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At least 1,621 records · Page 90Linked to original sources
PubMed2000

Structural genomics and its importance for gene function analysis.

Structural genomics projects aim to solve the experimental structures of all possible protein folds. Such projects entail a conceptual shift from traditional structural biology in which structural information is obtained on known proteins to one in which the structure of a protein is determined first and the function assigned only later. Whereas the goal of converting protein structure into function can be accomplished by traditional sequence motif-based approaches, recent studies have shown that assignment of a protein's biochemical function can also be achieved by scanning its structure for a match to the geometry and chemical identity of a known active site. Importantly, this approach can use low-resolution structures provided by contemporary structure prediction methods. When applied to genomes, structural information (either experimental or predicted) is likely to play an important role in high-throughput function assignment.

Animals↗
PubMed2004

Quantification and functional analysis of plasmacytoid dendritic cells in patients with chronic hepatitis C virus infection.

BACKGROUND: Plasmacytoid dendritic cells (PDCs) are the major producers of interferon (IFN)- alpha within peripheral blood mononuclear cells (PBMCs). METHODS: We analyzed whether chronic hepatitis C virus (HCV) infection could be linked to a defective function or number of PDCs. We evaluated the capacity of PBMCs from 5 cohorts of subjects to produce IFN- alpha after viral stimulation. We concomitantly analyzed the frequency of PDCs and the levels of IFN- alpha transcripts within the PBMCs from the same cohorts. RESULTS: PBMCs from patients with chronic HCV infection receiving antiviral therapy displayed a reduced capacity to release IFN- alpha, compared with those from healthy individuals, those from long-term responders to therapy, and those from nontreated patients. This defect was significantly correlated with the percentage of PDCs. In addition, PDCs from patients with chronic HCV infection receiving therapy displayed a reduced intrinsic capacity to produce IFN- alpha, which could be linked to the level of IFN- alpha transcripts. CONCLUSION: Our observations point to an effect of the therapy on either the survival or the localization of PDCs, rather than a direct detrimental effect due to the viral infection during chronic HCV infection.

Adolescent↗
PubMed2000

Structural and functional analysis of the gene cluster encoding carotenoid biosynthesis in Mycobacterium aurum A+.

The fragment containing the carotenoid gene cluster from Mycobacterium aurum A+, a 3,3'-dihydroxy-isoneriatene and 3-monohydroxy-isoneriatene accumulator, has been sequenced and the exposed eight genes are organised in two operons. The function of three of these genes, a phytoene desaturase (crtI), a phytoene cyclase (crtY) and a beta-carotene desaturase (crtU), was demonstrated by complementation of M. aurum carotenoid mutants. The eight genes of the carotenoid cluster are highly homologous to other carotenoid gene clusters and thus this cluster is a candidate for its introduction into mycobacteria as a non-antibiotic reporter gene(s) as well as a source of new regulated promoters.

Alkyl and Aryl Transferases↗
PubMed1992

Overexpression in Escherichia coli and functional analysis of a novel PPi-selective porin, oprO, from Pseudomonas aeruginosa.

Immediately upstream from and adjacent to the oprP gene, which codes for the phosphate-specific porin OprP of Pseudomonas aeruginosa, lies the PR region (oprO), which cross-hybridizes with oprP DNA. To determine the function of this region, the oprO gene was expressed behind the lactose promoter in Escherichia coli, and the resultant OprO protein was purified and reconstituted into planar lipid bilayers. OprO formed sodium dodecyl sulfate-stable trimers, cross-reacted immunologically with OprP, and, like OprP, formed an anion-specific, phosphate-selective porin. However, it demonstrated lower affinity for and higher maximal conductance of both chloride and phosphate than did the OprP channel. Examination by macroscopic conductance inhibition experiments of the affinity of OprO for phosphates of different lengths revealed a preference for PPi and tripolyphosphate over Pi, suggesting that OprO functioned as a PPi-selective polyphosphate channel, in contrast to OprP, which has a marked preference for Pi.

Bacterial Outer Membrane Proteins↗
PubMed2001

Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1.

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.

Bacterial Proteins↗
PubMed2003

Functional analysis and treatment of self-injurious behavior in young children a summary of 30 cases.

The assessment and treatment of self-injurious behavior (SIB) has received much attention in the literature; however, few studies have focused on early intervention for this behavior. In the current study, functional analyses with developmentally appropriate modifications were conducted in an outpatient clinic with 30 children (aged 10 months to 4 years 11 months) to assess SIB and problem behavior in its early stages. The reported mean age of SIB onset was 17 months, and head banging was the most prevalent topography. Functional analyses identified sources of reinforcement for SIB in 62.1% of cases; with the inclusion of all forms of problem behavior, sources of reinforcement were identified for 87.5% of cases. Function-based treatments were developed for 24 cases, with functional communication training prescribed most often (70.8% of cases). Implications of these findings for the development of early intervention programs for SIB are discussed.

Aggression↗
PubMed2003

Structures of phosphate and trivanadate complexes of Bacillus stearothermophilus phosphatase PhoE: structural and functional analysis in the cofactor-dependent phosphoglycerate mutase superfamily.

Bacillus stearothermophilus phosphatase PhoE is a member of the cofactor-dependent phosphoglycerate mutase superfamily possessing broad specificity phosphatase activity. Its previous structural determination in complex with glycerol revealed probable bases for its efficient hydrolysis of both large, hydrophobic, and smaller, hydrophilic substrates. Here we report two further structures of PhoE complexes, to higher resolution of diffraction, which yield a better and thorough understanding of its catalytic mechanism. The environment of the phosphate ion in the catalytic site of the first complex strongly suggests an acid-base catalytic function for Glu83. It also reveals how the C-terminal tail ordering is linked to enzyme activation on phosphate binding by a different mechanism to that seen in Escherichia coli phosphoglycerate mutase. The second complex structure with an unusual doubly covalently bound trivanadate shows how covalent modification of the phosphorylable His10 is accompanied by small structural changes, presumably to catalytic advantage. When compared with structures of related proteins in the cofactor-dependent phosphoglycerate mutase superfamily, an additional phosphate ligand, Gln22, is observed in PhoE. Functional constraints lead to the corresponding residue being conserved as Gly in fructose-2,6-bisphosphatases and Thr/Ser/Cys in phosphoglycerate mutases. A number of sequence annotation errors in databases are highlighted by this analysis. B. stearothermophilus PhoE is evolutionarily related to a group of enzymes primarily present in Gram-positive bacilli. Even within this group substrate specificity is clearly variable highlighting the difficulties of computational functional annotation in the cofactor-dependent phosphoglycerate mutase superfamily.

Amino Acid Sequence↗
PubMed1996

Functional analysis of the PUT3 transcriptional activator of the proline utilization pathway in Saccharomyces cerevisiae.

Proline can serve as a nitrogen source for the yeast Saccharomyces cerevisiae when preferred sources of nitrogen are absent from the growth medium. PUT3, the activator of the proline utilization pathway, is required for the transcription of the genes encoding the enzymes that convert proline to glutamate. PUT3 is a 979 amino acid protein that constitutively binds a short DNA sequence to the promoters of its target genes, but does not activate their expression in the absence of induction by proline and in the presence of preferred sources of nitrogen. To understand how PUT3 is converted from an inactive to an active state, a dissection of its functional domains has been undertaken. Biochemical and molecular tests, domain swapping experiments, and an analysis of activator-constitutive and activator-defective mutant proteins indicate that PUT3 is dimeric and activates transcription with its negatively charged carboxyterminus, which does not appear to contain a proline-responsive domain. A mutation in the conserved central domain found in many fungal activators interferes with activation without affecting DNA binding protein stability. Intragenic suppressors of the central domain mutation have been isolated and analyzed.

Base Sequence↗
PubMed2000

Functional analysis of a mutant sulfonylurea receptor, SUR1-R1420C, that is responsible for persistent hyperinsulinemic hypoglycemia of infancy.

The ATP-sensitive potassium (K(ATP)(+)) channel is crucial for the regulation of insulin secretion from the pancreatic beta-cell, and mutations in either the sulfonylurea receptor type 1 (SUR1) or Kir6. 2 subunit of this channel can cause persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We analyzed the functional consequences of the PHHI missense mutation R1420C, which lies in the second nucleotide-binding fold (NBF2) of SUR1. Mild tryptic digestion of SUR1 after photoaffinity labeling allowed analysis of the nucleotide-binding properties of NBF1 and NBF2. Labeling of NBF1 with 8-azido-[alpha-(32)P]ATP was inhibited by MgATP and MgADP with similar K(i) for wild-type SUR1 and SUR1-R1420C. However, the MgATP and MgADP affinities of NBF2 of SUR1-R1420C were about 5-fold lower than those of wild-type SUR1. MgATP and MgADP stabilized 8-azido-ATP binding at NBF1 of wild-type SUR1 by interacting with NBF2, but this cooperative nucleotide binding was not observed for SUR1-R1420C. Studies on macroscopic currents recorded in inside-out membrane patches revealed that the SUR1-R1420C mutation exhibits reduced expression but does not affect inhibition by ATP or tolbutamide or activation by diazoxide. However, co-expression with Kir6.2-R50G, which renders the channel less sensitive to ATP inhibition, revealed that the SUR1-R1420C mutation increases the EC(50) for MgADP activation from 74 to 197 microm. We suggest that the lower expression of the mutant channel and the reduced affinity of NBF2 for MgADP may lead to a smaller K(ATP)(+) current in R1420C-PHHI beta-cells and thereby to the enhanced insulin secretion. We also propose a new model for nucleotide activation of K(ATP)(+) channels.

ATP-Binding Cassette Transporters↗
PubMed2025

Genome-Wide Identification of the TIFY Family in Cannabis sativa L. and Its Potential Functional Analysis in Response to Alkaline Stress and in Cannabinoid Metabolism.

TIFY transcription factors play crucial regulatory roles in secondary metabolism and stress response. However, the expression patterns of the Cannabis sativa L. TIFY gene family under alkali stress, their involvement in cannabinoid metabolism, and their underlying genetic evolutionary mechanisms remain largely unexplored. In this study, we used bioinformatics approaches to conduct genome-wide identification and functional characterization of the C. sativa TIFY gene family. Fourteen TIFY genes were identified and mapped onto seven chromosomes. These genes were classified into four subfamilies: TIFY, JAZ, ZML, and PPD, with the JAZ subfamily further subdivided into five distinct branches. Collinearity analysis suggested that gene duplication events contributed to the expansion of the TIFY gene family in C. sativa. Weighted gene coexpression network analysis (WGCNA) revealed that CsJAZ2, CsJAZ3, and CsJAZ6 participated in the cannabinoid regulatory network. Cis-element analysis indicated that the promoter regions of TIFY genes were enriched in hormone- and stress-responsive elements. Furthermore, transcriptome and RT-qPCR analyses were conducted to examine gene expression patterns under alkaline stress (the RNA employed in RT-qPCR was extracted from the apical leaves of samples subjected to short-duration alkaline stress treatment). The results showed that CsJAZ5 and CsJAZ6 were downregulated, whereas CsPPD1, CsTIFY1, and CsZML1 were upregulated in response to alkali stress. In summary, CsJAZ5, CsPPD1, and CsTIFY1 may serve as candidate genes for the development of alkali-tolerant cultivars, while CsJAZ2 and CsJAZ3 may be valuable targets for enhancing cannabinoid production. This study provides important molecular insights and a theoretical basis for future research on the evolutionary dynamics and functional roles of TIFY transcription factors, particularly in stress adaptation and cannabinoid metabolism.

Cannabis↗
PubMed2004

Prediction and functional analysis of native disorder in proteins from the three kingdoms of life.

An automatic method for recognizing natively disordered regions from amino acid sequence is described and benchmarked against predictors that were assessed at the latest critical assessment of techniques for protein structure prediction (CASP) experiment. The method attains a Wilcoxon score of 90.0, which represents a statistically significant improvement on the methods evaluated on the same targets at CASP. The classifier, DISOPRED2, was used to estimate the frequency of native disorder in several representative genomes from the three kingdoms of life. Putative, long (>30 residue) disordered segments are found to occur in 2.0% of archaean, 4.2% of eubacterial and 33.0% of eukaryotic proteins. The function of proteins with long predicted regions of disorder was investigated using the gene ontology annotations supplied with the Saccharomyces genome database. The analysis of the yeast proteome suggests that proteins containing disorder are often located in the cell nucleus and are involved in the regulation of transcription and cell signalling. The results also indicate that native disorder is associated with the molecular functions of kinase activity and nucleic acid binding.

Databases, Genetic↗
PubMed2002

Solution structure and functional analysis of the cysteine-rich C1 domain of kinase suppressor of Ras (KSR).

Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to promote signal transmission from Raf-1 to MEK and MAPK. All KSR proteins contain a conserved cysteine-rich C1 domain, and studies have implicated this domain in the regulation of KSR1 subcellular localization and function. To further elucidate the biological role of the KSR1 C1 domain, we have determined its three-dimensional solution structure using nuclear magnetic resonance (NMR). We find that while the overall topology of the KSR1 C1 domain is similar to the C1 domains of Raf-1 and PKCgamma, the predicted ligand-binding region and the surface charge distribution are unique. Moreover, by generating chimeric proteins in which these domains have been swapped, we find that the C1 domains of Raf-1, PKCgamma, and KSR1 are not functionally interchangeable. The KSR1 C1 domain does not bind with high affinity or respond biologically to phorbol esters or ceramide, and it does not interact directly with Ras, indicating that the putative ligand(s) for the KSR1 C1 domain are distinct from those that interact with PKCgamma and Raf-1. In addition, our analysis of the chimeric proteins supports the model that Raf-1 is a ceramide-activated kinase and that its C1 domain is involved in the ceramide-mediated response. Finally, our findings demonstrate an absolute requirement of the KSR1 C1 domain in mediating the membrane localization of KSR1, a crucial feature of its scaffolding activity. Together, these results underscore the functional specificity of these important regulatory domains and demonstrate that the structural features of the C1 domains can provide valuable insight into their ligand-binding properties.

3T3 Cells↗
PubMed2004

Functional analysis of a 450-amino acid N-terminal fragment of phytochrome B in Arabidopsis.

Phytochrome, a major photoreceptor in plants, consists of two domains: the N-terminal photosensory domain and the C-terminal domain. Recently, the 651-amino acid photosensory domain of phytochrome B (phyB) has been shown to act as a functional photoreceptor in the nucleus. The phytochrome (PHY) domain, which is located at the C-terminal end of the photosensory domain, is required for the spectral integrity of phytochrome; however, little is known about the signal transduction activity of this domain. Here, we have established transgenic Arabidopsis thaliana lines expressing an N-terminal 450-amino acid fragment of phyB (N450) lacking the PHY domain on a phyB-deficient background. Analysis of these plants revealed that N450 can act as an active photoreceptor when attached to a short nuclear localization signal and beta-glucuronidase. In vitro spectral analysis of reconstituted chromopeptides further indicated that the stability of the N450 Pfr form, an active form of phytochrome, is markedly reduced in comparison with the Pfr form of full-length phyB. Consistent with this, plants expressing N450 failed to respond to intermittent light applied at long intervals, indicating that N450 Pfr is short-lived in vivo. Taken together, our findings show that the PHY domain is dispensable for phyB signal transduction but is required for stabilizing the Pfr form of phyB.

Arabidopsis↗
PubMed2002

Functional analysis of yeast snoRNA and snRNA 3'-end formation mediated by uncoupling of cleavage and polyadenylation.

Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3'-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3' portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

Base Sequence↗
PubMed2006

Single nucleotide polymorphisms and functional analysis of MxA promoter region in multiple sclerosis.

OBJECTIVE: Interferons (IFNs)-inducible myxovirus resistance protein A (MxA) has recently been used as an indirect marker of neutralizing antibody against IFN in patients with multiple sclerosis (MS). On the other hand, MxA inhibits the replication of viruses by means of modifying cellular function, including apoptotic pathway. Our objective is to investigate the genetic and pathological role of MxA in patients with MS. METHODS: We examined SNPs of MxA promoter region in 67 patients with MS. Moreover, to elucidate the functional roles of SNPs, we conducted Luciferase assay with pGL3-basic vector including patient-derived or artificially mutated MxA promoter region. RESULTS: A significantly higher frequency of the haplotype with -88T and -123A, which correlates with over-expression of MxA, was observed in MS. Moreover, we elucidated novel findings showing that nt -88 played a leading part with type I IFNs and that nt -123 played the same role independently without type I IFNs, respectively. CONCLUSION: SNPs on MxA promoter region may play an important role in the pathophysiology of MS and provide a novel strategy for the therapeutic resolutions of MS.

Adult↗
PubMed2006

Functional analysis of gammaretroviral vector transduction by quantitative PCR.

BACKGROUND: In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. METHODS: We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. RESULTS: Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the 'transcription index' (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. CONCLUSIONS: The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels.

3T3 Cells↗
PubMed1997

Functional analysis of four tetraspans, CD9, CD53, CD81, and CD82, suggests a common role in costimulation, cell adhesion, and migration: only CD9 upregulates HB-EGF activity.

Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other proteins such as beta 1 integrins and MHC antigens. Although their functions are not clear, they have been suggested to play a role in cell adhesion and migration, signal transduction, and costimulation. We have in this paper directly compared the functional properties of four tetraspans, CD9, CD53, CD81, and CD82. mAbs to any of these molecules were able to deliver a costimulatory signal for CD3-mediated activation of the T cell line Jurkat. CD82 mAbs were the most efficient in triggering this effect. Moreover, engagement of CD9, CD81, and CD82 induced the homotypic aggregation of the megakaryocytic cell line HEL, and inhibited the migration of this cell line. Similar results were obtained with the preB cell line NALM-6 using the CD9 and CD81 mAbs. The CD81 mAb 5A6 produced the strongest effects. Therefore, the tetraspans are recognized by mAbs which produce similar effects on the same cell lines. This is consistent with the tetraspans being included in large molecular complexes and possibly forming a tetraspan network (the tetraspan web). We also demonstrate that the tetraspans are likely to keep specific functional properties inside this network. Indeed, we have demonstrated that the human CD9 is able, like the monkey molecule, to upregulate the activity of the transmembrane precursor of heparin-binding EGF as a receptor for the diphtheria toxin when cotransfected in murine LM cells. Neither CD81, nor CD82 had such activity. By using chimeric CD9/CD81 molecules we demonstrate that this activity requires the second half of CD9, which contains the large extracellular loop, the fourth transmembrane region, and the last short cytoplasmic domain.

Animals↗