII-specific sites are present in M + DNA. Additional properties of sequences in the M+ compartment were investigated.

Binding Sites↗
PubMed1980

Sequence organization in nuclear deoxyribonucleic acid from Physarum polycephalum. Physical properties of foldback sequences.

An investigation was performed with the use of physical techniques, to determine the nature and organization of inverted repeat sequences in nuclear DNA fragments from Physarum polycephalum. From the average size of foldback duplexes (550 nucleotide pairs), and the foldback duplex yield as determined by treatment of DNA with S1 deoxyribonuclease followed by hydroxyapatite chromatography, it is estimated that there are at least 25000 foldback sequences in the Physarum genome. Foldback DNA molecules exhibit properties intermediate between single-stranded DNA and native duplexes on elution from hydroxyapatite with a salt gradient. In addition, thermal-elution chromatography of foldback DNA from hydroxyapatite crystals shows that foldback duplexes are less stable than native DNA. These properties can be explained on the basis that inverted repeat sequences are mismatched when in the foldback configuration. The results of experiments in which the binding of foldback DNA molecules to hydroxyapatite was determined, by using fragments of different single-chain size, agree with previous studies indicating that inverted repeat sequences are located, on average, every 7000 residues throughout the Physarum genome. The inverted repeats are derived from both the repetitive and single-copy components in Physarum nuclear DNA.

Base Sequence↗