Rifampicin binding as a probe for subunit interactions in Escherchia coli RNA polymerase.

The binding of the inhibitor rifampicin to RNA polymerase (alpha2betabeta') and its deficient subunit mixtures was investigated. The ability of beta to bind stoichiometric amounts of rifampicin was restored by formation of the alpha2beta subassembly. beta,beta' alpha, betabeta' and alpha2beta' were unable to bind rifampicin. RNA polymerase denatured with 6 M guanidine hydrochloride and dialysed against a renaturing buffer at 0degrees C ("renatured inactive enzyme") bound stoichiometric amounts of rifampicin but had lost the ability of bind dna. compared with native RNA polymerase "renatured inactive" enzyme possessed a markedly different tertiary structure as judged by limited proteolysis.

Importance of human gastric lipase for intestinal lipolysis: an in vitro study.

Using soybean triacylglycerols emulsified with egg lecithin we have studied, in vitro, the influence of substrate prehydrolysis by human gastric lipase upon subsequent degradation by the pancreatic lipase-co-lipase system. Fatty acids liberated by pure human gastric lipase or juice trigger immediate activity of human pancreatic lipase. Gastric lipolysis appears to be of prime importance for dietary lipid digestion in human.

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast.

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

Development of T-cell leukaemia in an ataxia telangiectasia patient following clonal selection in t(X;14)-containing lymphocytes.

Ataxia telangiectasia is a rare inherited and progressive neurological disorder in which patients show an unusual predisposition to T-cell leukaemia. We report here observations on a patient with a large cytogenetically abnormal clone showing a single t(X;14)(q28;q11) translocation which conferred a proliferative advantage on the cells. The further evolution of this clone to cytogenetically more complex clones of lymphocytes was seen in the patient. She subsequently developed a rapidly progressing T-cell leukaemia, with a CD4+CD8+ T-cell phenotype, about five years after the first appearance of additional chromosome translocations in the clone cells.

The nucleotide sequence of cDNA coding for the structural proteins of foot-and-mouth disease virus.

The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

An optical biosensor for real-time chromatography monitoring: breakthrough determination.

The use of an optical biosensor for immunorecognition of protein products during affinity chromatography is discussed to provide rapid data describing the loading and subsequent breakthrough, followed by elution and fraction collection. The optical biosensor works by following in real-time the interaction of soluble ligate with an appropriate ligand attached to the optically active surface. The initial rate of interaction between soluble ligate and immobilized ligand has been shown to correlate well with ligate concentration. This method of analysis has also been shown to agree well with ELISA, the traditionally employed technique for immunoassay of protein products lacking, for example, catalytic activity. Forward prediction, using models of the breakthrough fitted to the real-time data, has enabled the column saturation point to be determined before it has been reached, thus enabling appropriate action to ensure minimal loss of protein product while improving column utilization efficiency. The biosensor, operated within a flow injection analysis regime, has been demonstrated to provide concentration data within 10 s, with a total assay turnaround of 30 s.

Bioprocess monitoring: an optical biosensor for rapid bioproduct analysis.

The use of an optical biosensor for rapid bioproduct analysis is described. The biosensor, which is sensitive to changes in the concentration of bioproduct at its biologically active surface, has been shown to provide concentration data within 10 s of sample addition to the device. This has been achieved through the use of linear regression analysis to extract information from the early part of the biosensor interaction profiles. The system has been used to monitor both the production and purification of antibody fragments expressed during batch fermentation of recombinant Escherichia coli. Data obtained using the biosensor have been used to provide real-time profiles describing the location of antibody fragments during bioprocessing. Biosensor data have also been compared with those obtained from ELISA, the traditional method of retrospective analyses of samples collected during bioprocessing.

Analysis of kinetic data of antibody-antigen interaction from an optical biosensor by exponential curve fitting.

An optical biosensor system employing a resonant mirror (RM), with a stirred cuvette has been used to follow the interaction of a recombinant antibody fragment with its antigen, hen egg lysozyme. The data generated by the biosensor were analysed in order to determine the kinetic constants for the interaction using a linear transform (derivative analysis). For comparison the data were also analysed using an exponential curve fitting routine. It was demonstrated that the exponential curve fitting method produced results which were in agreement with the existing linear transform method. It was also shown that early fitting of the association phase response, using the exponential curve fitting routine between 0 and 70 s after sample addition, yielded sufficient information to provide a prediction of Kon. The potential use of the optical biosensor for the rapid monitoring of protein production and purification is discussed.

The nature of the stimulatory role of the supernatant fraction on triglyceride synthesis by the alpha-Glycerophosphate pathway.

Evidence is presented as to the nature and mechanism of the stimulatory effect of the supernatant fraction on the biosynthesis of triglycerides via the alpha-glycerophosphate pathway in the intestinal mucosa. When microsomes are employed as the enzyme source, the major lipid formed from either labeled palmitic acid orL: -alpha-glycerophosphate is phosphatidic acid and only a limited amount of triglyceride is synthesized. The addition of the supernatant fraction to microsomes results in a stimulation of triglyceride biosynthesis at the expense of phosphatidic acid. Employing the same microsomal fraction, the reaction sequence was followed step by step and the intermediates were isolated. The results suggest that the stimulatory role of the supernatant fraction can be attributed to the presence ofL: -alpha-phosphatidate phosphohydrolase (EC 3.1.3.4). The hydrolysis of the biosynthesized microsomal phosphatidic acid by the supernatant enzyme occurs at a faster rate than the hydrolysis of added phosphatidic acid prepared from egg lecithin. The initial acylation steps in the biosynthesis of triglycerides or phosphatidic acid via the glycerophosphate pathway occur only in the presence of fatty acid and the cofactors necessary for its activation. Under these conditions, fatty acyl-CoA will not substitute for the fatty acid activation system.

Age, gender, and education may have little influence on error patterns in the assessment of set-shifting and rule induction among normal elderly.

Error patterns have been found to be sensitive to cognitive status, but the relationship between aging and error patterns remains unclear, and may differ as a function of gender, education, and whether a task is verbal or nonverbal. The present study examined the error patterns of normal elderly individuals on a verbal measure of set-shifting and rule induction to determine whether demographic variables, that is, age, gender, and education, influenced test performance. The sample of 109 individuals, 38 males and 71 females, ranging in age from 54 to 89 years with 6 to 19 years of education, was assessed on the Classification subtest of the Test of Verbal Conceptualization and Fluency, a verbal measure of set-shifting and rule induction. Subjects' protocols were scored for perseverative, nonperseverative, and random errors, tabulated, and analyzed. Multivariate analysis of covariance with education as the covariate as well as other statistical tests revealed nonsignificant relationships between error scores and age, gender, and education. Years of education, however, showed a significant correlation with a reduction in random responses. Results are interpreted based on Horn's (1978) fluid-crystallized explanations of changes in intelligence with advancing age.

An evaluation of practice leaflets provided by general dental practitioners working in multi-racial areas.

AIM: The aim of this study was to assess the quality of information leaflets produced by NHS dental practices based in high density multi-racial areas within the city of Birmingham. METHOD: All of the 41 general dental practices based in ten Birmingham electoral wards with high concentrations of ethnic minorities were approached for a copy of their practice leaflet. Each leaflet was assessed in terms of: 1. overall presentation, 2. general information, and 3. information specifically relevant to the ethnic minorities. RESULTS: Seventy-eight per cent (32) of practices currently produce information leaflets. Compliance with specific NHS regulations ranged from 3% to 97%; 41% (13) of leaflets had one or more sections written in a minority language. Although ethnic minority languages were spoken by staff in three-quarters of the practices, less than one third specified this. Only one leaflet contained information on arrangements for non-English speaking patients. CONCLUSION: Recommendations are made concerning the quality and content of practice leaflets for practices based in high density multi-racial areas.

Structural properties of Escherichia coli RNA polymerase Subunits.

1. The surface of the RNA-polymerase-DNA complex possesses an exposed polypeptide loop. 2. Proteinases with differing specificities (trypsin, chymotrypsin, subtilisin and clostripain) preferentially cleave the exposed region. 3. The cleaved polypeptide is reassembled into RNA polymerase by renaturation from a solvent which promotes a random coil conformation. 4. Isolated beta subunit has a proteolytically resistant nucleus of approximately 70000 molecular weight. This resistant polypeptide may be generated by trypsin, chymotrypsin, subiilisin or clostripain. 5. Isolated alpha subunits are comparatively resistant to proteolysis. 6. Although of similar molecular weights beta and beta' appear to have unrelated primary sequences and markedly different conformations in free solution. 7. Digestion of the beta subunit may be blocked by formation of the alpha2beta subassembly. 8. Evidence is presented suggesting that beta' in the intact enzyme (alpha2beta beta') possesses the exposed polypeptide loop.

The dissociation of sigma-factor from ribonucleic acid polymerase.

The sigma-factor of Escherichia coli RNA polymerase was shown to dissociate from the core enzyme as a function of absolute concentration. The association constant is in the range 10(6)-10(8) litre/mol. This implies that the amount of holoenzyme, core enzyme and sigma-factor in RNA polymerase assays may vary according to the absolute concentration of the enzyme.

Ligand loading at the surface of an optical biosensor and its effect upon the kinetics of protein-protein interactions.

Optical biosensors are finding increasing use in the determination of kinetic and equilibrium constants for a variety of biomolecular interactions. Usually these biosensors require one biomolecule, the ligand, to be covalently attached to a hydrogel matrix which itself is bonded to the sensing surface. The ligands partner, the ligate, then binds from solution resulting in a measurable change in response which the instrument records as a function of time. Although in many cases, optical biosensors are used in order to obtain parameters that relate to interactions in solution, it is becoming clear that measurements involving the interaction of ligate with immobilized ligands on surfaces require careful experimental design. Here we report on how the density of ligand loading within the hydogel matrix affects the measured interaction kinetics. It is found that crowding of ligand within this matrix results in a significant reduction in the measured association rate constant, with a corresponding effect in the calculated overall affinity. However, measurements at low ligand loadings show association rate constants that are comparable to those measured in solution. Clearly, where this comparison is required, it is important to perform measurements under such conditions.

Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.

Denaturation studies on natural and recombinant bovine prochymosin (prorennin).

1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v.c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.

Altered chemical properties in three mutants of E. coli RNA polymerase sigma subunit.

We have analyzed some chemical properties of the sigma subunit of RNA polymerase from the sigma mutants: rpoD1 (Gross et al., 1978), rpoD2 (formerly known as alt-1) (Silverstone et al., 1972; Travers et al., 1978), and rpoD800 (Gross et al., 1979). Each of the three mutants is located at about 66 min on the E. coli genetic map and exhibits an alteration in the enzymatic properties of its sigma subunit. The tryptic peptides and isoelectric focusing behavior were analyzed for mutant and wild type sigma. A single, but different altered lysine tryptic peptide was observed for each mutant. No altered arginine tryptic peptides were observed. The rpoD800 mutant sigma showed an altered isoelectric point. These studies provide chemical evidence that the sigma polypeptide in all three mutants is altered and strongly support the conclusion that the mutations are in the structural gene for sigma.

In vitro thermal inactivation of a temperature-sensitive sigma subunit mutant (rpoD800) of Escherichia coli RNA polymerase proceeds by aggregation.

A temperature-sensitive mutant sigma subunit (rpoD800) purified from Escherichia coli was inactivated in vitro by temperatures in excess of 37 degrees C whereas wild type sigma remained stable up to 49 degrees C. Both temperature-sensitive and wild type sigma formed multimeric aggregates upon thermal inactivation which were visualized by electron microscopy as polymeric chains. Conditions favoring sigma monomer (low sigma concentration and binding to core polymerase) protected temperature-sensitive sigma from heat inactivation. Full activity was recovered from inactivated temperature-sensitive sigma aggregates by incubation in a buffer containing 6 M guanidine HCl and subsequent removal of denaturant by dilution. Both wild type and temperature-sensitive sigma recovered full activity levels, retaining their characteristic thermal inactivation temperatures after denaturation in 6 M guanidine HCl and renaturation. Transcription of T4 DNA by RNA polymerase containing the rpoD800 mutant sigma subunit remained undiminished for 10 min after shift up to 46 degrees C but was almost completely inhibited within the following 10 to 15 min.