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At least 361 records · Page 20Linked to original sources
PubMed2005

Genetic and functional interaction of evolutionarily conserved regions of the Prp18 protein and the U5 snRNA.

Both the Prp18 protein and the U5 snRNA function in the second step of pre-mRNA splicing. We identified suppressors of mutant prp18 alleles in the gene for the U5 snRNA (SNR7). The suppressors' U5 snRNAs have either a U4-to-A or an A8-to-C mutation in the evolutionarily invariant loop 1 of U5. Suppression is specific for prp18 alleles that encode proteins with mutations in a highly conserved region of Prp18 which forms an unstructured loop in crystals of Prp18. The snr7 suppressors partly restored the pre-mRNA splicing activity that was lost in the prp18 mutants. The close functional relationship of Prp18 and U5 is emphasized by the finding that two snr7 alleles, U5A and U6A, are dominant synthetic lethal with prp18 alleles. Our results support the idea that Prp18 and the U5 snRNA act in concert during the second step of pre-mRNA splicing and suggest a model in which the conserved loop of Prp18 acts to stabilize the interaction of loop 1 of the U5 snRNA with the splicing intermediates.

Alleles↗
PubMed2005

Genetic and functional analyses of the Actinobacillus actinomycetemcomitans AfeABCD siderophore-independent iron acquisition system.

The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions.

ATP-Binding Cassette Transporters↗
PubMed2006

Genetic and functional analyses of the lgtH gene, a member of the beta-1,4-galactosyltransferase gene family in the genus Neisseria.

Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Three galactosyltransferase genes, lgtB, lgtE and lgtH, responsible for the biosynthesis of LOS oligosaccharide chains, were analysed in five Neisseria species. The function of lgtH in Neisseria meningitidis 6,275 was determined by mutagenesis and chemical characterization of the parent and mutant LOS chains. The chemical characterization included SDS-PAGE, immunoblot, hexose and mass spectrometry analyses. Compared with the parent LOS, the mutant LOS lacked galactose, and its oligosaccharide decreased by three or four sugar units in matrix-assisted laser desorption ionization (MALDI)-MS analysis. The results show that lgtH encodes a beta-1,4-galactosyltransferase, and that the glucose moiety linked to heptose (Hep) in the alpha chain is the acceptor site in the biosynthesis of Neisseria LOS. To understand the sequence diversity and relationships of lgtB, lgtE and lgtH, the entire lgt-1 locus was further sequenced in three N. meningitidis strains and three commensal Neisseria strains, and compared with the previously reported lgt genes from Neisseria species. Comparison of the protein sequences of the three enzymes LgtB, LgtE and LgtH showed a conserved N-terminal region, and a highly variable C-terminal region, suggesting functional constraint for substrate and acceptor specificity, respectively. The analyses of allelic variation and evolution of 23 lgtB, 12 lgtE and 14 lgtH sequences revealed a distinct evolutionary history of these genes in Neisseria. For example, the splits graph of lgtE displayed a network evolution, indicating frequent DNA recombination, whereas splits graphs of lgtB and lgtH displayed star-tree-like evolution, indicating the accumulation of point mutations. The data presented here represent examples of the evolution and variation of prokaryotic glycosyltransferase gene families. These imply the existence of multiple enzyme isoforms for biosynthesis of a great diversity of oligosaccharides in nature.

Alleles↗
PubMed2002

Genetic and functional analysis of the von Hippel-Lindau (VHL) tumour suppressor gene promoter.

The VHL gatekeeper tumour suppressor gene is inactivated in the familial cancer syndrome von Hippel-Lindau disease and in most sporadic clear cell renal cell carcinomas. Recently the VHL gene product has been identified as a specific component of a SCF-like complex, which regulates proteolytic degradation of the hypoxia inducible transcription factors HIF-1 and HIF-2. pVHL is critical for normal development and mRNA expression studies suggest a role in nephrogenesis. Despite the importance of VHL in oncogenesis and development, little is known about the regulation of VHL expression. To investigate VHL promoter activity, we performed comparative sequence analysis of human, primate, and rodent 5' VHL sequences. We then proceeded to deletion analysis of regions showing significant evolutionary conservation between human and rat promoter sequences, and defined two positive and one negative regulatory regions. Analysis of specific putative transcription factor binding sites identified a functional Sp1 site, which was shown to be a regulatory element. Overlapping Sp1/AP2 sites were also identified and candidate E2F1 binding sites evaluated. Three binding sites for as yet unidentified transcription factors were mapped also. These investigations provide a basis for elucidating the regulation of VHL expression in development, the molecular pathology of epigenetic silencing of VHL in tumourigenesis, and suggest a possible link between Sp1, VHL, and nephrogenesis.

Animals↗
PubMed2001

Genetic and functional linkage of Kir5.1 and Kir2.1 channel subunits.

We have identified several cDNAs for the human Kir5.1 subunit of inwardly rectifying K(+) channels. Alternative splicing of exon 3 and the usage of two alternative polyadenylation sites contribute to cDNA diversity. The hKir5.1 gene KCNJ16 is assigned to chromosomal region 17q23.1-24.2, and is separated by only 34 kb from the hKir2.1 gene (KCNJ2). In the brain, Kir5.1 mRNA is restricted to the evolutionary older parts of the hindbrain, midbrain and diencephalon and overlaps with Kir2.1 in the superior/inferior colliculus and the pontine region. In the kidney Kir5.1 and Kir2.1 are colocalized in the proximal tubule. When expressed in Xenopus oocytes, Kir5.1 is efficiently targeted to the cell surface and forms electrically silent channels together with Kir2.1, thus negatively controlling Kir2.1 channel activity in native cells.

Alternative Splicing↗
PubMed1987

COOH-terminal propeptides of the major human procollagens. Structural, functional and genetic comparisons.

The sequences of the carboxy-terminal extensions (COOH-propeptides) of at least one chain of all of the major human procollagens have only recently been deduced, and include those of the interstitial (alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III)), basement membrane (alpha 1(IV)) and pericellular (alpha 2(V)) procollagens. Comparisons of DNA and protein sequences, corresponding to these COOH-propeptides domains, established the early divergence of the basement membrane alpha 1(IV) COOH-propeptide from the corresponding sequences of the interstitial and pericellular procollagens. The latter are relatively highly conserved and share 58% primary peptide sequence similarities, whereas sequence similarities relative to alpha 1(IV) are limited. Hydropathy profiles and secondary structure potentials further emphasize the clustering of conserved and variable regions among the interstitial and pericellular COOH-propeptides, and provided further evidence for significant structural differences between these sequences and the alpha 1(IV) COOH-propeptide. The most highly conserved sequences of the alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III) and alpha 2(V) COOH-propeptides include regions surrounding the carbohydrate attachment site, cysteine-containing regions and the COOH-terminal sequences. Cysteinyl, tyrosyl and tryptophanyl residues were found to be highly conserved as were most charged residues. Localization of variable regions, in general, occurs within hydrophilic sequences with high beta-turn potentials that are proximal to intron/exon splice junctions. The most variable sequences are associated with the telopeptides and adjoining NH2-terminal portions of the COOH-propeptides as demonstrated by predictive secondary structure analyses. These results, combined with similar analyses of abnormal alpha 2(I) COOH-propeptide (osteogenesis imperfecta) permitted the identification of subsequences that are likely to be a prerequisite for COOH-propeptide functions, namely procollagen chain recognition and nucleation sites for triple helix formation. These functions are also common to the alpha 1(IV) COOH-propeptide; however, the lack of cleavage of this region and its additional postulated structural role in extracellular matrix interactions likely account for its divergent primary and secondary structure.

Amino Acid Sequence↗
PubMed2005

Toward the evaluation of function in genetic variability: characterizing human SNP frequencies and establishing BAC-transgenic mice carrying the human CYP1A1_CYP1A2 locus.

Interindividual differences in human CYP1A1 and CYP1A2 expression appear to be associated with variability in risk toward various types of environmental toxicity and cancer. These two genes are oriented head-to-head on human chromosome 15; the 23.3-kb spacer region might contain distinct regulatory regions for CYP1A1 and distinct regulatory regions for CYP1A2, or the regulatory regions for the two genes might overlap one another. From 24 unrelated subjects of five major, geographically-isolated subgroups, we resequenced both genes (all exons and all introns) plus some 3' flanking sequences and the entire spacer region (39.6 kb total); 85 SNPs were found, 49 of which were not currently in the National Center for Biotechnology Information (NCBI) database. Of the 57 double-hit SNPs, we carried out SNP-typing in 94 Africans, 96 Asians, and 83 Caucasians and found striking ethnic differences in SNP frequencies and haplotype evolution; the two CYP1A1 SNPs and the one CYP1A2 SNP that are most commonly used in epidemiological studies were shown not to be representative haplotype tag SNPs across these three human subgroups. Four BAC-transgenic mouse lines, carrying the human CYP1A2 and 15,190 bp of 5' flank, expressed only negligible basal or inducible CYP1A2 mRNA. A fifth BAC-transgenic mouse line, carrying both the human CYP1A1 and CYP1A2 genes and ample amounts of 3' flanking sequences, plus all of the spacer region--in the absence of the mouse Cyp1a1 or Cyp1a2 genes--expressed the human CYP1A1 and CYP1A2 mRNA, protein and enzyme activities in liver and nonhepatic tissues very similar to that of the mouse. Comparison of this hCYP1A1_1A2 transgenic line with hCYP1A1_1A2 lines carrying other common human haplotypes will enable us to evaluate function in human CYP1A1_CYP1A2 locus variability, with regard to toxicity and cancer caused by combustion products.

Animals↗
PubMed1985

Influence of restricted food intake on brown adipose tissue function in genetically obese mice (genotype, ob/ob).

Measurements were made of cytochrome c oxidase activity and the GDP-binding capacity of mitochondria in brown adipose tissue of genetically obese mice and wild-type siblings, to estimate the thermogenic capacity of the tissue. The binding capacity was decreased in ad libitum fed obese animals compared with wild-type animals. Limited feeding of obese animals to restrict their body weight caused a large increase in the binding capacity of the tissue, which was greater than that in wild-type animals fed either ad limitum or on a limited diet. The decreased binding capacity of brown adipose tissue mitochondria in obese mice appears to be a consequence of ad libitum feeding and therefore not a cause of the obesity. Limit feeding of obese animals also corrected their characteristic hypothermia at low ambient temperature. The large increase in the thermogenic capacity of brown adipose tissue in obese animals, induced by limited feeding, may account for the vital improvement of their thermoregulation. However, close similarities were found between obesity hypothermia and hypothermia induced in wild-type animals by restraint. It is suggested that changes in posture caused by obesity, resulting in increased loss of body heat, may be important in the development of obesity hypothermia. Obese animals fed less than wild-type grained more weight than wild-type animals, indicating that the high thermogenic capacity of their brown adipose tissue did not function to regulate their calorie intake.

Adipose Tissue, Brown↗
PubMed2004

Genetic and functional diversification of small RNA pathways in plants.

Multicellular eukaryotes produce small RNA molecules (approximately 21-24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.

Arabidopsis↗
PubMed1994

Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis.

The steroid hormone ecdysone initiates metamorphosis in Drosophila melanogaster by activating a cascade of gene activity that includes primary response transcriptional regulators and secondary response structural genes. The Broad-Complex (BR-C) primary response gene is composed of several distinct genetic functions and encodes a family of related transcription factor isoforms. Our objective was to determine whether BR-C isoforms were components of the primary ecdysone response in all tissues and whether tissue-specific isoform expression is associated with tissue-specific metamorphic outcomes. We used specific antibody reagents that recognize and distinguish among the Z1, Z2 and Z3 BR-C protein isoforms to study protein expression patterns during the initial stages of metamorphosis. Western blot analyses demonstrated that BR-C isoforms are induced at the onset of metamorphosis, each with unique kinetics of induction and repression. Whole-mount immunostaining showed that the BR-C proteins accumulate in the nuclei of all larval and imaginal tissues indicating that the BR-C is induced as a primary response in many tissues. Several tissues express different levels and combinations of the BR-C isoforms suggesting that the BR-C is important in determining the tissue-specific outcome of many parallel ecdysone response cascades. For example, prepupal salivary glands (destined for histolysis during metamorphosis) express Z1 isoforms while imaginal discs (destined for cell differentiation and morphogenesis) shift from the synthesis of Z2 isoforms to the synthesis of Z1 isoforms. The prepupal central nervous system (destined for tissue remodeling) expresses all isoforms, with Z3 predominating. Salivary gland chromosome immunostaining indicated that BR-C proteins interact directly with numerous loci in the polytene genome. Finally, western blot analyses showed that distinct BR-C genetic functions can be correlated with single and specific BR-C protein isoforms.

Animals↗
PubMed1999

Genetic and functional characterization of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric tissue.

In this study, we isolated and characterized a chromosomal locus of Helicobacter pylori previously identified by transposon shuttle mutagenesis as being involved in the adhesion of the pathogen to gastric epithelial cells. Two closely homologous genes were identified, designated as alpA and alpB, encoding outer membrane (OM) proteins of 518 amino acids each. They are members of the outer membrane protein supergene family identified in the H. pylori 26695 complete genome sequence. AlpA carries a functional lipoprotein signal sequence. AlpB carries a putative standard N-terminal signal sequence and shows a strong amino-acid sequence identity to AlpA. Transposon insertion mutagenesis, immunoblotting and primer extension studies indicate that both genes are organized in an operon, but no obvious consensus promoter sequence was found upstream of the transcriptional start site. The C-terminal portion of both proteins is predicted to form a porin-like beta-barrel in the outer membrane, consisting of 14 transmembrane amphipathic beta-strands. Adhesion experiments with defined isogenic mutants indicate that both proteins are necessary for specific adherence of H. pylori to human gastric tissue. The pattern of AlpAB-dependent adherence of H. pylori to the gastric epithelial surface shows a clear difference to the BabA2-mediated adherence to Lewis, suggesting that a different receptor is involved.

Algorithms↗
PubMed2001

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster.

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of approximately 40 nitr genes are contiguous in the genome and span approximately 0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

Amino Acid Sequence↗
PubMed1992

Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin.

The 4.2-kb cryptic plasmid pVA380-1 has been used as a vector for the cloning of antibiotic resistance genes directly in streptococci, and in the construction of Escherichia coli/Streptococcus shuttle vectors. The results of subcloning experiments located the basic replicon of pVA380-1 within a 2.5-kb region. The nucleotide base sequence of this region was determined and contained a single complete open reading frame (ORF) encoding a 237-amino-acid peptide with a predicted size of 29 kDa. This peptide and a region of the DNA molecule 5' to the ORF encoding it shared homology with the replication protein and plus origin, respectively, of the Staphylococcus aureus plasmid pUB110. Data from Tn5 mutagenesis and complementation studies indicated that the protein product of the ORF was required for pVA380-1 replication in streptococci. Deletion of a region of the basic replicon distal to the plus origin and ORF produced an unstable derivative, and resulted in the accumulation of single-stranded replicative intermediates, consistent with the loss of a minus origin. All of these results suggest that pVA380-1 replicates by a rolling circle mode, and is most closely related to the pC194 family of single-stranded DNA plasmids.

Amino Acid Sequence↗
PubMed1995

The effect of selection for high-level vector expression on the genetic and functional stability of a single transcript vector derived from a low-leukemogenic murine retrovirus.

Single-gene murine leukemia virus-based retroviral vectors carrying the G418-resistance gene (neo) under transcriptional control of the long terminal repeat were used to study the effect of selection on long-term vector expression in a murine lymphoid cell line, L691. We used two isogenic vectors carrying either a strong or a weak transcriptional enhancer from low-leukemogenic Akv and high-leukemogenic SL3-3 murine leukemia virus, respectively. Effects of G418 selection were studied at the level of vector-transduced cell populations and at the level of single-vector-transduced cell clones obtained without selection for vector expression. Selection for vector expression prior to isolation of cell clones changed the range of vector expression for the two populations of cell clones. Cell clones harboring the Akv enhancer, isolated without selection and then subjected to prolonged growth under selective conditions, exhibited no mutations in the enhancer region or major vector rearrangements although showing increased vector expression in some cases. Our results are discussed in terms of retrovirus-mediated gene transfer strategies employing selection for expression of a selective marker in single-gene or bicistronic vectors with a low- or nonleukemogenic virus-derived backbone.

Animals↗
PubMed1999

Agonist-specific impairment of coronary vascular function in genetically altered, hyperlipidemic mice.

The objectives of the present study were to 1) examine mechanisms involved in endothelium-dependent responses of coronary arteries from normal mice and 2) determine whether vascular responses of coronary arteries are altered in two genetic models of hypercholesterolemia [apolipoprotein E (apoE)-deficient mice (apoE -/-) and combined apoE and low-density lipoprotein receptor (LDLR)-deficient mice (apoE + LDLR -/-)]. Plasma cholesterol levels were higher in both apoE -/- and apoE + LDLR -/- compared with normal mice on normal and high-cholesterol diets (normal chow: normal 110 +/- 5 mg/dl, apoE -/- 680 +/- 40 mg/dl, apoE + LDLR -/- 810 +/- 40 mg/dl; high-cholesterol chow: normal 280 +/- 60 mg/dl, apoE -/- 2,490 +/- 310 mg/dl, apoE + LDLR -/- 3,660 +/- 290 mg/dl). Coronary arteries from normal (C57BL/6J), apoE -/-, and apoE + LDLR -/- mice were isolated and cannulated, and diameters were measured using videomicroscopy. In normal mice, vasodilation in response to ACh and serotonin was markedly reduced by 10 microM Nomega-nitro-L-arginine (an inhibitor of nitric oxide synthase) or 20 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; an inhibitor of soluble guanylate cyclase). Vasodilation to nitroprusside, but not papaverine, was also inhibited by ODQ. Dilation of arteries from apoE -/- and apoE + LDLR -/- mice on normal diet in response to ACh was similar to that observed in normal mice. In contrast, dilation of arteries in response to serotonin from apoE -/- and apoE + LDLR -/- mice was impaired compared with normal. In arteries from both apoE -/- and apoE + LDLR -/- mice on high-cholesterol diet, dilation to ACh was decreased. In apoE + LDLR -/- mice on high-cholesterol diet, dilation of coronary arteries to nitroprusside was increased. These findings suggest that dilation of coronary arteries from normal mice in response to ACh and serotonin is dependent on production of nitric oxide and activation of soluble guanylate cyclase. Hypercholesterolemia selectively impairs dilator responses of mouse coronary arteries to serotonin. In the absence of both apoE and the LDL receptor, high levels of cholesterol result in a greater impairment in coronary endothelial function.

Acetylcholine↗
PubMed1978

Genetic and functional characterization of an antiserum to the lipid A-specific triggering receptor on murine B lymphocytes.

The inheritance of responsiveness to lipopolysaccharide (LPS), and of a marker recognized in LPS-reactive cells by a heterologous antiserum, was studied in crosses between C3H/HeJ (nonresponder) and C3H/Tif (high responder) mice. F1 hybrid mice show codominant expression of these traits: (a) LPS-reactive cells are only hlaf as frequent in the hybrids as in the high responder parent; (b) the serologically defined marker is expressed in half as many cells in the hybrids as in the high responder parent. In backcross generations, both LPS responsiveness and this serological marker segregated into high, intermediate, and nonresponders. LPS or free lipid A, but not two other B cell mitogens (lipoprotein, and purified protein derivative of tuberculin), compete with the antiserum for binding to the B cell surface membrane, and are capable of completely inhibiting such binding without interfering with the binding of a-ti-Ig antibodies or complexes to Fc receptors. The addition of an IgG fraction of the antiserum to B cell cultures results in exponetial growth of the cells and in maturation to antibody secretion. This mitogenic activity is dose-depedent and absorbable on spleen cells from LPS high responder mice. Taken together, these observations suggest that this antiserum contains antibodies to the lipid A-specific triggering receptor on B lymphocytes.

Animals↗
PubMed2006

Genetic and functional analysis of the Nkt1 locus using congenic NOD mice: improved Valpha14-NKT cell performance but failure to protect against type 1 diabetes.

Defective invariant natural killer T-cells (iNKT cells) have been implicated in the etiology of type 1 diabetes in nonobese diabetic (NOD) mice. In a genome scan of a cross between NOD and C57BL/6 mice, the most significant locus controlling the number of iNKT cells, referred to as Nkt1, was recently mapped to distal chromosome 1. Here, using congenic mice for this chromosomal segment, we definitively demonstrate the existence of Nkt1 and show that introgression of the C57BL/6 allele onto the NOD background improves both the number of iNKT cells and their rapid production of cytokines elicited by alpha-galactosylceramide treatment, explaining at least half of the difference between the NOD and C57BL/6 strains. Using new subcongenic lines, we circumscribed the Nkt1 locus to a 8.7-cM segment, between the NR1i3 and D1Mit458 markers, that notably includes the SLAM (signaling lymphocytic activation molecule) gene cluster, recently involved in murine lupus susceptibility. However, despite a significant correction of the iNKT cell defect, the Nkt1 locus did not alter the course of spontaneous diabetes in congenic mice. Our findings indicate a complex relationship between iNKT cells and autoimmune susceptibility. Congenic lines nonetheless provide powerful models to dissect the biology of iNKT cells.

Animals↗
PubMed2005

Genetic and functional characterization of the Escherichia coli BarA-UvrY two-component system: point mutations in the HAMP linker of the BarA sensor give a dominant-negative phenotype.

The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.

Dimerization↗