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H A Baylis

Publications and source records attributed to H A Baylis.

31 records · Page 2Linked to original sources
PubMed1994

Theileria annulata sporozoite surface antigen (SPAG-1) contains neutralizing determinants in the C terminus.

SPAG-1 is a surface antigen on Theileria annulata sporozoites that is a candidate both for inclusion in a subunit vaccine and as a ligand for host cell recognition. We have pinpointed major neutralizing epitopes to the C terminus. To facilitate this we expressed SPAG-1 as a series of defined fragments in the pGEX system. These constructs were validated by sequencing and by their spectrum of reactivity with monoclonal antibody (MoAb) BA4. This MoAb recognizes the elastin motif VGVAPG, that is predicted to occur three times in the N terminal half of SPAG-1. The recombinant proteins were then tested by Western blotting with a neutralizing MoAb (1A7) and two neutralizing bovine sera (10T and 34A). The results demonstrate that 1A7 and the bovine sera react with determinants unique to the C terminus. We mapped the neutralizing determinant recognized by MoAb 1A7 to a 16 residue sequence (residues 807-822) using synthetic peptides. Interestingly the bovine sera do not recognize the 1A7 epitope. The potential role of the C terminus as a ligand for host cell recognition and the implications for sub-unit vaccine production are discussed.

Amino Acid Sequence↗
PubMed1993

Characterisation of a glutamine- and proline-rich protein (QP protein) from Theileria parva.

We have isolated a clone from a Theileria parva infected lymphocyte cDNA library which has the potential to encode a protein of 480 amino acids. This protein is particularly rich in glutamine and proline and has some short repeated amino acid motifs based on the sequences QPXP and QPXQ. We have called it the 'QP protein'. Southern blotting suggests that the QP protein gene is present as a single copy in the T. parva Muguga genome. Northern blotting revealed that the gene is transcribed in both schizonts and piroplasms. We have expressed part of the QP protein as a fusion with glutathione S-transferase in Escherichia coli and used this product to raise an anti-QP protein serum. Western blots of T. parva lysates using this serum showed a major polypeptide of approximately 100 kDa and two further polypeptides of approximately 67 and 72 kDa. Indirect immunofluorescence assays using the anti-QP protein serum on infected cells showed that the protein is associated with the schizont. The pattern of staining in the indirect immunofluorescence assays and the structure of the protein suggest that it is a component of the schizont membrane.

Amino Acid Sequence↗
PubMed1993

Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences.

The complete small subunit ribosomal RNA (srRNA) gene of Theileria parva was cloned and sequenced. Two primers were designed which permitted the specific amplification of part of the Theileria srRNA gene from Theileria-infected cell line samples which were predominantly (> 95%) bovine DNA. The sequence of the central (variable) region of the srRNA genes of T. annulata, T. taurotragi, T. mutants and two unidentified parasites referred to as Theileria sp. (buffalo) and Theileria sp. (Marula) were obtained. An alignment of the sequences was generated from which 6 oligonucleotide probes, corresponding to species-specific regions, were designed. These probes were demonstrated to provide unequivocal identification of each of the 6 species either by direct detection of parasite srRNA or by hybridization to amplified parasite srRNA genes. The probes were not able to distinguish buffalo-derived T. parva, the causal agent of Corridor disease, from cattle-derived T. parva, the causal agent of East Coast fever.

Animals↗
PubMed1992

Theileria annulata-infected cells produce abundant proteases whose activity is reduced by long-term cell culture.

Lysates of Theileria annulata-infected bovine lymphoblastoid cells and their uninfected counterparts were tested for protease activity using gelatin substrate SDS-PAGE. The infected cells produced a number of extra activities at pH 8.0 in the presence of Ca2+. Calcium was found to enhance the activities but was not an absolute requirement. Studies using inhibitors, including E64, 3,4-dichloroisocoumarin, pepstatin and 1, 10-phenanthroline suggested that the activities were metalloproteases. We analysed two vaccine lines; the Ode line from India and the Ankara Pendik line from Turkey. In the Ode line the later passage had very much reduced levels of the enzyme activities. In the case of the Ankara Pendik line both stages analysed had very low protease activities, but a reduction from the early to late passage was also observed. The reduction in the level of protease activity was also observed as a gradual process during on-going culture of lines derived from the Hissar and Ode stocks. In the Ode line we demonstrated a parallel decrease in the production of microschizonts upon temperature shift in vitro.

Animals↗
PubMed1991

An unusual repetitive gene family in Theileria parva which is stage-specifically transcribed.

Tpr1 is a repetitive DNA element from Theileria parva which has previously been shown to be of value in strain characterisation. Further characterisation, described here, has shown that Tpr1 is present in long tandem arrays. The sequence of 8.1 kb from one end of an array has been determined. The sequence showed that Tpr1 is a 1.44-kb element which contains an ORF extending from its 5' end to the 3' end. The sequenced region contains 4 large ORFs; 2 of these consisted only of Tpr1 whilst the third consisted of Tpr1 and a 0.55-kb element (Tpr2) located 5' of Tpr1. The largest ORF consisted of Tpr1 plus Tpr2 as well as an additional 420-bp element (Tpr3) 5' of Tpr2, thus a continuous ORF arranged 5'-Tpr3-Tpr2-Tpr1-3' was formed. This ORF potentially encodes a 795 amino acid polypeptide commencing at an ATG close to the 5' end. In contrast the first in frame ATGs in the other 3 ORFs are at least 417 bp from the 5' end. Southern analysis confirmed that the sequenced region was typical of the rest of the Tpr array(s). Transcripts containing both Tpr3 and Tpr1 were detected in the piroplasm but not the schizont stages of the life cycle.

Amino Acid Sequence↗
PubMed1991

The stringent response in Streptomyces coelicolor A3(2).

The stringent response was elicited in the antibiotic producer Streptomyces coelicolor A3(2) either by amino acid depletion (nutritional shiftdown) or by the addition of serine hydroxamate; both led to increased levels of ppGpp and to a reduction in transcription from the four promoters of the rrnD rRNA gene set. Analysis of untreated batch cultures revealed elevated ppGpp levels at the end of exponential growth, preceding the onset of antibiotic production. The effect of provoking the stringent response on antibiotic production in exponentially growing cultures was assessed by S1 nuclease mapping of actIII, an early gene of the actinorhodin biosynthetic cluster. Expression of actIII occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis alone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2).

Amino Acids↗
PubMed1988

Organisation of the ribosomal RNA genes in Streptomyces coelicolor A3(2).

Using Southern hybridisation of radiolabelled purified ribosomal RNAs to genomic DNA the ribosomal RNA genes of Streptomyces coelicolor A3(2) were shown to be present in six gene sets. Each gene set contains at least one copy of the 5 S, 16 S and 23 S sequences and in at least two cases these are arranged in the order 16 S - 23 S - 5 S. Three gene sets, rrnB, rrnD and rrnF, were isolated by screening a lambda library of S. coelicolor A3(2) DNA. The restriction map of one of these, rrnD, was determined and the nucleotide sequences corresponding to the three rRNAs were localised by Southern hybridisation. The gene order in rrnD is 16 S - 23 S - 5 S.

DNA Restriction Enzymes↗
PubMed1988

Transcriptional analysis of the 16S rRNA gene of the rrnD gene set of Streptomyces coelicolor A3(2).

The nucleotide sequence of 2.5 kb of the Streptomyces coelicolor A3(2) rRNA gene set rrnD, extending from upstream of the 16S rRNA gene to the putative 5' end of the 23S rRNA gene, has been determined (Baylis and Bibb, 1987; this paper). In addition to locating the 5' end of the 16S rRNA gene, nuclease S1 mapping identified seven RNA 5' end-points upstream of the 16S rRNA gene; four of these were coincident with transcriptional initiation points for S. coelicolor A3(2) RNA polymerase in vitro and were consequently regarded as in vivo transcription start points for promoters p1 to p4. One end-point identified by nuclease S1 mapping localized a putative processing site analogous to those found upstream of 16S rRNA genes in other eubacteria. Sequence motifs similar to those discovered in low G+C Gram-positive bacteria were found associated with two of the promoters and the processing site. A probable protein coding region was observed upstream of the promoter region.

Base Sequence↗
PubMed1987

Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2).

In Streptomyces coelicolor, bldA mutants are defective in antibiotic production and the development of aerial hyphae and spores. Subcloning analysis showed that sequences spanning an NcoI site in cloned bldA+ DNA were needed to allow complementation of a bldA mutant. Nucleotide sequencing revealed a tRNA-like sequence 9 bp downstream from the NcoI site. Five independent bldA mutations all fell in a 16-bp region in the tRNA-like sequence, one of them changing the putative anticodon. In RNA dot-blot analysis, hybridization was detected with a probe specific for the tRNA-like transcript but not with a probe for "anti-tRNA-like" transcripts. The transcripts detected were all in the salt-soluble RNA fraction and accumulated relatively late in growth. It is postulated that bldA specifies a tRNA that would recognize the codon UUA (for leucine). This codon is very rare in Streptomyces genes [which generally contain greater than 70 mole% (G + C)], suggesting a possible role for bldA in translational control of development.

Base Sequence↗
PubMedDate not supplied

Polycyclic dinitriles: a novel class of potent GABAergic insecticides provides a new radioligand, [3H]BIDN.

The polycyclic dinitriles are a potent class of insecticides which are non-competitive GABA (gamma-aminobutyric acid) antagonists acting at the convulsant site. Comparison with other classes of GABA convulsant site ligands using molecular modelling has shown significant structural similarities. We have developed a pharmacophore model which unifies this class and some previous classes of GABA convulsants. Key pharmacophore elements are a polarizable functionality separated by a fixed distance from two H-bond accepting elements. This model is based on information from X-ray crystal structures and Sybyl using the Tripos force field. Using this pharmacophore model, numerous structural modifications were explored to enhance understanding of structure-activity relationships at the GABA receptor convulsant site of insects and mammals. A radiolabelled bicyclic dinitrile, [3H]BIDN [3H]3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile+ ++), was prepared from this area of chemistry and was used as a probe for the interaction of polycyclic dinitriles at the target site.

Animals↗