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At least 199 records · Page 11Linked to original sources
PubMed2005

Investigation of B-branch electron transfer by femtosecond time resolved spectroscopy in a Rhodobacter sphaeroides reaction centre that lacks the Q(A) ubiquinone.

The dynamics of electron transfer in a membrane-bound Rhodobacter sphaeroides reaction centre containing a combination of four mutations were investigated by transient absorption spectroscopy. The reaction centre, named WAAH, has a mutation that causes the reaction centre to assemble without a Q(A) ubiquinone (Ala M260 to Trp), a mutation that causes the replacement of the H(A) bacteriopheophytin with a bacteriochlorophyll (Leu M214 to His) and two mutations that remove acidic groups close to the Q(B) ubiquinone (Glu L212 to Ala and Asp L213 to Ala). Previous work has shown that the Q(B) ubiquinone is reduced by electron transfer along the so-called inactive cofactor branch (B-branch) in the WAAH reaction centre (M.C. Wakeham, M.G. Goodwin, C. McKibbin, M.R. Jones, Photo-accumulation of the P(+)Q(B)(-) radical pair state in purple bacterial reaction centres that lack the Q(A) ubiquinone, FEBS Letters 540 (2003) 234-240). In the present study the dynamics of electron transfer in the membrane-bound WAAH reaction centre were studied by femtosecond transient absorption spectroscopy, and the data analysed using a compartmental model. The analysis indicates that the yield of Q(B) reduction via the B-branch is approximately 8% in the WAAH reaction centre, consistent with results from millisecond time-scale kinetic spectroscopy. Possible contributions to this yield of the constituent mutations in the WAAH reaction centre and the membrane environment of the complex are discussed.

Electron Transport↗
PubMed1996

Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.

The NF-kappaB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-kappaB/Rel proteins are coupled to inhibitory molecules, collectively termed IkappaB, which are responsible for cytoplasmic retention of NF-kappaB. Cell activation leads to the phosphorylation and degradation of IkappaBalpha, permitting NG-kappaB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IkappaBalpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IkappaBalpha in an affinity chromatography step and phosphorylated IkappaBalpha with high specificity in vitro. By using an in-gel kinase assay with recombinant IkappaBalpha as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IkappaBalpha delta1 to delta4) localized phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IkappaBalpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IkappaBalpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IkappaBalpha resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha.

Base Sequence↗
PubMed2001

Molecular analysis of the prostate-specific antigen upstream gene enhancer.

BACKGROUND: Our objective was to identify factors other than androgen receptor that bind to and regulate the prostate-specific antigen (PSA) upstream gene enhancer (PSE). METHODS: DNAse I footprinting and electromobility shift assays (EMSA) were performed over the PSE using lysates from PSA-producing cell lines, LNCaP and LAPC4, and nonproducing PSA cell lines, PC-3 cells, U937 monocytes, and Namalwa B cells. Mutational analysis and transient transfection assays were used to determine the contributions made by different elements towards the regulation of the enhancer. RESULTS: Three distinct regions surrounding androgen response elements of the PSE were found to bind unknown ubiquitous and cell type-specific proteins. These regions, when mutated in a PSE reporter construct, were shown to be required for maximal activation in LNCaP cells. CONCLUSIONS: These results correlate unknown sequence-specific DNA binding proteins with androgen-mediated regulation of a prostate-specific gene, thus providing further insight into the mechanism of PSA gene expression.

Base Sequence↗
PubMed1996

Regulation of a human cytomegalovirus immediate-early gene (US3) by a silencer-enhancer combination.

The US3 open reading frame of human cytomegalovirus (HCMV) is transcribed at immediate-early (IE) times after infection. Upstream of the US3 promoter, between -84 and -259 bp relative to the transcription start site, there are five copies of an 18-bp repeat, referred to as 5R2. Between -340 and -560 bp there are seven copies of a 10-bp dyad repeat, referred to as 7R1. We investigated the roles of these repeats in transcription from the US3 promoter in human foreskin fibroblast or HeLa cells. In transient transfection assays, the region containing 5R2 up-regulated transcription and was responsive to the p65 subunit of NF-kappa B. The DNA region containing 7R1 down-regulated transcription from either the US3 promoter or a heterologous promoter in a position- and orientation-independent manner. Mutational analysis and transient transfections indicated that DNA containing the 10-bp dyad or one-half of the dyad was sufficient to cause repression of downstream gene expression. DNA probes containing one or more copies of the pentanucleotide sequence TGTCG specifically bound cellular proteins, as demonstrated by electrophoretic mobility shift assays and cold-competition electrophoretic mobility shift assays. Two different DNA-protein complexes were detected with DNA probes containing one or two copies of the pentanucleotide. In HCMV-infected cell nuclear extracts, one of the DNA-protein complexes was present in amounts inversely proportional to the amount of US3 transcription. Its formation was affected by dephosphorylation of the DNA-binding protein(s). Transient dephosphorylation of the cellular repressor protein may occur during HCMV infection. Repression of US3 transcription may relate to the number of pentanucleotides and the cellular proteins that bind to it. Twenty-one copies of a TRTCG motif (R = purine) were found clustered upstream of the US3 gene and also in the modulator upstream of the HCMV IE1 and IE2 genes.

Base Sequence↗
PubMed1988

The effect of the reproductive system on mutation load.

J. B. S. Haldane (Amer. Nat. 71, 337-349, 1937) argued that, in equilibrium populations, the effect of deleterious mutation on average fitness depends primarily on the mutation rate and is independent of the severity of the mutations. Specifically, the equilibrium population fitness is e-microH, where microH is the haploid genomic mutation rate. Here we extend Haldane's result to a variety of reproductive systems. Using an analysis based on the frequency of classes of individuals with a specified number of mutations, we show that Haldane's principle holds exactly for haploid sex, haploid apomixis, and facultative haploid sex. In the cases of diploid automixis with terminal fusion, diploid automixis with central fusion, and diploid selfing, Haldane's principle holds exactly for recessive mutations and approximately for mutations with some heterozygous effect. In the cases of K-ploid apomixis, diploid endomitosis, and haplodiploidy, we show that Haldane's principle holds exactly for recessive lethal mutations. In addition we extend Haldane's result to various mixtures of the above-mentioned reproductive systems. In the case of diploid out-crossing sexuals, we do not obtain an exact analytic result, but present arguments and computer simulations which show that Haldane's result extends to this case as well in the limit as the number of loci becomes large. Although diverse reproductive systems are equally fit at equilibrium, different reproductive systems harbor vastly different numbers of recessive genes at equilibrium and we provide estimates of these numbers. These different numbers of mutations may create transient selective pressures on individuals with reproductive systems different from that of the equilibrium population.

Animals↗
PubMed2004

Importance of transient structures during post-transcriptional refolding of the pre-23S rRNA and ribosomal large subunit assembly.

An important step of ribosome assembly is the folding of the rRNA into a functional structure. Despite knowledge of the folded state of rRNA in the ribosomal subunits, there is very little information on the rRNA folding pathway. We are interested in understanding how the functional structure of rRNA is formed and whether the rRNA folding intermediates have a role in ribosome assembly. To this end, transient secondary structures around both ends of pre-23S rRNA were analyzed by a chemical probing approach, using pre-23S rRNA transcripts. Metastable hairpin loop structures were found at both ends of 23S rRNA. The functional importance of the transient structures around the ends of 23S rRNA was tested by mutations that alter only the transient structure. The effect of mutations on 23S rRNA folding was tested in vitro and in vivo. It was found that both stabilization and destabilization of the transient structure around the 5' end of 23S rRNA inhibits post-transcriptional refolding in vitro and ribosome formation in vivo. The data suggest that the transient structure of rRNA has a function during 23S rRNA folding and thereby in ribosome assembly.

Base Sequence↗
PubMed1999

The human KDR/flk-1 gene contains a functional initiator element that is bound and transactivated by TFII-I.

KDR/flk-1, the receptor for vascular endothelial growth factor, is required for normal vascular development. KDR/flk-1 is a TATA-less gene, containing four upstream Sp1 sites and a single transcription start site, although analysis of the start site sequence discloses only weak similarities with the consensus initiator element (Inr) sequence. In vitro transcription assays, however, demonstrate that the region from -10 to +10 relative to the start site contains Inr activity that is orientation- and position-dependent, and mutagenesis of the KDR/flk-1 Inr reduces promoter activity to 28% of the wild-type promoter in transient transfection assays. Gel shift assays confirm that nuclear proteins specifically bind the Inr, and competition experiments demonstrate that TFII-I, a multifunctional Inr-binding nuclear protein, is a component of these DNA-protein complexes. TFII-I transactivates the wild-type KDR/flk-1 promoter, but not a promoter containing a mutated Inr, in transient transfection assays. Immunodepletion of TFII-I from nuclear extracts prior to in vitro transcription assays abolishes transcription from the KDR/flk-1 Inr, an effect that can be rescued by adding back purified TFII-I, reflecting the importance of TFII-I in KDR/flk-1 Inr activity. These experiments demonstrate that the KDR/flk-1 gene contains a functional Inr that is bound by TFII-I and that both the functional Inr and TFII-I activity are essential for transcription.

Animals↗
PubMed2005

Pituitary homeobox 1 (Pitx1) stimulates rat LHbeta gene expression via two functional DNA-regulatory regions.

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH beta-subunit gene (LHbeta). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHbeta gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHbeta gene promoter. While investigating the role of Pitx1 in regulating rat LHbeta gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions -73 to -52 in the rat LHbeta gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5'Pitx1 site as well as this putative 3'Pitx1 region. In transient transfection analysis, mutation of the LHbeta-3'Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5'Pitx1 site with further loss following mutation of the 3'Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHbeta gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

Animals↗
PubMed1998

Characterization of a factor VII molecule carrying a mutation in the second epidermal growth factor-like domain.

A missense mutation at codon 100 in the second epidermal growth factor-like domain, resulting in Gln100-->Arg, was detected in 19 out of 21 available severely factor VII (FVII) deficient patients in Norway. Seventeen patients were homozygous, and the two remaining were compound heterozygotes. In the homozygous patients, FVII antigen was measured to 10-28%, and activity to 0.6-6.5% of that in normal pooled plasma. Recombinant FVII containing the mutation was expressed transiently in CHO cells to a mean antigen level of 57% of the wild type FVII protein, and with a specific activity of 6% of wild type. The mutant protein had a 14-fold reduction in affinity for tissue factor (TF), whereas binding of FX seemed unaffected. In line with the experimental data, molecular modelling of the mutation based on the coordinates of the tissue factor/FVIIa complex showed that substituting arginine for glutamine disrupts the interface between the catalytic and second epidermal growth factor-like domains.

Adolescent↗
PubMed1990

Mutations induced at the hypoxanthine-guanine phosphoribosyltransferase locus of human T-lymphoblasts by perturbations of purine deoxyribonucleoside triphosphate pools.

Chronic perturbations of intracellular deoxyribonucleoside triphosphate (dNTP) pools have been associated with a mutator phenotype and increased mutation rates at several genetic loci. We have examined the specific effects of transient pharmacological purine dNTP pool perturbations on mutations induced at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in a cultured human T-lymphoblast cell line. Incubation of CEM cells with 50 microM 2'-deoxyguanosine for 6 h increased intracellular dGTP levels 43-fold and induced a 40-fold increase in mutation frequency at the HPRT locus. Six-h incubations with 5, 10, and 20 microM 2'-deoxyadenosine increased dATP pools 4.8-, 8-, and 14.5-fold, respectively, with 59-, 34-, and 43-fold increases in HPRT mutant fractions. In contrast, 24-h incubations with hydroxyurea at concentrations which inhibited cell growth to similar extents did not induce HPRT mutations. Sequencing of HPRT complementary DNA derived from mutant cell lines revealed that the mutations induced by transient purine dNTP pool perturbations exhibited no significant misincorporation of the nucleotide in excess or next-nucleotide effect, and were similar in nature and location to spontaneous HPRT mutations. We conclude that mutations caused by transient purine dNTP pool elevations in these dividing cells are most likely induced by inhibition of DNA repair processes.

Base Sequence↗
PubMed2000

Transforming growth factor beta inhibitory element in the rabbit matrix metalloproteinase-1 (collagenase-1) gene functions as a repressor of constitutive transcription.

Transforming growth factor beta (TGF-beta) is a potent modulator of the extracellular matrix, enhancing collagen synthesis and regulating expression of several genes that encode the matrix metalloproteinases (MMPs), enzymes that degrade the extracellular matrix. In this study, we explored the mechanisms whereby TGF-beta inhibits expression of the MMP-1 (collagenase 1) gene. We used transient transfection and gel mobility shift assays to characterize a TGF beta inhibitory element (TIE) at -249 bp in the rabbit MMP-1 promoter, which is also conserved at -246 bp in the human gene. This sequence shares homology to a previously identified TIE in the rat stromelysin-1 (MMP-3) promoter, where it is located at -709 bp. Mutational analyses and transient transfections indicate that MMP-1 TIE functions both as a constitutive repressor of MMP-1 gene expression and, in the presence of TGF-beta, as an antagonist of transcriptional induction by phorbol esters. c-Fos binds to the TIE in the rabbit MMP-1 promoter, along with other nuclear proteins, even in the absence of treatment with TGF-beta. However, the pattern of proteins binding to the TIE is altered in the presence of nuclear extracts from TGF-beta-treated cells, suggesting that TGF-beta leads to an alteration in protein/DNA interaction, with subsequent modulation of MMP-1 gene expression. We conclude that in the rabbit MMP-1 promoter, the TIE has dual functions as a repressor of basal transcription and as a mediator of the biologic effects of TGF-beta. Furthermore, these dual functions provide additional and subtle mechanisms for regulating MMP-1 gene expression under a variety of biological and pathological conditions.

Animals↗
PubMed2004

Triple heterozygosity in the integrin alphaIIb subunit in a patient with Glanzmann's thrombasthenia.

We report triple heterozygosity in the integrin alpha(IIb) subunit in a 5-year-old Canadian girl with Glanzmann's thrombasthenia. The patient has a severe bleeding history possibly aggravated by low VWF suggestive of associated type 1 von Willebrand's disease. Platelet aggregation was absent or severely reduced for all physiologic agonists. Flow cytometry showed an approximately 4% residual surface expression of alpha(IIb)beta(3). Western blotting confirmed a low platelet expression of both subunits. PCR-SSCP and direct sequencing showed no abnormalities in the beta(3) gene, but revealed a G-->A transition at a splice site [IVS 19 (+1)] of exon 19 in the alpha(IIb) gene. Of maternal inheritance, the splice site mutation was associated with intermediate levels of alpha(IIb)beta(3) in carriers. Unexpectedly, two G-->A transitions were detected in exon 29 of the alpha(IIb) gene and led to V(951)-->M and A(958)-->T amino acid substitutions. Family studies using restriction enzymes showed that both exon 29 mutations were paternal in origin and cosegregated across three generations. Transient expression in which mutated alpha(IIb) was cotransfected with wild-type beta(3) in COS-7 cells showed that V(951)-->M gave a much reduced surface expression of alpha(IIb)beta(3) and a block in the maturation of pro-alpha(IIb). In contrast, the A(958) substitution appeared to be a novel polymorphism. Our studies highlight an unusual mixture of defects giving rise to severe bleeding in a child and describe the first pathological missense mutation affecting a C-terminal residue of the calf-2 domain of alpha(IIb).

Child, Preschool↗
PubMed1990

Point mutations in the Moloney murine leukemia virus enhancer identify a lymphoid-specific viral core motif and 1,3-phorbol myristate acetate-inducible element.

The transcriptional enhancer of the Moloney murine leukemia virus (MoMLV) is organized as a 75-base-pair repeat, and in each copy of the repeat there are multiple binding sites for nuclear factors. We have introduced point mutations into each of the known nuclear factor-binding sites in the MoMLV enhancer, in both copies of the direct repeat, and have analyzed the transcriptional activity conferred by the mutated enhancers by transient-expression assays in both hematopoietic and nonhematopoietic cell lines. Mutation of individual binding sites in the MoMLV enhancer has moderate effects (less than 2-fold to 20-fold) on transcription in six independent cell lines. Several mutations decreased transcription from the MoMLV enhancer ubiquitously (the leukemia virus factor b site and the glucocorticoid response element), whereas others affected transcription specifically in lymphoid cell lines (core motif) or, more significantly, in fibroblasts (nuclear factor 1 site). The transcriptional activity of the MoMLV enhancer can be induced 8- to 10-fold by 1,3-phorbol myristate acetate in Jurkat T cells. Mutations in any of three adjacent binding sites (leukemia virus factor b and c sites and the core motif) within a 28-base-pair region in the center of the direct repeat sequence of the MoMLV enhancer completely attenuate the response to 1,3-phorbol myristate acetate.

Animals↗
PubMed2001

Compound heterozygosity of the GPIbalpha gene associated with Bernard-Soulier syndrome.

We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibalpha and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbalpha in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbalpha gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution chan-ino Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbbeta and GPIX failed to detect surface expression of the GPIbalpha mutants. Cells transfected with [1418insT]GPIbalpha-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast. transfection of [T715A]GPIbalpha-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbalpha more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.

Adult↗
PubMed1990

Maximal stress-induced transcription from the human HSP70 promoter requires interactions with the basal promoter elements independent of rotational alignment.

Transcription of the human HSP70 gene is regulated by a complex array of cis-acting promoter elements that respond to conditions that include normal conditions of cell growth and induction following physiological stress. We have examined the requirements of the basal and inducible promoter elements by using promoter mutations and a transient transfection assay. Multiple forms of stress-induced transcription, including heat shock and incubation with heavy metals or amino acid analogs, are mediated by a single heat shock element (HSE) between -105 and -91 consisting of three contiguous 5-base-pair units, NGAAN, that are inverted relative to adjacent units. Maximal inducible expression requires a fully functional basal promoter. Spacing mutations which alter the relative helical orientation of adjacent genetic elements have only minimal effects on basal and stress-inducible expression and show no effects of periodicity. In addition, placement of the HSE adjacent to the basal promoter removes the requirements for a fully functional basal promoter for maximal stress-inducible expression. These results suggest that factors bound at the HSE and the basal promoter can function through multiple interactions.

Base Sequence↗