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PubMed2004

GOToolBox: functional analysis of gene datasets based on Gene Ontology.

We have developed methods and tools based on the Gene Ontology (GO) resource allowing the identification of statistically over- or under-represented terms in a gene dataset; the clustering of functionally related genes within a set; and the retrieval of genes sharing annotations with a query gene. GO annotations can also be constrained to a slim hierarchy or a given level of the ontology. The source codes are available upon request, and distributed under the GPL license.

Animals↗
PubMed1988

Functional analysis of the interaction of a tissue-specific factor with an upstream enhancer element of the rat prolactin gene.

A nuclear factor which binds to an upstream element of the rat PRL gene has been identified and the functional properties of the factor-DNA interaction have been assessed by mutagenesis of the factor binding sites. Gel mobility shift assays have been used to identify a factor which binds to a fragment from the -1712 to -1494 region of the rat PRL gene. The DNA binding factor is present in nuclear extracts from PRL-producing GH3 cells, but not in nuclear extracts from several other cell lines. Although previous studies have shown that the estrogen receptor binds to this region of DNA, chromatography on heparin-agarose demonstrated that the factor detected by mobility shift assay is probably not the estrogen receptor. Nuclease protection experiments demonstrate that the factor binds to a discrete region at positions -1666 to -1652. The protected region includes half of a palindrome, TCATTAT ... ATAATGA. Mutagenesis by T to G transversions of either both halves of this symmetrical sequence, or only the upstream portion shown to interact with the factor substantially reduced factor binding as assessed by gel mobility shift assay. Transfer of fusion genes containing this region of DNA into GH3 cells demonstrated that the -1712 to -1494 region has a basal enhancer activity which is reduced severalfold by T to G mutagenesis of the complete dyad symmetry at positions -1665 to -1644. The results suggest that the -1712 to -1494 region of the rat PRL gene contains two relatively independent elements. One element, located at positions -1582 to -1569, interacts with the estrogen receptor and mediates estrogenic stimulation of transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Phenotypic and functional analysis of oral mucosal macrophages.

Oral mucosal macrophages were prepared in single cell suspensions from the oral mucosa of BALB/c mice and isolated by adherence to glass. Adherent cells were identified on the basis of morphological, cytochemical, phenotypical, and functional characteristics and compared with peritoneal adherent cells. Adherent cells were found to be esterase positive and could perform non immune phagocytosis. From immunofluorescence studies it was found that about 30% of oral adherent cells bear macrophage differentiation (Mac 1) and Ia antigens, whereas the F4/80 marker could not be detected. The presence of Ig receptors was assessed by the binding of IgA and IgG-coated fluorescent microspheres; distinct populations of mucosal macrophages were shown to display IgA and IgG receptors (25% each) with a significant percentage of double-bearers (15%). The effector functions of these Ig receptors was verified by opsono-phagocytosis of live lactobacilli. Murine oral mucosal tissue appears to host a heterogeneous population of resident macrophages including IgA-dependent phagocytic cells which would be particularly well suited for immune defense functions at secretory sites.

Animals↗
PubMed2004

Functional analysis of genetic variation in catechol-O-methyltransferase (COMT): effects on mRNA, protein, and enzyme activity in postmortem human brain.

Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3' flanking region (rs165599)--both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val--had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3' SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a predominant factor that determines higher COMT activity in the prefrontal cortex, which presumably leads to lower synaptic dopamine levels and relatively deleterious prefrontal function.

Analysis of Variance↗
PubMed2003

Cloning and function analysis of full-length cDNA sequence of Schistosoma japonicum eukaryotic translation initiation factor 2 alpha subunit.

OBJECTIVE: To subclone the novel gene screened from the Schistosoma japonicum cercariae cDNA library through expressed sequence tag (EST) strategy and analyze its functions. METHOD: The cDNA fragment inserted in pTriplEx2 vector was sequenced and the result retrieved with BLASTn program. It was found that this cDNA was highly homologous to Schistosoma mansoni eukaryotic translation initiation factor 2 alpha subunit (eIF2 alpha) mRNA. According to the known EST sequence, the 3'-terminal primer that matched the sequencing primer for the 5'-terminal in pTriplEx2 plasmid was designed and used to amplify the full-length open reading frame (ORF) sequence of the eIF2 alpha from the cDNA Library. After proper purification, the PCR product was linked to pGEM-T vector and the recombinant T-vector was sequenced to obtain the full length ORF, which was retrieved for homologue identification using NCBI blast program. The sequences that were highly homologous underwent comparison at the levels of amino acids and nucleotides using BLAST 2 Sequence program on NCBI BLAST site. The motif and conserved domain were also retrieved with the software available online. RESULT: A novel cDNA sequence coding for a eIF2 alpha was found from the cDNA library of Schistosoma japonicum cercariae, which was highly homologous to the known Schistosoma mansoni eIF2 alpha mRNA, with the homology of 87% at the nucleotide level and 79% at the amino acid level. CONCLUSION: The novel gene found by EST strategy may encode a eIF2 alpha which is highly homologous to Schistosoma mansoni eukaryotic eIF2 alpha mRNA.

Amino Acid Sequence↗
PubMed2006

Phenotypical and functional analysis of memory and effector human CD8 T cells specific for mycobacterial antigens.

Mycobacterium tuberculosis infects one-third of the global population and claims two million lives every year. Because memory CD8 T cells exhibit a high heterogeneity in terms of phenotype and functional characteristic, we investigated the frequency, phenotype, and functional properties of Ag85A epitope-specific HLA-A*0201 CD8 T cells in children affected by tuberculosis (TB) before and 4 mo after chemotherapy and healthy contact children. Using Ag85A peptide/HLA-A*0201 pentamer, we found a low frequency of blood peptide-specific CD8 T cells in tuberculous children before therapy, which consistently increased after therapy to levels detected in healthy contacts. Ex vivo analysis of the expression of CD45RA and CCR7 surface markers indicated a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of T central memory cells and a decrease of terminally differentiated T cells, which was reversed after therapy. Accordingly, pentamer-specific CD8 T cells from tuberculous patients produced low levels of IFN-gamma and had low expression of perforin, which recovered after therapy. The finding of an elevated frequency of pentamer-specific CD8 T cells with T effector memory and terminally differentiated phenotypes in the cerebrospinal fluid of a child with tuberculous meningitis strongly indicates compartmentalization of such CD8 effectors at the site of disease. Our study represents the first characterization of Ag-specific memory and effector CD8 T cells during TB and may help to understand the type of immune response that vaccine candidates should stimulate to achieve protection.

Antigens, Bacterial↗
PubMed1999

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site.

The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the attB site of the host during lysogenization of Lactobaccillus delbrueckii subsp. bulgaricus. The mv4 integrase also functions in a wide variety of gram-positive bacteria and in Escherichia coli. In this report, in vitro and in vivo recombination assays were developed and the integrase was purified in order to study in greater detail the mv4 attP x attB recombination event. In a cell-free extract of E. coli at 42 degrees C, the mv4 integrase promotes efficient in vitro recombination between a supercoiled attP-containing plasmid and a linear attB fragment. The integrase, which was purified to apparent homogeneity, showed no absolute requirement for accessory factors, unlike the majority of the lambda Int family of recombinases. Deletion derivatives of the attP site were constructed and tested for recombination with the attB site in vitro. A 234-bp DNA fragment containing five scattered putative mv4 Int-binding sites was sufficient for function of the attP site. In contrast to the right arm of attP, most of the left arm could be deleted without drastically reducing the recombination efficiency. In vivo in E. coli, mv4 Int catalyzed recombination in trains between attP and attB sites present on two separate plasmids. This property was used to confirm in vivo the results of the deletion analysis of the attP site performed in vitro.

Bacteriophages↗
PubMed2001

Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8.

The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8(S87L) partially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8(R107P) is defective in interactions with both VirB9 and VirB10. A second mutant VirB8(S87L) is defective in interaction with VirB9.

Agrobacterium tumefaciens↗
PubMed2001

Identification and functional analysis of an interaction between domains of the 126/183-kDa replicase-associated proteins of tobacco mosaic virus.

The Tobacco mosaic virus (TMV) 126-kDa and read-through 183-kDa replicase-associated proteins have been shown to interact [Watanabe, T., Honda, A., Iwata, A., Ueda, S., Hibi, T., Ishihama, A. (1999). J. Virol. 73, 2633-2640]. To identify and investigate the sequence required for this interaction, five segments covering different portions of the 126/183-kDa open reading frame, including the methyl-transferase, intervening region (IR), helicase-like (HEL), and polymerase domains, were screened via the yeast two-hybrid system against a library of TMV protein segments. Only one specific interaction between the HEL domain clone and a TMV library clone, IRnHEL, encoding the C-terminal half of the IR and the N-terminal portion of the HEL domain was identified. Sequence and deletion analysis revealed that the interacting clones share a region containing the helicase NTP-binding motif and that this region was essential for the interaction. To determine the functional significance of this interaction, mutants of the HEL domain segment that conferred a temperature-sensitive (ts) defect in the yeast interaction were identified and cloned into a recombinant TMV strain. Of the five selected mutants, three (V823I/S824N/V1042M, A877V, V1087I) produced a ts replication phenotype in protoplasts while the other two (A1073V, T884I) abolished TMV replication at both the permissive and the nonpermissive temperatures. An additional mutation, K839S, designed to disrupt the shared NTP-binding motif, nearly abolished the two-hybrid interaction and prevented virus replication, suggesting that NTP-binding and/or the structure of this motif is a contributing factor in the interaction. Taken together, these results provide support for an interaction between TMV replicase-associated proteins that involves specific structural features of the HEL and IR domains.

Amino Acid Motifs↗
PubMed1994

Functional analysis of the zinc cluster domain of the CYP1 (HAP1) complex regulator in heme-sufficient and heme-deficient yeast cells.

CYP1 determines the expression of several genes whose transcription is heme-dependent in yeast. It exerts regulatory functions even in the absence of heme, usually considered to be its effector. It mediates both positive and negative effects, depending on the target gene and on the redox state of the cell. In the presence of heme, it binds through a cysteine-rich domain in which a histidine residue occupies the position of the sixth and essential cysteine of the otherwise classical zinc cluster DNA-binding domain exemplified by GAL4. We constructed specific missense mutations in the potential CYP1 zinc cluster domain by site-directed mutagenesis and looked for regulatory effects of the mutated proteins under specific physiological conditions. We show that CYP1 does belong to the zinc cluster regulatory family since a sixth essential cysteine residue is indeed present, albeit at a modified position when compared to the consensus sequence. We also show that the amino acid preceding the first cysteine residue of the DNA-binding domain critically affects the efficiency of regulation both in the presence and in the absence of heme: mutations known to affect DNA binding under heme-sufficient conditions also affect regulation under heme-deficient conditions. We therefore surmise that regulation under heme-deficient conditions is dependent upon DNA binding.

Amino Acid Sequence↗
PubMed2006

Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae.

In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO4, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use.

Animals↗
PubMed2001

Functional analysis of TamA, a coactivator of nitrogen-regulated gene expression in Aspergillus nidulans.

The tam A gene of Aspergillus nidulans encodes a 739-amino acid protein with similarity to Uga35p/Dal81p/DurLp of Saccharomyces cerevisiae. It has been proposed that TamA functions as a co-activator of AreA, the major nitrogen regulatory protein in A. nidulans. Because AreA functions as a transcriptional activator under nitrogen-limiting conditions, we investigated whether TamA was also present in the nucleus. We found that a GFP-TamA fusion protein was predominantly localised to the nucleus in the presence and absence of ammonium, and that AreA was not required for this distribution. As the predicted DNA-binding domain of TamA is not essential for function, we have used a number of approaches to further define functionally important regions. We have cloned the tamA gene of A. oryzae and compared its functional and sequence characteristics with those of A. nidulans tamA and S. cerevisiae UGA35/DAL81/DURL. The Aspergillus homologues are highly conserved and functionally interchangeable, whereas the S. cerevisiae gene does not complement a tamA mutant when expressed in A. nidulans. Uga35p/Dal81p/DurLp was also found to be unable to recruit AreA. The sequence changes in a number of tamA mutant alleles were determined, and altered versions of TamA were tested for tamA complementation and interaction with AreA. Changes in most regions of TamA appeared to destroy its function, suggesting that the overall conformation of the protein may be critical for its activity.

Alleles↗
PubMed2004

BODIL: a molecular modeling environment for structure-function analysis and drug design.

BODIL is a molecular modeling environment geared to help the user to quickly identify key features of proteins critical to molecular recognition, especially (1) in drug discovery applications, and (2) to understand the structural basis for function. The program incorporates state-of-the-art graphics, sequence and structural alignment methods, among other capabilities needed in modern structure-function-drug target research. BODIL has a flexible design that allows on-the-fly incorporation of new modules, has intelligent memory management, and fast multi-view graphics. A beta version of BODIL and an accompanying tutorial are available at http://www.abo.fi/fak/mnf/bkf/research/johnson/bodil.html.

Algorithms↗
PubMed2006

Magnetic resonance imaging of arrhythmogenic right ventricular dysplasia: sensitivity, specificity, and observer variability of fat detection versus functional analysis of the right ventricle.

OBJECTIVES: The purpose of this study was to determine interobserver agreement for interpretation of magnetic resonance imaging (MRI) examinations of arrhythmogenic right ventricular dysplasia (ARVD) and to determine sensitivity and specificity of fat detection versus functional parameters measured by MRI. BACKGROUND: The interobserver variability of MRI and the relative importance of different MRI parameters (fat detection, regional and global right ventricular [RV] function) for ARVD diagnosis is unknown. METHODS: Two experienced observers blinded to the clinical history independently analyzed MRI datasets obtained from 40 patients evaluated for ARVD. Twenty normal subjects underwent MRI and served as control subjects. The MRI scans were performed according to a standard protocol on a 1.5-T scanner. The observers reported on fat infiltration, global and regional RV function, myocardial thinning, and chamber dilatation qualitatively. The RV volumes were measured on the cine sequences. RESULTS: Interobserver kappa scores for fat infiltration, global and regional RV function, wall thinning, and RV outflow dilatation were 0.74, 0.94, 0.89, 0.93, and 0.93, respectively. Correlation coefficients between observers for RV end-diastolic volume, end-systolic volume, and ejection fraction were 0.93, 0.94, and 0.95, respectively (p < 0.001). Fifteen patients were diagnosed with ARVD using Task Force criteria. Sensitivity of fat infiltration, RV enlargement, and regional RV dysfunction for diagnosing ARVD was 84%, 68%, and 78%, and specificity was 79%, 96%, and 94%, respectively. CONCLUSIONS: Qualitative assessment of RV structure and function is highly reproducible for experienced observers. Among the qualitative parameters, fat infiltration is less reproducible and lacks specificity compared with RV kinetic abnormalities.

Adipose Tissue↗
PubMed2003

Cloning and functional analysis of ascidian Mitf in vivo: insights into the origin of vertebrate pigment cells.

The microphthalmia-associated transcription factor (Mitf) is a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) transcription factor essential for the development and function of all melanin-producing pigment cells in vertebrates. To elucidate the evolutionary history of Mitf and the antiquity of its association with pigment cells, we have isolated and characterized HrMitf, a sole member of the Mitf-TFE bHLH-ZIP subfamily in the ascidian Halocynthia roretzi. Maternal HrMitf mRNA is detected in the fertilized egg and in the animal hemisphere from 4-cell stage through the gastrula stage. From the neurula through the early tailbud stage, HrMitf is preferentially expressed in the pigment-lineage cells that express the lineage-specific melanogenesis genes tyrosinase (HrTyr) and Tyrp. Overexpression of HrMitf induced ectopic expression of HrTyr enzyme activity in mesenchymal cells where the same enzyme activity was induced by overexpression of HrPax3/7, suggesting that a part(s) of the Pax3-Mitf-tyrosinase gene regulatory cascade seen in vertebrate melanocytes is operative during ascidian embryogenesis. We also show HrMitf and mouse Mitf-A, a Mitf isoform abundantly expressed in pigmented epithelial cells, share similar functional characteristics. These results suggest antiquity of the association of the Mitf-TFE subfamily with pigment cells and may support the idea that acquisition of multiple promoters (isoforms) by an ancestral Mitf gene has allowed the evolution of multiple pigment cell types.

Amino Acid Sequence↗
PubMed2003

Comparative functional analysis of the Rac GTPases.

Small GTPases of the Rho family including Rac, Rho and Cdc42 regulate different cellular processes like reorganization of the actin cytoskeleton by acting as molecular switches. The three distinct mammalian Rac proteins share very high sequence identity but how their specificity is achieved is hitherto unknown. Here we show that Rac1 and Rac3 are very closely related concerning their biochemical properties, such as effector interaction, nucleotide binding and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis support the idea that altered dynamics of Rac2 at the switch I region may be responsible for different biochemical properties. These results provide insight into the individual functionalities of the Rac isoforms.

Amino Acid Motifs↗