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At least 721 records · Page 40Linked to original sources
PubMed2005

Evidence for essential catalytic determinants for human erythrocyte pyrimidine 5'-nucleotidase.

Human erythrocyte pyrimidine 5'-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.

5'-Nucleotidase↗
PubMed2004

Molecular identification and chromosomal localization of genes encoding Triticum aestivum xylanase inhibitor I-like proteins in cereals.

TAXI ( Triticum aestivum xylanase inhibitor) proteins are present in wheat flour and are known to inhibit glycosyl hydrolase family 11 endoxylanases, enzymes which are commonly applied in grain processing. Here, we describe the PCR-based molecular identification of genes encoding endoxylanase inhibitors HVXI and SCXI, the TAXI-like proteins from barley ( Hordeum vulgare) and rye ( Secale cereale) respectively. The HVXI coding sequence encodes a mature protein of 384 amino acids preceded by a 19 amino acid long signal sequence. SCXI-II/III has an open reading frame encoding a signal peptide of 21 amino acids and a mature protein of 375 amino acids. As for TAXI-I, no introns were detected in the untranslated regions and coding sequences identified. These newly identified sequences allowed us to perform a multiple sequence alignment with TAXI-I and similar proteins. Rice TAXI-type proteins clustered together with the cereal endoxylanase inhibitors. Dicotyledonous proteins with sequence similarity to TAXI-I, including the tomato xyloglucan-specific endoglucanase inhibiting protein, formed a different clade. The TAXI-type proteins may hence be part of a superfamily of proteins all involved in plant responses to biotic or abiotic stress and for which a function as glycosyl hydrolase inhibitors can be suggested. The chromosomal localization of the TAXI-I gene identified on wheat chromosome 3B, of the SCXI-II/III gene identified on rye chromosome 6R, and the presence of a cluster of TAXI-like genes on rice chromosome 1, allowed us to assign the location of TAXI-like genes to the wheat-rye translocation area 3BL/6RL characterized by RFLP markers XGlb33 and Xpsr454 and isozyme Est-5. In rice, RFLP marker C1310S corresponds to a TAXI-like protein encoding sequence.

Amino Acid Sequence↗
PubMed2005

Resistance gene analogue markers are mapped to homeologous chromosomes in cultivated tetraploid cotton.

Degenerate primers designed from conserved motifs of known plant resistance gene products were used to amplify genomic DNA sequences from the root-knot nematode (Meloidogyne incognita) resistance genetic source, Upland cotton (Gossypium hirsutum) cultivar Auburn 634 RNR. A total of 165 clones were isolated, and sequence analysis revealed 57 of the clones to be novel nucleotide sequences, many containing the resistance (R)-protein nucleotide-binding site motif. A cluster analysis was performed with resistance gene analogue (RGA) nucleotide sequences isolated in this study, in addition to 99 cotton RGA nucleotide sequences already deposited in GenBank, to generate a phylogenetic tree of cotton R genes. The cotton RGA nucleotide sequences were arranged into 11 groups and 56 sub-groups, based on genetic distances. Multiple sequence alignments were performed on the RGA sequences of each sub-group, and either the consensus sequences or individual RGA sequences were used to design 61 RGA-sequence-tagged site primers. A recombinant inbred line (RIL) population of cultivated tetraploid cotton was genotyped using RGA-specific primers that amplified polymorphic fragments between the two RIL parents. Nine RGA markers were mapped to homeologous chromosomes 12 and 26, based on linkage to existing markers that are located on these chromosomes.

Amino Acid Sequence↗
PubMed2003

A zinc-containing mannitol-2-dehydrogenase from Leuconostoc pseudomesenteroides ATCC 12291: purification of the enzyme and cloning of the gene.

Mannitol-2-dehydrogenase (EC 1.1.1.67) of Leuconostoc pseudomesenteroides ATCC 12291 catalyzing the NADH-dependent reduction of d-fructose to d-mannitol was purified to homogeneity. Native mannitol-2-dehydrogenase has a molecular mass of 155 kDa as determined by gel filtration chromatography. In SDS-PAGE, a single band appeared corresponding to a molecular mass of 43 kDa which indicated that the enzyme was composed of four identical subunits. Enzyme activity was completely inhibited by EDTA and could be restored by zinc ions, but not by Mn(2+) or Mg(2+) which demonstrated that zinc is a cofactor. Purified mannitol-2-dehydrogenase exhibited a maximal specific activity of 400 micromol fructose reduced min(-1) x (mg protein)(-1), using NADH as electron donor. The enzyme showed a high substrate specificity for d-fructose and d-mannitol, however it accepted NADPH as a cofactor with 32% activity ( V(max)) relative to NADPH (100%). The mdh gene, encoding mannitol-2-dehydrogenase, was identified by hybridization with a degenerate gene probe complementary to the nucleotide sequence encoding the first eight N-terminal amino acids of the enzyme. The mdh gene was cloned on a 4.2-kb DNA fragment, subcloned, and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 1017 bp, encoding a protein of 338 amino acids with a predicted molecular mass of 36.0 kDa. Plasmid-encoded mdh was functionally expressed, with 70 U/mg of cell-free protein in E. coli. Multiple sequence alignments showed that mannitol-2-dehydrogenase was affiliated with members of the Zn(2+)-containing medium-chain alcohol/polyol dehydrogenase/reductase protein family (MDR).

Alcohol Oxidoreductases↗
PubMed2006

Characterization of a mercury-reducing Bacillus cereus strain isolated from the Pulicat Lake sediments, south east coast of India.

Pulicat Lake sediments are often severely polluted with the toxic heavy metal mercury. Several mercury-resistant strains of Bacillus species were isolated from the sediments and all the isolates exhibited broad spectrum resistance (resistance to both organic and inorganic mercuric compounds). Plasmid curing assay showed that all the isolated Bacillus strains carry chromosomally borne mercury resistance. Polymerase chain reaction and southern hybridization analyses using merA and merB3 gene primers/probes showed that five of the isolated Bacillus strains carry sequences similar to known merA and merB3 genes. Results of multiple sequence alignment revealed 99% similarity with merA and merB3 of TnMERI1 (class II transposons). Other mercury resistant Bacillus species lacking homology to these genes were not able to volatilize mercuric chloride, indicating the presence of other modes of resistance to mercuric compounds.

Anti-Bacterial Agents↗
PubMed2006

Thrombin-like effect of an important green pit viper toxin, albolabrin: a bioinformatic study.

The green pit viper venom has a major effect on the hematological system. Clinical features of venomous snakebites vary from asymptomatic to fatal bleeding. The venom is found to have a thrombin-like effect in vitro. Here, the author performs a bioinformatic analysis on the green pit viper venom focusing on its thrombin-like effect. Sequence comparison between green pit viper venom, albolabrin and thrombin was performed. In addition, the author performed a search for other human proteins closely relating to the thrombin and created a multiple sequence alignment phylogenetic tree to present the family tree of the thrombin, albolabrin and those proteins recorded in the genomic database. In conclusion, the comparative sequence analysis between green pit viper venom and thrombin gives several identities. The reported relationship on the phylogenetic tree can match with the reported in vivo function of green pit viper. Explanations on the effect of green pit viper toxin on the hemostasis can be derived from this study. Furthermore, future researches based on the reported identities can be expected.

Amino Acid Sequence↗
PubMed2002

Structure prediction and phylogenetic analysis of a functionally diverse family of proteins homologous to the MT-A70 subunit of the human mRNA:m(6)A methyltransferase.

MT-A70 is the S-adenosylmethionine-binding subunit of human mRNA:m(6)A methyl-transferase (MTase), an enzyme that sequence-specifically methylates adenines in pre-mRNAs. The physiological importance yet limited understanding of MT-A70 and its apparent lack of similarity to other known RNA MTases combined to make this protein an attractive target for bioinformatic analysis. The sequence of MT-A70 was subjected to extensive in silico analysis to identify orthologous and paralogous polypeptides. This analysis revealed that the MT-A70 family comprises four subfamilies with varying degrees of interrelatedness. One subfamily is a small group of bacterial DNA:m(6)A MTases. The other three subfamilies are paralogous eukaryotic lineages, two of which have not been associated with MTase activity but include proteins having substantial regulatory effects. Multiple sequence alignments and structure prediction for members of all four subfamilies indicated a high probability that a consensus MTase fold domain is present. Significantly, this consensus fold shows the permuted topology characteristic of the b class of MTases, which to date has only been known to include DNA MTases.

Amino Acid Sequence↗
PubMed2004

cDNA sequence, protein structure, and evolution of the single hemocyanin from Aplysia californica, an opisthobranch gastropod.

By protein immunobiochemistry and cDNA sequencing, we have found only a single hemocyanin polypeptide in an opisthobranch gastropod, the sea hare Aplysia californica, which contrasts with previously studied prosobranch gastropods, which express two distinct isoforms of this extracellular respiratory protein. We have cloned and sequenced the cDNA encoding the complete polypeptide of Aplysia californica hemocyanin (AcH). The cDNA comprises 11,433 bp, encompassing a 5'UTR of 77 bp, a 3'UTR of 1057 bp, and an open reading frame for a signal peptide of 20 amino acids plus a polypeptide of 3412 amino acids (Mr ca. 387 kDa). This polypeptide is the subunit of the cylindrical native hemocyanin (Mr ca. 8 MDa). It comprises eight different functional units (FUs: a, b, c, d, e, f, g, h) that have been identified immunobiochemically after limited proteolysis of AcH purified from the hemolymph. Each FU shows a highly conserved copper-A and copper-B site for reversible oxygen binding. FU AcH-h carries a specific C-terminal extension of ca. 100 amino acids that include two cysteines that may be utilized for disulfide bridge formation. Potential N-glycosylation sites are present in six FUs but lacking in AcH-b and AcH-c. On the basis of multiple sequence alignments, phylogenetic trees and a statistically firm molecular clock were calculated. The latter suggests that the last common ancestor of Haliotis and Aplysia lived 373+/-47 million years ago, in convincing agreement with fossil records from the early Devonian. However, the gene duplication yielding the two distinct hemocyanin isoforms found today in Haliotis tuberculata occurred 343+/-43 million years ago.

Amino Acid Sequence↗
PubMed2006

Multiple gene duplication and rapid evolution in the groEL gene: functional implications.

The chaperonins, GroEL and GroES, are present ubiquitously and provide a paradigm in the understanding of assisted protein folding. Due to its essentiality of function, GroEL exhibits high sequence conservation across species. Complete genome sequencing has shown the occurrence of duplicate or multiple copies of groEL genes in bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. Monophyly of each bacterial clade in the phylogenetic tree generated for the GroEL protein suggests a lineage-specific duplication. The duplicated groEL gene in Actinobacteria is not accompanied by the operonic groES despite the presence of upstream regulatory elements. Our analysis suggests that in these bacteria the duplicated groEL genes have undergone rapid evolution and divergence to function in a GroES-independent manner. Evaluation of multiple sequence alignment demonstrates that the duplicated genes have acquired mutations at functionally significant positions including those involved in substrate binding, ATP binding, and GroES binding and those involved in inter-ring and intra-ring interactions. We propose that the duplicate groEL genes in different bacterial clades have evolved independently to meet specific requirements of each clade. We also propose that the groEL gene, although essential and conserved, accumulates nonconservative substitutions to exhibit structural and functional variations.

Adenosine Triphosphate↗
PubMed2000

Phylogeny of the restriction endonuclease-like superfamily inferred from comparison of protein structures.

To date all attempts to derive a phyletic relationship among restriction endonucleases (ENases) from multiple sequence alignments have been limited by extreme divergence of these enzymes. Based on the approach of Johnson et al. (1990), I report for the first time the evolutionary tree of the ENase-like protein superfamily inferred from quantitative comparison of atomic coordinates of structurally characterized enzymes. The results presented are in harmony with previous comparisons obtained by crystallographic analyses. It is shown that lambda-exonuclease initially diverged from the common ancestor and then two "endonucleolytic" families branched out, separating "blunt end cutters" from "5' four-base overhand cutters." These data may contribute to a better understanding of ENases and encourage the use of structure-based methods for inference of phylogenetic relationship among extremely divergent proteins. In addition, the comparison of three-dimensional structures of ENase-like domains provides a platform for further clustering analyses of sequence similarities among different branches of this large protein family, rational choice of homology modeling templates, and targets for protein engineering.

Catalytic Domain↗
PubMed2006

Identification and characterization of porcine mannan-binding lectin A (pMBL-A), and determination of serum concentration heritability.

Mannan-binding lectin (MBL) is an innate immune collectin present in the serum of humans and many farm animals. This oligomeric pattern-recognition protein effectively binds to the glycoconjugate arrays present on the surfaces of microorganisms and activates the complement system to enhance pathogen killing and clearance. MBL deficiency is often associated with immunodeficiency in humans. Although two MBLs (MBL-A and MBL-C) have been characterized in various species, the identity of porcine MBL (pMBL) was not clearly defined. In this study, we purified an MBL from porcine serum by mannose affinity, ion exchange, and size exclusion chromatography and determined many of its characteristics. Based on the N-terminal sequence, multiple sequence alignment, and relative affinities to various carbohydrate ligands, we propose that the MBL purified in this study is pMBL-A. We have generated antibodies to this protein and established an immunoassay to quantify pMBL-A in serum. Using this assay, we found breed differences in pMBL-A concentration distributions and heritability estimates. In the Duroc breed (n=588), pMBL-A concentrations show a unimodal distribution with a mean of 9,125 ng/ml. In contrast, the pMBL-A concentration distributions in the Landrace breed (n=533) show three distinct mean values: 301, 2,385, and 11,507 ng/ml. Furthermore, heritability calculations based on an additive genetic variance model with no fixed effects indicate that serum pMBL-A concentration is highly heritable in the Landrace (h (2)=0.8) but not in the Duroc breed (h (2)=0.15). These genetic differences may be useful in selecting breeding pigs for improved disease resistance.

Amino Acid Sequence↗
PubMed2001

Replacement of arginine-171 and aspartate-453 in Streptomyces coelicolor malate synthase A by site-directed mutagenesis inactivates the enzyme.

Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA. Escherichia coli is known to possess two forms of malate synthase, A and G respectively. The recent elucidation of the E. coli malate synthase G crystal structure suggested two residues, Arg338 and Asp631, are essential for catalysis. Multiple sequence alignment of 26 known malate synthase enzymes revealed that the two proposed sites are highly conserved, despite the low homologies between the two distinct forms of the enzyme (13-18%). The conservation of these residues in both forms of malate synthase suggests that they possess a similar catalytic strategy. Thus, despite the absence of a three-dimensional structure for malate synthase A, the significance of this enzyme in the primary metabolic pathway has prompted the investigation of the involvement of the corresponding residues, Arg171 and Asp453, in Streptomyces coelicolor malate synthase A by site-directed mutagenesis. Heterologous expression in E. coli followed by purification of the constructed mutant proteins, Arg171Leu and Asp453Ala, were performed and subsequent enzyme assays of the purified mutant proteins indicated a significant loss of catalytic activity, thus attesting to the need for the corresponding conserved residues to maintain malate synthase functionality.

Amino Acid Sequence↗
PubMed2007

Porin isolated from the outer membrane of Erwinia amylovora and its encoding gene.

A major Erwinia amylovora outer-membrane protein (Omp-EA) and the gene encoding for this protein (omp-EA) were isolated and characterized. The native Omp-EA protein forms a trimeric structure of approximately 114 kDa. This protein demonstrated high resistance to detergents such as SDS and octyl-glucopyranoside, but disaggregated to monomers with a molecular weight (MW) of approximately 39 kDa after heating at 95 degrees C for 10 minutes in sample buffer. The pore-forming ability of the oligomeric Omp-EA was determined by the liposome swelling assay, demonstrating that the oligomeric protein formed nonspecific channels with an exclusion limit of approximately 660 Da. On dissociation, the monomers did not exhibit pore-forming ability. The omp-EA gene was cloned and sequenced (GenBank Accession No. DQ184680). Sequence analysis revealed an open reading frame of 1152 bases. The deduced amino-acid sequence had 383 amino acids. The mature protein consisted of 362 amino acids and had a calculated MW of 39,210 Da. Multiple-sequence alignment of Omp-EA with other porins from the Enterobacteriaceae family revealed 51% to 63% identity. The first 16 amino acids from the N-terminal exhibited the highest identity (100%) to the porins OmpC, OmpF, and PhoE of Escherichia coli. Two methods were used to predict the secondary structure: APSSP2 and Hidden and Markov's model. The monomers of Omp-EA porin presented a topology of 16 transmembranal beta-strands. The area of the loops between the beta -strands was proposed. It is suggested that further research on the porin and its loops may be important for understanding the mechanism of E. amylovor to invade plant tissues.

Amino Acid Sequence↗
PubMed2006

Characterization of the 16S-23S internal transcribed spacer among 34 higher plants: suitability for interspecific plastid transformation.

Biomanufacturing by chloroplast transgene expression has the potential to produce significant amounts of biopharmaceuticals, endow plants with novel commercial or humanitarian capabilities, enhance phytoremediation methods and harden plants against adverse environments. Plastid bioengineering exploits the phenomenon of homologous recombination to specifically integrate heterologous sequences into the plastid genome. Previous research suggests the plastid genome 16S-23S internal transcribed spacer provides an advantageous integration site for transgene expression. To characterize the suitability of the 16S-23S region for interspecific recombination, we developed primers against conserved plastid sequences and amplified approximately 2.6 kb from 25 plant species. We analyzed the amplicons with nine species from Genbank for homeology, phylogenetic relationships, potential to form chimeric rDNA elements disruptive to translational/replication systems, and the potential number of recombination events for various minimal essential processing segments (MEPS) lengths. Multiple sequence alignment of the 34 species revealed considerable conservation, with identities exceeding 95% among the angiosperms. Substitutions were statistically clustered, generally in noncoding sites, although proposed functional elements such as the OriA region and 3' terminus of the 16S rRNA exhibited unexpected variation. The nonrandom distribution of substitutions undermines the established, statistical method of estimating the number of recombination initiation sites. This finding is further substantiated by comparing statistical estimates of the number of MEPS sites to a direct count at three different MEPS lengths. We frame this in silico analysis in terms of the potential of the 16S-23S region as a target for interspecific transformation, and describe a 'primer-to-plastid' system to rapidly generate species-specific flanking regions for transformation vectors.

Base Sequence↗
PubMed1997

Cloning of the homogentisate 1,2-dioxygenase gene, the key enzyme of alkaptonuria in mouse.

We determined 48 amino acid residues from five peptides from the homogeneous monomer of homogentisate 1,2-dioxygenase (HGO; E.C. 1.13. 11.15) of mouse liver. After digestion with trypsin, peptides were separated by reversed phase chromatography and amino acid sequenced. The deduced codon sequence of three peptides was used to derive degenerated oligomeres. By combining these oligos, we were able to amplify fragments from 100 to 300 bases (b) from mouse liver cDNA by polymerase chain reaction after reverse transcription (RT-PCR). A fragment of 200 b was cloned and used as a probe to screen a mouse liver cDNA library. One clone from this library contained the complete cDNA-insert for HGO as determined by sequencing. The cDNA encodes for a protein of 50 kDa, as predicted. The cDNA of mouse HGO has an overall identity of 41% to the corresponding gene hmgA from Aspergillus. Sequence similarities to human expressed sequence tags (EST) clones ranged from 70% to 20%. The positions of 122 conserved amino acids could be determined by multiple sequence alignment. We identified one first intron of 928 b in the mouse gene. The gene for HGO seems to be expressed in various tissues, as shown by RT-PCR on different cDNAs. FISH experiments with the whole murine cDNA as probe clearly revealed signals at the human chromosomal band 3q13. 3-q21. This corresponds well to the previous assignment of the locus for the human alkaptonuria gene (AKU) to the same chromosomal region by multipoint linkage analysis. We therefore conclude that the HGO cDNA encodes the gene responsible for alkaptonuria.

Alkaptonuria↗
PubMed1998

High sequence similarity within ras exons 1 and 2 in different mammalian species and phylogenetic divergence of the ras gene family.

We have determined the canine and feline N-, K-, and H-ras gene sequences from position +23 to +270 covering exons I and II which contain the mutational hot spot codons 12, 13, and 61. The results were used to assess the degree of similarity between ras gene DNA regions containing the critical domains affected in neoplastic disorders in different mammalian species. The comparative analyses performed included human, canine, feline, murine, rattine, and, whenever possible, bovine, leporine (rabbit), porcelline (guinea pig), and mesocricetine (hamster) ras gene sequences within the region of interest. Comparison of feline and canine nucleotide sequences with the corresponding regions in human DNA revealed a sequence similarity greater than 85% to the human sequence. Contemporaneous analysis of previously published ras DNA sequences from other mammalian species showed a similar degree of homology to human DNA. Most nucleotide differences observed represented synonymous changes without effect on the amino acid sequence of the respective proteins. For assessment of the phylogenetic evolution of ras gene family, a maximum parsimony dendrogram based on multiple sequence alignment of the common region of exons I and II in the N-, K-, and H-ras genes was constructed. Interestingly, a higher substitution rate among the H-ras genes became apparent, indicating accelerated sequence evolution within this particular clade. The most parsimonious tree clearly shows that the duplications giving rise to the three ras genes must have occurred before the mammalian radiation.

Animals↗
PubMed2002

Ancestry and diversity of BEL1-like homeobox genes revealed by gymnosperm ( Gnetum gnemon) homologs.

BEL1-like homeobox genes encode plant-specific transcription factors, at least some of which are important for ovule development. Here we report MELBEL1-MELBEL4,the first BEL1-like genes from a non-flowering plant, the gymnosperm Gnetum gnemon. Our analyses suggest that there was already at least one BEL1-like gene present in the most recent common ancestor of extant seed plants about 300 million years ago. Multiple sequence alignments revealed that since this time, not only the DNA-binding homeodomain, but also a protein-protein interaction domain upstream of the homeodomain, termed the BEL domain, has been highly conserved. Sequence comparison of the BEL domain with upstream domains that have been conserved in other TALE homeodomain proteins, i.e. MEIS, KNOX, and PBC, revealed only weak sequence similarity. However, since homology has been shown for MEIS, KNOX, and PBC domains and since KNOX and BEL domains directly interact in vivo, it appears likely that the BEL domain was also derived from an ancestral upstream (MEINOX) domain.

Amino Acid Sequence↗
PubMed2006

The putative telomerase reverse transcriptase component of Leishmania amazonensis: gene cloning and characterization.

The Leishmania amazonensis telomerase gene was cloned by a polymerase chain reaction-based strategy using primers designed from a Leishmania major sequence that shared similarities with conserved telomerase motifs. The genes from three other species were cloned for comparative purposes. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania telomerase gene was probably in single copy and located in the largest chromosomes. A single messenger ribonucleic acid transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases.

Amino Acid Sequence↗