Detection of 81 of 81 known mouse beta-globin promoter mutations with T4 endonuclease VII--the EMC method

The enzyme mismatch cleavage (EMC) method relies on the use of the resolvase T4 Endonuclease VII to cleave and thus detect mismatches in heteroduplex DNA formed by annealing normal DNA with mutant DNA. Detection is based on cleavage 3' to the mismatch within a few nucleotides. We report the detection of all 81 different homozygous single-basepair changes tested and present in the mouse beta-globin promoter by using the EMC method with a single set of conditions. Efficiency of cleavage was rated as strong, medium, or weak based on the intensity of the cleavage product(s) compared with background bands on autoradiography. We expect this method to detect near 100% of mutations.     

Association of holliday-structure resolving endonuclease VII with gp20 from the packaging machine of phage T4

Endonuclease VII (endo VII) is the product of gene 49 (gp49) of bacteriophage T4. It is a Holliday-structure resolvase (X-solvase) responsible for clearing branched replicative DNA prior to packaging. Consequently, mutations in gene 49 are unable to fill heads to completion because unresolved branches stop translocation of DNA. A likely association of gp49 with heads or proheads, however, could not be shown in the past. We have investigated whether gp49 could be part of the transiently assembled packaging machine (the "packasome") located at the base of proheads. Using purified proteins gpl6, gpl7 and gp20, which are constituents of the packasome, we found that gp49 binds tightly to gp20 and does not bind to gpl6 or gpl7. Quantification revealed that one dimer of gp49 binds one monomer of gp20. Notably, dimerisation of gp49 was an essential prerequisite for complex formation with gp20, and the dimerisation-deficient point mutation His-EVII-W87R showed only residual affinity to gp20. Furthermore, truncated peptides of gp49 deficient in dimer formation to various degrees were found to be impaired in binding to gp20. In contrast, the cleavage-deficient mutation EVII-N62D bound normally to gp20. The cruciform DNA (cf-DNA) resolving activity typical of endo VII is maintained in gp20-gp49 complexes. Furthermore, the complexes bind cf-DNA in the absence of Mg2+ as demonstrated by electromobility shift assays. The binding of the complexes to cf-DNA occurs via gp49, since gp20 alone does not bind cf-DNA. In conclusion, these findings are consistent with a model in which gp49 is an integral part of the packaging machine of phage T4.     

Epitope mapping of T4 endonuclease VII with monoclonal antibodies reveals importance of both ends of the protein for target binding

Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure. The protein has a molecular mass of 18 kDa and exists as a dimer in solution. Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme. From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established. The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays. Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII. The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization. Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs.     

Education for the nurse of tomorrow: a community-focused curriculum

A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC50) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor.     

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine-to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.     

An apyrimidinic site kinks DNA and triggers incision by endonuclease VII of phage T4

Apurinic/apyrimidinic lesions (AP-sites) occur frequently in DNA, generated by physically and chemically induced or spontaneous loss of bases. Repair mechanisms have evolved in organisms to deal efficiently with AP-sites by first incising the DNA at the lesion, followed by excision and resynthesis of the damaged strand. Here we report that endonuclease VII (endo VII) of phage T4, which was originally classified as a debranching and Holliday structure resolving enzyme, also recognizes AP-sites with high efficiency. The enzyme cleaves both strands of double-stranded DNA in a stepwise fashion a few nucleotides 3' of the lesion. In a search for a recognition signal shared by all known endo VII substrates, kinking of DNA has earlier been suggested as such a signal. In support of this hypothesis, we demonstrate here that AP-sites induce distinct kinks in synthetic oligonucleotides allowing efficient intramolecular ring closure by ligation.     

The influence of buffer composition on separation efficiency and resolution in capillary electrophoresis of 8-aminonaphthalene-1,3,6-trisulfonic acid labeled monosaccharides and complex carbohydrates

The effect of buffer conditions -- varying in salt type, pH, and concentration -- on the separation of 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled monosaccharides and complex-type carbohydrates was investigated. Different buffer systems for high and low electroosmotic flow conditions were chosen: a phosphate and a citrate background electrolyte, each at pH 2.5, a phosphate buffer, pH 9.0, and a borate buffer at pH 9.5. All buffer systems displayed differences in resolution and selectivity. Phosphate and borate buffer demonstrated the greatest selectivity changes for ANTS-labeled carbohydrates. While separation in the phosphate system relies mainly on differences in the charge-to-mass-ratio, additional selectivity can be achieved with borate complexation of glycoconjugates. The use of borate buffers improved monosaccharide separations whereas complex carbohydrates showed a loss in resolution. The citrate background electrolyte at low pH caused no significant changes in the separation performance. The pH 9.0 phosphate buffer showed a reversed migration order of the ANTS conjugates with a decreased resolution, compared to the pH 2.5 phosphate buffer, due to the strong electroosmotic flow generated under high pH conditions. An ovalbumin-derived oligosaccharide library demonstrates the significance of buffer selectivity for complex carbohydrate separations. The separation in the acidic phosphate and the alkaline borate buffer generates a different pattern and only the combination of both buffer systems allows an appropriate assessment of sample complexity.     

Acute and chronic effects of gallium chloride (GaCl3) on tilapia (Oreochromis mossambicus) larvae

PURPOSE: The synthesis of chemically stable triazene prodrugs capable of hydrolysing under physiological conditions to liberate cytotoxic monomethyltriazene alkylating agents. METHODS: A series of 3-aminoacyl-1-aryl-3-methyltriazenes was synthesised through reaction of 1-aryl-3-methyltriazenes with N-BOC protected amino acids using the DCC method of activation, followed by deprotection of the amino function using HCl in nitromethane. Half-lives for the hydrolysis of these compounds to the corresponding monomethyltriazenes at 37 degrees C in isotonic phosphate buffer and in 80% human plasma containing 20% phosphate buffer were determined by HPLC. RESULTS: The aminoacyltriazene prodrugs hydrolyse in isotonic phosphate buffer with t1/2 values ranging from 26 to 619 minutes. In human plasma, several decompose at the same rate as in phosphate buffer whereas those containing more lipophilic groups decompose more slowly. A beta-alanyl derivative was found to be more stable in phosphate buffer (t1/2 = 180 minutes) than in plasma (t1/2 = 53 minutes). An N-acetylated alpha-alanyl derivative was found to be chemically stable in phosphate buffer (t1/2 = 10 hours) but liberated the cytotoxic drug in t1/2 = 41 minutes in plasma, demonstrating its ability to act as a substrate for plasma enzymes. CONCLUSIONS: Aminoacyltriazenes are prodrugs of the antitumour monomethyltriazenes hydrolysing in human plasma with a range of reactivities. The acylation of the alpha-amino group seems to be an effective and simple means of reducing the chemical reactivity of the alpha-aminoacyl derivatives while retaining a rapid rate of enzymatic hydrolysis.     

Specificity of hammerhead ribozyme cleavage

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.     

The persistence of leptospiral agglutinins titers in human sera diagnosed by the microscopic agglutination test

Human serum albumin (HSA) is used in large amounts as an excipient in many biopharmaceutical formulation to prevent loss of the active ingredient through adsorption and/or degradation. Traditionally, iso-electric focusing has been used to demonstrate charge heterogeneity in HSA preparations. In an effort to develop new methods for the analysis of formulation components, a capillary zone electrophoresis method was developed for the analysis of HSA. Under initial separation conditions using untreated silica capillaries and 20 mM sodium phosphate, pH 6.0 as electrophoretic buffer, HSA migrated as a single peak. Addition of 1,4-diaminobutane allowed separation of several components which could be further resolved by varying the buffer pH. Optimal separation conditions were attained at 5 mM 1,4-diaminobutane and pH 8.5. The reproducibility of the separation conditions was verified by using capillaries from a different manufacturer. A comparative analysis of HSA preparations from different manufacturers provided evidence that the method may be used to qualitatively differentiate individual preparations. The analysis of rhEPO formulations, composed largely of HSA, showed levels of heterogeneity comparable to that of HSA preparations. Electrospray ionisation mass spectrometry (ESA-MS) was used as an independent method to confirm the heterogeneous nature of HSA.     

Calorimetric studies of carbon monoxide and inositol hexaphosphate binding to hemoglobin A

Heats of CO and IHP binding to hemoglobin A have been determined under a variety of buffer and pH conditions. From these data heats of ion binding linked to hemoglobin oxygenation have been estimated. For IHP binding to deoxyhemoglobin the buffer-corrected enthalpies are surprisingly large, reaching -25 kcal/mol of IHP at pH 7.4. These values correspond to approximately -11 kcal/mol of proton absorbed upon IHP binding and may rise largely from the protonation of hitidine and NH2-terminal groups in the binding site (Arnone, A., and Perutz, M.F. (1974) Nature 249, 34-36). The decreased magnitude of delta HIHP observed at low pH parallels the decreased proton uptake at low pH. In 0.1 M chloride (pH 7.4) the reaction Hb(aq) + IHP leads to Hb x IHP(aq) has a standard free energy change (Edalji, R., Benesch, R.E., and Benesch, R. (1976) J. Biol. Chem. 251, 7720-7721) of -10 kcal and an enthalpy change of -25 kcal. Therefore, enthalpic forces provide the dominant driving force of this process. The origin of these large negative enthalpy changes is attributed to the exothermic protonation of protein basic groups induced by the proximity of phosphate negative charges. The importance of protonation in the binding of organic phosphates to hemoglobin may well extend to the specific binding of other phosphate substrates to enzyme reaction sites.     

Local tolerance of subcutaneous injections

Human insulin-like growth factor I (hIGF-I) has several possible clinical applications. Because subcutaneous administration of the drug can cause pain, local tolerance to injection of different formulations with or without hIGF-I has been investigated in man using isotonic saline solution as reference. The formulations, made isotonic with NaCl, ranged in pH from 6 to 7 with phosphate buffer concentrations of 5 to 50 mM. The local tolerance after injection was assessed as injection pain on a visual analogue scale, pain duration and local tolerance (redness, paleness and oedema). The discomfort at the injection site was lowest with 10 mM phosphate, pH 7. Injection of buffer at pH 6 (50 mM phosphate) caused significantly more pain than using 10 mM phosphate, whereas the pain at pH 6 using 10 mM phosphate did not differ significantly from that experienced on injection of the solution at pH 7 using either 10 or 50 mM phosphate. hIGF-I itself did not seem to cause pain. We concluded that for subcutaneous injections at non-physiological pH, the buffer strength should be kept as low as possible to avoid pain upon injection. We also hypothesize that when a non-physiological pH must be used for stability reasons, a lower buffer strength enables more rapid normalization of the pH at the injection site.     

Genetic mapping of the alpha-galactosidase MEL gene family on right and left telomeres of Saccharomyces cerevisiae

The alpha-galactosidase MEL2-MEL10 genes have been genetically mapped to right and left telomere regions of the following chromosomes of Saccharomyces cerevisiae: MEL2 at VII L, MEL3 at XVI L, MEL4 at XI L, MEL5 at IV L, MEL6 at XIII R, MEL7 at VI R, MEL8 at XV R, MEL9 at X R and MEL10 at XII R. A set of tester strains with URA3 inserted into individual telomeres and no MEL genes was used for mapping.     

Effects of steady-state versus stochastic exercise on subsequent cycling performance

Mutations in the type VII collagen gene (COL7A1) have been shown to underlie dystrophic epidermolysis bullosa (DEB). The dominantly inherited forms of DEB have been divided into two clinical subcategories, the Pasini (DDEB-P) and the Cockayne-Touraine (DDEB-CT) variants, on the basis of the presence or absence of albopapuloid lesions. In this study, we have examined the molecular basis of DDEB in two Japanese families, one with DDEB-P and the other with DDEB-CT. Mutation detection strategy consisted of polymerase chain reaction amplification of COL7A1 from genomic DNA, followed by heteroduplex analysis and direct nucleotide sequencing. The results revealed heterozygous glycine substitution mutations, G2076D and G2034R, in these families, respectively. Thus, these two variants of DDEB are allelic, and subtle differences in the clinical presentation may reflect the precise position of the mutation along the type VII collagen molecule. Alternatively, the nature of the substituting amino acid (D versus R) may influence the clinical phenotype. This is the first demonstration of a COL7A1 mutation in DDEB-P, and brings the total number of dominant DEB variants with underlying glycine substitutions in COL7A1 to five, including the pretibial and localized variants as well as the Bart's syndrome, in addition to DDEB-P and DDEB-CT.     

Factor VIIa's first epidermal growth factor-like domain's role in catalytic activity

Factor VIIa-tissue factor complex formation initiates the extrinsic blood coagulation pathway. We investigated factor VIIa's first epidermal growth factor-like (egf1) domain's role in the catalytic activity increase caused when factor VIIa binds tissue factor. Starting with a factor VIIa with factor IX's egf1 domain (factor VII(IXegf1)a), we made 4 proteins with egf1 residues changed to those in factor VIIa, including E51A, D64Q, FG74-75PA, and K79R. We measured each enzyme's affinity for tissue factor and determined the enzymes' kinetic constants with and without tissue factor. The Kd for factor VII(IXegf1)a binding to tissue factor was 60-200-fold higher than that of factor VIIa depending on the assay employed. Only factor VII(IXegf1)a with the K79R (K79Ra) mutation, among all the mutants, had an effect on binding with a Kd 3-8-fold lower than that of factor VII(IXegf1)a. In kinetic analyses with a small peptide substrate, in the absence of tissue factor, factor VIIa, factor VII(IXegf1)a, and K79Ra had similar kcat's and Km's. With tissue factor, due to a kcat decrease, factor VII(IXegf1)a's catalytic efficiency (kcat/Km) was 2-fold lower than factor VIIa's. K79Ra's catalytic efficiency was intermediate between those of factor VIIa and factor VII(IXegf1)a. With factor X as substrate, in the absence of tissue factor, K79Ra and factor VII(IXegf1)a had catalytic efficiencies 1.5-fold and 2-fold lower than that of factor VIIa. In contrast, with tissue factor and with factor X as substrate, due to higher Km's, factor VII(IXegf1)a and K79Ra had only 9% and 33% of factor VIIa's catalytic efficiency. Our results suggest the egf1 domain's role in tissue factor binding involves critical alignment of tissue factor with factor VIIa's catalytic domain. Proper alignment in turn promotes optimal catalytic activities.     

The interaction of acetoxy-dimethylnitrosamine, a proximate metabolite of the carcinogenic amine, and bacteriophages R17 and T7

The biological and physicochemical effects of reacting bacteriophages R17 and T7 with acetoxy-dimethylnitrosamine (ADMN) have been studied. The rate-determining step in the reactions appeared to be the loss of the acetoxy group by hydrolysis, the hydroxymethyl-methylnitrosamine generated decomposing rapidly to give a methyldiazonium ion and formaldehyde. In experiments with bacteriophage suspended in phosphate buffer the biological inactivation observed was the sum of the effects of the formaldehyde and of alkylation by the methylcarbonium ion produced from the diazonium ion. In experiments with bacteriophage suspended in Tris--HCl buffer the effects of formaldehyde were eliminated by its reaction with the buffer component. Alkylation by the carbonium ion produced unstable phosphotriesters in the bacteriophage RNA which on hydrolysis led to degradation of the molecule. In phosphate buffer the formaldehyde cross-linked the protein coat of the bacteriophage blocking the extraction of the RNA. Estimates of the mean lethal dose and of the extent of degradation of the RNA following reaction in Tris--HCl buffer were fairly close to those observed in experiments with N-methyl-N-nitrosourea (MNUA).     

Specific mismatch recognition in heteroduplex intermediates by p53 suggests a role in fidelity control of homologous recombination

We demonstrate that wild-type p53 inhibits homologous recombination. To analyze DNA substrate specificities in this process, we designed recombination experiments such that coinfection of simian virus 40 mutant pairs generated heteroduplexes with distinctly unpaired regions. DNA exchanges producing single C-T and A-G mismatches were inhibited four- to sixfold more effectively than DNA exchanges producing G-T and A-C single-base mispairings or unpaired regions of three base pairs comprising G-T/A-C mismatches. p53 bound specifically to three-stranded DNA substrates, mimicking early recombination intermediates. The KD values for the interactions of p53 with three-stranded substrates displaying differently paired and unpaired regions reflected the mismatch base specificities observed in recombination assays in a qualitative and quantitative manner. On the basis of these results, we would like to advance the hypothesis that p53, like classical mismatch repair factors, checks the fidelity of homologous recombination processes by specific mismatch recognition.     

Functional overlap in mismatch repair by human MSH3 and MSH6

Three human genes, hMSH2, hMSH3, and hMSH6, are homologues of the bacterial MutS gene whose products bind DNA mismatches to initiate strand-specific repair of DNA replication errors. Several studies suggest that a complex of hMSH2 x hMSH6 (hMutSalpha) functions primarily in repair of base x base mismatches or single extra bases, whereas a hMSH2 x hMSH3 complex (hMutSbeta) functions chiefly in repair of heteroduplexes containing two to four extra bases. In the present study, we compare results with a tumor cell line (HHUA) that is mutant in both hMSH3 and hMSH6 to results with derivative clones containing either wild-type hMSH3 or wild-type hMSH6, introduced by microcell-mediated transfer of chromosome 5 or 2, respectively. HHUA cells exhibit marked instability at 12 different microsatellite loci composed of repeat units of 1 to 4 base pairs. Compared to normal cells, HHUA cells have mutation rates at the HPRT locus that are elevated 500-fold for base substitutions and 2400-fold for single-base frameshifts. Extracts of HHUA cells are defective in strand-specific repair of substrates containing base x base mismatches or 1-4 extra bases. Transfer of either chromosome 5 (hMSH3) or 2 (hMSH6) into HHUA cells partially corrects instability at the microsatellite loci and also the substitution and frameshift mutator phenotypes at the HPRT locus. Extracts of these lines can repair some, but not all, heteroduplexes. The combined mutation rate and mismatch repair specificity data suggest that both hMSH3 and hMSH6 can independently participate in repair of replication errors containing base x base mismatches or 1-4 extra bases. Thus, these two gene products share redundant roles in controlling mutation rates in human cells.     

Initial-phase optimization for bioremediation of munition compound-contaminated soils

We examined the bioremediation of soils contaminated with the munition compounds 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine by a procedure that produced anaerobic conditions in the soils and promoted the biodegradation of nitroaromatic contaminants. This procedure consisted of flooding the soils with 50 mM phosphate buffer, adding starch as a supplemental carbon substrate, and incubating under static conditions. Aerobic heterotrophs, present naturally in the soil or added as an inoculum, quickly removed the oxygen from the static cultures, creating anaerobic conditions. Removal of parent TNT molecules from the soil cultures by the strictly anaerobic microflora occurred within 4 days. The reduced intermediates formed from TNT and hexahydro-1,3,5-trinitro-1,3,5-triazine were removed from the cultures within 24 days, completing the first stage of remediation. The procedure was effective over a range of incubation temperatures, 20 to 37 degrees C, and was improved when 25 mM ammonium was added to cultures buffered with 50 mM potassium phosphate. Ammonium phosphate buffer (50 mM), however, completely inhibited TNT reduction. The optimal pH for the first stage of remediation was between 6.5 and 7.0. When soils were incubated under aerobic conditions or under anaerobic conditions at alkaline pHs, the TNT biodegradation intermediates polymerized. Polymerization was not observed at neutral to slightly acidic pHs under anaerobic conditions. Completion of the first stage of remediation of munition compound-contaminated soils resulted in aqueous supernatants that contained no munition residues or aminoaromatic compounds.