RESUMEN
We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. which rely on PCR.
Asunto(s)
Etiquetas de Secuencia Expresada , Expresión Génica , Adulto , ADN Complementario , Escherichia coli , Genoma Humano , Humanos , Pulmón/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Programas InformáticosRESUMEN
A fusion protein construct consisting of the short form of human fibroblast growth factor (FGFR) fused to the heavy chain of mouse IgG1 was used to screen four phage display libraries displaying 8, 13, 38 and 43 amino acids at the amino terminus of the bacteriophage M13 gene III minor coat protein. Phage with specific FGFR binding activity were isolated from the 13, 38 and 43 mer libraries. One of the highest affinity phage clones from the 13mer library was chosen to be further evolved by oligonucleotide saturation mutagenesis. We have isolated evolved sequences that have approximately 8 times the relative binding affinity of the parent sequence. The phage clones have a minimum consensus sequence of CR/SXLLXGAPFXXXXC, where X represents positions tolerant of several amino acids. A synthetic peptide based on this sequence specifically inhibits FGF from binding to its receptor in an in vitro ELISA.
Asunto(s)
Bacteriófagos/genética , Receptores de Factores de Crecimiento de Fibroblastos/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Clonación Molecular , ADN , Ensayo de Inmunoadsorción Enzimática , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Inmunoglobulina G/genética , Ratones , Datos de Secuencia Molecular , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Cyclic peptides capable of activating the erythropoietin receptor (EPOR) were isolated from phage display libraries by screening with a novel EPOR-IgG fusion protein reagent. A parental clone ERB1 (EPO Receptor Binder 1) was first isolated from a phage display library displaying 38 random amino acids as an N-terminal fusion with the M13 minor capsid protein, pill. An evolved library was then produced from the parental sequence using an oligonucleotide saturation mutagenesis strategy which yielded EPOR binding sequences with 20 times the relative affinity of ERB1. Two synthetic peptides were constructed from these sequences both of which bind the EPO receptor in specific ELISA, and act as full agonists in EPO dependent cell proliferation assays. These peptides are 18 amino acids in length, disulfide-bonded, and have a minimum consensus sequence of CXXGWVGXCXXW, where X represents positions tolerant of several amino acids.
Asunto(s)
Bacteriófago M13/genética , Péptidos/aislamiento & purificación , Receptores de Eritropoyetina/agonistas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Clonación Molecular , Eritropoyetina/química , Eritropoyetina/metabolismo , Humanos , Imitación Molecular , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Receptores de Eritropoyetina/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
We have developed a cloning vector for the expression of type I cytokine receptor, NO, extracellular domain (ECD)-mouse IgG1 Fc fusion proteins. The vector has a versatile polylinker that allows in-frame cloning of the receptor ECD with the mouse IgG1 sequence to encode a receptor ECD-IgG1 fusion construct. The receptor-IgG1 fusion proteins are transiently expressed in useful amounts following transfection of the expression vector into COS7 cells and G418 selection. The mouse IgG1 portion of the fusion protein provides a universal handle for purification on an affinity matrix and detection by anti-mouse IgG antibodies in ELISA or Western blot formats. The expressed receptor ECD-IgG1 fusion proteins bind their cognate ligands. In order to demonstrate that the fusion proteins have similar ligand binding affinities as the native receptors, the affinity constants (Kd) for EPOR, TNFR, IL-4R, and IL-6R-IgG1 fusion proteins were measured by surface plasmon resonance and shown to be in good agreement with published values. The TNFR-IgG1 fusion protein was employed in a demonstration of a novel ELISA format for detecting cytokine receptor binding to cytokine.
Asunto(s)
Inmunoglobulina G/genética , Receptores de Citocinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Técnicas Biosensibles , Células COS , Cartilla de ADN/genética , Expresión Génica , Vectores Genéticos , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/metabolismo , Técnicas In Vitro , Cinética , Ligandos , Ratones , Datos de Secuencia Molecular , Receptores de Citocinas/aislamiento & purificación , Receptores de Citocinas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
We have constructed two phage display libraries expressing N-terminal pIII fusions in M13 composed of 37 and 43 random amino acid domains, respectively. The D38 library expresses 37 random amino acids with a central alanine residue, and the DC43 library contains 43 random amino acids with a central cysteine flanked by two glycine residues, giving the displayed peptide the potential to form disulfide loops of various sizes. We demonstrate that the majority of random sequences in both libraries are compatible in pentavalent display with phage viability. The M13 phage display vector itself has been engineered to contain a factor Xa protease cleavage site to provide an alternative to acid elution during affinity selection. An in-frame amber mutation has been inserted between the pIII cloning sites to allow for efficient selection against nonrecombinant phage in the library. These libraries have been panned against mAb 7E11-C5, which recognizes the prostate-specific membrane antigen (PSM). Isolated phage display a consensus sequence that is homologous to a region in the PSM molecule.
Asunto(s)
Bacteriófago M13/genética , Evolución Molecular Dirigida/métodos , Biblioteca de Péptidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , ADN/genética , Cartilla de ADN/genética , Variación Genética , Vectores Genéticos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
M proteins, the major virulence factor of group A streptococci, have been implicated in the pathogenesis of acute rheumatic fever (ARF) and other streptococcal related autoimmune diseases. A 22-kD fragment of M type 5 protein is a potent stimulant of human T cells and has recently been shown by our laboratory to belong to the newly designated family of superantigens. Using flow cytometry and the polymerase chain reaction, we demonstrate that this molecule reacts with subsets of human T cells expressing specific T cell receptor (TCR) V beta elements, namely V beta 2, 4, and 8. We employed similar techniques to analyze the TCR V alpha usage of pep M5-stimulated T cells. These studies revealed that the preferential usage of particular V alpha elements is not specific for the superantigen; rather, it may reflect the repertoire of the individual being tested. The expansion of a large number of T cells bearing specific TCR V beta sequences by M protein may account for its role in mediating the pathogenesis of post-streptococcal diseases. Furthermore, the preferential usage of TCR V alpha elements in certain individuals may be an important factor that predisposes them to development of self-reactivity.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/farmacología , Proteínas Portadoras , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Antígenos Bacterianos , Secuencia de Bases , Células Cultivadas , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN/genética , ARN/aislamiento & purificación , Linfocitos T/efectos de los fármacosRESUMEN
The nucleotide sequence of the human procollagen alpha 1(II) collagen gene extending from within the first intron through exon 15, and part of the 15th intron has been determined. This sequence analysis (7056 bases) identifies the intron/exon organization of the region of this gene encoding the N-propeptide and part of the triple-helical domain. Structural comparison of this with the genes of other human fibrillar collagens shows considerable diversity in terms of size and number of introns and exons that encodes the N-propeptide domain. Although the genomic structure of the human procollagen alpha 1(II) gene is quite different from the rat procollagen alpha 1(II) gene, the nucleotide coding sequences are 89% identical.
Asunto(s)
Genes , Procolágeno/genética , Secuencia de Bases , Clonación Molecular , Colágeno/genética , Exones , Vectores Genéticos , Humanos , Intrones , Datos de Secuencia Molecular , Plásmidos , Mapeo RestrictivoRESUMEN
Collagen-induced arthritis (CIA) is a rodent arthritis model in which immunization with heterologous type II collagen induces an inflammatory polyarthritis. Susceptibility to the disease is mediated by major histocompatibility complex (MHC) genes as well as genes at other loci. Previous studies of the SWR/J mouse strain, which is resistant to CIA despite bearing the susceptible H-2q haplotype, have suggested that this resistance is the result of a deletion of T-cell receptor (Tcr) Vb gene segments which is carried by this strain. Other studies have implicated a deficiency in complement component C5 as the cause for the resistance. In order to assess the relative importance of these two genes in susceptibility to CIA, and to provide an estimate of the number of independent genes involved in the disease, we analyzed 196 F2 progeny of a (DBA/1 x SWR/J) cross for arthritis susceptibility, and expression of both C5 and Tcr genes. Thirty of the F2 progeny developed arthritis. All of the arthritic mice had at least one copy of the wild-type C5 allele, while the Tcr-Vb haplotypes were distributed in Mendelian fashion. These results demonstrate that C5 sufficiency is an absolute requirement for CIA, but that Tcr-Vb genes located within the SWR deletion have little influence. Genetic analysis of the incidence rate suggests that there is polygenic control of susceptibility to CIA and that in addition to H-2, 5-6 other independent loci (including C5) may be involved.
Asunto(s)
Artritis/genética , Complemento C5/genética , Receptores de Antígenos de Linfocitos T/genética , Análisis de Varianza , Animales , Artritis/inducido químicamente , Artritis/inmunología , Deleción Cromosómica , Colágeno , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Predisposición Genética a la Enfermedad , Genotipo , RatonesRESUMEN
Strains of laboratory mice bearing a germline deletion of some T cell receptor V beta genes have proven useful in a variety of studies of T cell receptor function. Analysis of genetic crosses between deleted and wild type strains can provide information about the relevance of genes located within the deletion to specific T cell responses. Existing techniques for genotyping offspring of such crosses usually involve flow cytometric analysis which may not be available to all laboratories. Recent nucleotide sequence data indicate the presence of two restriction enzyme site polymorphisms in the closely linked V beta 1 gene which discriminate between deleted and wild-type strains. Amplification of a DNA segment containing the diagnostic sites by polymerase chain reaction followed by restriction enzyme digestion of the product offers a simple and rapid method for genotyping animals.
Asunto(s)
Técnicas Genéticas , Genotipo , Haplotipos/genética , Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T/genética , Animales , Secuencia de Bases , Sangre , ADN , Electroforesis en Gel de Agar , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo RestrictivoRESUMEN
We have identified and sequenced a cDNA encoding human neutrophil collagenase from a lambda gt11 cDNA library constructed from mRNA extracted from the peripheral leukocytes of a patient with chronic granulocytic leukemia. The library was screened with an oligonucleotide probe constructed from the putative zinc-binding region of fibroblast collagenase. Eleven positive clones were identified, of which the one bearing the largest insert (2.2 kilobases (kb)) was sequenced. From the nucleotide sequence of the 2.2-kb cDNA clone we have deduced a 467-amino acid sequence representing the entire coding sequence of the enzyme. The deduced protein was confirmed as neutrophil collagenase by conformity with the amino-terminal sequence analyses of three tryptic peptides of purified neutrophil collagenase. The cDNA clone hybridizes to a 3.3-kb mRNA present in RNA extracted from human bone marrow but did not hybridize with RNA isolated from U937 cells induced to differentiate with phorbol myristate acetate. Neutrophil collagenase was found to possess 57% identity with the deduced protein sequence for fibroblast collagenase with 72% chemical similarity. Certain regions of the molecule, including the putative zinc-binding region, are highly conserved. When compared with the published sequence for fibroblast collagenase, neutrophil collagenase contains four additional sites for glycosylation. Medium from COS-7 cells transfected with a pcDNA1 eucaryotic expression vector containing cDNA for neutrophil collagenase degraded type I collagen into the three-quarter, one-quarter fragments characteristic of mammalian interstitial collagenase activity. Thus, definitive evidence based on the cDNA sequence confirms the neutrophil collagenase is a distinct gene product and a member of the family of matrix metalloproteinases.
Asunto(s)
Genes , Metaloendopeptidasas/genética , Colagenasa Microbiana/genética , Neutrófilos/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Biblioteca de Genes , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Colagenasa Microbiana/sangre , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
We have reviewed the relationship between C4 and its related blood group and discussed the mechanisms whereby a fragment of C4 could become attached to erythrocytes (E). We hypothesize that there is chronic fluid-phase activation of C4 by either C1 to form C4b or spontaneous cleavage of the thioester to form iC4. These activated molecules bind to E. Proteolytic degradation of the bound C4b or iC4 would leave a covalently attached fragment of C4 on E and thereby give rise to the Ch and Rg blood group antigens. This system is of further immunopathologic interest since this 'normal' activation or turnover of C4 is closely regulated. In patients deficient in regulatory proteins, this spontaneous or normal turnover of C4 and C3 may initiate a pathologic condition.
Asunto(s)
Antígenos de Grupos Sanguíneos , Complemento C4 , Animales , Antígenos de Grupos Sanguíneos/inmunología , Activación de Complemento , Complemento C4/genética , Complemento C4/inmunología , Complemento C4/metabolismo , Eritrocitos/inmunología , Humanos , Fragmentos de Péptidos/metabolismo , Receptores de Complemento/metabolismo , Receptores de Complemento 3bRESUMEN
The molecular analysis of crossing-over within the mouse major histocompatibility complex provides a useful approach for the study of the structural characteristics of meiotic recombination. In this study five intra-I-region recombinants, each derived from Ik/Ib heterozygotes, were characterized for restriction-fragment length polymorphisms (RFLPs) characteristic of the I region of the two parental strains. Southern blot analysis of intra-I recombinant strains A.TBR2, A.TBR3, A.TBR5, A.TBR13, and A.TBR17 using six I-region DNA probes revealed that the point of crossing-over in all five recombinants occurred within a 6.2-kb KpnI-EcoRI segment located within the E beta gene. The segments of DNA containing the crossover point from each of the recombinant chromosomes were cloned by screening partial genomic libraries constructed in lambda gt7 bacteriophage. Construction of partial restriction maps of the cloned segments from the parental and recombinant chromosomes permitted the boundaries of the area containing the crossover site to be narrowed to a 4.0-kb segment located almost entirely within an intron of the E beta gene. The recognition that the points of crossing-over in all five recombinants studied are clustered in a relatively small area of the I region provides further evidence for a hot spot of recombination associated with the E beta gene.
Asunto(s)
Intercambio Genético , Genes MHC Clase I , Meiosis , Animales , Mapeo Cromosómico , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Hibridación de Ácido Nucleico , Polimorfismo de Longitud del Fragmento de Restricción , Recombinación GenéticaRESUMEN
To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.
Asunto(s)
Insulina/inmunología , Ratones Endogámicos C57BL/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Línea Celular , Ratones , Ratones Endogámicos C57BL/genética , Fragmentos de Péptidos/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta , Homología de Secuencia de Ácido NucleicoRESUMEN
Fifteen independently isolated complementary DNA clones that contain T-cell receptor (TCR) V beta genes were sequenced and found to represent 11 different V beta genes. When compared with known sequences, 14 different V beta genes could be defined from a total of 25 complementary DNA's; 11 clones therefore involved repeated usage of previously identified V beta's. Based on these data, we calculate a maximum likelihood estimate of the number of expressed germline V beta genes to be 18 with an upper 95 percent confidence bound of 30 genes. Southern blot analysis has shown that most of these genes belong to single element subfamilies which show very limited interstrain polymorphism. The TCR beta-chain diversity appears to be generated from a limited V beta gene pool primarily by extensive variability at the variable-diversity-joining (V-D-J) junctional site, with no evidence for the involvement of somatic hypermutation.
Asunto(s)
Variación Genética , Receptores de Antígenos de Linfocitos T/genética , Alelos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN , Pool de Genes , Humanos , Hibridomas , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C/genética , Ratones Endogámicos C57BL/genética , Ratones Endogámicos/genética , Especificidad de la Especie , Bazo , Linfocitos T , TimoAsunto(s)
Antígenos H-2/genética , Complejo Mayor de Histocompatibilidad , Ratones/genética , Alelos , Animales , Mapeo Cromosómico , Conversión Génica , Ligamiento Genético , Variación Genética , Antígenos de Histocompatibilidad Clase II/genética , Ratones/inmunología , Ratones Endogámicos , Recombinación GenéticaRESUMEN
A C4-specific monoclonal antibody has been developed that allows the rapid purification of C4 from whole serum or plasma by affinity chromatography. 1- to 2-ml volumes of serum are loaded onto a small column of antibody-coupled agarose beads and washed through with buffer. C4 is eluted by adjusting the pH to 11.2. The purified C4 is free of extraneous proteins as detected by SDS-polyacrylamide gel electrophoresis and retains high activity as assessed by hemolytic assay and incorporation of [14C]-methylamine. Columns are reusable, and the entire procedure can be adapted for large-scale purifications.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Complemento C4/inmunología , Cromatografía de Afinidad/métodos , Complemento C4/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Hemólisis , Humanos , Técnicas de InmunoadsorciónAsunto(s)
Formación de Anticuerpos , Complemento C4/genética , Complemento C4b , Antígenos H-2/genética , Alotipos de Inmunoglobulinas/genética , Fragmentos de Péptidos/genética , Animales , Cruzamientos Genéticos , Ensayo de Inmunoadsorción Enzimática , Variación Genética , Pruebas de Hemaglutinación , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Endogámicos , Especificidad de la EspecieRESUMEN
A previous study has shown that prolonged immunization of strain A.TBR16 mice (H-2 haplotype at16) with lymphoid tissue derived from A.TBR13 (at13) donors results in an antiserum that defines a new H-2-associated allotype with a strain distribution antithetical to H-2.7. The present report describes a sensitive ELISA that demonstrated that this specificity represents an allotypic determinant(s) on the fourth component of complement (C4). Studies with Sephadex G-200 fractionated plasma and serum proteins suggest that, as is the case for H-2.7, the new specificity appears to reside on the C4d fragment after C activation. We therefore propose the tentative nomenclature C4d.2 for this specificity and suggest revision of the nomenclature for H-2.7 to C4d.1. Also described in this report is the formal mapping of the genetic control of the C4d.2 allotype to the H-2S region.