A second site-specific recombination event in the lambdoid bacteriophage HK022

 An in vitro site-specific recombination reaction of the lambdoid phage HK022 has revealed two supercoiled products that proved to be Holliday intermediates. One of them is the Holliday intermediate which has resulted from an attP×attB reaction. The other is an intermediate which has resulted from a recombination reaction between attP and the attL site of the product from the first reaction. The preferential attL×attP over attR×attP reaction was confirmed in vitro and in vivo by challenging attP sites with attL and attR sites. The biased attP×attL over attP×attR reaction in phage HK022 is discussed. Received: 25 April 1996 / Accepted: 11 July 1996     

Escherichia coli tRNAs Are Resistant to the Hyperprocessing Reaction of Homologous E. coli Ribonuclease P Ribozyme

Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs. The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA. Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes. In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E. coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction. Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E. coli.     

Photosystem I reaction centers fromChlamydomonas and higher plant chloroplasts

A photosystem I reaction center has been isolated fromChlamydomonas chloroplasts and compared with the photosystem I reaction center from higher plants. While the higher plant reaction center is active in cytochrome 552 photooxidation, theChlamydomonas preparation was not active unless salts were included in the assay medium or the pH was lowered to 5. Subunit III-depleted photosystem I reaction center from higher plants is also inactive in cytochrome 552 photooxidation in the absence of salts. As with theChlamydomonas reaction center, salts induced its activity. Subunit I of the photosystem I reaction center has tentatively been identified as the binding site of cytochrome 552.     

Blood group association with severity and speed of the halothane reaction1

The second generation (n= 227) of British Landrace pigs from selected halothane-positive parents (36 litters) were blood-typed for the S(A-0), H and Phi loci and subjected to four 5-minute halothane tests at 21, 35, 49 and 63 days of age. Cumulative scores based on seventy and speed of reaction were analysed in relation to single-locus blood group genotypes and linkage group sequences at two and three loci. A highly significant negative correlation (r = -0.79) was found between severity and speed of reaction. Significant differences occurred between blood group genotypes and linkage groups in both severity and speed of reaction. Genotypes S s/s, H a/a or H a/- and Phi B/B and linkage groups involving these three types had the highest cumulative reaction score and the fastest reaction time, whereas genotypes Phi A/B, S S/S or S S/s and H a/cd and linkage groups with these types had the Iowest and slowest reaction scores. Some differences between genotypes and linkage groups were attributed to phenotypically halothane-positive parents and offspring being genotypically Hal N/n. These effects could result from linkage with heterozygous types such as H a/cd and S S/s. The possible role of the H ^cd allele acting as a genetic marker for a suppressor gene to the halothane reaction is discussed.     

Kinetics and Mechanism of the Peptide Synthesis in Solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: = kC _N ^a C _AE ^b , where k is the reaction rate constant, C _N is the concentration of leucinamide, and C _AE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates.     

Effects of external diffusion and design geometry on the performance of immobilized glucose isomerase reactor system

Using immobilized glucose isomerase, the effects of superficial velocity of the reaction solution flowing through a packed-bed reactor on the apparent kinetic constants of reversible reaction system were studied. The results showed that the apparent kinetic constants, both V_m″ and K_m″, of the forward reaction varied significantly as the superficial velocity is changed, whereas those of the reverse reaction varied only slightly. Using the kinetic data determined experimentally, computer simulation of the enzyme reactor performance was carried out, and the importance of the external mass transfer in the proximity of immobilized-enzyme particles was recognized. The reactor performance, expressed in terms of productivity, was examined as a function of the reactor height-to-diameter ratio, H/D. The productivity of the reactor system goes through a maximum value at a H/D ratio of about 1.6. and decreases as the H/D ratio increases. Theoretical analysis of the reaction kinetics of immobilized-enzyme system that has reversible reaction kinetics is also presented. The experimental results showed good agreement with the results found from the theoretical analysis and the computer simulation studies. Based on the principles of the methods and the results presented in this paper, it is anticipated that one can predict the optimal design and operating conditions for the glucose isomerase reactor system and that application of the results could be extended to other enzyme systems with reversible reaction kinetics.     

Possibility of the Nonenzymatic Browning (Maillard) Reaction in the ISM

The possibility of the occurrence of the nonenzymatic browning reaction in the gaseous phase in the interstellar medium has been investigated by using Density Functional Theory computations. Mechanisms for the reactions between formaldehyde (Fald) + glycine (Gly), Fald + NH ₃ and Fald + methylamine (MeAm) have been proposed, and the possibility of the formation of different compounds in the proposed mechanisms has been evaluated through calculating the Gibb's free energy changes for different steps of the reaction, by following the total mass balance. The Fald + Gly reaction under basic conditions is found as the most favorable for producing 1-methyl-amino methene or 1-methyl-amino methelene (MAM). The reaction under acidic conditions is found to be the least favorable for producing MAM. The Fald + NH ₃ reaction is found to be plausible for the production of MeAm, which can participate by reaction with Fald, resulting in the formation of MAM.     

Enantiomer selection in the reaction of N-methyl-α-amino acid N-carboxyanhydride and 3-methyl-5-substituted hydantoin: A model reaction for the stereoselective polymerization of α-amino acid N-carboxyanhydride

The addition reaction to N-methyl-(S)-alanine or N-methyl-(S)-phenylalanine N-car-boxyanhydride (NCA) of 3-methyl-5-substituted hydantoin (HDT) catalyzed by a tertiary amine was investigated as a model reaction for the propagation reaction of NCA according to the activated-NCA mechanism. Several activated HDTs having the (S)-configuration of the asymmetric carbon atom were found to react more rapidly than their activated enantiomers. This experimental result indicates that the enantiomer selection by terminal-unit control takes place in the propagation reaction according to the activated-NCA mechanism in which an activated NCA is added to a terminal acylated NCA ring of the growing chain. The enantiomer excess of the HDT recovered from the reaction mixture of N-methyl-(S)-phenylalanine NCA and racemic HDTs activated by a tertiary amine was determined. The extent of the enantiomer selection in the polymerization was found to be 3–10 times as large as that in the model reaction. From these results, it was concluded that the chirality of the penultimate unit, as well as that of the terminal NCA ring, plays an important role in determining the enantiomer selection in the NCA polymerization.     

Peptide Thioester Synthesis via an Auxiliary-Mediated N–S Acyl Shift Reaction in Solution

The 4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group attached to a main chain amide in a peptide is easily transformed into an S-peptide via an intramolecular N–S acyl shift reaction under acidic conditions, and the S-peptide produces a peptide thioester through an intermolecular thiol–thioester exchange reaction. In order to develop a method for efficiently preparing peptide thioesters based on the N–S acyl shift reaction, the factors involved in this process were analyzed in detail. The general features of the transformation at the Dmmb group attached amide bond in a trifluoroacetic acid (TFA) solution and the generation of a peptide thioester were examined by ¹³C-NMR spectral measurements, reversed-phase (RP) HPLC analyses, mass measurements, and amino acid analyses. The methoxy group of the Dmmb group was not essential for the N–S acyl shift reaction, but played a role in stabilizing the thioester form. The addition of water to the TFA solution accelerated the N–S acyl shift reaction mediated by the Dmmb group and also suppressed the acid-catalyzed cleavage of the Dmmb group. A peptide thioester was produced from the S-peptide via an intermolecular thiol–thioester exchange reaction with minimal epimerization of the amino acid residue that constituted the thioester bond. Undesirable side reactions, such as the hydrolysis of the thioester bond and an S–N acyl shift reaction occurred during the synthetic process, which is a subject of further investigation.     

Enzymatic Production of Pyrimidine Nucleotides Using Corynebacterium ammoniagenes Cells and Recombinant Escherichia coli Cells: Enzymatic Production of CDP-Choline from Orotic Acid and Choline Chloride (Part I)

Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Corynebacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mm) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY13505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95 g/liter (15.1 mm) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CKIase; encoded by the CKI gene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mm) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.     

Serological difference betweenErwinia herbicola strains of plant and human origin

FiftyErwinia herbicola isolates obtained from host plants were examined in an agglutination reaction with antiserum prepared againstE. ananas (E. herbicola) strain CCM 2407 antigen of plant origin and with antiserum prepared againstEnterobacter agglomerans strain CNCTC M 269 antigen of human origin. In tests with strain CCM 2407 antiserum, 56% isolates showed a positive reaction, while in tests with strain CNCTC M 269 antiserum only 14 % isolates showed a positive reaction. AmongE. herbicola isolates which showed a positive reaction with CCM 2407 antiserum 18 % showed a positive reaction with the CNCTC M 269 antiserum too. Our results confirmed the serological heterogeneity ofE. herbicola population. In spite of the difference in the origin of the two antigens used for the preparation of antisera (plant, human; Japan, Czech Republic) our results indicate that some of ourE. herbicola strains andE. agglomerans strain CNCTC M 269 are serologically identical.     

Characterization of the Aminoacylation of Cytoplasmic Transfer Ribonucleic Acids from Germinating Soybeans

Extracts from germinating soybean [Glycine max (L.) Merr.] cotyledons were assayed for aminoacyl-tRNA formation. This esterification reaction depends on several factors, which must be determined and controlled for each system investigated. The reaction was maximum when the Mg: ATP ratio was 12.5 and concentrations were 9 and 0.72 mM, respectively. The optimum temperature for the in vitro reaction was 25°C. In addition, the reaction was inhibited by inorganic pyrophosphate and magnesium could be replaced by spermidine.     

Batch kinetic studies of glucose isomerization to fructose by Arthrobacter species

Glucose can be isomerized to fructose by the catalytic action of the enzyme, glucose isomerase. This enzyme is synthesized by a variety of micro-organisms, predominantly by bacteria. Arthrobacter species cells are grown in a medium standardized specifically to synthesize the enzyme and are then used to isomerize glucose under conditions of no further cell growth. Effect of metal ions on the isomerization is studied and it is found that magnesium promoted the reaction, sodium had no effect and calcium and manganese inhibited the reaction. Rate of reaction per unit of catalyst is found to be constant. Michaelis-Menten model modified for the reversibility of the reaction is suitable to describe the isomerization kinetics and the kinetic parameters are determined and reported.List of Symbols k ₁ rate constant (Glucose to intermediate complex) - k _–1 rate constant (Intermediate complex to glucose) - k ₂ rate constant (Intermediate complex to fructose) - k _–2 rate constant (Fructose to intermediate complex) - v _mf maximum reaction velocity of the forward (GF) reaction - v _mb maximum reaction velocity of the reverse (FG) reaction - K _f Michaelis-Menten constant for the forward (GF) reaction - K _b Michaelis-Menten constant for the reverse (FG) reaction - K _eq equilibrium constant - r _G rate of glucose consumption     

Detection of Rhopalosiphum insertum (apple-grass aphid) predation by the predatory mite Anystis baccarum using molecular gut analysis

Abstract 1 A simple, yet sensitive polymerase chain reaction based technique was developed for the detection of the apple‐grass aphid Rhopalosiphum insertum in the gut of Anystis baccarum, a predatory mite. 2 A range of conserved polymerase chain reaction primers for insect mitochondrial and ribosomal DNA were tested in order to amplify R. insertum DNA. The mitochondrial DNA primers LrRNAR2 + N1F1, amplified a region between the ND1 and large subunit RNA genes. 3 DNA sequencing of the R. insertum ND1‐LRNA polymerase chain reaction product allowed aphid‐specific polymerase chain reaction primers to be designed. These amplified a 283‐bp product from individual aphids. No polymerase chain reaction product was amplified from individual A. baccarum. 4 Using the aphid‐specific primers against A. baccarum fed on R. insertum, the diagnostic 283‐bp product was amplified. 5 Two restriction enzymes (RsaI and AluI) produced patterns that allowed unambiguous identification of R. insertum DNA from that of Macrosiphum euphorbiae and Myzus persicae.     

An unusual form of reaction wood in Koromiko [<Emphasis Type="Italic">Hebe salicifolia</Emphasis> G. Forst. (Pennell)], a southern hemisphere angiosperm

Koromiko [Hebe salicifolia G. Forst. (Pennell)] is a woody angiosperm native to New Zealand and Chile. Hebe spp. belong to the otherwise herbaceous family Plantaginaceae in the order Lamiales. Reaction wood exerting expansional forces was found on the lower side of leaning H. salicifolia stems. Such reaction wood is atypical for angiosperms, which commonly form contracting reaction wood on the upper side of leaning stems. Reaction wood typical for angiosperms is formed by species in other families in the order Lamiales. This suggests that the form of reaction wood is specific to the family level. Functionally the reaction wood of H. salicifolia is similar to that found in gymnosperms, which both act by pushing. However, their chemical, anatomical and physical characteristics are different. Typical features of reaction wood present in gymnosperms such as high density, thick-walled rounded cells and the presence of (1 → 4)-β-galactan in the secondary cell wall layer are absent in H. salicifolia reaction wood. Reaction wood of H. salicifolia varies from normal wood in having a higher microfibril angle, which is likely to determine the direction of generated maturation stresses.     

Comparing bivariate reaction norms among species: time and size at metamorphosis in three species of Hyla (Anura: Hylidae)

Summary Univariate and bivariate methods for comparing norms of reaction among species are discussed and illustrated with an example using North American hylid treefrogs. Norms of reaction for size at metamorphosis (SM) and length of larval period (LP) were compared among treefrog species raised at different food levels (Hyla cinerea vs H. gratiosa) and at different temperatures (H. cinerea vs H. gratiosa vs H. squirella). Hyla cinerea and Hyla gratiosa show parallel norms of reaction across food levels and temperatures. Across temperatures, H. squirella shows a much smaller change in SM relative to change in LP than do H. cinerea and H. gratiosa. This difference in shape of reaction norms may reflect different histories of selection resulting from these species' use of different larval habitats.     

β-galactosidase-catalyzed synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol: Optimization by response surface methodology

In this study, the synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol (GG) was performed by the reverse hydrolysis of D-galactose and glycerol using β-galactosidase from Kluyveromyces lactis. Four process variables, reaction temperature (30.0–45.0?°C), reaction time (24–48?h), enzyme concentration (150.00–350.00?U/mL), and substrate molar ratio (glycerol:D-galactose, 7.5:12.5?mmol/mmol) were investigated and optimized via response surface methodology (RSM) for optimal GG synthesis. Both quadratic equations and the optimal reaction conditions were established. Results showed that the four variables, i.e., reaction temperature, reaction time, enzyme concentration, and substrate molar ratio had significant (p?β-galactosidase concentration and 8.65:1.00 of substrate molar concentration ratio (glycerol: D-galactose) at 39.8?°C and 48?h of reaction. Under these conditions, the GG concentration was 140.03?g/L and GG yield was 55.71%, which both were close to the predicted values (143.26?g/L and 56.73%). This finding proves the RSM to be a useful tool in optimizing process conditions for GG synthesis.     

Comparison of a Molecular and an Immobilized Gadolinium(III)‐tris[(1R,4S)‐3‐heptafluorobutanoyl‐camphor] as Catalyst in the Asymmetric Danishefsky‐Hetero‐Diels‐Alder‐Reaction

On‐column reaction gas chromatography combines the power of separation and rapid analysis of reactants and reaction products with screening of reactions in a single step. Not only conversions but the reaction rates at various temperatures can be obtained from single measurements, making this approach superior to the time‐consuming measurements typically performed in reaction progress analysis. However, this approach has only been used in the investigation of interconversion processes, rearrangement reactions, and only a few examples of higher‐order reactions are known. Here we present the screening of immobilized gadolinium(III)‐tris[(1R,4S)‐3‐heptafluorobutanoyl‐camphor] in the Danishefsky‐hetero‐Diels‐Alder‐reaction by enantioselective on‐column reaction gas chromatography utilizing cryogenic focusing to achieve catalytic conversions in this higher‐order reaction and subsequent separation of the enantiomeric product mixture to determine the enantiomeric ratio. The results obtained by this approach could be transferred to the conventional batch reaction at a larger scale, demonstrating that on‐column reaction chromatography provides reliable results in the screening of enantioselective reactions. Chirality 26:243–248, 2014. © 2014 Wiley Periodicals, Inc.     

Asymmetric Synthesis of (S)-α-Methylbenzylamine by Recombinant Escherichia coli Co-Expressing Omega-Transaminase and Acetolactate Synthase

To produce (S)-α-methylbenzylamine (MBA) from acetophenone, recombinant Escherichia coli co-expressing ω-transaminase and acetolactate synthase was used as a whole-cell biocatalyst. The solvent-bridge reaction system increased the yield of the whole-cell reaction by 2.5-fold, and the inhibitory (S)-α-MBA produced in the ω-transaminase reaction solution (pH 8.0) moved into the extraction solution (pH 3.0) via an organic solvent.     

Changes in Sapwood of Roots of Norway Spruce, Attacked by Fomes annosus. Part I

Chemical and physiological changes occurring in root sapwood of Norway spruce (Picea abies [L.] Karst.), when attacked by Fomes annosus (Fr.) Cke, were studied. The transformation of sapwood to reaction zone, induced by the fungal attack, implies a very sharp increase of carbonate content, correlated with higher amounts of potassium, calcium and magnesium. The buffer capacity of the reaction zone is strong, especially in the pH range 6–9. The high peroxidase activity in the rays of the sapwood is almost totally absent in the reaction zone, probably due to inactivating phenolic compounds. o-Diphenol oxidase was detectable only in the presence of microorganisms. p-Diphenol oxidase was active in connection with decaying wood but not in the reaction zone.