Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution

The three-dimensional structure of goose-type lysozyme (GEWL), determined by x-ray crystallography and refined at high resolution, has similarities to the structures of hen (chicken) egg-white lysozyme (HEWL) and bacteriophage T4 lysozyme (T4L). The nature of the structural correspondence suggests that all three classes of lysozyme diverged from a common evolutionary precursor, even though their amino acid sequences appear to be unrelated (Grütter et al. 1983). In this paper we make detailed comparisons of goose-type, chicken-type, and phage-type lysozymes. The lysozymes have undergone conformational changes at both the global and the local level. As in the globins, there are corresponding alpha-helices that have rigid-body displacements relative to each other, but in some cases corresponding helices have increased or decreased in length, and in other cases there are helices in one structure that have no counterpart in another. Independent of the overall structural correspondence among the three lysozyme backbones is another, distinct correspondence between a set of three consecutive alpha-helices in GEWL and three consecutive alpha-helices in T4L. This structural correspondence could be due, in part, to a common energetically favorable contact between the first and the third helices. There are similarities in the active sites of the three lysozymes, but also one striking difference. Glu 73 (GEWL) spatially corresponds to Glu 35 (HEWL) and to Glu 11 (T4L). On the other hand, there are two aspartates in the GEWL active site, Asp 86 and Asp 97, neither of which corresponds exactly to Asp 52 (HEWL) or Asp 20 (T4L). (The discrepancy in the location of the carboxyl groups is about 10 A for Asp 86 and 4 A for Asp 97.) This lack of structural correspondence may reflect some differences in the mechanisms of action of the three lysozymes. When the amino acid sequences of the three lysozyme types are aligned according to their structural correspondence, there is still no apparent relationship between the sequences except for possible weak matching in the vicinity of the active sites.     

An estimate on the effect of point mutation and natural selection on the rate of amino acid replacement in proteins

We outline a method for estimating quantitatively the influence of point mutations and selection on the frequencies of codons and amino acids. We show how the mutation rate, i.e., the rate of amino acid replacement due to point mutation, can be affected by the codon usage as well as by the rates of the involved base exchanges. A comparison of the mutation rates calculated from reliable values of codon usage and base exchange probabilities with those that would be expected on the basis of chance reveals a notable suppression of replacements leading to tryptophan, glutamate, lysine, and methionine, and particularly of those leading to the termination codons. If selection constraints are neglected and only mutations are taken into account, the best agreement between expected and observed frequencies of both codons and amino acids is obtained for alpha = 1.13-1.15, where (Formula: see text). The "selection values" of codons and amino acids derived by our method show a pattern that partially deviates from others in the literature. For example, the selection pressure on methionine and cysteine turns out to be much more pronounced than expected if only the discrepancies between their observed and expected occurrences in proteins are considered. To estimate to what extent randomly occurring amino acid replacements are accepted by selection, we constructed an "acceptability matrix" from the well-established matrix of accepted point mutations. On the basis of this matrix "acceptability values" of the amino acids can be defined that correlate with their selection values. We also examine the significance of mutations and selection of amino acids with respect to their physicochemical properties and functions in proteins. The conservatism of amino acid replacements with respect to certain properties such as polarity can be brought about by the mutational process alone, whereas the conservatism with respect to other relevant properties--among them all measures of bulkiness--obviously is the result of additional selectional constraints on the evolution of protein structures.     

Sucrose density gradient centrifugation of human myometrial and myomal progestin receptors

Progestin receptors present in cytosols of myomal and myometrial origin were analyzed on sucrose density gradients of low ionic strength using a vertical tube rotor. Short term incubations (90 min) contained mainly 8S receptors. Most myometrial but few myomal preparations contained an additional 4S component, the presence of which was hormone dependent. None could be detected if the serum concentration of estradiol was high. Myometrial receptors incubated over night exhibited only a 4S peak, whereas many myomal ones still sedimented as 8S peaks. The 8 to 4S shift represented a transformation of the receptors and not a sequential interaction of the hormone with first an 8S and then a 4S entity. The transforming activity contained in the myometrial cytosol could also attack the myomal receptor as shown by mixing experiments. Only the 8S peak was observed, whenever the cytosol was first fractionated and then incubated with the hormone. Thus the transformation occurred only in presence of [3H]promegestone. It could be suppressed with protease inhibitors. It is concluded that these tissues contain a receptor processing protease which (1) is much less prominent in myomal than myometrial cytosol, which (2) attacks only the occupied receptors, and (3) the activity of which is hormone dependent.     

An RP4-prime containing a 285 kb fragment of rhizobium meliloti pSym megaplasmid: Structural characterization and utilization for genetic studies of symbiotic functions controlled by pSym

Summary We integrated the RP4 plasmid into a selected region of the pSym megaplasmid of Rhizobium meliloti 2011 by homologous recombination between pSym and a cloned fragment of pSym present in the RP4. This cointegrate was used to mobilize into Escherichia coli a Tn5 transposon located on pSym in the vicinity of the site of integration of the RP4. By this technique we obtained a series of RP4-primes that contained large fragments of the pSym megaplasmid and that were most probably generated by IS8 promoted deletions in the RP4-pSym cointegrate. One of them, pGMI42, which carries nitrogenase genes nifD and H as well as nodulation genes, was used for mutagenesis of the corresponding region of pSym after insertion of the Mu prophage into the tet gene. When various (pGMI-42:: Mu)::Tn7 were introduced into R. meliloti 2011 by conjugation, homologous recombination allowed insertion of Tn7 into pSym whereas the pGMI42::Mu was lost due to the suicide effect of Mu. In this way we obtained several symbiotic mutants deficient in either nodulation (Nod^-) or nitrogen fixation (Fix^-) in association with the host plant Medicago sativa.This paper is affectionately dedicated to the memory of Jean-Simon Julliot who initiated and inspired this work and who was killed by an avalanche on February 21, 1982     

The effect of ampicillin and tylosin on the faecal enterococci of healthy young chickens

Enterococcal isolates from young chickens were differentiated into one of three species, namely Enterococcus faecalis, Ent. faecium and Ent. gallinarum . The proportion of each species among the enterococcal population changed with time in birds not dosed with antibiotics. This pattern of change was modified in birds dosed with either tylosin or ampicillin even though ampicillin did not select for ampicillin resistance among the enterococcal population. A gradual increase in tylosin resistance was recorded with time among the enterococci of the 'undosed' control birds. This was associated with an increase in the proportion of Ent. faecium , a species commonly resistant to tylosin, among the enterococci of the birds as they grew older.     

Penicillin acylase production by E. coli

Enzyme production with E. coli ATCC 11105, in a complex medium using phenylacetic acid as inducer is carried out in a stirred-tank reactor of 10 dm³ and an airlift tower-loop reactor of 60 dm³ with outer loop at a temperature of 27 °C. The optimum inducer concentration was 0.8 kg/m³, which was kept constant by fed-batch operation. The optimum of the relative dissolved O₂-concentration with regard to saturation is below 10% in a stirred-tank reactor and at 35% in a tower-loop reactor. It was kept constant by parameter-adaptive control of the aeration rate. In a stirred-tank enzyme productivity is slightly higher than in a tower-loop reactor, and much higher than in a bubble column reactor.List of Symbols CPR kg/(m³ h) CO₂-production rate - OTR kg/(m³ h) O₂-transfer rate - OUR kg/(m³ h) O₂-utilization rate - PAA phenylacetic acid (inducer) - RQ = CPR/OUR respiratory quotient - X kg/m³ cell mass concentration - _m h^–1 maximum specific growth rate     

Growth of E. coli in a stirred tank and in an air lift tower reactor with an outer loop

E. coli ATCC 11105 was cultivated in a 10-1 stirred tank reactor and in a 60-1 tower loop reactor in batch and continuous operation. By on-line measurements of O₂ and CO₂ concentrations in the outlet gas, pH, temperature, cell mass concentration X as well as dissolved O₂ concentration along the tower in the broth, gas holdup, broth recirculation rate through the loop and by offline measurements of substrate concentration DOC and cell mass concentration along the tower, the maximum specific growth rate _m , yield coefficients Y _X/S. Y _X/DOC and were evaluated in stirred tank and tower loop in batch and continuous cultures with and without motionless mixers in the tower and at different broth circulation rates through the loop. To control the accuracy of the measurements the C balance was calculated and 95% of the C content was covered.The biological parameters determined depend on the mode of operation as well as on the reactor used. Furthermore, they depend on the recirculation rate of the broth and built-ins in the tower. The unstructured cell and reactor models are unable to explain these differences. Obviously, structured cell and reactor models are needed. The cell mass concentration can be determined on line by NADH fluorescence in balanced growth, if the model parameters are determined under the same operational conditions in the same reactor.List of Symbols a, b empirical parameters in Eq. (1) - CPR kg/(m³ h) CO₂ production rate - C kg/m³ concentration - D l/h dilution rate - DOC kg/m³ dissolved organic carbon - I net. fluorescence intensity - K _S kg/m³ Monod constant - k _L a l/h volumetric mass transfer coefficient - OTR kg/(m³ h) oxygen transfer rate - OUR kg/(m³ h) oxygen utilization rate - RQ = CPR/OUR respiratory quotient - S kg/m³ substrate concentration - t h,min, s time - t _u min recirculation time - t _M min mixing time - v m³/h volumetric flow rate through the loop - X kg/m³ (dry) cell mass concentration - Y _X/S yield coefficient of cell mass with regard to the consumed substrate - Y _X/DOC yield coefficient of the cell mass with regard to the consumed DOC - Y _X/O yield coefficient of the cell mass with regard to the consumed oxygen - Z relative distance in the tower from the aerator with regard to the height of the aerated broth - l/h specific growth rate - _m l/h maximum specific growth rate Indices f feed - e outlet     

Vaccination against schistosomiasis in mice with killed schistosomula without adjuvant

Attempts to develop a killed vaccine against schistosomiasis have generally resulted in failure. There are two recent reports, but unfortunately, harsh adjuvants were used in conjunction with the antigenic materials. In our laboratory, a killed vaccine was developed by freezing (-196 degrees C) and thawing the schistosomula of S. mansoni. The use of such a preparation without adjuvant was effective in vaccinating mice. A worm reduction of 36.4-41.1% was achieved by one vaccinating injection, a 60.2% worm reduction by 3 injections, and a 63.7-66.0% reduction by 5 injections. The sequence of the development and the expression of the immune reactions were similar to those previously found in hosts immunized with highly X-irradiated schistosome organisms. Delayed hypersensitivity was demonstrated in histological sections of the skin in the challenged mice after one vaccination, showing that an adjuvant was not necessary to initiate the induction of cellular immunity.     

Multiline varieties and disease control

Summary Existing theoretical models have led to conflicting predictions concerning the likely effect of the widespread use of dirty crop multilines on the evolution of virulence in pathogen populations. Here we attempt to clarify these problems by extending existing models to include selection against unnecessary genes for virulence at two different stages in the life cycle of the pathogen. The results of these studies indicate that the stage of the life cycle at which selection occurs can significantly influence the evolution of virulence in pathogen populations growing on multiline varieties.