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PubMed1993

Enzyme electrode based on oxalate oxidase immobilized in gelatin for specific determination of oxalate.

A biosensor for the specific determination of oxalate was developed using oxalate oxidase (EC 1.2.3.4) from barley (Hordeum vulgare) seedling roots in combination with a dissolved oxygen probe. Oxalate oxidase immobilized with gelatin using glutaraldehyde and fixed on pretreated teflon membrane served as an enzyme electrode. The electrode response was maximum when 50 mM succinate buffer was used at pH 3.2 and 35 degrees C. The biosensor response depends linearly on oxalate concentration between 5 x 10(-6)-2 x 10(-4) M with response time 30 sec and substrate specificity of the oxalate oxidase electrode of 100%. The sensor is stable for more than 3 months during which time more than 400 assays can be performed.

Biosensing Techniques↗
PubMed2000

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway.

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B. subtilis.

Amino Acid Sequence↗
PubMed1988

Characterization of the cell type-specific determinant in the genome of minute virus of mice.

Two strains of minute virus of mice (MVM) show different host cell specificities. The prototype strain MVM(p) grows in fibroblasts, whereas the immunosuppressive variant MVM(i) grows in T lymphocytes. In this study, we have mapped on the viral genome a cell type-specific determinant: it is located between 69 and 85 map units in a region coding for the viral capsid proteins. The DNA of MVM(p) does not replicate in lymphocytes. MVM(i) cannot help MVM(p) grow in lymphocytes; thus the determinant acts in a cis fashion. We did not detect viral mRNA during a restrictive infection of lymphocytes with MVM(p). However, when the same cells were transfected with cloned DNA, both MVM(p) and MVM(i) DNAs were transcribed with the same efficiency from both promoters and the RNA was processed normally. Therefore, the specificity determinant is not a cell type-specific enhancer.

Cell Line↗
PubMed2005

Prediction of functional specificity determinants from protein sequences using log-likelihood ratios.

MOTIVATION: A number of methods have been developed to predict functional specificity determinants in protein families based on sequence information. Most of these methods rely on pre-defined functional subgroups. Manual subgroup definition is difficult because of the limited number of experimentally characterized subfamilies with differing specificity, while automatic subgroup partitioning using computational tools is a non-trivial task and does not always yield ideal results. RESULTS: We propose a new approach SPEL (specificity positions by evolutionary likelihood) to detect positions that are likely to be functional specificity determinants. SPEL, which does not require subgroup definition, takes a multiple sequence alignment of a protein family as the only input, and assigns a P-value to every position in the alignment. Positions with low P-values are likely to be important for functional specificity. An evolutionary tree is reconstructed during the calculation, and P-value estimation is based on a random model that involves evolutionary simulations. Evolutionary log-likelihood is chosen as a measure of amino acid distribution at a position. To illustrate the performance of the method, we carried out a detailed analysis of two protein families (LacI/PurR and G protein alpha subunit), and compared our method with two existing methods (evolutionary trace and mutual information based). All three methods were also compared on a set of protein families with known ligand-bound structures. AVAILABILITY: SPEL is freely available for non-commercial use. Its pre-compiled versions for several platforms and alignments used in this work are available at ftp://iole.swmed.edu/pub/SPEL/

Algorithms↗
PubMed2001

Identification of specificity determinants and generation of alleles with novel specificity at the het-c heterokaryon incompatibility locus of Neurospora crassa.

The capacity for nonself recognition is a ubiquitous and essential aspect of biology. In filamentous fungi, nonself recognition during vegetative growth is believed to be mediated by genetic differences at heterokaryon incompatibility (het) loci. Filamentous fungi are capable of undergoing hyphal fusion to form mycelial networks and with other individuals to form vegetative heterokaryons, in which genetically distinct nuclei occupy a common cytoplasm. In Neurospora crassa, 11 het loci have been identified that affect the viability of such vegetative heterokaryons. The het-c locus has at least three mutually incompatible alleles, termed het-c(OR), het-c(PA), and het-c(GR). Hyphal fusion between strains that are of alternative het-c specificity results in vegetative heterokaryons that are aconidial and which show growth inhibition and hyphal compartmentation and death. A 34- to 48-amino-acid variable domain, which is dissimilar in HET-C(OR), HET-C(PA), and HET-C(GR), confers allelic specificity. To assess requirements for allelic specificity, we constructed chimeras between the het-c variable domain from 24 different isolates that displayed amino acid and insertion or deletion variations and determined their het-c specificity by introduction into N. crassa. We also constructed a number of artificial alleles that contained novel het-c specificity domains. By this method, we identified four additional and novel het-c specificities. Our results indicate that amino acid and length variations within the insertion or deletion motif are the primary determinants for conferring het-c allelic specificity. These results provide a molecular model for nonself recognition in multicellular eucaryotes.

Alleles↗
PubMed2005

An evolution-based analysis scheme to identify CO2/O2 specificity-determining factors for ribulose 1,5-bisphosphate carboxylase/oxygenase.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) catalyzes a rate-limiting step in photosynthetic carbon assimilation (reacting with CO2) and its competitive photo-respiratory carbon oxidation (reacting with O2). RuBisCo enzyme with an enhanced CO2/O2 specificity would boost the ability to make great progress in agricultural production and environmental management. RuBisCos in marine non-green algae, resulting from an earlier endo-symbiotic event, diverge greatly from those in green plants and cyanobacteria and, further, have the highest CO2/O2 specificity whereas RuBisCos in cyanobacteria have the lowest. We assumed that there exist different levels of CO2/O2 specificity-determining factors, corresponding to different evolutionary events and specificity levels. Based on this assumption, we devised a scheme to identify these substrate-determining factors. From this analysis, we are able to discover different categories of the CO2/O2 specificity-determining factors that show which residue substitutions account for (relatively) small specificity changes, as happened in green plants, or a tremendous enhancement, as observed in marine non-green algae. Therefore, the analysis can improve our understanding of molecular mechanisms in the substrate specificity development and prioritize candidate specificity-determining surface residues for site-directed mutagenesis.

Amino Acid Sequence↗
PubMed2001

Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein Avr4.

The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9, Cf-4 lacks two LRRs and differs in 78 amino acid residues. To investigate the relevance of these differences for specificity, we exchanged domains between Cf-4 and Cf-9, and mutant constructs were tested for mediating the hypersensitive response by transient coexpression with either Avr4 or Avr9. We show that the number of LRRs is essential for both Cf-4 and Cf-9 function. In addition, Cf-9 specificity resides entirely in the LRR domain and appears to be distributed over several distant LRRs. In contrast, Cf-4 specificity determinants reside in the N-terminal LRR-flanking domain and three amino acid residues in LRRs 13, 14, and 16. These residues are present at putative solvent-exposed positions, and all are required for full Cf-4 function. Finally, we show that Cf-9 carrying the specificity determinants of Cf-4 has recognitional specificity for AVR4. The data indicate that diversifying selection of solvent-exposed residues has been a more important factor in the generation of Cf-4 specificity than has sequence exchange between Cf-4 progenitor genes. The fact that most variant residues in Cf-4 are not essential for Cf-4 specificity indicates that the diverse decoration of R proteins is not fully adapted to confer recognition of a certain avirulence determinant but likely provides a basis for a versatile, adaptive recognition system.

Amino Acid Sequence↗
PubMed1980

The interaction of Escherichia coli core RNA polymerase with specificity-determining subunits derived from unmodified and SP82-modified Bacillus subtilis RNA polymerase.

the ability of the core isolated from Escherichia coli RNA polymerase to interact with specificity-determining subunits isolated from Bacillus subtilis RNA polymerase has been determined by measuring the transcription of "early" and "middle" genes of phage SP82. Two specificity-determining subunits were tested: the sigma subunit and a 28,000 dalton (28 K) peptide isolated from a modified polymerase produced at approximately 8 min after infection of B. subtilis with SP82. Earlier experiments (Spiegelman, G. B. and Whiteley, H. R. (1978) Biochem. Biophys. Res. Commun. 81, 1058-1065) demonstrated that sigma and the 28K peptide are required for the recognition of early and middle gene promoters, respectively, by the B. subtilis core assembly. The present investigation showed that E. coli core interacted more efficiently with the B. subtilis sigma than with the 28K peptide, as judged by the rate of RNA synthesis. Early RNA was produced by the E. coli and B. subtilis holoenzymes and by E. coli core supplenented with B. subtilis sigma and only minor differences were found in comparisons of transcripts by hybridization and by electrophoretic analysis. Measurements of template specificity, the formation of stable enzyme . DNA complexes, and the hybridization of transcripts to fragments of SP82 DNA produced by digestion with restriction endonuclease Hha indicated that E. coli core supplemented with the 28K-supplemented E. coli core with those synthesized by the modified polymerase extracted from B. subtilis 8 min after infection with SP82 suggest that both preparations recognized the same initiation and termination sequences.

Bacillus subtilis↗
PubMed1983

Monoclonal antibodies specific for Leishmania tropica. I. Characterization of antigens associated with stage- and species-specific determinants.

Four monoclonal antibodies (T-1 through T-4), which were produced to membrane-enriched preparations of Leishmania tropica major promastigotes, reacted specifically with the members of L. tropica complex. The antibodies T-1 and T-4 react exclusively with L. t. major and L. t. minor. The remaining two monoclonal antibodies bind, in addition, to L. t. aethiopica and weakly to L. mexicana amazonensis. No significant cross-reactivity was observed with L. donovani, L. braziliensis braziliensis, and Trypanosoma cruzi. Antibodies T-1, T-2, and T-4 were found to be specific for the promastigote stage (insect) of L. tropica. Antibody T-3 reacts with both the amastigote and promastigote stages of the parasite. All of the monoclonal antibodies react with cell surface components on intact promastigotes. The protein antigens containing the species-specific determinants recognized by each of the four antibodies were identified by radioimmunoprecipitation of solubilized 125I-labeled L. t. major promastigotes. A single 50 kilodalton protein is recognized by clone T-4. T-1 recognized two high m.w. proteins (100 and 200 kilodaltons). These two antigens plus an additional protein of lower m.w. (70 kilodaltons) are also immunoprecipitated by the antibodies T-2 and T-3, demonstrating that species-specific determinants are present on several different cell surface proteins of L. t. major.

Animals↗
PubMed1977

The demonstration of subtype (D or Y)-specific determinants on the surface of the presumed hepatitis B virus.

Dane particles isolated from the sera of HBsAg/ad and HBsAg/ay carriers were reacted with monospecific antibodies to the d and y subtype-specific determinants of HBsAg/. Dane particles from HBsAg/ad expressed the d determinant on their surfaces and those from HBsAg/ay sera contained the y specificity. Both complete (DNA-P and HBcAg) and defective (HBcAg alone) Dane particles expressed the subtype-specific determinants.

Epitopes↗
PubMed1987

An enzymic assay for the specific determination of methanol in serum.

This method for the specific determination of methanol in serum is based on the following two reactions: (formula; see text) Alcohol oxidase is not specific: it converts all lower alcohols to their corresponding aldehydes; however, formaldehyde dehydrogenase is specific and thus the transformation of NAD+ to NADH (which is used to monitor the reaction) proceeds only if methanol is originally present in the sample. The method was automated with a Roche COBAS FARA centrifugal analyzer. The calibration curve is linear between 0.6 and 12 mmol/L. The detection limit is about 0.6 mmol/L. The CV is 4.6% for a concentration of 3 mmol/L. When 55 serum specimens known to be free of methanol were supplemented with known amounts of methanol and analyzed by the enzymatic method, the results correlated well (r = 0.987) with the true values, the regression equation being: y = 1.016x + 0.661, where x represents the true values. Results are not affected by other alcohols that may be present in serum, by methanol metabolites, or by some commonly prescribed drugs. The major advantage of this new assay is that it can be used 24 h a day in any clinical chemistry laboratory.

Alcohol Oxidoreductases↗
PubMed1986

Monoclonal antibodies to the v-fes product and to feline leukemia: virus P27 interspecies-specific determinants encoded by feline sarcoma viruses.

Monoclonal antibodies to p27 gag and v-fes specific determinants on the gag-onc poly-protein encoded by Snyder-Theilen feline sarcoma virus (ST-FeSV) were prepared. In order to obtain hybridoma clones specific to the antigenic determinants encoded by the FeSV genome, Lou rats were immunized with ST-FeSV-transformed, virus-nonproducing syngeneic cells, and boosted with either the same cells or affinity-purified feline leukemia virus (FeLV) p27. Three distinct clones reactive to both FeLV p27 and p85gag-fes, and one clone specific for a p85fes determinant were established. The anti-p27 monoclonal antibodies also reacted with the polyproteins p95gag-fes and p83gag-fgr, from Gardner-Arnstein (GA) and Theilen-Pedersen (TP1) FeSV, respectively. The anti-p27 monoclonal antibodies reacted with MuLV p30 and RD114 p28 but not with RSV, MMTV, or BLV. These results indicated that the part of the p27 gag gene that is preserved in ST-, GA, and TP1-FeSV encodes interspecies-specific p27 determinants.

Animals↗
PubMed2005

Predicting specificity-determining residues in two large eukaryotic transcription factor families.

Certain amino acid residues in a protein, when mutated, change the protein's function. We present an improved method of finding these specificity-determining positions that uses all the protein sequence data available for a family of homologous proteins. We study in detail two families of eukaryotic transcription factors, basic leucine zippers and nuclear receptors, because of the large amount of sequences and experimental data available. These protein families also have a clear definition of functional specificity: DNA-binding specificity. We compare our results to three other methods, including the evolutionary trace algorithm and a method that depends on orthology relationships. All of the predictions are compared to the available mutational and crystallographic data. We find that our method provides superior predictions of the known specificity-determining residues and also predicts residue positions within these families that deserve further study for their roles in functional specificity.

Algorithms↗
PubMed2004

A species-specific determinant on beta2-microglobulin required for Ly49A recognition of its MHC class I ligand.

The mouse inhibitory NK cell receptor Ly49A recognizes the mouse MHC class I molecule H-2D(k). The present study focuses on the species specificity of beta(2)-microglobulin (beta(2)m), an invariant component of MHC class I, in the interaction between Ly49A and H-2D(k). Transfection of the beta(2)m-defective mouse cell line R1E/TL8x.1 with human (h) beta(2)m induced cell-surface expression of H-2D(k), but failed to protect the cells from killing by Ly49A(+) NK cells. In contrast, the cells transfected with mouse (m) beta(2)m were protected from killing by Ly49A(+) NK cells. These data indicate that Ly49A distinguishes mbeta(2)m from hbeta(2)m when it recognizes the H-2D(k) complexes. To identify the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k), we prepared a panel of mbeta(2)m mutants and tested the H-2D(k) that included each of the beta(2)m mutants for its capacity to engage Ly49A on NK cells. Ly49A failed to functionally recognize the H-2D(k) that included the mbeta(2)m with K3R and Q29G mutations. Moreover, Ly49A was able to recognize the H-2D(k) that included the hbeta(2)m with R3K and G29Q mutations. These data indicate that Lys3 and Gln29 consist of the central part of the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k). The two residues are conserved in the mouse and the rat, in which NK cells use Ly49 family molecules as the receptors specific for MHC class I. These results suggest functional importance of beta(2)m in NK cell recognition of target cells.

Amino Acid Sequence↗
PubMed1990

[A method for determining specific circulating immune complexes in patients with acute Sonne dysentery].

The antigen-specific method for the determination of specific immune complexes in the blood serum of patients with acute Sonne dysentery has been developed on the basis of the enzyme-linked immunosorbent assay with the use of reagents manufactured in the USSR. The possibility of the early prognostication of prolonged carrier state by using the proposed method for the determination of the level of specific circulating immune complexes has been shown.

Acute Disease↗
PubMed2002

Using orthologous and paralogous proteins to identify specificity determining residues.

BACKGROUND: Concepts of orthology and paralogy are become increasingly important as whole-genome comparison allows their identification in complete genomes. Functional specificity of proteins is assumed to be conserved among orthologs and is different among paralogs. We used this assumption to identify residues which determine specificity of protein-DNA and protein-ligand recognition. Finding such residues is crucial for understanding mechanisms of molecular recognition and for rational protein and drug design. RESULTS: Assuming conservation of specificity among orthologs and different specificity of paralogs, we identify residues which correlate with this grouping by specificity. The method is taking advantage of complete genomes to find multiple orthologs and paralogs. The central part of this method is a procedure to compute statistical significance of the predictions. The procedure is based on a simple statistical model of protein evolution. When applied to a large family of bacterial transcription factors, our method identified 12 residues that are presumed to determine the protein-DNA and protein-ligand recognition specificity. Structural analysis of the proteins and available experimental results strongly support our predictions. Our results suggest new experiments aimed at rational re-design of specificity in bacterial transcription factors by a minimal number of mutations. CONCLUSIONS: While sets of orthologous and paralogous proteins can be easily derived from complete genomic sequences, our method can identify putative specificity determinants in such proteins.

Amino Acids↗
PubMed2000

Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination.

Multiple alleles controlling different gene-for-gene flax rust resistance specificities occur at the L locus of flax. At least three distinct regions can be recognized in the predicted protein products: the Toll/interleukin-1 receptor homology (TIR) region, a nucleotide binding site (NBS) region, and a leucine-rich repeat (LRR) region. Replacement of the TIR-encoding region of the L6 allele with the corresponding regions of L2 or LH by recombination changed the specificity of the allele from L6 to L7. Replacement of the TIR and most of the NBS-encoding region of L10 with the equivalent region of L2 or L9 generated recombinant alleles having a novel specificity. However, replacement of the L10 TIR-encoding region with the TIR-encoding region of L2 gave rise to an allele with no detectable specificity. These data indicate that non-LRR regions can determine specificity differences between allelic gene products and that functional specificity involves interactions between coadapted polymorphic regions in the protein products of the alleles. Evidence for the action of diversifying selection on the TIR region is observed.

Adaptation, Physiological↗
PubMed2006

Gender-specific determinants of goiter.

Despite the strong implications of differences between females and males in the risk of goiter, gender-specific issues have not been extensively addressed in investigations of goiter prevalence. The objective of our analysis was to investigate the gender-specific determinants of goiter. Between April 2001 and April 2002. A total of 853 healthy employees from 4 institutions in the western part of Germany between 18 and 68 yr of age were examined by ultrasound of the neck to determine the thyroid volume. Information on sex, age, daily use of iodized salt, the history of goiter in the first-degree relatives, type and amount of smoking, oral contraceptives, and number of pregnancies were assessed by standardized questionnaires. Gender-specific predictors of goiter prevalence were assessed by multivariate logistic regression. The overall prevalence of goiter among study subjects was 204/853 (23.9%). Goiter was present in 80 out of 370 females (21.6%) vs 124/483 (25.7%) in males. In general, smoking (p < 0.0001), increasing age (p < 0.0001), and lack of daily intake of iodized salt (p = 0.004) were associated with goiter prevalence, but not sex (p = 0.39) and family history of goiter (p = 0.16). In 370 females, parity (p = 0.004) and lack of daily intake of iodized salt (p = 0.01) were the major determinants for goiter, whereas age (p = 0.18), oral contraceptives (p = 0.82), family history of goiter (p = 0.33), and smoking (p = 0.09) did not affect goiter prevalence. In 483 males, smoking (p < 0.0001) and age (p < 0.001) affected goiter prevalence, but not family history of goiter (p = 0.39), and the iodine status failed just to reach the significant level (p = 0.08) in this analysis. Gender-specific determinants of goiter are parity and iodine status in females and smoking and increasing age in males.

Adolescent↗