Polypeptide chain elongation factors have been purified from an extreme thermophile, Thermus thermophilus HB8. By chromatography on a DEAE-Sephadex column, the factors were separated into two peaks; peak I contained a complex of EF-Tu and EF-Ts, while peak I1 was composed of EF-Tu . G D P and EF-G. These factors were subsequently purified to homogeneous states and crystallized.The EF-Tu . EF-Ts complex could be resolved into EF-Tu and EF-Ts by chromatography on a Sephadex G-200 column in the presence of 8 M guanidine-HC1. The complex could be reconstituted from EF-Tu and the renatured EF-Ts.No immunological cross-reaction was detected between EF-Tu, EF-Ts, and EF-G from T. thermophilus and the antibodies to their corresponding Escherichia coli factors.

The gene encoding the citric acid cycle enzyme, citrate synthase, has been cloned from the thermoacidophilic archaebacterium, Thermoplasma acidophilum. We report the sequencing of this gene and its flanking regions, and the derived amino acid sequence of the enzyme is compared by multiple-sequence alignment analysis with those of citrate synthases from eubacterial and eukaryotic organisms. The similarity is < 30% between the archaebacterial and non-archaebacterial sequences, although the majority of residues implicated in the catalytic action of the enzyme have been conserved across all three kingdoms. The cloned archaebacterial gene has been expressed in Escherichia coli to produce catalytically active citrate synthase. This is the first reported sequence of citrate synthase from the archaebacteria.Citrate synthase, the first enzyme of the citric acid cycle, has been studied from organisms that represent all three primary kingdoms : the eukaryotes, the eubacteria and the archaebacteria [l -31. Two oligomeric forms of citrate synthase have been identified: a dimeric form found in eukaryotes and Gram-positive eubacteria, and a hexameric form found in Gram-negative eubacteria. Both types are made up of identical subunits, with M , values of approximately 50 000, the hexameric form appearing to behave functionally as a trimer of the basic dimer [4]. However, the two forms of the enzyme differ in their regulatory sensitivities: the dimer is inhibited isosterically by ATP whereas the hexameric form is inhibited allosterically by NADH and, in some cases, additionally by 2-oxoglutarate [l -31.The primary amino acid sequences of citrate synthase from the eukaryotes pig heart [5] and kidney [6], Arabidopsis thaliana [7] and Saccharomyces cerevisiae [8], and from the eubacteria Escherichia coli [9, lo], Rickettsia prowazekii [l 11, Acinetobacter anitratum [12] and Pseudomonas aeruginosa [ 131 have been determined, either by amino acid sequencing of the protein or from the DNA sequence encoding the gene. In addition, a high-resolution X-ray crystallographic structure is available for the pig heart enzyme [14, 151. This has allowed identification of 12 residues involved in the active site; multiple sequence comparisons [16] have indicated that the majority of these 12 active site residues are conserved between all citrate synthases.To extend our studies on the diversity of citrate synthases and the correlation of oligomeric structure and function, we have initiated investigations on the enzyme from the archaebacteria. These organisms are thought to constitute a phylogenetically distinct evolutionary group, in addition to Correspondence to M. J. Danson, Department of Biochemistry, Enzyme. Citrate synthase (EC 4.1.3.7). Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X5.5282.

The majority of Halicephalobus gingivalis-infections in horses have been fatal and are usually not diagnosed before necropsy. Therefore, knowledge about the nematode and the pathogenesis of infection in horses is limited. This has resulted in an on-going discussion about the port of entry and subsequent dissemination of H. gingivalis within the host. The present case of H. gingivalis-infection in a horse was diagnosed ante mortem. Post mortem findings, the distribution pattern of H. gingivalis nematodes in the brain, a high prevalence of inflammation in close relation to blood vessels, and the presence of the nematode in multiple organs with a disseminated pattern of distribution strongly suggested a haematogenous spread of the nematode in the horse.

A protein designed de novo to fold into the Greek key jellyroll structural motif has been studied. Theoretical analyses have indicated that the designed sequence should adopt the beta-strand arrangement of the Greek key jellyroll rather than any other arrangement. A synthetic gene was constructed and the protein expressed in Escherichia coli. Circular dichroism spectroscopy is consistent with the protein folding into the designed conformation and also suggests the presence of tertiary structure. Fluorescence spectroscopy showed the single tryptophan to be partially buried, while denaturation studies showed changes in fluorescence to precede alterations in secondary structure.

The alignment of Escherichia coli citrate synthase to pig heart citrate synthase and the multiple alignment of the known sequences of the citrate synthase family of enzymes have been performed using six different amino acid similarity scoring matrices and a large range of gap penalty ratios for insertions and deletions of amino acids. The alignment studies have been performed as the first step in a project aimed at homology modelling E. coli citrate synthase (a hexamer) from pig heart citrate synthase (a dimer) in a molecular modelling approach to the study of multi-subunit enzymes. The effects of several important variables in producing realistic alignments have been investigated. The difference between multiple alignment of the family of enzymes versus simple pairwise alignment of the pig heart and E. coli proteins was explored. The effects of initial separate multiple alignments of the most highly related or most homologous species of the family of enzymes upon a subsequent pairwise alignment between species was evaluated. The value of 'fingerprinting' certain residues to bias the alignment in favour of matching those residues, as well as the worth of the computerized approach compared to an intuitive alignment technique, were assessed.