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At least 901 records · Page 50Linked to original sources
PubMed2001

Molecular identification of a mechanosensitive channel in archaea.

The TM1 domain of the large conductance mechanosensitive (MS) channel of Escherichia coli was used as a genetic probe to search the genomic database of the archaeon Methanoccoccus jannashii for MscL homologs. We report that the hypothetical protein MJ0170 of M. jannashii exhibited 38.5% sequence identity with the TM1 domain of Eco-MscL. Moreover, MJ0170 was found to be a conserved homolog of MscS, the second type of E. coli MS channel encoded by the yggB gene. Furthermore, we identified a cluster of charged residues KIKEE in the C-terminus of MJ0170 that strikingly resembled the charged C-terminal amino acid cluster present in Eco-MscL (RKKEE). We cloned and expressed MJ0170 in E. coli, which when reconstituted into liposomes or expressed in the cell membrane of giant E. coli spheroplasts, exhibited similar activity to the bacterial MS channels. Our study suggests that the M. jannashii MS channel and its homologs evolved as a result of gene duplication of the ancestral MscL-like molecule with the TM1 domain remaining the most conserved structural motif among prokaryotic MS channels.

Amino Acid Sequence↗
PubMed2002

Novel human G protein-coupled receptors with long N-terminals containing GPS domains and Ser/Thr-rich regions.

We report eight novel members of the superfamily of human G protein-coupled receptors (GPCRs) found by searches in the human genome databases, termed GPR97, GPR110, GPR111, GPR112, GPR113, GPR114, GPR115 and GPR116. Phylogenetic analysis shows that these are additional members of a family of GPCRs with long N-termini, previously termed EGF-7TM, LNB-7TM, B2 or LN-7TM. Five of the receptors form their own phylogenetic cluster, while three others form a cluster with the previously reported HE6 and GPR56 (TM7XN1). All the receptors have a GPS domain in their N-terminus and long Ser/Thr-rich regions forming mucin-like stalks. GPR113 has a hormone binding domain and one EGF domain. GPR112 has over 20 Ser/Thr repeats and a pentraxin domain. GPR116 has two immunoglobulin-like repeats and a SEA box. We found several human EST sequences for most of the receptors showing differential expression patterns, which may indicate that some of these receptors participate in reproductive functions while others are more likely to have a role in the immune system.

Amino Acid Sequence↗
PubMed2003

The use of peptidomics in endocrine research.

In 2002, the Nobel Prize for chemistry was awarded to the inventors of two novel ionization techniques in mass spectrometry, MALDI and ESI. These techniques, often in combination with data from genomic databases, represent an extremely powerful tool in analytical (bio)chemistry, with many applications, e.g., in the field of proteomics. Peptides, which are small proteins, have, despite their importance as controlling agents in numerous physiological processes, as yet been much less intensively studied by these novel techniques than larger proteins. The term peptidomics, i.e., the study of all peptides expressed by a certain cell, organ or organism was only introduced in 2001. In neuroendocrinology, spectacular progress could already be realized and the future looks bright. In this minireview we discuss the different methodologies that are used in peptidomics and give an overview of the wide range of applications.

Animals↗
PubMed2002

Expression of a novel HsMCAK mRNA splice variant, tsMCAK gene, in human testis.

Identification of specifically expressed genes in the adult or fetal testis is very important for the study of genes related to the development and function of the testis. In this study, a human adult testis cDNA microarray was constructed and hybridized with 33P-labeled human adult and embryo testis cDNA probes, respectively. After differential display analyzing, a number of new genes related to the development of testis and spermatogenesis had been identified. One of these new genes is tsMCAK. tsMCAK was expressed 2.62 folds more in human adult testis than fetal testis. The full length of tsMCAK is 2401 bp and contains a 2013 bp open reading frame, encoding a 671-amino-acid protein. Sequence analysis showed that it has a central kinesin motor domain and is homologous to HsMCAK gene of the somatic cells. Blasting human genome database localized tsMCAK to human chromosome 1P34 and further investigation showed that it is a splice variant of HsMCAK. The tissue distribution of tsMCAK was determined by RT-PCR and it is expressed highly and specifically in the testis. Southern blot studies of its expression in patients with infertility indicated its specific expression in spermatogenic cells and its correlation with male infertility. The above results suggested that tsMCAK is a candidate gene for the testis-specific KRPs and its specific expression in the testis was correlated with spermatogenesis and may be correlated with male infertility.

Adult↗
PubMed2002

Identification and characterization of SnrA, an inducible oxygen-insensitive nitroreductase in Salmonella enterica serovar Typhimurium TA1535.

The biological activity of many nitrosubstituted compounds, many of which are produced commercially or have been identified as environmental contaminants, is dependent on metabolic activation catalyzed by nitroreductases. In the current study, we have cloned a nitroreductase gene, Salmonella typhimurium nitroreductase A (snrA), from S. enterica serovar Typhimurium strain TA1535, and characterized the purified gene product. SnrA is 240 amino acids in length and shares 87% sequence identity to the Escherichia coli homolog, E. coli nitroreductase A (NfsA). SnrA is the major nitroreductase in S. enterica serovar Typhimurium strain TA1535 and catalyzes nitroreduction through a ping-pong bi-bi mechanism in a NADPH and flavine mononucleotide (FMN) dependent manner. SnrA exhibits extremely low levels of FMN reductase activity but the nitroreductase activity of SnrA is competitively inhibited by exogenously added FMN. Treatment of TA1535 with paraquat resulted in induction of nitroreductase activity, suggesting that SnrA is a member of the S. enterica serovar Typhimurium SoxRS regulon associated with cellular defense against oxidative damage. Examination of the microbial genomes databases shows that SnrA homologs are widely distributed in the microbial world, being present in isolates of both Archea and Eubacteria. Southern hybridization and PCR failed to detect the snrA gene in the closely related S. enterica serovar Typhimurium strain TA1538. S. enterica serovar Typhimurium strains TA1535 and TA1538 and their derivatives are commonly used in mutagenicity testing. Differences in metabolic capacity between these two strains may have implications for the interpretation of mutagenicity data.

Amino Acid Sequence↗
PubMed2001

Complex inheritance: the 21st century.

At least for the early years of the twenty-first century we can anticipate some of the advances to be made in mapping, positional cloning, pooling of evidence over samples for linkage and allelic association, and fully parametric methods that combine the latter with segregation analysis. This preoccupation with problems the twentieth century failed to solve is not grounds for pessimism if the new century provides solutions and applies them to problems of biological interest. We may hope that genetic epidemiology will be part of a community that addresses the needs of geneticists for international communication, a stable nomenclature, genome databases, and a consensus on ethical, legal, and social issues transcending regional prejudices.

Chromosome Mapping↗
PubMed2005

Sulfur-containing amino acid metabolism in parasitic protozoa.

Sulfur-containing amino acids play indispensable roles in a wide variety of biological activities including protein synthesis, methylation, and biosynthesis of polyamines and glutathione. Biosynthesis and catabolism of these amino acids need to be carefully regulated to achieve the requirement of the above-mentioned activities and also to eliminate toxicity attributable to the amino acids. Genome-wide analyses of enzymes involved in the metabolic pathways of sulfur-containing amino acids, including transsulfuration, sulfur assimilatory de novo cysteine biosynthesis, methionine cycle, and degradation, using genome databases available from a variety of parasitic protozoa, reveal remarkable diversity between protozoan parasites and their mammalian hosts. Thus, the sulfur-containing amino acid metabolic pathways are a rational target for the development of novel chemotherapeutic and prophylactic agents against diseases caused by protozoan parasites. These pathways also demonstrate notable heterogeneity among parasites, suggesting that the metabolism of sulfur-containing amino acids reflects the diversity of parasitism among parasite species, and probably influences their biology and pathophysiology such as virulence competence and stress defense.

Amino Acids, Sulfur↗
PubMed2002

Sequence alignments, variabilities, and vagaries.

It seems as if the algorithms and weighting matrices for multiple sequence alignments of the highly divergent members of the P450 gene superfamily have advanced to the point that unknown proteins can be aligned to structurally known members with reasonable accuracy. As stated earlier, the alignment tends to break down at gaps in the sequence alignments, but these regions can be improved manually. This type of alignment and analysis is especially useful for extracting and analyzing the various genome databases. Variations of the conservation analysis can be used to identify charged and uncharged residues that may be important in domain/domain interactions with redox partners or effector molecules (e.g., cytochrome b5). From these alignments and with comparative analysis within families and across P450 families, one can readily obtain an estimation of those residues that might be involved in substrate binding, in redox partner interaction, and in the catalytic mechanism.

Amino Acid Sequence↗
PubMed2003

Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation.

The use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria. PhaJ provides monomer units for PHA synthesis from the fatty acid ss-oxidation cycle. Previously, two phaJ genes (phaJ1(Pa) and phaJ2(Pa)) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3(Pa) and phaJ4(Pa)) in P. aeruginosa through a genomic database search. The abilities of the four PhaJ(Pa) proteins and the (R)-3-hydroxyacyl-acyl carrier protein [(R)-3HA-ACP] dehydrases, FabA(Pa) and FabZ(Pa), to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1(Pa) or PhaJ4(Pa) in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36-41 wt.% in dry cells) consisting of mainly short- (C4-C6) and medium-chain-length (C6-C10) 3HA units, respectively. Furthermore, detailed characterizations of PhaJ1(Pa) and PhaJ4(Pa) were performed using purified samples. Kinetic analysis revealed that only PhaJ4(Pa) exhibits almost constant maximum reaction rates (V(max)) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1(Pa), PhaJ4(Pa) has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria.

Alkanes↗
PubMed2001

Isolation and analysis of expression mechanisms of a rice gene, EL5, which shows structural similarity to ATL family from Arabidopsis, in response to N-acetylchitooligosaccharide elicitor.

Two rice cDNAs, EL5 and RRF1, were isolated and characterized. EL5 was responsive to N-acetylchitooligosaccharide, a biotic elicitor active in suspension-cultured rice cells. The structural specificity of the elicitor required for the expression of EL5 was consistent with other defense reactions observed in the experimental system, indicating that the elicitor signal to EL5 is transmitted through a single class of receptor-mediated recognition events. However, the intracellular signaling pathway to EL5 was distinct from those to other elicitor-responsive genes. Sequence analysis and alignment showed that a genomic sequence stored in rice genome databases in addition to EL5 and RRF1 belongs to the ATL family of RING-H2 finger motif proteins first isolated from Arabidopsis.

Journal Article↗
PubMed2002

Characterization of a functional neuropeptide F receptor from Drosophila melanogaster.

Potential receptors for Drosophila neuropeptide F (DmNPF) were identified in the genome database. One receptor (DmNPFR1) sequence resembled the Lymnaea NPY receptor, an invertebrate homolog of the vertebrate Y-receptor family. DmNPFR1 was cloned and tested for functionality in stably transfected mammalian CHO cells. In whole cell binding assays, DmNPF displaced 125I-NPF in a concentration-dependent manner (IC(50) = 65 nM). DmNPF inhibited forskolin-stimulated adenylyl cyclase activity similarly (IC(50) = 51 nM). Whole-mount in situ hybridization revealed that DmNPFR1 RNA is expressed in CNS and midgut of Drosophila larvae. DmNPFR1, a new invertebrate Y-receptor homolog, apparently is a functional receptor for DmNPF.

Adenylyl Cyclases↗
PubMed2002

Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A.

Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding to a 93% homology at the amino acid level. Based on available data from human genome databases, we localized the gene to chromosome 9. The transcript could be detected by in situ hybridization in human fetal spinal cord and forebrain. Two splice variants of NR3A have been reported in rat brain, the longer of the two containing a 60 bp insert in the intracellular domain. We were unable to detect this 60 bp insert in fetal or adult human brain, suggesting that only the short variant is expressed in humans.

Amino Acid Sequence↗
PubMed2003

Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis.

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.

Archaeal Proteins↗
PubMed2000

Identification of mammalian-like purple acid phosphatases in a wide range of plants.

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.

Acid Phosphatase↗
PubMed2000

Putative telomerase catalytic subunits from Giardia lamblia and Caenorhabditis elegans.

Eukaryotic chromosomes end in short nucleotide repeats that are added by the enzyme telomerase. The catalytic subunit of telomerase has been shown to be most closely related in sequence to reverse transcriptases encoded by eukaryotic retrotransposable elements. This raises the question as to whether the telomerase subunit was present in the first eukaryotes or was derived during early eukaryote evolution from the replication machinery of a retrotransposable element. We present the sequence of a putative telomerase catalytic subunit from the diplomonad parasite, Giardia lamblia. The G. lamblia subunit appears to have most of the characteristics of other sequenced telomerases, except that it lacks the conserved telomerase-specific 'T' motif previously identified in other eukaryotic genes. Searching genomic databases with the G. lamblia sequence, we also identified a potential telomerase catalytic subunit from Caenorhabditis elegans. The C. elegans subunit is uncharacteristically short, and lacks several motifs found in all other telomerases. The identification of a G. lamblia telomerase similar to that of most other eukaryotes suggests that telomerase dates back to the earliest extant marker of eukaryotic evolution. The atypical C. elegans telomerase, on the other hand, raises intriguing biochemical questions concerning sub-domains of the telomerase catalytic subunit previously considered indispensable. The enzymatic machinery for telomere formation in C. elegans is likely to differ substantially from that of other eukaryotes.

Amino Acid Sequence↗
PubMed2000

Genomic organization and chromosomal localization of the murine 2 P domain potassium channel gene Kcnk8: conservation of gene structure in 2 P domain potassium channels.

A 2 P domain potassium channel expressed in eye, lung, and stomach, Kcnk8, has recently been identified. To initiate further biochemical and genetic studies of this channel, we assembled the murine Kcnk8 cDNA sequence, characterized the genomic structure of the Kcnk8 gene, determined its chromosomal localization, and analyzed its activity in a Xenopus laevis oocyte expression system. The composite cDNA has an open reading frame of 1029 bp and encodes a protein of 343 amino acids with a predicted molecular mass of 36 kDa. Structure analyses predict 2 P domains and four potential transmembrane helices with a potential single EF-hand motif and four potential SH3-binding motifs in the COOH-terminus. Cloning of the Kcnk8 chromosomal gene revealed that it is composed of three exons distributed over 4 kb of genomic DNA. Genome database searching revealed that one of the intron/exon boundaries identified in Kcnk8 is present in other mammalian 2 P domain potassium channels genes and many C. elegans 2P domain potassium channel genes, revealing evolutionary conservation of gene structure. Using fluorescence in situ hybridization, the murine Kcnk8 gene was mapped to chromosome 19, 2B, the locus of the murine dancer phenotype, and syntenic to 11q11-11q13, the location of the human homologue. No significant currents were generated in a Xenopus laevis oocyte expression system using the composite Kcnk8 cDNA sequence, suggesting, like many potassium channels, additional channel subunits, modulator substances, or cellular chaperones are required for channel function.

Amino Acid Sequence↗
PubMed2000

Cloning and characterization of the Caenorhabditis elegans CeCRMP/DHP-1 and -2; common ancestors of CRMP and dihydropyrimidinase?

The vertebrate CRMP (collapsin-response-mediator protein) gene family comprises at least four members. These CRMPs exhibit about 60% amino acid identity with vertebrate dihydropyrimidinase (DHP), an amidohydrolase involved in the pyrimidine degradation pathway. CRMP is also referred to as DRP (DHP-related protein), TOAD-64 (turned on after division, 64 kDa) and Ulip (Unc-33-like phosphoprotein). These vertebrate CRMPs are expressed mainly in early neuronal differentiation, which suggests that they play a role in neuronal development. In this study we isolated two cDNA clones from nematode C. elegans based on their sequence homology to vertebrate CRMPs and DHP. These two molecules, termed CeCRMP/DHP-1 and -2, turned out to be Ulip-B and -A, respectively, which were previously identified in the C. elegans genomic database by Byk et al. (1998). These newly isolated molecules were believed to represent a common ancestral state before the gene duplication between CRMPs and DHP. CeCRMP/DHP-1 and -2 protein retained all putative zinc-binding residues thought to be essential for the amidohydrolase activity of DHP and exhibited a weak amidohydrolase activity when 5-bromo-dihydrouracil was used as a substrate. Whole-mount in situ hybridization and expression analysis using GFP fusions revealed that CeCRMP/DHP-1 was transiently expressed in the hypodermis of C. elegans during the early larva stage. CeCRMP/DHP-1 was also expressed in a single nerve cell between the pharynx and ring neuropil. On the other hand, expression of CeCRMP/DHP-2 was observed in the body wall muscle throughout the lifespan of C. elegans. These results indicate that a major site of CeCRMP/DHP-1 and -2 expression is non-neuronal. Targeted gene disruption of CeCRMP/DHP-2 caused no particular difference in appearance or movement phenotype.

Amidohydrolases↗
PubMed2001

A DNA polymorphism specific to Candida albicans strains exceptionally successful as human pathogens.

A large proportion of infection-causing isolates of the yeast Candida albicans belong to a general-purpose genotype, identifiable by fingerprinting with the moderately repetitive sequence Ca3. The high prevalence of this group -- up to 70% in some patient categories -- suggests that its members possess genetic determinants, which enhance their success as pathogens compared to other strains. To find such determinants we are comparing the genomes of representatives of the general-purpose genotype cluster with the genomes of other strains. In this paper we describe the identification of a 985 bp HpaII fragment (MU13-4) specific to general-purpose genotype strains. The fragment was present in 90% of these strains, but only in 10% of other strains. The fragment did not hybridize with probe Ca3, used to define the general-purpose cluster. It contains elevated levels of repetitive DNA. Sequences homologous to MU13-4 are dispersed throughout the chromosomes of general-purpose strains but are rarer or absent in other strains, as judged by Southern hybridization. Using the Stanford C. albicans genome database, we have placed the MU13-4 fragment next to a CARE-1 element. We also found 79 significant homologies between parts of MU13-4 and 19 other contigs. Attempts to amplify the region surrounding the polymorphic fragment in non-general-purpose genotype strains suggest, as do the hybridization data, that the polymorphism is created by a deletion in non-cluster strains. These results show that it is possible to identify polymorphisms specific to general-purpose genotype strains. Primers against the fragment will allow PCR-based discrimination between general-purpose genotype strains and other strains, facilitating investigations aimed at determining morbidity and mortality caused by general-purpose genotype strains compared to other strains.

Base Sequence↗