An androgen-inducible expression system for Saccharomyces cerevisiae.

A novel controllable expression system for Saccharomyces cerevisiae has been developed. Expression of the gene encoding the human androgen receptor, from a strong yeast promoter, results in transactivation of a hybrid promoter carrying androgen-responsive sequences such that a target gene may be expressed in an androgen-dependent manner. By selection of an appropriate combination of androgen receptor level, target-gene copy number and concentration of the androgenic ligand, dihydrotestosterone, the expression level can be set within a 1400-fold range with no detectable effect on normal cell growth.

Characterization of three non-peptide endothelin receptor ligands using human cloned ETA and ETB receptors.

1. A number of putative endothelin (ET) receptor ligands were synthesized with a view to assessing their relative affinity for human recombinant ET receptors. 2. Human (h) and endothelin ETA and ETB receptor open reading frames were cloned by reverse transcription-polymerase chain reaction into the mammalian expression vector pcDNA1 and stable cell lines were created by transfection of Chinese hamster ovary cells. 3. Scatchard analyses of saturation isotherms for the specific binding of [125I]-endothelin-1 ([125I]-ET-1) to membranes, prepared from Chinese hamster ovary cells transfected with hETA or hETB receptors, yielded values for equilibrium dissociation constants (Kd) of 20.5 +/- 1.8 pM and 25.5 +/- 5.5 pM, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogeneous, non-interacting receptor populations. 4. Pharmacological characterization of the transfected hETA and hETB receptors was undertaken by measuring the relative abilities of ETA and ETB receptor-selective peptide ligands to inhibit binding of [125I]ET-1. For interaction with hETA receptors, the relative order of potency was ET-1 > ET-3 = FR139317 = BQ123 >[Ala1,3,11,15]-ET-1 = sarafotoxin S6c (S6c). In contrast, the relative order of potency, at hETB receptors, was ET-1 = ET-3 = [Ala1,3,11,15]-ET-1 = S6c >> FR139317 = BQ123. 5. The novel non-peptide ligands, Ro 46-2005, SB 209670 and BMS 182874, were found to inhibit [125I]-ET-1 binding to human recombinant ETA and ETB receptors. At hETA receptors, the calculated pIC50 values were 6.7 (Ro 46-2005), 8.7 (SB 209670) and 5.8 (BMS 182874), while at hETB receptors, the corresponding pIC50 values were 6.8, 7.5 and <5, respectively.6. In conclusion, we have characterized the pharmacology of human cloned ETA and ETB receptors and used these in membrane binding assays to determine the affinity and selectivity of three structurally diverse non-peptide ET receptor ligands. SB 209670 is, to date, the highest affinity non-peptide ligand to be described for ET receptors. As such, it may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of endothelins.

The first cleavage site in pepsinogen activation.

By incubation of porcine, bovine, canine, or chicken pepsinogens and calf prochymosin with pepstatin at pH 2.5, the first active protein generated on activation is trapped in an inactive complex. The first activation peptide liberated from porcine pepsinogen has been identified as residues 1-16 whereas that from prochymosin is derived from residues 1-27. This suggests that pepsin and chymosin are not formed by one-step conversions from their zymogens, but by (different) sequential, activation mechanisms.

Genes, disease and medicine.

1. Information and technologies derived from genomic research are beginning to revolutionize the study of disease. It is now being predicted that the human genome sequencing project will be more than 90% complete by the end of this decade and that most of the major genes involved in common diseases will have been identified by that time. Correlations between genetic mutations, disease susceptibility, and adverse reactions to drugs are already being established and it seems inevitable that this will lead to the development of novel therapies accurately targeted at subsets of patients most likely to show a favourable response. 2. Targeted therapies present a significant challenge to the pharmaceutical industry because the potential market for such drugs is likely to be smaller than for current 'phenotypic' treatments. However, application of molecular genetic technologies may allow parallel reductions in development costs since it should be more straightforward to demonstrate efficacy and safety in accurately selected patient groups, reducing the requirement for large clinical trials. 3. Gene-based diagnostics have the potential to radically improve medical practice, and progress in genetic testing technology has been impressive. Yet there is growing concern that commercial and other pressures may result in genetic testing being made widely available before the biological consequences of mutations in disease susceptibility genes are fully understood and before the legal, ethical and psychological consequences of testing have been fully debated.

Interaction of pepstatin with pig pepsinogen.

In contrast with pepsin, pepsinogen does not bind pepstatin at pH values between 5.3 and 2.5. Pepsinogen is not retarded by pepstatin immobilized on to aminohexyl-Sepharose at pH5.3 or 4.1, whereas at pH3.0 activation takes place during the chromatography, with retardation of the resultant pepsin.

Conversion of pepsinogen into pepsin is not a one-step process.

By incubation of pepsinogen with pepstatin at pH2.5, the first 'active' protein generated on activation is trapped in an inactive complex. The first activation peptide liberated has been identified as residues 1-16 from the pepsinogen sequence. This suggests a sequential mechanism rather than a one-step formation of pepsin.

Incorporation of choline and ethanolamine into phospholipids in germinating soya bean.

1. Incorporation of [Me-14C]choline and [2-14C]ethanolamine into lipids was studied in germinating soya bean (Glycine max L.) seeds. The precursors are only incorporated into phosphatidylcholine and into phosphatidylethanolamine respectively. 2. Base-labelling via a phospholipase-D type of reaction was eliminated as a significant factor. 3. Cyclo heximide inhibited labelling of phosphatidylcholine from [Me-14C]choline but did not affect labelling of the aqueous choline pool. It had no effect on [2-14C]ethanolamine uptake or incorporation into phosphatidylethanolamine. 4. Hemicholinium-15 at 10mM concentrations decreased uptake and lipid labelling from the both bases. 5. There was no evidence for base competition. 6. The endogenous pool of choline was much larger than that of ethanolamine, which resulted in higher specific radioactivities for phosphatidyl-ethanolamine than for phosphatidylcholine. 7. The results can be interpreted as indicating that the kinase and phosphoryltransferase enzymes of the CDP-base pathways are separate for each phospholipid.

Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene.

In vitro mutagenesis of the LTA gene, encoding the A subunit of the Escherichia coli heat-labile enterotoxin, has been used to obtain A subunits deficient in enzymic activity. One inactive A-subunit mutant which contained two amino acid substitutions, was shown to associate with native B subunits to form a holotoxoid lacking toxin activity. A serine to phenylalanine mutation appears to be responsible for the loss of toxicity.

Nucleotide sequences of four variants of the K88 gene of porcine enterotoxigenic Escherichia coli.

The nucleotide sequences of four variants of the Escherichia coli K88 antigen gene, K88ab1, K88ab2, K88ac, and K88ad, have been determined. The K88ab2 and K88ac sequences have not been reported previously. The K88ab1 sequence is very similar to that determined by other workers, but the K88ad sequence differs considerably from that described in a previous report. Comparison of the amino acid sequences inferred from the gene sequences revealed certain clusters of amino acid substitutions which have been correlated with areas of potential antigenicity in the mature proteins.

M13 clones carrying point mutations: identification by solution hybridization.

A solution hybridization procedure for the rapid identification of M13 clones carrying a particular sequence is described. The method, which employs a radiolabeled oligonucleotide probe, can discriminate between sequences which differ by only a single base, and can therefore be used for the identification of mutant sequences created by oligonucleotide-directed mutagenesis. Samples of phage-containing supernatant from cultures of M13-infected Escherichia coli are incubated with radiolabeled probe in the presence of sodium dodecyl sulfate. The mixtures are then subjected to agarose gel electrophoresis to separate hybrid molecules from unbound probe and hybridization is detected by autoradiography. This solution hybridization procedure is quicker and more convenient than membrane hybridization and has the added advantage that more than one probe can be used on a given gel.

Expression of an autoprocessing CAT-HIV-1 proteinase fusion protein: purification to homogeneity of the release 99 residue proteinase.

The 99 residue human immunodeficiency virus type 1 proteinase has been expressed in Escherichia coli as part of an autocleaving fusion protein. Expression of the fusion protein is toxic to the host cells, however yields of the released proteinase have been improved by optimising induction nad harvest times to increase culture biomass, and decrease degradation of the proteinase. Soluble proteinase was extracted from these cells by a simple and highly efficient three step process. N-terminal sequence analysis confirms that the enzyme preparation is highly pure and correctly autoprocessed. The proteinase cleaves peptide substrate IGCTLNFPISPIETV between F and P at pH 6.0 with a Km of 310 microM and a Kcat of 14s-1. The enzyme is sensitive to its ionic environment, showing stimulation of activity at high salt concentrations, and shows a pH optimising 5.5.

Structure-activity relationships of recombinant human interleukin 2.

Structure-activity relationships of recombinant human interleukin 2 were investigated by preparation, purification, and characterization of 21 missense mutants. A key role for residue Phe42 in the high-affinity interaction with receptor was indicated by (a) the reduction of 5-10-fold in binding affinity and bioactivity upon mutation of this residue to Ala and (b) the lack of evidence for conformational perturbation in Phe42----Ala in comparison with the wild-type protein as investigated by intrinsic fluorescence, second-derivative UV spectroscopy, electrophoresis, and reversed-phase HPLC, suggesting that the drop in binding is a direct effect of removal of the aromatic ring. In contrast, the conservative mutations Phe42----Tyr and Phe42----Trp did not cause significant reductions in bioactivity. UV and fluorescence spectra indicated approximately 60% overall exposure to solvent of tyrosines in the wild-type molecule, the tryptophan (residue 121) being buried; fluorescence data also showed that Trp42 in Phe42----Trp is likely to be within 1 nm of Trp121 and about 50% exposed to solvent. Phe44----Ala, Cys105----Ala, and Trp121----Tyr also exhibited reduced bioactivity, but these mutants are conformationally perturbed relative to wild type. None of the remaining mutants had detectably reduced bioactivity, even though several showed signs of altered conformation. Four mutants were recovered in very low yield, probably because of defective refolding.

Expression of atrial natriuretic factor as a cleavable fusion protein with chloramphenicol acetyltransferase in Escherichia coli.

Recombinant fusion proteins containing human atrial natriuretic factor, ANF(1-28) joined to chloramphenicol acetyltransferase (CAT) via cleavable linker sequences have been produced in Escherichia coli. The linker sequences were designed to allow the release of authentic ANF(1-28) following proteolytic cleavage by enterokinase or thrombin, or chemical cleavage with 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine. Proteins, containing ANF(1-28) fused to the carboxyl-terminal region of CAT (using the ScaI restriction site in the cat gene), were largely soluble in E. coli and were obtained in higher yield than analogues containing ANF(1-28) linked to shorter CAT sequences. The longer derivatives also retained CAT activity allowing subsequent purification by affinity chromatography.

Renin: structural features of active enzyme and inactive precursor.

To determine the structural basis for the unique catalytic mechanism of renin and the mechanism of activation of inactive renin, renin and inactive renin were isolated in pure form. The active site of renin consists of two aspartyl residues, two tyrosyl residues, and one arginyl residue, analogous to pepsin and other acid proteases. The complete amino acid sequence of mouse submaxillary gland renin was determined. Of the amino acids, 43% were identical to those in porcine pepsin. Combination of various chromatographic techniques permitted the separation of inactive renin from active renin in human plasma and kidney. Inactive renin of hog kidney was completely purified. Inactive renin consists of a single polypeptide chain and is activated by proteolysis but not by dissociative reagents such as 4 M NaCl or detergent. Thus it was concluded that the inactive renin in these tissues is renin zymogen rather than a renin-inhibitor complex.

Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)

The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.