RESUMO
Immersive, interactive and mHealth technologies are increasingly being used in clinical research, healthcare and rehabilitation solutions. Leveraging technology solutions to derive new and novel clinical outcome measures is important to the ongoing assessment of clinical interventions. While demonstrating statistically significant changes is an important element of intervention assessment, understanding whether changes detected reflect changes of a magnitude that are considered meaningful to patients is equally important. We describe methodologies used to determine meaningful change and recommend that these techniques are routinely included in the development and testing of clinical assessment and rehabilitation technology solutions.
RESUMO
The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.
Assuntos
Efeito Fundador , Predisposição Genética para Doença , Hipertensão/genética , Adolescente , Adulto , Canadá , Feminino , França/etnologia , Ligação Genética , Variação Genética , Humanos , Escore Lod , Masculino , Pessoa de Meia-Idade , Fenótipo , Característica Quantitativa Herdável , População Branca/genéticaRESUMO
Genomewide linkage analysis has been extremely successful at identification of the genetic variation underlying single-gene disorders. However, linkage analysis has been less successful for common human diseases and other complex traits in which multiple genetic and environmental factors interact to influence disease risk. We hypothesized that a highly heritable complex trait, in which the contribution of environmental factors was relatively limited, might be more amenable to linkage analysis. We therefore chose to study stature (adult height), for which heritability is approximately 75%-90% (Phillips and Matheny 1990; Carmichael and McGue 1995; Preece 1996; Silventoinen et al. 2000). We reanalyzed genomewide scans from four populations for which genotype and height data were available, using a variance-components method implemented in GENEHUNTER 2.0 (Pratt et al. 2000). The populations consisted of 408 individuals in 58 families from the Botnia region of Finland, 753 individuals in 183 families from other parts of Finland, 746 individuals in 179 families from Southern Sweden, and 420 individuals in 63 families from the Saguenay-Lac-St.-Jean region of Quebec. Four regions showed evidence of linkage to stature: 6q24-25, multipoint LOD score 3.85 at marker D6S1007 in Botnia (genomewide P<.06), 7q31.3-36 (LOD 3.40 at marker D7S2195 in Sweden, P<.02), 12p11.2-q14 (LOD 3.35 at markers D12S10990-D12S398 in Finland, P<.05) and 13q32-33 (LOD 3.56 at markers D13S779-D13S797 in Finland, P<.05). In a companion article (Perola et al. 2001 [in this issue]), strong supporting evidence is obtained for linkage to the region on chromosome 7. These studies suggest that highly heritable complex traits such as stature may be genetically tractable and provide insight into the genetic architecture of complex traits.
Assuntos
Estatura/genética , Mapeamento Cromossômico , Ligação Genética/genética , Característica Quantitativa Herdável , Mapeamento Cromossômico/métodos , Mapeamento Cromossômico/estatística & dados numéricos , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 7/genética , Meio Ambiente , Feminino , Finlândia , Genótipo , Humanos , Escore Lod , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Quebeque , Software , SuéciaRESUMO
Expression of the polyoma virus middle T antigen in HL-60 cells accelerates their differentiation in response to both monocytic and granulocytic differentiation-inducing agents. Middle T-expressing cells treated with the granulocytic inducer retinoic acid or the monocytic inducer 1,25-dihydroxy vitamin D3 differentiated 24 h earlier than parental, mock-electroporated, or vector control cell lines. The rapid onset of differentiation correlated with an increase in the cellular level of the middle T protein as well as two known retinoic-acid-inducible markers in HL-60 cells: the paxillin and transglutaminase gene products. The accelerated functional differentiation response and expression of retinoic-acid-inducible markers indicate that middle T played a causal role in differentiation. Thus, expression of the polyoma middle T antigen in HL-60 cells enhanced a variety of molecular changes associated with cellular differentiation.
Assuntos
Antígenos Transformantes de Poliomavirus/biossíntese , Diferenciação Celular/fisiologia , Granulócitos/citologia , Monócitos/citologia , Calcitriol/farmacologia , Moléculas de Adesão Celular/biossíntese , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/biossíntese , Células HL-60 , Humanos , Cinética , Paxilina , Fosfoproteínas/biossíntese , Proteínas Recombinantes/biossíntese , Fatores de Tempo , Transfecção , Transglutaminases/biossíntese , Tretinoína/farmacologiaRESUMO
The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins for the Rho- and Rab-subtype GTP-binding proteins. As a first step toward identifying the key functional domain(s) on the Rho-subtype GDI, truncations of the amino and carboxyl termini were performed. Deletion of the final four amino acids from the carboxyl terminus of Rho GDI or the removal of 25 amino acids from the amino terminus had no significant effect on the ability of the GDI to inhibit GDP dissociation from the Rho-like protein Cdc42Hs or on its ability to release Cdc42Hs from membrane bilayers. However, the deletion of 8 amino acids from the carboxyl terminus of Rho GDI eliminated both activities. To further test the importance of the carboxyl-terminal domain of the Rho GDI molecule, chimeras were constructed between this GDI and a related protein designated LD4, which is 67% identical to Rho GDI but is less potent by a factor of 10-20 than Rho GDI in functional assays with the Cdc42Hs protein. Two sets of chimeras were constructed that together indicated that as few as 6 amino acids near the carboxyl terminus of Rho GDI could impart full GDP dissociation inhibition and membrane dissociation activities on the LD4 molecule. Further analysis of this region by site-directed mutagenesis showed that a single change at residue 174 of LD4 to the corresponding residue of Rho GDI (i.e., Asp-174-->Ile) could impart nearly full (70%) Rho GDI activity on the LD4 molecule.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Guanosina Difosfato/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico , Bovinos , Desenho de Fármacos , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/isolamento & purificação , Glutationa Transferase/metabolismo , Humanos , Isoleucina , Camundongos , Dados de Sequência Molecular , Mutagênese , Mutação Puntual , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Lrp (leucine-responsive regulatory protein) is a global regulator of metabolism in Escherichia coli (J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). The lrp genes from three other enteric microorganisms, Enterobacter aerogenes, Klebsiella aerogenes, and Salmonella typhimurium, were cloned and sequenced. An analysis of these sequences and of the previously determined sequence from E. coli indicated that the vast majority of changes were synonymous rather than nonsynonymous changes. Nucleotide changes occurred at 89 of 492 positions but resulted in amino acid changes at only 2 of 164 positions. This analysis suggests that the Lrp amino acid sequence is highly adapted for function and that almost all amino acid changes lead to a protein that functions less well than the wild-type protein.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Enterobacteriaceae/genética , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Enterobacter/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Klebsiella pneumoniae/genética , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Salmonella typhimurium/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Expression of an activated raf transgene accelerated the terminal myeloid differentiation of HL-60 human promyelocytic leukemia cells induced by retinoic acid. A similar result was obtained when 1,25-dihydroxyvitamin D3 was used to induce monocytic differentiation. The stable transfectants were derived by transfecting HL-60 cells with DNA encoding an N-terminal truncated raf-1 protein. In normal HL-60 cells retinoic acid is known to induce a colony-stimulating factor-1 (CSF-1)-dependent metabolic cascade culminating in G0 arrest and phenotypic conversion. Early in this cascade, expression of the RB tumor suppressor gene product is down-regulated. A progressive redistribution of the form of the protein from largely hyperphosphorylated protein to the hypophosphorylated form begins later with G0 arrest and differentiation. In the activated raf-transfected cells, RB down regulation occurred more rapidly, consistent with accelerated differentiation. But the conversion to the hypophosphorylated form was not accelerated and occurred after G0 arrest and phenotypic conversion to myeloid differentiated cells. Thus raf activation appears to be a component of the induced metabolic cascade culminating in terminal differentiation. In this cascade raf activation promotes RB down-regulation. The data are consistent with a model in which raf is an effector of the CSF-1-dependent metabolic cascade which culminates in terminal cell differentiation, and RB downregulation is one of the downstream consequences of RAF action. Furthermore, they indicate that RB down-regulation may be an essential component of the cellular processes causing G0 arrest and differentiation, but RB hypophosphorylation is more likely a consequence thereof and not a cause.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Proteína do Retinoblastoma/metabolismo , Tretinoína/farmacologia , Diferenciação Celular/genética , Divisão Celular/efeitos dos fármacos , Regulação para Baixo/genética , Humanos , Fosforilação , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity.
Assuntos
Receptores ErbB/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/biossíntese , Receptores ErbB/isolamento & purificação , Glutationa Transferase/metabolismo , Humanos , Insetos , Cinética , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/isolamento & purificação , Receptor ErbB-3 , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
ErbB3 is a member of the epidermal growth factor (EGF) receptor subfamily of receptor tyrosine kinases and is believed to be a receptor for an unknown ligand. We have tested the possibility that heregulin, a growth factor possessing an EGF-like domain, is a ligand for ErbB3. We have found that the iodinated recombinant EGF-like domain of heregulin-beta 1 (125I-rHRG beta 1(177-244) bound specifically to insect cell-expressed bovine ErbB3 with a dissociation constant of 0.85 nM. Moreover, 125I-rHRG beta 1(177-244) bound to NIH3T3 fibroblasts stably transfected with bovine erbB3 with a dissociation constant of 60 pM, but did not bind to parental cells. 125I-rHRG beta 1(177-244) could be chemically cross-linked to a 170-180 kDa protein in erbB3-transfected fibroblasts, and the cross-linked product could be immunoprecipitated with antibodies specific for ErbB3. Finally, rHRG beta 1 stimulated the tyrosine phosphorylation of both ErbB3 and endogenous p185erbB2/neu in transfectants but not in parental cells. We conclude that ErbB3 is a receptor for HRG and is capable of mediating HRG-stimulated tyrosine phosphorylation of itself and p185erbB2/neu in cells that express both receptors.
Assuntos
Proteínas de Transporte/metabolismo , Receptores ErbB/metabolismo , Glicoproteínas/metabolismo , Neuregulina-1 , Proteínas Proto-Oncogênicas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sítios de Ligação , Bovinos , Células Cultivadas , Clonagem Molecular , Escherichia coli/genética , Radioisótopos do Iodo , Camundongos , Dados de Sequência Molecular , Mariposas , Fosforilação , Receptor ErbB-3 , Transfecção , Tirosina/metabolismoRESUMO
The dependence of induced myelomonocytic cell differentiation, and regulation of the RB tumor suppressor gene during this process, on the c-fms gene product, the CSF-1 lymphokine receptor, was determined in HL-60 promyelocytic leukemia cells. Adding a monoclonal antibody with specificity for the c-fms gene product to cells treated with various inducers of myelomonocytic or macrophage differentiation, including retinoic acid and 1,25-dihydroxy vitamin D3, inhibited the rate of differentiation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greater inhibition of differentiation. In a stable HL-60 transfectant overexpressing the CSF-1 receptor at the cell surface due to a constitutively driven c-fms trans gene, the rate of differentiation was enhanced compared to the wild type cell, consistent with a positive regulatory role for the CSF-1 receptor. The anti-fms antibody caused much less inhibition of differentiation in the transfectants than in wild type cells, consistent with a larger number of receptors causing reduced sensitivity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB expression per cell, which offset the decrease normally caused by differentiation inducers (1,25-dihydroxy vitamin D3 and retinoic acid). The changes in RB expression preceded changes in the RB protein to the hypophosphorylated state. Most of the RB protein in proliferating cells was phosphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of G0 arrest. Thus the metabolic cascade leading to myelomonocytic differentiation of HL-60 cells appears to be driven by a function of the c-fms protein. Inhibiting that process by attacking this receptor impedes differentiation and also compromises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide additional support for a potential role for down regulating RB expression in promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF-1 actions.
Assuntos
Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica/genética , Genes do Retinoblastoma/genética , Leucemia Promielocítica Aguda/patologia , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Western Blotting , Calcitriol/farmacologia , Transformação Celular Neoplásica/genética , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes do Retinoblastoma/fisiologia , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Macrófagos/química , Macrófagos/patologia , Macrófagos/ultraestrutura , Monócitos/química , Monócitos/patologia , Monócitos/ultraestrutura , Receptor de Fator Estimulador de Colônias de Macrófagos/análise , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Fatores de Tempo , Transfecção , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced increased expression of the retinoblastoma (RB) tumor suppressor gene product in the course of megakaryocytic differentiation of the K562 human leukemia cell line, a differentiatively multipotent hematopoietic precursor cell. The induced increase in RB protein per cell occurred early, by 8 h of treatment, preceding any significant phenotypic differentiation evidenced by cellular expression of the CD41 differentiation-specific megakaryocytic cell surface marker, but not inhibition of cell cycle transit, leading to a cell population arrested with 2 n, 4 n, and 8 n DNA content. The increase in RB protein per cell occurred for cells in all cell cycle phases. Staurosporine (STSP) was found to induce a similar course of cell cycle arrest and differentiation. Furthermore, STSP caused an up-regulation of RB expression similar to that caused by TPA. Almost all of the RB protein is phosphorylated in untreated cells, but TPA and STSP both caused the late appearance of hypophosphorylated RB protein following cell cycle arrest. The STSP-caused hypophosphorylation was much later than the TPA effect. Hypophosphorylation of RB is, thus, not necessarily a prerequisite for cell cycle arrest but may be a consequence of G0. Given that TPA can be an activator and STSP an inhibitor of protein kinase C, it appears that the induced processes of tumor suppressor gene regulation and growth and differentiation control are not necessarily protein kinase C dependent in K562 cells. Furthermore, the findings that these two presumably divergent inducing agents caused a similar increase in RB gene expression suggests that the up-regulation of RB associated with differentiation is not a coincidence of just one specific inducer but may be a common essential feature of the induced differentiation. The amount of RB protein per cell increased within hours of exposure to TPA or STSP and may have a role in the induced metabolic cascade producing the new phenotype.
Assuntos
Alcaloides/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes do Retinoblastoma/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Megacariócitos/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , DNA de Neoplasias/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/genética , Genes do Retinoblastoma/efeitos dos fármacos , Humanos , Cinética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Megacariócitos/citologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Estaurosporina , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Lrp is a regulatory protein in Escherichia coli that increases expression of some operons and decreases expression of others. Mutations in Lrp were isolated on the basis of their effects on ilvIH, one of the operons regulated positively by Lrp. The ilvIH operon encodes an enzyme involved in the biosynthesis of leucine, valine, and isoleucine, and expression of this operon is repressed when cells are grown in the presence of leucine. Three groups of mutants were isolated. Mutant strains that were resistant to the repressive effects of leucine were termed leucine response mutants. These mutants had changes in the Lrp amino acid sequence between amino acid residues 108 and 149. Mutant strains having low expression of ilvIH in vivo were identified as colonies having reduced expression of a reporter gene. For some of these mutants, called DNA-binding mutants, binding to ilvIH DNA in vitro was markedly reduced. The mutations in these strains caused changes in Lrp between amino acids 16 and 70. Six of ten of these mutations were within a region having a putative helix-turn-helix motif. A third group of mutants had low ilvIH expression in vivo but apparently normal DNA binding in vitro. These mutants were called activation mutants since they affected the ability of Lrp to activate expression. Lrp from these strains had changes in amino acids between residues 76 and 125. This study suggests that Lrp has separate domains responsible for binding DNA, activating transcription, and responding to leucine.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Leucina/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Genes Bacterianos , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The ras-related protein, CDC42Hs, is a 22-kDa GTP-binding protein which is the human homolog of a Saccharomyces cerevisiae yeast-cell-division cycle protein. In attempting to isolate and biochemically characterize mammalian proteins capable of regulating various activities of CDC42Hs, we have identified an activity in bovine brain cytosol which effectively inhibits the dissociation of [3H]GDP from the platelet- or the Spodoptera frugiperda-expressed CDC42Hs protein. The purification of this activity was achieved by a series of steps which included ammonium sulfate fractionation, DEAE-Sephacel, Mono-Q, and Mono-S chromatographies. The purified CDC42Hs regulatory protein has an apparent molecular weight of 28,000, and cyanogen bromide-generated peptide sequences of this protein were identical to sequences from the carboxyl-terminal portion of rho-GDP-dissociation inhibitor (rho-GDI) (Fukumoto, Y., Kaibuchi, K., Hori, Y., Fujioka, H., Araki, S., Ueda, T., Kikuchi, A., and Takai, Y. (1990) Oncogene 5, 1321-1328). In addition, an Escherichia coli-expressed, glutathione S-transferase-rho-GDI fusion protein fully substitutes for the GDI which we have purified from bovine brain in its ability to inhibit GDP dissociation from CDC42Hs. These findings suggest either that a common regulatory protein (GDI) is capable of inhibiting GDP dissociation from the rho and CDC42Hs proteins or that these two GTP-binding proteins interact with GDI proteins of very similar structure. The purified brain GDI protein shows little ability to inhibit GDP dissociation from the E. coli-expressed CDC42Hs and is capable of only a very weak inhibition of the dissociation of [35S]guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) from the Spodoptera frugiperda-expressed CDC42. However, brain GDI very effectively inhibits the ability of the human dbl oncogene product to catalyze GDP dissociation from CDC42Hs. In addition to influencing guanine nucleotide association with CDC42Hs, the purified brain GDI protein also appears to catalyze the dissociation of CDC42Hs from the plasma membranes of human placenta and human epidermoid carcinoma (A431) cells. This effect by the GDI protein is observed whether the membrane-associated CDC42Hs is preincubated with GDP, GTP gamma S, or no guanine nucleotides, and occurs over a similar concentration range as that necessary for the inhibition of the intrinsic GDP dissociation.
Assuntos
Plaquetas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Guanosina Difosfato/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia por Troca Iônica , Clonagem Molecular , Citosol/metabolismo , Escherichia coli/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Cinética , Dados de Sequência Molecular , Mariposas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Transfecção , Proteína cdc42 de Ligação ao GTP , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Fímbrias Bacterianas/fisiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genes Reguladores , Óperon , Fatores de Transcrição , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/fisiologia , Proteínas de Escherichia coli , Leucina/farmacologia , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , RNA Mensageiro/genética , Mapeamento por Restrição , Transcrição Gênica/efeitos dos fármacosRESUMO
We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Escherichia coli/metabolismo , Leucina/metabolismo , Aminoácidos de Cadeia Ramificada/genética , Transporte Biológico , Clonagem Molecular , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Óperon Lac , Leucina/genética , Óperon , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes de FusãoRESUMO
Exogenous leucine affects the expression of a number of different operons in Escherichia coli. For at least some of these operons, the leucine-related effect is mediated by a protein called Lrp (Leucine-responsive regulatory protein). The purification of Lrp to near homogeneity is described. Lrp is a moderately abundant, basic protein composed of two subunits of molecular mass 18.8 kDa each. In addition, the corresponding protein was purified from a strain having a mutation within the gene that encodes Lrp (lrp). This mutation (lrp-1) causes high constitutive expression of ilvIH, one of the operons controlled by Lrp (Platko, J. V., Willins, D.A., and Calvo, J.M. (1990) J. Bacteriol. 172, 4563-4570). The Lrp-1 and Lrp proteins have similar physical properties, but they show some differences in the characteristics with which they bind DNA upstream of the ilvIH promoter. The nucleotide sequences of the lrp and lrp-1 genes differ by only a single nucleotide, a C to G change that would substitute a Glu for an Asp at amino acid 114. Lrp has some amino acid sequence similarity to AsnC, a protein that regulates asnA expression (Kolling, R., and Lother, H. (1985) J. Bacteriol. 164, 310-315).
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Leucina/farmacologia , Óperon/efeitos dos fármacos , Fatores de Transcrição , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Western Blotting , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Genes Bacterianos , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido NucleicoRESUMO
The ilvIH operon of Escherichia coli (located near min 2) encodes acetohydroxyacid synthase III, an isozyme involved in branched-chain amino acid biosynthesis. A strain with lacZ fused to the ilvIH promoter was constructed. Transposon Tn10 was introduced into this strain, and tetracycline-resistant derivatives were screened for those in which ilvIH promoter expression was markedly reduced. In one such derivative, strain CV1008, beta-galactosidase expression was reduced more than 30-fold. The transposon giving rise to this phenotype inserted near min 20 on the E. coli chromosome. Extract from a wild-type strain contains a protein, the IHB protein, that binds to two sites upstream of the ilvIH promoter (E. Ricca, D. A. Aker, and J. M. Calvo, J. Bacteriol. 171:1658-1664, 1989). Extract from strain CV1008 lacks IHB-binding activity. These results indicate that the IHB protein is a positive regulator of ilvIH operon expression. The gene that encodes the IHB protein, ihb, was cloned by complementing the transposon-induced mutation. Definitive evidence that the cloned DNA encodes the IHB protein was provided by determining the sequence of more than 17 amino acids at the N terminus of the IHB protein and comparing it with the nucleotide sequence. A mutation that prevents repression of the ilvIH operon by leucine in vivo and that alters the DNA-binding characteristics of the IHB protein in vitro was shown to be an allele of the ihb gene. The ihb gene is identical to oppI, a gene that regulates the oppABCDF operon (E. A. Austin, J. C. Andrews, and S. A. Short, Abstr. Mol. Genet. Bacteria Phages, p. 153, 1989). Thus, oppI/ihb encodes a protein that regulates both ilvIH, an operon that is repressed by leucine, and oppABCDF, an operon involved in peptide transport that is induced by leucine. We propose that the designation lrp be used in the future instead of oppI or ihb and that Lrp (leucine-responsive regulatory protein) be used in place of IHB.