RESUMO
Bone-cancer-related pain is one of the most disabling factors in patients suffering from primary bone cancer or bone metastases. Recent studies point toward an important role of proinflammatory cytokines, example tumor necrosis factor-alpha (TNF), for tumor growth and bone-cancer-associated pain. Mechanisms by which TNF, through its receptor subtypes, TNF receptor 1 (TNFR1) and -2 (TNFR2), elicits altered sensation and pain behavior, are still incompletely understood. To look for a potential role of TNF in bone cancer pain, cancer-related pain was analyzed in fibrosarcoma-bearing C57Bl/6J wild type mice after systemic antagonism of TNF. To further clarify the role of TNF receptor (TNFR) in bone-cancer pain, naive and fibrosarcoma-bearing C57Bl/ 6J wild type and transgenic mice with a deficiency of TNFR1 (TNFR1ko), TNFR2 (TNFR2ko), and TNFR1+2 (TNFR1+2ko) were compared regarding cancer-related pain and hyperalgesia, tumor growth, osteoclast activation, and spinal astrogliosis. Systemic antagonism of TNF significantly alleviated tactile hypersensitivity and spontaneous bone-cancer-related pain behavior. Most interestingly, combined deletion of the TNFR1 and TNFR2, but not of either gene alone, almost completely inhibited the development of tactile hypersensitivity, whereas spontaneous pain behavior was transiently increased. Accordingly, spinal astrogliosis was markedly reduced, whereas tumor growth was significantly increased in TNFR1+2ko mice. In contrast, deletion of the TNFR1 or TNFR2 gene alone did not change tumor growth or spinal astrogliosis. Our findings suggest that the combined absence of TNFR1 and TNFR2 is necessary for the attenuation of cancer-related tactile hypersensitivity and concomitant spinal astrogliosis, whereas tumor growth seems to be inhibited by combined TNFR activation. These findings support the hypothesis of cytokine-dependent pain development in cancer pain. Differential targeting of TNFR activation could be an interesting strategy in bone-cancer-related pain conditions.
Assuntos
Neoplasias Ósseas/secundário , Fibrossarcoma/secundário , Gliose/fisiopatologia , Proteínas de Neoplasias/fisiologia , Dor/fisiopatologia , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Medula Espinal/patologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Astrócitos/patologia , Comportamento Animal , Neoplasias Ósseas/fisiopatologia , Etanercepte , Fibrossarcoma/fisiopatologia , Gliose/etiologia , Gliose/prevenção & controle , Hiperalgesia/fisiopatologia , Hiperalgesia/prevenção & controle , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Coxeadura Animal/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Dor/etiologia , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/uso terapêutico , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Tato , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
The aim of the present study was to develop a screening tool to aid non-headache specialists, like general practitioners, in deciding whether migraine prophylaxis in the individual migraine patient is useful or not. The first step was the development of a questionnaire, consisting of 10 items, which was filled in by 132 migraineurs who called on neurologists or headache experts. Independently, the physicians filled in another questionnaire to answer the question of whether they decided to prescribe migraine prophylaxis and if they had, to give their reasons for doing so. Using logistic regression analysis, we identified the three questions which had the most influence on the decision regarding prophylaxis in the data set. As results, we identified the following three questions: 1. Do you suffer from migraine on more than 3 days/month? 2. Do you have to rest in bed while experiencing a migraine attack? 3. Do you have to take medication against migraine on more than 5 days/month? Validation of this reduced questionnaire is currently ongoing and involves 150 migraine patients of general practitioners.
Assuntos
Transtornos de Enxaqueca/prevenção & controle , Humanos , Estudos Prospectivos , Reprodutibilidade dos Testes , Inquéritos e Questionários , Fatores de TempoRESUMO
let-502 rho-binding kinase and mel-11 myosin phosphatase regulate Caenorhabditis elegans embryonic morphogenesis. Genetic analysis presented here establishes the following modes of let-502 action: (i) loss of only maternal let-502 results in abnormal early cleavages, (ii) loss of both zygotic and maternal let-502 causes elongation defects, and (iii) loss of only zygotic let-502 results in sterility. The morphogenetic function of let-502 and mel-11 is apparently redundant with another pathway since elimination of these two genes resulted in progeny that underwent near-normal elongation. Triple mutant analysis indicated that unc-73 (Rho/Rac guanine exchange factor) and mlc-4 (myosin light chain) act in parallel to or downstream of let-502/mel-11. In contrast mig-2 (Rho/Rac), daf-2 (insulin receptor), and age-1 (PI3 kinase) act within the let-502/mel-11 pathway. Mutations in the sex-determination gene fem-2, which encodes a PP2c phosphatase (unrelated to the MEL-11 phosphatase), enhanced mutations of let-502 and suppressed those of mel-11. fem-2's elongation function appears to be independent of its role in sexual identity since the sex-determination genes fem-1, fem-3, tra-1, and tra-3 had no effect on mel-11 or let-502. By itself, fem-2 affects morphogenesis with low penetrance. fem-2 blocked the near-normal elongation of let-502; mel-11 indicating that fem-2 acts in a parallel elongation pathway. The action of two redundant pathways likely ensures accurate elongation of the C. elegans embryo.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/embriologia , Proteínas de Helminto/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor de Insulina/fisiologia , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas Contráteis/genética , Proteínas Contráteis/fisiologia , Transtornos do Desenvolvimento Sexual , Embrião não Mamífero/enzimologia , Fertilidade , Proteínas de Helminto/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Morfogênese , Fosfatase de Miosina-de-Cadeia-Leve , Fenótipo , Fosfoproteínas Fosfatases/genética , Proteínas Serina-Treonina Quinases/genética , Receptor de Insulina/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Processos de Determinação Sexual , Quinases Associadas a rhoRESUMO
Germ line mutations of the multiple endocrine neoplasia type 1 (MEN1) tumour suppressor gene cause MEN1, a rare familial tumour syndrome associated with parathyroid hyperplasia, adenoma and hyperparathyroidism (HP). Here we investigated the role of the MEN1 gene in isolated sporadic and familial HP. Using RT-PCR single-strand conformational polymorphism screening, somatic (but not germ line) mutations of the MEN1 coding sequence were identified in 6 of 31 (19.3%) adenomas from patients with sporadic primary HP, but none in patients (n=16) with secondary HP due to chronic renal failure. MEN1 mutations were accompanied by a loss of heterozygosity (LOH) for the MEN1 locus on chromosome 11q13 in the adenomas as detected by microsatellite analysis. No DNA sequence divergence within the 5' region of the MEN1 gene, containing the putative MEN1 promoter, was detectable in HP adenomas. Clinical characteristics were not different in HP patients with or without MEN1 mutation. Heterozygous MEN1 gene polymorphisms were identified in 9.6% and 25% of patients with primary and secondary HP respectively. In a large kindred with familial isolated familial HP, MEN1 germ line mutation 249 del4 and LOH was associated with the HP phenotype and a predisposition to non-endocrine malignancies. We suggest that the bi-allelic somatic loss of MEN1 wild-type gene expression is involved in the pathogenesis of a clinically yet undefined subset of sporadic primary HP adenomas. MEN1 genotyping may further help define the familial hyperparathyroidism-MEN1 disease complex, but it seems dispensable in sporadic primary HP.
Assuntos
Adenoma/genética , Genes Supressores de Tumor , Hiperparatireoidismo/genética , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasias das Paratireoides/genética , Adulto , Idoso , Cromossomos Humanos Par 11 , Feminino , Mutação em Linhagem Germinativa , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mutations of the MEN1 tumor suppressor gene cause the multiple endocrine neoplasia type 1 (MEN1) syndrome in humans, and they are involved in a variety of sporadic human endocrine tumors. We here characterize the MEN1 gene homologs of the mouse and rat. cDNA was isolated from a mouse phage library, and two alternative MEN1 mRNA transcripts containing variant 5' untranslated regions were identified by RT-PCR in several mouse and rat tissues. When compared to the human molecule, mouse and rat MEN1 (611 and 610 amino acids, respectively) show an overall identity of 96.5% and 97.0% at the protein level, delimiting four conservational domains (A-D). Mouse and rat MEN1 mRNA, as studied by template-calibrated quantitative RT-PCR, is non-exclusively expressed in hematopoietic and endocrine cells, with similar expression patterns found in fetal and adult tissues. Fluorescent in situ hybridization maps the single murine MEN1 locus to chromosome 19, region B. No MEN1 gene mutations were identified in endocrine islet tumor cell lines RIN 5AH (rat) and NIT-1 (mouse) as compared to wild type cDNA. Our data define mouse and rat MEN1 as widely expressed and highly conserved homologs of the human MEN1 tumor suppressor gene whose role in biology and endocrine tumorigenesis is due for experimental study.
Assuntos
Genes Supressores de Tumor , Neoplasia Endócrina Múltipla Tipo 1/genética , Fatores de Transcrição , Animais , Mapeamento Cromossômico , Sondas de DNA , DNA Complementar/química , Regulação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
Caenorhabditis elegans embryonic elongation is driven by cell shape changes that cause a contraction of the epidermal cell layer enclosing the embryo. We have previously shown that this process requires a Rho-associated kinase (LET-502) and is opposed by the activity of a myosin phosphatase regulatory subunit (MEL-11). We now extend our characterization and show that mel-11 activity is required both in the epidermis during embryonic elongation and in the spermatheca of the adult somatic gonad. let-502 and mel-11 reporter gene constructs show reciprocal expression patterns in the embryonic epidermis and the spermatheca, and mutations of the two genes have opposite effects in these two tissues. These results are consistent with let-502 and mel-11 mediating tissue contraction and relaxation, respectively. We also find that mel-11 embryonic inviability is genetically enhanced by mutations in a Rac signaling pathway, suggesting that Rac potentiates or acts in parallel with the activity of the myosin phosphatase complex. Since Rho has been implicated in promoting cellular contraction, our results support a mechanism by which epithelial morphogenesis is regulated by the counteracting activities of Rho and Rac.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Epiderme/embriologia , Gônadas/embriologia , Proteínas de Helminto/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Proteínas Serina-Treonina Quinases/genética , Animais , Northern Blotting , Embrião não Mamífero/anatomia & histologia , Feminino , Viabilidade Fetal , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica , Genótipo , Proteínas de Fluorescência Verde , Proteínas de Helminto/metabolismo , Infertilidade , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/metabolismo , Modelos Genéticos , Mutagênese , Fosfatase de Miosina-de-Cadeia-Leve , Proteínas do Tecido Nervoso/metabolismo , Oviductos/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Temperatura , Fatores de Tempo , Útero/metabolismo , Proteínas rac de Ligação ao GTP , Quinases Associadas a rhoRESUMO
INTRODUCTION: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the development of multiple endocrine adenomas, typically in the pancreas, anterior pituitary, and parathyroid glands. The disease is associated with germ-line mutations of the menin gene, a putative tumor-suppressor gene located on human chromosome 11q13. METHODS: To facilitate the diagnosis and prediction of MEN1 in patients and their relatives, we developed a molecular two-step strategy to screen for menin gene mutations. DNA fragments covering the entire menin coding sequence are generated from patient cDNA by polymerase reaction (PCR) and subsequently analyzed by single-strand conformational polymorphism electrophoresis (SSCP). Fragments with aberrant SSCP migration are DNA-sequenced to directly characterize menin mutations. In a second diagnostic step, genomic DNA of healthy relatives of the corresponding MEN1 index patient is analyzed by PCR, with only the specific exon amplified harboring the family-specific mutation. Mutation-specific restriction enzyme digestion of this PCR product finally allows the identification of mutation carriers through pathological restriction fragment patterns. RESULTS: Using this approach, we identified an in-frame deletion mutation (delta Tyr Met) located in menin exon 4 (codon 227-228) that co-segregates with the disease phenotype in a large MEN1 family from Southern Germany. CONCLUSION: It is likely that the direct molecular analysis of menin gene mutations will replace the genetic and biochemical screening tests currently used in the clinical management of MEN1 families. In addition, these studies may provide clues to the tumor biology of both sporadic and MEN1-associated endocrine adenomas.
Assuntos
Neoplasia Endócrina Múltipla Tipo 1/genética , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas , Adulto , Aberrações Cromossômicas/genética , Deleção Cromossômica , Transtornos Cromossômicos , Cromossomos Humanos Par 11 , Análise Mutacional de DNA , Feminino , Genes Dominantes/genética , Triagem de Portadores Genéticos , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla Tipo 1/diagnóstico , Neoplasia Endócrina Múltipla Tipo 1/prevenção & controle , Linhagem , Reação em Cadeia da PolimeraseRESUMO
We have identified two genes associated with the hypodermal cell shape changes that occur during elongation of the Caenorhabditis elegans embryo. The first gene, called let-502, encodes a protein with high similarity to Rho-binding Ser/Thr kinases and to human myotonic dystrophy kinase (DM-kinase). Strong mutations in let-502 block embryonic elongation, and let-502 reporter constructs are expressed in hypodermal cells at the elongation stage of development. The second gene, mel-11, was identified by mutations that act as extragenic suppressors of let-502. mel-11 encodes a protein similar to the 110- to 133-kD regulatory subunits of vertebrate smooth muscle myosin-associated phosphatase (PP-1M). We suggest that the LET-502 kinase and the MEL-11 phosphatase subunit act in a pathway linking a signal generated by the small GTP-binding protein Rho to a myosin-based hypodermal contractile system that drives embryonic elongation. LET-502 may directly regulate the activity of the MEL-11 containing phosphatase complex and the similarity between LET-502 and DM-kinase suggests a similar function for DM-kinase.
Assuntos
Caenorhabditis elegans/genética , Tamanho Celular , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Alelos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans , Clonagem Molecular , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Fosfatase de Miosina-de-Cadeia-Leve , Miotonina Proteína Quinase , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Quinases Associadas a rhoRESUMO
We have investigated whether the RNA polymerase III-driven transcription of eukaryotic tRNA genes can be regulated by the prokaryotic tetracycline operator-repressor system. The bacterial tet operator (tetO) was inserted at two different positions (-7 and -46) upstream of a tRNA(Glu) (amber) suppressor gene. Both constructs are transcribed in Saccharomyces cerevisiae and yield functional tRNAs as scored by suppression of an amber nonsense mutation in the met8-1 allele. Controlled expression of Tet repressor was achieved by fusing the bacterial tetR gene to the yeast gal1 promoter. This leads to expression of Tet repressor in yeast on galactose--but not on glucose--containing media. Regulation of the su-tRNA gene with the tetO fragment inserted at position -7 has been demonstrated. Under conditions which allow tetR expression, cells exhibit a met- phenotype. This methionine auxotrophy can be conditionally reverted to prototrophy by adding tetracycline. However, a su-tRNA gene with the tetO fragment inserted at position -46 cannot be repressed. Our results demonstrate clearly that the bacterial repressor protein binds to its operator in the yeast genome. Formation of this complex in the vicinity of the pol III transcription initiation site reduces the level of su-tRNA at least 50-fold as concluded from quantitative primer extension analyses. This indicates for the first time that class III gene expression can be regulated by a DNA binding protein with its target site in the 5'-flanking region and that a prokaryotic repressor can confer regulation of a suitably engineered tRNA gene.
Assuntos
Regulação Fúngica da Expressão Gênica , Genes Bacterianos , Genes Supressores , Óperon , RNA Polimerase III/metabolismo , RNA de Transferência de Ácido Glutâmico/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Tetraciclina/farmacologia , Transcrição Gênica , Alelos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Dictyostelium/genética , Dados de Sequência Molecular , Fenótipo , Plasmídeos , RNA de Transferência/isolamento & purificação , Mapeamento por Restrição , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologiaRESUMO
Each of 22 amino acids in the proposed alpha-helix-turn-alpha-helix operator binding motif of the Tn10 encoded Tet repressor was replaced by alanine and one residue was replaced by valine to determine their role in tet operator recognition by a 'loss of contact' analysis with 16 operator variants. One class of amino acids consisting of T27 and R28 in the first alpha-helix and L41, Y42, W43 and H44 in the recognition alpha-helix are quantitatively most important for wild-type operator binding. These residues are probably involved in the structural architecture of the motif. A second class of residues is quantitatively less important for binding, but determines specificity by forming base pair specific contacts to three positions in tet operator. This property is most clearly demonstrated for Q38 and P39 and to a lesser extent for T40 at the N-terminus of the recognition alpha-helix. The contacted operator base pairs indicate that the N-terminus of the recognition alpha-helix is located towards the palindromic center in the repressor-operator complex. Although the orientation of the recognition alpha-helix in the Tet repressor-tet operator complex is inversed as compared with the lambda- and 434 repressor-operator complexes, the reduced operator binding of the TA27 mutation in the first alpha-helix suggests that the hydrogen bonding networks connecting the two alpha-helices may be similar in these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aminoácidos/genética , Regiões Operadoras Genéticas , Proteínas Repressoras/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , DNA Bacteriano/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Mutação , Plasmídeos , Conformação Proteica , Fatores R/genética , Especificidade por SubstratoRESUMO
We have constructed a genetic assay which selects positively for a functional interaction between Tet repressor and its cognate operator in Escherichia coli. In this strain Tet repressor blocks expression of lacI and lacZ. This leads to derepression of a lacPO controlled galK gene. The strain can be selected by growth on galactose as the sole carbon source and screened for the beta-galactosidase phenotype. These features allow the identification of one candidate among 10(8) false clones on a single plate. The assay was applied to select mutants with a ts DNA binding phenotype and to screen oligonucleotide generated Tet repressor mutants. Analysis of these mutations revealed that they affect DNA and inducer binding and possibly the dimerization domains. These mutations are located at residues 21, 48, 49, 89 and at the C terminus of the protein (193), respectively.
Assuntos
Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Escherichia coli/genética , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Dados de Sequência Molecular , Mutagênese , Plasmídeos , Proteínas Repressoras/genética , TemperaturaAssuntos
DNA Bacteriano/metabolismo , DNA/metabolismo , Fatores R , Proteínas Repressoras/metabolismo , Alquilação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/química , Indicadores e Reagentes , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , ÓperonRESUMO
A saturating oligonucleotide-directed mutagenesis of both tet operators in the tet regulatory sequence was performed yielding mutants with four identical base pair exchanges at equivalent positions in the four tet operator half sides. The mutants were cloned between bipolar lacZ and galK indicator genes on a multicopy plasmid allowing the quantitative analysis of their effects in vivo. In the absence of Tet repressor the mutations lead to considerably different expression levels of both genes. They are discussed with respect to the promoter consensus sequences. In particular, the -10 region of the in vivo active tetPR2 promoter is unambiguously defined by these results. In the presence of Tet repressor most of the mutants exhibit a lower affinity for that protein as determined quantitatively by their reduced expression levels. In general, tet operator recognition is most strongly affected by alterations of base pairs near the center of the palindromic sequence. The most important position is the third base pair, followed by base pairs two, four, five and six, the latter showing similar effects as base pair one. At each position, the four possible base pairs show different affinities for Tet repressor. They are discussed according to their exposure of H-bond donors and -acceptors in the major and minor grooves of the B-DNA. The results are in agreement with major groove contacts at positions two, three and five. At position four a low potential correlation of efficiencies with the H-bonding in the minor groove is found, while mutations at position six seem to influence repressor binding by other mechanisms.
Assuntos
Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido NucleicoRESUMO
Saturation mutagenesis of Tn10-encoded tet operator O1 was performed by chemical synthesis of 30 sequence variants yielding all possible point mutations of an operator half side. Their effect on Tet repressor binding was scored by an in-vivo repressor titration system. Tet repressor affinities of selected operator mutants were further characterized in vitro by dissociation rate measurements. The O1 sequence spans 19 base-pairs. Out of these, all 18 palindromic base-pairs are involved in Tet repressor recognition. The central base-pair does not contribute to sequence-specific binding of Tet repressor. At position 1 a pyrimidine residue is sufficient for maximal affinity to the repressor. At positions 2, 3 and 4, each mutation reduces repressor binding at least tenfold. Mutations at positions 5, 6, 7, 8 and 9 result in less drastic reductions of Tet repressor binding. Differential effects of mutations at a given position are used to deduce the chemical functions contacted by Tet repressor. The T.A to A.T transversion at position 9 increases Tet repressor affinity slightly, while all other mutations decrease repressor binding. The increased affinity of the wild-type tet operator O2 compared to wild-type O1 results from the addition of two favorable transversions at positions +/- 9 and an unfavorable T.A to C.G transition at position -7. Deletion or palindromic doubling of the central base-pair of the O1 palindrome reveals that the wild-type spacing of both operator half sides is crucial for efficient Tet repressor binding.
Assuntos
Mutação , Regiões Operadoras Genéticas , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Bases , Elementos de DNA Transponíveis , DNA Bacteriano , Escherichia coli/genética , Dados de Sequência MolecularRESUMO
The effect of single base pair alterations of the Tn10 encoded tet operator on recognition of Tet repressor was studied in vivo using a repressor titration system and in vitro by dissociation rate determinations of the respective complexes. Both methods reveal that the two operators, O1 and O2, which are in a tandem arrangement in the wild type, are recognized with a two-fold different affinity when separated. Studies on synthetic operator sequences indicate that the Tet repressor binds with higher affinity to the non-palindromic O2 wildtype than to the respective palindromic sequences. The in vivo repressor titration system links the expression of lacZ to the affinity of tet operator to Tet repressor. It was used to isolate tet operator mutations with reduced affinity to the repressor. The in vivo and in vitro obtained results with these mutants agree quantitatively and indicate, that the GC base pairs at positions 2, 6, and 8 are involved in interaction with the Tet repressor. Their importance for recognition decreases in that order. Transitions at position 7 of the tet operator show smaller effects on recognition than transversions.