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PubMed2004

Annotation of post-translational modifications in the Swiss-Prot knowledge base.

High-throughput proteomic studies produce a wealth of new information regarding post-translational modifications (PTMs). The Swiss-Prot knowledge base is faced with the challenge of including this information in a consistent and structured way, in order to facilitate easy retrieval and promote understanding by biologist expert users as well as computer programs. We are therefore standardizing the annotation of PTM features represented in Swiss-Prot. Indeed, a controlled vocabulary has been associated with every described PTM. In this paper, we present the major update of the feature annotation, and, by showing a few examples, explain how the annotation is implemented and what it means. Mod-Prot, a future companion database of Swiss-Prot, devoted to the biological aspects of PTMs (i.e., general description of the process, identity of the modification enzyme(s), taxonomic range, mass modification) is briefly described. Finally we encourage once again the scientific community (i.e., both individual researchers and database maintainers) to interact with us, so that we can continuously enhance the quality and swiftness of our services.

Computational Biology↗
PubMed1996

Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups.

L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.

Alcohol Oxidoreductases↗
PubMed1993

Single-site modifications of half-ligated hemoglobin reveal autonomous dimer cooperativity within a quaternary T tetramer.

The patterns of energetic response elicited by single-site hemoglobin mutations and chemical modifications have been determined in order to probe the dimer-dimer interface of the half-ligated tetramer (species [21]) that was previously shown to behave as allosterically distinct from both the unligated and fully ligated molecules. In this study the free energies of quaternary assembly (dimers to tetramers) were determined for a series of 24 tetrameric species in which one dimeric half-molecule is ligated (cyanomet hemes) while the adjacent alpha beta dimer is unligated and contains a single amino acid modification. Assembly energies have also been determined for tetramers bearing the same amino acid modifications but where the hemesites were completely vacant and additionally where they were fully occupied. A total of 72 molecular species were thus characterized. It was found that mutationally induced perturbations to the free energy of quaternary assembly were identical for the half-ligated tetramers and the unligated tetramers over the entire spatial distribution of altered sites, but exhibited a radically different pattern from that of the fully ligated molecules. These results indicate that the dimer-dimer interface of the half-ligated tetramer (species [21]) has the same quaternary structure as that of the unligated molecule, i.e., "quaternary T." This quaternary structure assignment of species [21] strongly supports the operation of a Symmetry Rule which translates changes in hemesite ligation into six T-->R quaternary switchpoints. Analysis of the observed Symmetry Rule behavior in relation to the measured distribution of cooperative free energies for the partially ligated species reveals significant cooperativity between alpha and beta subunits of the dimeric half-tetramer within quaternary T. The mutational results indicate that these interactions are not "paid for" by breaking or making noncovalent bonds at the dimer-dimer interface (alpha 1 beta 2). They arise from structural and energetic changes that are "internal" to the ligated dimer even though its association with the unligated dimer is required for the cooperativity to occur. Free energy of "tertiary constraint" is thus generated by the first binding step and is propagated to the second hemesite while the dimer-dimer interface alpha 1 beta 2 serves as a constraint. The "sequential" cooperativity that occurs within the half-molecule is thus preconditioned by the constraint of a quaternary T interface; release of this constraint by dissociation produces only noncooperative dimers.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed2001

Evaluation of protein modification during anti-viral heat bioprocessing by electrospray ionization mass spectrometry.

During the preparation of therapeutic plasma and recombinant protein biopharmaceuticals heat-treatment is routinely applied as a means of viral inactivation. However, as most proteins denature and aggregate under heat stress, it is necessary to add thermostabilizing excipients to protein formulations destined for anti-viral heat-treatment in order to prevent protein damage. Anti-viral heat-treatment bioprocessing therefore requires that a balance be found between the bioprocessing conditions, virus kill and protein integrity. In this study we have utilized a simple model protein, beta-lactoglobulin, to investigate the relationship between virucidal heat-treatment conditions (protein formulation and temperature) and the type and extent of protein modification in the liquid state. A variety of industrially relevant heat-treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) we show here that protein modifications do occur with increasingly harsh heat-treatment. The predominant modification under these conditions was protein glycation by either glucose or fructose derived from hydrolyzed sucrose. Advanced glycation end products and additional unidentified products were also present in beta-lactoglobulin protein samples subjected to extended heat-treatment. These findings have implications for the improvement of anti-viral heat-treatment bioprocesses to ensure the safety and efficacy of protein biopharmaceuticals. CopyrightCopyright 2001 John Wiley & Sons, Ltd.

Blood Proteins↗
PubMed2001

Modification of a commercial electrospray nebulizer for operation in a liquid chromatography/mass spectrometry system at flow rates in the low microL/min range.

A simple and inexpensive approach to convert the electrospray nebulizer of a commercial liquid chromatography/mass spectrometry (LC/MS) system (HP 1100) to accommodate lower flow rates has been proposed and evaluated. This modification consists of simply replacing the nebulizer needle by a commercially available stainless steel needle with a smaller internal diameter. Experiments were conducted in order to optimize operational parameters. Using two different internal diameter needle sizes, flow rates ranging from 1 to 250 microL/min could be accommodated. The modification presented allows an extension of the range of compatible flow rates without major modification of the standard design of the interface.

Chromatography, High Pressure Liquid↗
PubMed2005

Small-sample performance of the robust score test and its modifications in generalized estimating equations.

The sandwich variance estimator of generalized estimating equations (GEE) may not perform well when the number of independent clusters is small. This could jeopardize the validity of the robust Wald test by causing inflated type I error and lower coverage probability of the corresponding confidence interval than the nominal level. Here, we investigate the small-sample performance of the robust score test for correlated data and propose several modifications to improve the performance. In a simulation study, we compare the robust score test to the robust Wald test for correlated Bernoulli and Poisson data, respectively. It is confirmed that the robust Wald test is too liberal whereas the robust score test is too conservative for small samples. To explain this puzzling operating difference between the two tests, we consider their applications to two special cases, one-sample and two-sample comparisons, thus motivating some modifications to the robust score test. A modification based on a simple adjustment to the usual robust score statistic by a factor of J/(J - 1) (where J is the number of clusters) reduces the conservativeness of the generalized score test. Simulation studies mimicking group-randomized clinical trials with binary and count responses indicated that it may improve the small-sample performance over that of the generalized score and Wald tests with test size closer to the nominal level. Finally, we demonstrate the utility of our proposal by applying it to a group-randomized clinical trial, trying alternative cafeteria options in schools (TACOS).

Adolescent↗
PubMed1994

The effect of p-(chloromercuri)benzoate modification of cytosolic aldehyde dehydrogenase from sheep liver. Evidence for a second aldehyde binding site.

p-Chloromercuribenzoate (PCMB) at stoichiometric levels reacts with a thiol group of the binary NAD+ complex of sheep liver cytoplasmic aldehyde dehydrogenase (E.NAD+) faster than with the corresponding thiol group of either the free enzyme or the binary enzyme. NADH complexes. High concentrations of propionaldehyde have a protective effect against modification of the enzyme with PCMB in steady-state assays. This protection arises from a reduction in the concentration of the E.NAD+ binary complex rather than competition for a common binding site. PCMB has three major effects on aldehyde dehydrogenase. First, rapid reaction with a high-affinity thiol group in the E.NAD+ binary complex causes activation of the steady-state rate. The activation results from an increase in the rate of NADH release from the enzyme. This modification simultaneously protects against dilution-induced dissociation of enzyme tetramers. Second, premodification of the high-affinity thiol group leads to inhibition of the steady-state rate at high propionaldehyde concentrations, because of the increased affinity of the free enzyme for propionaldehyde with the resultant formation of an enzyme-aldehyde dead-end complex. Third, when higher ratios of PCMB to enzyme (> 3:1) are used, one or more other thiol groups are also modified, causing enzyme dissociation and subsequent inactivation. Since modification of the high-affinity thiol by PCMB causes activation, clearly it cannot be the active site acylation center involved in propionaldehyde oxidation. The different amplitudes of the proton burst at high and low propionaldehyde concentrations for the PCMB modified enzyme provide support for a second binding site for propionaldehyde on the enzyme.

Aldehyde Dehydrogenase↗
PubMed1995

Farnesylation and proteolysis are sequential, but distinct steps in the CaaX box modification pathway.

Membrane localization of Ras proteins requires posttranslational modification of a conserved C-terminal sequence motif known as the CaaX box (C is Cys, a is any aliphatic amino acid, and X is the carboxyl terminal residue). The modification steps include farnesylation, removal of the three C-terminal amino acids, carboxyl-methylation, and palmitoylation. In yeast, the farnesyltransferase (FTase) is encoded by the RAM1(DPR1) and RAM2 genes, and the methyltransferase is the product of STE14. The gene encoding the protease(s) that is responsible for modification of the CaaX has not been identified. We have used in vitro-synthesized Ras2p and synthetic peptide substrates to investigate the relationship between farnesylation and proteolysis. Addition of yeast cytosolic extracts to rabbit reticulocyte extracts programmed to synthesize Ras2p led to prenylation of Ras2p and a change in electrophoretic mobility similar to that observed during Ras maturation in vivo. However, it was not possible to determine if the mobility shift is the result of prenylation, proteolysis or a combination of both steps. Therefore, we examined the relationship between farnesylation and proteolysis directly using extracts prepared from bacteria overexpressing the genes for the yeast FTase (RAM1 and RAM2) and synthetic CaaX box peptides. Extracts from bacteria expressing RAM1/RAM2 efficiently prenylate CaaX box peptides, but do not proteolyze the -aaX residues. However, addition of yeast extracts from wild type, ram1, or ste14 mutants resulted in the removal of the -aaX residues from prenylated CaaX box peptides.

Alkyl and Aryl Transferases↗
PubMed1995

Role of carbohydrates in oxidative modification of fibrinogen and other plasma proteins.

Oxidative stress is related to the mechanism of oncogenesis, cell death, and the pathogenesis of many human diseases. Proteins are important targets for oxidative modification, and a Western blot assay that can identify individual oxidized proteins in whole tissue extracts has been described. Using that assay, it was found that plasma proteins show different susceptibilities to oxidative modification. Here, we examine the possibility that the carbohydrate groups of glycoproteins may contribute to the assessment of protein oxidation by carbonyl assays. We used fibrinogen as a model because it is highly susceptible to oxidative modification and contains subunits that are differentially glycosylated. When oxidation-induced carbonyls were measured in fibrinogen subunits by Western blot immunoassay, it was found that the A alpha-chains, which contain no associated carbohydrate groups, were most highly oxidized while the B beta- and gamma-chains, which are glycosylated, were oxidized far less. However, no major difference in the oxidation pattern was obtained when fibrinogen was deglycosylated prior to or after exposure to oxidants. This argues against a possible protective role of the carbohydrate moieties in oxidation of the different fibrinogen subunits. Similar results were obtained with purified human immunoglobulin G and transferrin as well as whole plasma. The results show that carbohydrate moieties are not good targets for oxidative attack by metal-catalyzed oxidation systems. Oxidant-induced carbonyl formation in glycoproteins derives largely, if not entirely, from amino acid oxidation and not from oxidation of carbohydrate groups.

Blood Proteins↗
PubMed1997

Reactivity of nitrogen monoxide species with NADH: implications for nitric oxide-dependent posttranslational protein modification.

Nitric oxide (NO.) and NO. donors incite NAD- [i.e., mono(ADP-ribosylation)] and NADH-dependent posttranslational protein modifications by an as yet unknown mechanism. A route of pyridine nucleotide-dependent, NO.-stimulated protein modification has recently been hypothesized [S. Dimmeler, and B. Brune, (1992) Eur. J. Biochem. 210, 305-310; J. S. Stamler (1994) Cell 78, 931-936]. An essential feature of this proposed mechanism is NADH nitrosation, for a nitroso-NADH adduct is considered to be a key reactant in the generation of pyridine nucleotide-modified protein. To evaluate at the molecular level the ability of NADH to act as a nitrosation substrate, the potential effects of NO., the nitrosothiols S-nitrosoglutathione and S-nitrosocysteine, the nitrosating agent tert-butyl-nitrite, and the NO. metabolite peroxynitrite on the molecular and functional (i.e., hydride-transfer) properties of NADH have been directly assessed at physiological pH. Exposure of NADH to NO. or nitrosothiol altered neither the hydride-transfer capability of the pyridine nucleotide nor its ultraviolet spectrum in ways suggestive of NADH nitrosation. As determined by NMR spectroscopy, NADH was refractory to the well-recognized nitrosating agent tert-butyl nitrite. Consequently, it appears that NADH is an unfavorable substrate for nitrosation under physiological conditions. These data are inconsistent with the proposal that NO. or a NO.-derived nitrosating agent interacts with NADH to generate the nitroso-NADH hypothesized to be essential to NO.-stimulated, pyridine nucleotide-dependent protein modification. Peroxynitrite, a possible source of nitrosating compounds, readily oxidized NADH to NAD, but demonstrated no potential to form a nitroso-NADH adduct. The facility with which NADH is oxidized to NAD has implications for peroxynitrite-mediated tissue damage.

Magnetic Resonance Spectroscopy↗
PubMed1998

Chemical modification of barley root oxalate oxidase shows the presence of a lysine, a carboxylate, and disulfides, essential for enzyme activity.

Oxalate oxidase (OXO) was chemically modified using amino acid-specific reagents. The modification reactions were monitored spectrophotometrically, to follow the progress of labeling, and catalytically, to assess the effect of labeling on the enzyme function. The enzyme does not bear arginines essential for activity, since 2,3-butanedione and cyclohexanodione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Incubation of urea-pretreated OXO with N-acetylimidazole leads to labeling all 10 tyrosines without affecting the enzyme activity, thus suggesting that OXO does not have tyrosines essential for activity. However, OXO modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide followed by kinetic analysis, leads to the conclusion that the enzyme possesses one carboxylate essential for activity. When using the modifier 2,4, 6-trinitrobenzene sulfonic acid (TNBS), while 28 of the total 45 lysines are labeled within 3 h (the first 5 reacting lysines of the homopentametic enzyme are modified at a faster rate than the others), the enzyme rapidly loses 90% of its activity in the first 2 min, a period during which only one lysine is being labeled. Complete enzyme inactivation with TNBS is observed after approximately 8 min, when 5 lysines are being labeled. The modification of the first lysine also triggers the dissociation of native OXO to its subunits (after SDS-PAGE analysis), a phenomenon not observed with the other modifiers. These findings indicate that OXO bears a lysine per monomer, essential for enzyme activity. When using 5, 5-dithio-bis-(2-nitrobenzoic)acid to determine the number of disulfide bonds, in the presence of NaBH4, 10 sulfhydryls are determined, but in the absence of reducing agent, none are determined. Further, chloro-mercuribenzoate does not inactivate OXO but beta-mercaptoethanol does. Therefore, the sulfhydryls in OXO are not free but form disulfide bonds essential for activity. Furthermore, the metallo-chelating agents HgCl2 and 8-hydroxychinolin inactivate the enzyme, suggesting that barley root oxalate oxidase is a metalloenzyme. It is possible that the metal(s) are involved in the oxidative mechanism since the enzyme does not bear prosthetic groups such as FAD and FMN.

Arginine↗
PubMed1998

Disulfide isomerization within the C-terminus of cobrotoxin decelerates by thiol compounds and trinitrophenylation, but accelerates by modification of carboxyl groups.

A disulfide isomerization at the C-terminus of cobrotoxin occurred spontaneously by dissolving in alkali buffer. Irreversible conversion of cobrotoxin into its isomers was completely achieved within 4 days. The isomerization reaction was decelerated by thiol compounds including GSSG, GSH, cystamine, and cysteamine in a pseudo-first-order kinetic, and GSSG was the most effective one among the thiol compounds used. Moreover, the oxidized thiol compounds were always superior to reduced ones in decreasing the rate of disulfide interchange. To further assess the intrinsic elements essential for the occurrence of disulfide isomerization of cobrotoxin, the toxin molecule was subjected to modification on its Arg, Lys, Trp, Tyr, and carboxyl groups. In sharp contrast to other modified derivatives, the isomerization reaction was decelerated by trinitrophenylation on Lys-26, Lys-27, and Lys-47, whereas it was rapidly completed after modification of carboxyl groups. Neither chemical modification nor the toxin's conformation affected the irreversibility of isomerization reaction. Thus, the observed change in the rate of disulfide isomerization reflects the involvement of Lys residues and carboxyl groups in this reaction. Although thiol compounds further decelerated the conversion of trinitrophenylated cobrotoxin into its isomers, they did not exert a notable effect on the isomerization of carboxyl groups-modified derivative. These results clearly indicate that disulfide isomerization of cobrotoxin is, in part, driven by the positively charged Lys residues at positions 26, 27, and 47 of the toxin molecule, and that the thiol compounds are coordinated with the negatively charged groups of cobrotoxin to exert their inhibitory action.

Animals↗
PubMed1999

Modification of leukotriene A(4) hydrolase/aminopeptidase by sulfhydryl-blocking reagents: differential effects on dual enzyme activities by methyl-methane thiosulfonate.

The presence of a cysteine residue at or near the active site of leukotriene A(4) hydrolase (EC 3.3.2.6) was suggested by inactivation of the enzyme with sulfhydryl-blocking reagents and by protection against inactivation afforded by substrates and competitive inhibitors. The aminopeptidase activity was more susceptible to inactivation than the epoxide hydrolase activity. The sulfhydryl-modifying reagent methyl-methane thiosulfonate reacted with one thiol as judged by kinetic data and titration with 5, 5'-dithiobis-2-nitrobenzoate. Inactivation was a time- and dose-dependent process of apparent pseudo-first-order and maximal at 80-85%. The inactivation rate was nonsaturable and strongly influenced by ion strength. The second-order rate constant increased from 0.9 to 4.3 M(-1) s(-1) in the presence of 0.2 M NaCl. Albumin, a stimulator of the aminopeptidase activity, increased apparent inactivation rates by shifting pK(a) for the modification from 8.2 to 7.8. The inactivated enzyme partially regained activity upon treatment with beta-mercaptoethanol. Peptide substrates and competitive inhibitors protected against inactivation. Bestatin, a competitive inhibitor, afforded complete protection with a K(D) = 0.15 microM, similar to K(i) = 0.17 microM for inhibition of peptidase activity. Treated enzyme had an unchanged K(m) but a reduced V(max). The epoxide hydrolase activity was only weakly affected by methyl-methane thiosulfonate with a maximal inactivation of 15-20% after prolonged treatment. Pretreatment of leukotriene A(4) hydrolase with the reagent did not protect against mechanism-based inactivation by its lipid substrate, leukotriene A(4). On the other hand, leukotriene B(4) was a competitive inhibitor of aminopeptidase activity and protected against modification by methyl-methane thiosulfonate. Our results suggest the presence of a cysteine at or close to subsite S'(1) of the active site of leukotriene A(4) hydrolase and that modification of this residue interferes with the function of the aminopeptidase activity, but not the epoxide hydrolase activity. This is the first report to distinguish the two catalytic activities of leukotriene A(4) hydrolase by chemical means.

Binding, Competitive↗
PubMed2001

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences.

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.

Blotting, Western↗
PubMed2002

Posttranslational modification of human alphaA-crystallin: correlation with electrophoretic migration.

alphaA-crystallin is a major protein component of the human lens. It is known to undergo posttranslational modification. This study was done to further elucidate the temporal and spatial nature of these posttranslational modifications and to correlate the modified forms with electrophoretic migration. We dissected normal human lenses into concentric shells of fiber cells, separated the proteins by two-dimensional electrophoresis, and identified modified forms by mass spectrometry. We found that alphaA-crystallin migrated as a major spot and in over 20 additional protein spots. The extent of modification correlated with the age of the fiber cells and the depth within a lens. A correlation was also seen between these parameters and the concentration of modified forms that had full-length sequences but migrated at more acidic positions. These proteins were phosphorylated, acetylated, and/or deamidated. A few proteins migrated to a more basic position than the major form of alphaA-crystallin. The locations of several species that were truncated after C-terminal residues Ser172 and Ser162 were identified. Each of these species had intact N termini. The similarity of the C-terminal cleavage sites found in alphaA- and alphaB-crystallins was noted.

Amino Acid Sequence↗
PubMed1993

Identification of chemical modification sites on metalloproteins by capillary electrophoresis.

Capillary electrophoresis (CE) has been used to separate the peptides obtained from tryptic digestion of ruthenium-modified cytochrome c. The modified peptide was identified from a comparison of the elution profile and absorbance characteristics of the native and modified proteins. Automatic fraction collection on the CE instrument provides sufficient amounts of this modified peptide for amino acid sequencing. Capillary electrophoresis has also been used to monitor the modification reaction and to optimize the modification efficiency. The methodologies have been extended to myoglobin in order to monitor the modification of multiple surface sites.

Animals↗
PubMed1997

Comparison of results of various methods used to determine the extent of modification of methoxy polyethylene glycol 5000-modified bovine cupri-zinc superoxide dismutase.

The protein bovine cupri-zinc superoxide dismutase (SOD) was modified by the reaction of lysine residues with an active ester of methoxy polyethylene glycol 5000 (PEG). The extent of modification was determined by capillary zone electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry, Fourier-transform infrared spectroscopy, and Raman spectroscopy and after removing PEG by alkaline hydrolysis of the linkages to SOD followed by quantification of the released PEG using gel-permeation chromatography. There was generally good agreement among the results obtained by these techniques on a typical sample of the PEG-modified protein. The results, extent of protein modification, determined by the preceding methods were compared to that found from the classical trinitrobenzene sulfonic acid (TNBS) procedure. In addition to providing alternatives to the TNBS procedure, results from the described methodologies strongly suggest that the extent of modification determined from the TNBS procedure is overestimated.

Animals↗
PubMed2001

Selection-marker-free modification of the murine beta-casein gene using a lox2272 [correction of lox2722] site.

Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site. DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the beta-casein gene in murine embryonic stem cells. The incompatibility of the heterospecific lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification. This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene.

Animals↗