RESUMO
We have constructed a new generation yeast artificial chromosome (YAC) library from female C57BL/10 mice in a recombination-deficient strain of Saccharomyces cerevisiae carrying a mutation in the RAD52 gene. The YAC library contains 41,568 clones with an average insert size of 240 kilobases, representing a greater than threefold coverage of the mouse genome. Currently, the library can be screened by polymerase chain reaction and we have isolated positive clones at a number of loci in the mouse genome. This rad52 library should enable a long-term assessment of the effect of one of the yeast recombination pathway genes on both, genome-wide YAC clone stability and the frequency of chimaeric clones.
Assuntos
Clonagem Molecular/métodos , Biblioteca Gênica , Saccharomyces cerevisiae/genética , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Recombinação Genética , Transformação GenéticaRESUMO
Although the tandem pore potassium channel TASK-3 is thought to open and shut at its selectivity filter in response to changes of extracellular pH, it is currently unknown whether the channel also shows gating at its inner, cytoplasmic mouth through movements of membrane helices M2 and M4. We used two electrode voltage clamp and single channel recording to show that TASK-3 responds to voltage in a way that reveals such gating. In wild-type channels, P(open) was very low at negative voltages, but increased with depolarisation. The effect of voltage was relatively weak and the gating charge small, 0.17. Mutants A237T (in M4) and N133A (in M2) increased P(open) at a given voltage, increasing mean open time and the number of openings per burst. In addition, the relationship between P(open) and voltage was shifted to less positive voltages. Mutation of putative hinge glycines (G117A, G231A), residues that are conserved throughout the tandem pore channel family, reduced P(open) at a given voltage, shifting the relationship with voltage to a more positive potential range. None of these mutants substantially affected the response of the channel to extracellular acidification. We have used the results from single channel recording to develop a simple kinetic model to show how gating occurs through two classes of conformation change, with two routes out of the open state, as expected if gating occurs both at the selectivity filter and at its cytoplasmic mouth.
Assuntos
Ativação do Canal Iônico , Potenciais da Membrana , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Citoplasma/fisiologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Técnicas de Patch-ClampRESUMO
The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity-including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes.
Assuntos
Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Interações Medicamentosas , Humanos , Modelos Moleculares , Especificidade por SubstratoRESUMO
Three CYP6Z genes are linked to a major pyrethroid resistance locus in the mosquito Anopheles gambiae. We have expressed CYP6Z2 in Escherichia coli and produced a structural model in order to examine its role in detoxification. E. coli membranes co-expressing CYP6Z2 and An. gambiae P450 reductase (AgCPR) catalysed the dealkylation of benzyloxyresorufin with kinetic parameters K(m) = 0.13 microM; K(cat) = 1.5 min(-1). The IC(50) values of a wide range of compounds were measured. Pyrethroids cypermethrin and permethrin produced low IC(50) values, but were not metabolized. Plant flavanoids were the most potent inhibitors. Several compounds were shown to be substrates, suggesting that CYP6Z2 has broad substrate specificity and plays an important chemo-protective role during the herbivorous phase of the life-cycle.
Assuntos
Anopheles/enzimologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Insetos Vetores/enzimologia , Inseticidas/farmacologia , Piretrinas/farmacologia , Laranja de Acridina , Sequência de Aminoácidos , Animais , Anopheles/genética , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , DNA/química , DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Concentração Inibidora 50 , Insetos Vetores/genética , Resistência a Inseticidas , Inseticidas/farmacocinética , Isoenzimas , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Piretrinas/farmacocinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
Chromosome aberrations have a major role in pediatric acute lymphoblastic leukemia (ALL) risk assignment. The Children's Cancer Group (CCG) and the Pediatric Oncology Group (POG) independently assessed the significance of trisomy for chromosomes 4, 10, and 17 in National Cancer Institute (NCI) Standard- and High-Risk ALL. Data from 1582 (CCG) and 3902 (POG) patients were analyzed. Eight-year event-free survivals (EFS) of 91% (CCG) and 89% (POG) (P < 0.001) were achieved in patients assigned to NCI Standard Risk whose leukemic cells had simultaneous trisomies 4, 10, and 17. Both groups showed the degree of favorable prognostic importance increased with the actual number of favorable trisomies. POG analyses also demonstrated hyperdiploidy (> or =53 chromosomes) was less of an independently significant prognostic factor in the absence of these key trisomies. This finding supported conclusions from previous CCG and POG studies that specific trisomies are more important than chromosome number in predicting outcome in pediatric B-precursor ALL. In NCI Higher Risk patients, the number of favorable trisomies was not prognostically significant, but showed the same trend. Moreover, specific trisomies 4, 10, and 17 remain associated with favorable prognosis in Standard-Risk B-precursor ALL, even in the context of very different treatment approaches between the groups.
Assuntos
Linfoma de Burkitt/genética , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 4/genética , Trissomia/genética , Anormalidades Múltiplas/genética , Adolescente , Adulto , Linfoma de Burkitt/diagnóstico , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/mortalidade , Criança , Pré-Escolar , Aberrações Cromossômicas , Intervalo Livre de Doença , Humanos , Lactente , National Institutes of Health (U.S.) , Prognóstico , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Sociedades Médicas , Trissomia/diagnóstico , Estados UnidosRESUMO
The structure of the 129-residue protein hen lysozyme has been determined in solution by two-dimensional 1H nuclear magnetic resonance methods. 1158 NOE distance restraints, and 68 phi and 24 chi 1 dihedral angle restraints were employed in conjunction with distance geometry and simulated annealing procedures. The overall C alpha root-mean-square deviation from the average for 16 calculated structures is 1.8(+/- 0.2) A, but excluding 14 residues in exposed disordered regions, this value reduces to 1.3(+/- 0.2) A. Regions of secondary structure, and the four alpha-helices in particular, are well defined (C alpha root-mean-square deviation 0.8(+/- 0.3) A for helices). The main-chain fold is closely similar to structures of the protein in the crystalline state. Furthermore, many of the internal side-chains are found in well-defined conformational states in the solution structures, and these correspond well with the conformational states found in the crystal. The general high level of definition of mainchain and many internal side-chains in the solution structures is reinforced by the results of an analysis of coupling constants and ring current shifts. Many side-chains on the surface, however, are highly disordered amongst the set of solution structures. In certain cases this disorder has been shown to be dynamic in origin by the examination of 3J alpha beta coupling constants.
Assuntos
Muramidase/química , Animais , Galinhas , Simulação por Computador , Cristalização , Feminino , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , SoluçõesRESUMO
We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints. A protocol which involved distance geometry, simulated annealing and restrained molecular dynamics was used to determine ensembles of 40 structures consistent with these restraints. The structures are found to consist of an alpha-helix packed against a four-stranded antiparallel-parallel-antiparallel beta-sheet. The structures of the two domains are compared to each other and to the reported structure of a similar domain from a protein G from a different strain of Streptococcus. We conclude that the difference in affinity of domains II and III for IgG is due to local changes in amino acid side-chains, rather than a more extensive change in conformation, suggesting that one or more of the residues which differ between them are directly involved in interaction with IgG.
Assuntos
Proteínas de Bactérias/química , Receptores de IgG/química , Streptococcus/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Hidrogênio/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Computação Matemática , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Estrutura Secundária de Proteína , Receptores de IgG/biossíntese , Receptores de IgG/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Software , Soluções/química , Streptococcus/genéticaRESUMO
The antibiotic fusidic acid and certain closely related steroidal compounds are potent competitive inhibitors of the type I variant of chloramphenicol acetyltransferase (CATI). In the absence of crystallographic data for CATI, the structural determinants of steroid binding were identified by (1) construction in vitro of genes encoding chimaeric enzymes containing segments of CATI and the related type III variant (CATIII) and (2) site-directed mutagenesis of the gene encoding CATIII, followed by kinetic characterisation of the substituted variants. Replacement of four residues of CATIII (Gln92, Asn146, Tyr168 and Ile172) by their equivalents from CATI yields an enzyme variant that is susceptible to competitive inhibition by fusidate with respect to chloramphenicol (Ki = 5.4 microM). The structure of the complex of fusidate and the Q92C/N146F/Y168F/I172V variant, determined at 2.2 A resolution by X-ray crystallography, reveals the inhibitor bound deep within the chloramphenicol binding site and in close proximity to the side-chain of His195, an essential catalytic residue. The aromatic side-chain of Phe146 provides a critical hydrophobic surface which interacts with non-polar substituents of the steroid. The remaining three substitutions act in concert both to maintain the appropriate orientation of Phe 146 and via additional interactions with the bound inhibitor. The substitution of Gln92 by Cys eliminates a critical hydrogen bond interaction which constrains a surface loop (residues 137 to 142) of wild-type CATIII which must move in order for fusidate to bind to the enzyme. Only two hydrogen bonds are observed in the CAT-fusidate complex, involving the 3-alpha-hydroxyl of the A-ring and both hydroxyl of Tyr25 and NE2 of His195, both of which are also involved in hydrogen bonds with substrate in the CATIII-chloramphenicol complex. In the acetyl transfer reaction catalysed by CAT, NE2, of His195 serves as a general base in the abstraction of a proton from the 3-hydroxyl of chloramphenicol as the first chemical step in catalysis. The structure of the CAT-inhibitor complex suggests that deprotonation of the 3-alpha-hydroxyl of bound fusidate by this mechanism could produce an oxyanion nucleophile analogous to that seen with chloramphenicol, but one which is incorrectly positioned to attack the thioester carbonyl of acetyl-CoA, accounting for the observed failure of CAT to acetylate fusidate.
Assuntos
Cloranfenicol O-Acetiltransferase/metabolismo , Ácido Fusídico/metabolismo , Esteroides/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/genética , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
The usefulness of representing an ensemble of NMR-derived protein structures by a single structure has been investigated. Two stereochemical properties have been used to assess how a single structure relates to the ensemble from which it was derived, namely the distribution of phi psi torsion angles and the distribution of chi 1 torsion angles. The results show that the minimized average structure derived from the ensemble (a total of 11 ensembles from the Brookhaven Protein Data Bank were analyzed) does not always correspond well with this ensemble, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue. An alternative method that selects the member of the ensemble which is closest to the "average" of the ensemble has been investigated (a total of 23 ensembles from the Brookhaven Protein Data Bank were analyzed). Although this method selected a structure that on the whole corresponded more closely to the ensemble than did the minimized average structure, this is still not a totally reliable means of selecting a single structure to represent the ensemble. This suggests that it is advisable to study the ensemble as a whole. A study has also been made of the practice of selecting the "best" rather than the most representative member of the ensemble. This too suggests that the ensemble should be studied as a whole. A study of the conformational space occupied by the ensemble also suggests the need to consider the ensemble as a whole, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue.
Assuntos
Proteínas/química , Animais , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Conformação Proteica , Estereoisomerismo , Termodinâmica , Difração de Raios XRESUMO
In cases where the structure of a single protein is represented by an ensemble of conformations, there is often a need to determine the common features and to choose a "representative" conformation. This occurs, for example, with structures determined by NMR spectroscopy, analysis of the trajectory from a molecular dynamics simulation, or an ensemble of structures produced by comparative modeling. We reported previously automatic methods for (1) defining the atoms with low spatial variance across an ensemble (i.e., the "core" atoms) and the domains in which these atoms lie, and (2) clustering an ensemble into conformationally related subfamilies. To extend the utility of these methods, we have developed a freely available server on the World Wide Web at http:/(/)neon.chem.le.ac.uk/olderado/. This (1) contains an automatically generated database of representative structures, core atoms, and domains determined for 449 ensembles of NMR-derived protein structures in the Protein Data Bank (PDB) in May 1997, and (2) allows the user to upload a PDB-formatted file containing the coordinates of an ensemble of structures. The server returns in real time: (1) information on the residues constituting domains: (2) the structures that constitute each conformational subfamily; and (3) an interactive java-based three-dimensional viewer to visualise the domains and clusters. Such information is useful, for example, when selecting conformations to be used in comparative modeling and when choosing parts of structures to be used in molecular replacement. Here we describe the OLDERADO server.
Assuntos
Bases de Dados Factuais , Espectroscopia de Ressonância Magnética , Proteínas/química , Redes de Comunicação de Computadores , Modelos Moleculares , Conformação Proteica , Troponina C/químicaRESUMO
We report on a girl with a de novo 6q1 interstitial deletion. To our knowledge, this is the second reported case with a deletion of 6q11-q15. We review the phenotype of monosomy 6q1. Our patient has manifestations similar to others with monosomy 6q1 including mental deficiency, growth retardation, short neck, and minor facial anomalies.
Assuntos
Anormalidades Múltiplas/genética , Deleção Cromossômica , Cromossomos Humanos Par 6 , Adulto , Pré-Escolar , Citogenética , Feminino , Humanos , Masculino , Monossomia , Gravidez , SíndromeRESUMO
We report on a 3.5-year-old girl with a mosaic karyotype including full trisomy 18, normal cells and a majority of cells with partial trisomy involving an extra chromosome 18 deleted at band q22. She had cardiac and CNS anomalies, dysmorphic facial features failure to thrive and developmental delay. A gastrostomy tube was placed at 2 years of age. The combination of improved nutrition and optimal developmental therapy has led to her sitting supported, attempting to stand and enhancement of her cognitive and non-verbal communication abilities. Molecular investigation of the patient and her parents using microsatellite analysis has led to the conclusion that, as expected, the additional copy of chromosome 18 constituting the full trisomic cell line is maternal meiosis I in origin. The data, however, indicate that in the trisomic cell line containing the deleted chromosome 18q, the structurally abnormal 18 was of paternal origin. We think this case is the first described with both structural and numerical trisomic mosaicism involving chromosome 18 in a liveborn infant. We propose a mechanism of origin and review the literature, comparing the clinical presentation of this case with individuals having full or partial trisomy 18.
Assuntos
Aberrações Cromossômicas/genética , Cromossomos Humanos Par 18/genética , Mosaicismo , Células Cultivadas , Pré-Escolar , Aberrações Cromossômicas/patologia , Bandeamento Cromossômico , Quebra Cromossômica , Deleção Cromossômica , Transtornos Cromossômicos , Análise Citogenética , Feminino , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Modelos Genéticos , TrissomiaRESUMO
Two brothers with presumed Baller-Gerold syndrome, one of whom was previously diagnosed with the association of vertebral, cardiac, renal, limb anomalies, anal atresia, tracheo-esophageal fistula (VACTERL) association with hydrocephalus, were evaluated for chromosome breakage because of severe thrombo cytopenia in one of them. Spontaneous and clastogen-induced breakage was markedly increased in both patients as compared to control individuals. Clinical manifestations and chromosome breakage, consistent with Fanconi anemia, in patients with a prior diagnosis of either Baller-Gerold syndrome, reported earlier in one other patient [Farrell et al., 1994: Am J Med Genet 50:98-99], or with VACTERL association with hydrocephalus, recently reported in 3 patients [Toriello et al., 1991: Proc Greenwood Genet Center 11:142; Porteus et al., 1992: Am J Med Genet 43:1032-1034], underline the clinical heterogeneity of Fanconi anemia and raise the question of whether these syndromes are distinct disorders or phenotypic variations of the same disease.