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At least 433 records · Page 24Linked to original sources
PubMed2003

Structural, genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori.

Mass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are post-translationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno -n o n-ulosonic acid). Unlike Campylobacter, flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution, although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter. Bioinformatic analysis revealed five genes (HP0840, HP0178, HP0326A, HP0326B, HP0114) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologues in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non-motile phenotype, no structural flagella filament and only minor amounts of flagellin protein detectable by Western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using capillary electrophoresis-electrospray mass spectrometry (CE-ESMS) and precursor ion scanning. HP0326A, HP0326B and the HP0178 gene products are directly involved in the biosynthesis of the nucleotide-activated form of Pse, CMP-Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono- and a diacetamido trideoxyhexose UDP sugar nucleotide precursor.

Amino Acid Sequence↗
PubMed2001

Expression of pathogen-like Opa adhesins in commensal Neisseria: genetic and functional analysis.

Several species of commensal Neisseriae (Cn) may colonize the human nasopharynx, but little is known about their adhesion mechanisms. We have investigated structural and functional similarities between adhesins of Cn and of Neisseria meningitidis (Nm), also a frequent colonizer of the nasopharynx. In this study, we demonstrate the expression of Opa-like proteins in nine strains of Cn. Phylogenetic analysis segregated the majority of the Cn Opa in a cluster separated from the pathogenic cluster with a few exceptions. One Opa, which located within the pathogenic cluster, was strikingly similar (74%) to an Opa of a Neisseria gonorrhoeae (Ng) strain and, like Ng, it lacked the extra Y11 or the 136DKF138 triplet insert, which are conserved among many N. meningitidis Opa proteins. Most importantly, the majority of the Cn Opa proteins were able to interact with human CEACAM1 (CD66a) molecules, previously identified as receptors for pathogenic Opa proteins. By the use of CEACAM1 N-domain mutants, we demonstrate that Cn Opa target the same region of the N-domain of the receptor as that used by Nm. Furthermore, Cn strains bound to cell-expressed human CEACAM1. In competition assays, adherent Cn strain C450, exhibiting high affinity for CEACAM1, was not displaced by a Nm isolate and vice versa. But in simultaneous incubation, Nm out-competed the Cn strain. This is the first study to demonstrate the expression of adhesins in Cn that are structurally and functionally closely related to pathogenic adhesins. The studies imply that some Cn have the potential to occupy and thus compete with the pathogens for receptors on human mucosa, their common and exclusive niche.

Adhesins, Bacterial↗
PubMed1996

Functional and genetic characterization of the (methyl)ammonium uptake carrier of Corynebacterium glutamicum.

Under nitrogen starvation conditions, Corynebacterium glutamicum was found to take up methylammonium at a rate of 20 +/- 5 nmol.min-1.(mg dry weight)-1. The specific activity of this uptake was 10-fold lower when growing the cells under sufficient nitrogen supply, indicating a tight regulation on the expression level. The methylammonium uptake showed Michaelis-Menten kinetics with an Km of 44 +/- 7 microM and was completely inhibited by the addition of 10 microM ammonium. This finding and the fact that methylammonium was not metabolized by C. glutamicum strongly suggests that the uptake carrier actually represents an ammonium uptake system. Methylammonium uptake was strictly dependent on the membrane potential. From the pH optimum and the accumulation of methylammonium in equilibrium, it could be deduced that only one net charge is transported and, thus, that methylammonium is taken up in its protonated form via an uniport mechanism. The amt gene encoding the (methyl)ammonium uptake system was isolated and characterized. The predicted gene product of amt consists of 452 amino acids (Mr = 47,699) and shows 26-33% identity to ammonium transporter proteins from Saccharomyces cerevisiae and Arabidopsis thaliana. According to the hydrophobicity profile, it is an integral membrane protein containing 10 or 11 membrane-spanning segments.

Amino Acid Sequence↗
PubMed1998

Aquaporins in Saccharomyces. Genetic and functional distinctions between laboratory and wild-type strains.

Aquaporin water channel proteins mediate the transport of water across cell membranes in numerous species. The Saccharomyces genome data base contains an open reading frame (here designated AQY1) that encodes a protein with strong homology to aquaporins. AQY1 from laboratory and wild-type strains of Saccharomyces were expressed in Xenopus oocytes to determine the coefficients of osmotic water permeability (Pf). Oocytes injected with wild-type AQY1 cRNAs exhibit high Pf values, whereas oocytes injected with AQY1 cRNAs from laboratory strains exhibit low Pf values and have reduced levels of Aqy1p due to two amino acid substitutions. When the AQY1 gene was deleted from a wild-type yeast and cells were cultured in vitro with cycled hypo-osmolar or hyper-osmolar stresses, the AQY1 null yeast showed significantly improved viability when compared with the parental wild-type strain. We conclude that Saccharomyces cerevisiae contains at least one aquaporin gene, but it is not functional in laboratory strains due to apparent negative selection pressures resulting from in vitro methods.

Amino Acid Sequence↗
PubMed2002

Genetic and functional analysis of single nucleotide polymorphisms in the beta2-neuronal nicotinic acetylcholine receptor gene (CHRNB2).

The beta2-neuronal nicotinic acetylcholine receptor gene (CHRNB2) is a logical candidate for influencing smoking behavior and nicotine dependence. We discovered six single nucleotide polymorphisms (SNPs) in the CHRNB2 gene by surveying 15.4 kb of genomic sequence including a previously undescribed 3' untranslated region that extends 4.0 kb downstream of the coding region. One of the SNPs causes an amino acid substitution in exon 5, one occurs in the promoter region, one changes an intronic base, and three occur in the 3' untranslated region. The ethnically dependent allele frequencies and the marker-to-marker linkage disequilibrium patterns of five of these polymorphisms were determined. The SNPs were assayed in 743 individuals for whom information on smoking history and lifelong nicotine dependence was available. No significant associations of the individual markers or their haplotypes to smoking behavior or level of nicotine dependence were found.

DNA Primers↗
PubMed1992

Genetic and functional analysis of a set of HIV-1 envelope genes obtained from biological clones with varying syncytium-inducing capacities.

To study HIV-1 envelope-mediated syncytium formation we have amplified, cloned, expressed, and sequenced individual envelope genes from a set of eight biological HIV-1 clones. These clones were obtained from two patients and display either a syncytium-inducing (SI) or nonsyncytium-inducing (NSI) phenotype. Upon expression through recombinant vaccinia virus, individual envelope gene products display heterogeneous syncytium-inducing capacities which reflect the phenotype of the parental biological HIV-1 clones in all cases. For the eight biological HIV-1 clones presented here, variation of the envelope gene alone is sufficient to explain the observed variable syncytium-inducing capacity of the respective parental viruses. In addition we determined the complete nucleotide sequence of these envelope genes. The predicted amino acid sequence revealed a considerable amount of variation located mainly in the previously denominated variable regions. In various regions of envelope genes obtained from the same patient, phenotype associated amino acid variation was found. This phenotype associated amino acid variation however, is not conserved between the two sets of envelope genes derived from different patients. Four envelope sequences derived from clones obtained from one patient showed phenotype-associated amino acid variation in the fusion domain. Sequencing of 12 additional fusion domains revealed that this same variation is found in four additional clones. However, a functional test performed on recombinant vaccinia expressing mutant envelope genes showed that this observed fusion domain variation does not contribute to the variation in syncytium-inducing capacity of the envelope gene product.

Amino Acid Sequence↗
PubMed1991

A mouse monoclonal antibody specific for an allotypic determinant of the Igha allele of murine IgM: genetic and functional analysis of Igh-6a epitopes using anti-IgM monoclonal antibodies.

An IgG1 mouse monoclonal antibody (MAb) specific for a mouse IgM allotypic determinant in the a, c, f, g, h, and j haplotypes was derived from a fusion of SP2/O-Ag14 mouse myeloma cells with C57BL/6 mouse spleen cells (Igh-Cb) immune to TC31, a MAb of the IgMa allotype. MAb from one hybridoma derived from this fusion (designated DS1) was demonstrated to bind in an ELISA to immunoglobulin bearing the IgMa allotype (TC31, MOPC104E), but not to immunoglobulin bearing the IgMb allotype (C.BPC112). Fluorescein-conjugated DS1 was shown to bind to the surface of BALB/cByJ splenic B cells, but was shown to have negligible binding on C57BL/6J cells. Similarly, DS1-conjugated Sepharose beads were able to stimulate in vitro proliferation of BALB/c, but not C57BL/6 splenic B cells. DS1 was unable to bind to spleen cells from BALB/c allotype congenic strains, BAB/14 (Igh-Cb) and C.AL-20 (Igh-Co), demonstrating that DS1 recognizes a determinant under the control of a gene linked to the Igh-C gene complex. Using sera from recombinant inbred lines, the determinant defined by DS1 was shown to be linked to the Igh-1 locus. Furthermore, the determinant was localized to the CH1 domain of the mu heavy chain. Sera from BALB/cByJ, NMRI, CBA/J, SEA/GnJ, RIIIs/J, and CE/J mouse strains were shown to bind to DS1 in an ELISA, while sera from A/J, SJL/J, NZB/B1NJ, AKR/J, C57BL/6J, and C57BL/10SnJ mouse strains did not bind to DS1. From these data we propose that DS1 is reactive with specificity Igh-6.1, which was originally defined by an allotypic antiserum developed by Black et al. (Immunogenetics 7:213, 1978).

Alleles↗
PubMedDate not supplied

Structure, function, and genetics of streptococcal M protein.

Streptococcal M protein is a coiled-coil fibrillar structure extending about 60 nm from the cell wall. From DNA sequencing of the M6 protein gene, it has been determined that the C-terminal end contains a membrane anchor and an adjacent cell wall stabilization domain, both of which are similar to C-terminal regions of surface proteins in other gram-positive organisms. Studies with monoclonal antibodies revealed that the C-terminal half of the protein is conserved among M proteins of different serotypes, whereas the N-terminal half varies. The M protein contains tandem repeats, which, through homologous recombination, are responsible for the observed size variation of the M proteins from different streptococcal strains. M protein size mutants occur in a laboratory-grown culture at a frequency of 1/2,000 colony-forming units. DNA sequence analysis of the M gene in size mutants derived from a single strain suggests that sequence changes which result from homologous recombination may play a role in the antigenic variation of M protein.

Antigenic Variation↗
PubMed1993

Chiasma interference as a function of genetic distance.

For many organisms, meiotic double crossing over is less frequent than expected on the assumption that exchanges occur at random with respect to each other. This "interference," which can be almost total for nearby intervals, diminishes as the intervals in which the double crossovers are scored are moved farther apart. Most models for interference have assumed, at least implicitly, that the intensity of interference depends inversely on the physical distance separating the intervals. However, several observations suggest that interference depends on genetic distance (Morgans) rather than physical distance (base pairs or micrometers). Accordingly, we devise a model in which interference is related directly to genetic distance. Its central feature is that recombinational intermediates (C's) have two fates--they can be resolved with crossing over (Cx) or without (Co). We suppose that C's are distributed at random with respect to each other (no interference); interference results from constraints on the resolution of C's. The basic constraint is that each pair of neighboring Cx's must have between them a certain number of Co's. The required number of intervening Co's for a given organism or chromosome is estimated from the fraction of gene conversions that are unaccompanied by crossover of flanking markers. The predictions of the model are compared with data from Drosophila and Neurospora.

Animals↗
PubMed2005

Exploring the role of galectin 3 in kidney function: a genetic approach.

Galectin 3 belongs to a family of glycoconjugate-binding proteins that participate in cellular homeostasis by modulating cell growth, adhesion, and signaling. We studied adult galectin 3 null mutant (Gal 3-/-) and wild-type (WT) mice to gain insights into the role of galectin 3 in the kidney. By immunofluorescence, galectin 3 was found in collecting duct (CD) principal and intercalated cells in some regions of the kidney, as well as in the thick ascending limbs at lower levels. Compared to WT mice, Gal 3-/- mice had approximately 11% fewer glomeruli (p < 0.04), associated with kidney hypertrophy (p < 0.006). In clearance experiments, urinary chloride excretion was found to be higher in Gal 3-/- than in WT mice (p < 0.04), but there was no difference in urinary bicarbonate excretion, in glomerular filtration, or urinary flow rates. Under chronic low sodium diet, Gal 3-/- mice had lower extracellular fluid (ECF) volume than WT mice (p < 0.05). Plasma aldosterone concentration was higher in Gal 3-/- than in WT mice (p < 0.04), which probably caused the observed increase in alpha-epithelial sodium channel (alpha-ENaC) protein abundance in the mutant mice (p < 0.001). Chronic high sodium diet resulted paradoxically in lower blood pressure (p < 0.01) in Gal 3-/- than in WT. We conclude that Gal 3-/- mice have mild renal chloride loss, which causes chronic ECF volume contraction and reduced blood pressure levels.

Animals↗
PubMed2005

Comparative evolutionary analysis of VPS33 homologues: genetic and functional insights.

VPS33B protein is a homologue of the yeast class C vacuolar protein sorting protein Vps33p that is involved in the biogenesis and function of vacuoles. Vps33p homologues contain a Sec1 domain and belong to the family of Sec1/Munc18 (SM) proteins that regulate fusion of membrane-bound organelles and interact with other vps proteins and also SNARE proteins that execute membrane fusion in all cells. We demonstrated recently that mutations in VPS33B cause ARC syndrome (MIM 208085), a lethal multisystem disease. In contrast, mutations in other Vps33p homologues result in different phenotypes, e.g. a mutation in Drosophila melanogaster car gene causes the carnation eye colour mutant and inactivation of mouse Vps33a causes buff hypopigmentation phenotype. In mammals two Vps33p homologues (e.g. VPS33A and VPS33B in humans) have been identified. As comparative genome analysis can provide novel insights into gene evolution and function, we performed nucleotide and protein sequence comparisons of Vps33 homologues in different species to define their inter-relationships and evolution. In silico analysis (a) identified two homologues of yeast Vps33p in the worm, fly, zebrafish, rodent and human genomes, (b) suggested that Carnation is an orthologue of VPS33A rather than VPS33B and (c) identified conserved candidate functional domains within VPS33B. We have shown previously that wild-type VPS33B induced perinuclear clustering of late endosomes and lysosomes in human renal cells. Consistent with the predictions of comparative analysis: (a) VPS33B induced significantly more clustering than VPS33A in a renal cell line, (b) a putative fly VPS33B homologue but not Carnation protein also induced clustering and (c) the ability to induce clustering in renal cells was linked to two evolutionary conserved domains within VPS33B. One domain was present in VPS33B but not VPS33A homologues and the other was one of three regions predicted to form a t-SNARE binding site in VPS33B. In contrast, VPS33A induced significantly more clustering of melanosomes in melanoma cells than VPS33B. These investigations are consistent with the hypothesis that there are two functional classes of Vps33p homologues in all multicellular organisms and that the two classes reflect the evolution of organelle/tissue-specific functions.

Amino Acid Sequence↗
PubMed1997

Functional and genetic studies of the angiotensin II type 1 receptor in pre-eclamptic and normotensive pregnant women.

OBJECTIVE: To examine and compare angiotensin II type 1 receptor genotype and its relationship to platelet angiotensin II binding for pre-eclamptic and normotensive pregnant women. DESIGN: In a case-control study, 43 pre-eclamptic women and 83 normotensive women were genotyped at the angiotensin II type 1 receptor gene locus. Platelet angiotensin II binding was measured for a subset of 11 pre-eclamptic and 57 normotensive pregnant women. We genotyped 162 healthy blood donors also, to examine the allelic background and patterns of linkage disequilibrium in the Nottingham population. METHODS: Patients were recruited during pregnancy using a rigorous definition of pre-eclampsia. DNA was extracted from peripheral venous blood and genotyped at six previously described diallelic polymorphisms in the angiotensin II type 1 receptor gene, using competitive allele-specific oligonucleotide hybridization, and at a dinucleotide repeat polymorphism in the 3' flanking region of the gene. Platelet angiotensin II binding and plasma angiotensin II concentrations were determined for peripheral venous blood. RESULTS: Normotensive pregnant women homozygous for cytosine at nucleotide 573 had significantly higher levels of platelet angiotensin II binding than did heterozygous women and women homozygous for thymidine at this site. Pre-eclamptic women had significantly higher levels of platelet angiotensin II binding than did normotensive pregnant women. The frequencies of allelic variants did not differ significantly between normotensive and pre-eclamptic women. CONCLUSION: The physiological regulation of platelet angiotensin II type 1 receptor expression in normal pregnancy is determined in part by angiotensin II type 1 receptor genotype. There was no evidence that the polymorphisms in the angiotensin II type 1 receptor gene were associated with pre-eclampsia.

Adult↗
PubMed1996

Primate brain evolution: genetic and functional considerations.

Functionally distinct regions of the brain to which maternal and paternal genomes contribute differentially (through genomic imprinting) have developed differentially over phylogenetic time. While certain regions of the primate forebrain (neocortex, striatum) have expanded relative to the rest of the brain, other forebrain regions have contracted in size (hypothalamus, septum). Areas of relative expansion are those to which the maternal genome makes a substantial developmental contribution. This may be significant with respect to the importance of primate forebrain expansion in the development of complex behavioural strategies and the way in which these are deployed, especially by the matriline. In many primate societies the maintenance of social cohesion and group continuity over successive generations is dependent on the matriline, with high ranking females producing high ranking daughters that stay within the group. Regions of relative contraction are those to which the paternal genome makes a differential contribution and these are target areas for gonadal hormones, which is congruent with the diminished role for gonadal hormones in the emancipation of primate reproductive behaviour.

Animals↗