RESUMEN
A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.
Asunto(s)
Comovirus/inmunología , Infecciones por Parvoviridae/prevención & control , Parvovirus Canino/inmunología , Proteínas Recombinantes/uso terapéutico , Proteínas Virales/uso terapéutico , Vacunas Virales/uso terapéutico , Secuencia de Aminoácidos , Animales , Cápside/uso terapéutico , Proteínas de la Cápside , Comovirus/efectos de la radiación , Enfermedades de los Perros/prevención & control , Enfermedades de los Perros/virología , Perros , Esquemas de Inmunización , Datos de Secuencia Molecular , Infecciones por Parvoviridae/mortalidad , Infecciones por Parvoviridae/veterinaria , Parvovirus Canino/efectos de la radiación , Rayos Ultravioleta , Vacunas de Productos Inactivados/uso terapéutico , Vacunas Sintéticas/uso terapéuticoRESUMEN
alpha 4 beta 1 integrin (VLA-4) is crucial for the adhesion of leukocytes to human vascular cell adhesion molecule-1 (VCAM-1) on inflamed endothelium. This cell adhesion event is the first step in leukocyte extravasation across the blood-brain barrier in inflammatory diseases of the central nervous system (CNS) such as experimental autoimmune encephalomyelitis (EAE). Prevention of leukocyte infiltration by antibodies against the alpha 4 integrin, which block the alpha 4 beta 1 integrin/VCAM-1 interaction, have been shown to suppress clinical and pathological features of EAE. In this study, two mouse monoclonal antibodies (MAb) directed against human alpha 4 integrin were analyzed in vitro for their ability to block the interaction of leukocytes with VCAM-1 under different assay conditions. The best blocking MAb, AN100226m, was humanized by complementarily-determining region grafting, associated with human C regions and expressed. We found that modification of two structural determinants (H27 and H29) for the heavy chain CDR1 loop in one hand, and modification of framework amino acid H38, H40 and H44 in the other hand, had no effect on antigen binding. In contrast, modification of a structural determinant (H71) for the heavy chain CDR2 loop resulted in loss of binding. The humanized antibody. AN100226, was equivalent to the murine antibody. AN100226m, in binding to alpha 4 beta 1 integrin and in blocking cell adhesion. More importantly, AN100226 was as effective as AN100226m in the reversal of active EAE in guinea pigs and thus may be useful in the treatment of autoimmune diseases such as multiple sclerosis. AN100226 is currently in phase II clinical trials in the UK for the treatment of multiple sclerosis exacerbations.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Inmunoterapia , Esclerosis Múltiple/terapia , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Modelos Animales de Enfermedad , Encefalitis/inmunología , Encefalitis/terapia , Citometría de Flujo , Cobayas , Humanos , Integrina alfa4 , Células Jurkat , Células L , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Homología de Secuencia de AminoácidoRESUMEN
Mouse monoclonal antibody AUK12-20 binds to human IL-6 receptor and inhibits IL-6 functions. It has been humanized by CDR-grafting for therapeutic use. In the design of reshaped human AUK12-20 VL region, the human framework regions (FRs) from the human Bence-Jones protein REI were used. The reshaped human AUK12-20 light chain, in combination with chimeric AUK12-20 heavy chain, bound to antigen as well as chimeric AUK12-20 antibody. In the design of reshaped human AUK12-20 VH region, two sets of the human FRs were chosen and compared. One set was from the consensus amino acid sequence for human VH regions subgroup (HSG)-I and the other set was from human antibody HAX, the most similar human VH region found in a database of human immunoglobulin sequences. The HSG-I-based and the HAX-based reshaped human AUK12-20 heavy chains in combination with the reshaped human AUK12-20 light chain, showed approximately 90 and 100% antigen-binding and competition-binding activities as compared to the chimeric or mouse AUK12-20 heavy chains. Most importantly, these humanized antibodies inhibited the IL-6-dependent tumor cell growth as well as the original mouse antibody suggesting that these humanized antibodies could be efficacious in human patients. Our results show that both approaches for the design of reshaped human antibodies can be used for successful humanization. The approach based on FRs from the most similar individual human antibody, however, seemed to be best for designing a reshaped human antibody that mimicked as closely as possible the original mouse antibody.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Receptores de Interleucina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Humanos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Interleucina-6/antagonistas & inhibidores , Ratones , Datos de Secuencia Molecular , Receptores de Interleucina-6 , Células Tumorales CultivadasRESUMEN
Enhanced expression of epidermal growth factor receptor (EGFR) occurs on a variety of malignant tissues thus making anti-EGFR antibodies possible agents for the diagnosis and therapy of human tumors. Standard hybridoma technology has been used successfully to isolate anti-EGFR antibodies from immunized mice and rats. This report demonstrates that phage-antibody libraries are an alternative, and more versatile, method for isolating antibodies from immunized mice. Anti-EGFR antibodies were isolated from phage-antibody libraries constructed not only from the spleen of an immunized mouse but also from the draining lymph node of an immunized mouse and from in vitro immunized mouse cells. Two of the single-chain Fv isolated from the phage-antibody libraries were engineered to create partially humanized whole antibody molecules.
Asunto(s)
Bacteriófagos/genética , Receptores ErbB/inmunología , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Neoplasias/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Bacteriófagos/inmunología , Secuencia de Bases , Biblioteca de Genes , Humanos , Inmunización , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos CBA , Datos de Secuencia MolecularRESUMEN
Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and, therefore, has potential as a therapeutic agent in patients with IgE-mediated allergies such as hay fever, food and drug allergies and extrinsic asthma. The clinical usefulness of mouse antibodies is limited, however, due to their immunogenicity in humans. Mouse C21 antibody was humanized by complementarity determining region (CDR) grafting with the aim of developing an effective and safe therapeutic for the treatment of IgE-mediated allergies. The CDR-grafted, or reshaped human, C21 variable regions were carefully designed using a specially constructed molecular model of the mouse C21 variable regions. A key step in the design of reshaped human variable regions is the selection of the human framework regions (FRs) to serve as the backbones of the reshaped human variable regions. Two approaches to the selection of human FRs were tested: (i) selection from human consensus sequences and (ii) selection from individual human antibodies. The reshaped human and mouse C21 antibodies were tested and compared using a biosensor to measure the kinetics of binding to human IgE. Surprisingly, a few of the reshaped human C21 antibodies exhibited patterns of binding and affinities that were essentially identical to those of mouse C21 antibody.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Inmunoglobulina E/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Secuencia de Bases , Células Cultivadas , Humanos , Hipersensibilidad/tratamiento farmacológico , Regiones Constantes de Inmunoglobulina/biosíntesis , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/inmunología , Inmunoterapia Adoptiva/métodos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
We have characterized the development and expansion of CD5+ B cells in interleukin-5 (IL-5) transgenic mice in terms of autoantibody production and immunoglobulin gene usage. CD5+IL-5R alpha+ B cells maintained in the presence of IL-5 secreted fewer autoantibodies and had fewer N nucleotides at the 3' end of the D elements compared with CD5- B cells. The reduction in nucleotides, along with the finding that CD5+IL-5R alpha+ B cells in IL-5 transgenic mice use Q52 families more frequently than age-matched control B cells, also suggests that these cells have the characteristics of fetus-type B cells and represent an early stage of B-cell development. All of the VH11 families were expressed with JH1 and the Q52 families were frequently expressed with JH1. Furthermore, JH proximal DQ52 was frequently used in IL-5 transgenic mice. All of these characteristics in terms of immunoglobulin gene usage have been described for CD5+ B cells. These results suggest that IL-5 maintains CD5+ B cells that have a fetus-type of immunoglobulin gene usage. This cytokine could be responsible for prolonging the life span of immature CD5+ B cells, which subsequently mature to CD5- B cells that secrete polyreactive natural antibodies.
Asunto(s)
Linfocitos B/citología , Cadenas Pesadas de Inmunoglobulina/genética , Interleucina-5/fisiología , Animales , Antígenos CD , Autoanticuerpos/biosíntesis , Secuencia de Bases , Antígenos CD5 , Diferenciación Celular , ADN de Cadena Simple/inmunología , Regulación de la Expresión Génica , Inmunoglobulina M/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia MolecularRESUMEN
The mouse PM-1 monoclonal antibody binds to the human interleukin 6 receptor, inhibits IL-6 functions, and shows strong antitumor cell activity against multiple myeloma cells. In order to be effective as a therapeutic agent administered to human patients in repeated doses, reshaped human PM-1 antibodies consisting of human REI-based light chain and NEW-based heavy chain variable regions were designed and constructed with the assistance of a structural model of the mouse PM-1 variable regions. The best reshaped human PM-1 antibody is equivalent to mouse or chimeric PM-1 antibody in terms of antigen binding and growth inhibition against multiple myeloma cells. Only a few minor changes in the human framework regions were required to recreate the mouse PM-1 antigen-binding site within a human antibody. The reshaped human PM-1 antibody, therefore, could be efficacious in human multiple myeloma patients.
Asunto(s)
Anticuerpos Monoclonales/química , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Interleucina-6/inmunología , Receptores Inmunológicos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , División Celular , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Inmunoterapia , Interleucina-6/uso terapéutico , Ratones , Datos de Secuencia Molecular , Mieloma Múltiple/terapia , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-6 , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
We have optimized primers for cloning libraries of murine heavy and light chain variable regions using the polymerase chain reaction. Since we are interested in cloning murine Fab fragments for expression in bacterial cells, the heavy chain primers were designed to clone Fd fragments comprising the heavy chain variable domain and the first domain of the IgG constant region. The light chain primers were designed to clone the entire murine kappa chain. Using ten degenerate 5' primers and a degenerate 3' primer to amplify murine Fd and seven degenerate 5' primers with a single 3' primer to amplify kappa chains, a diverse repertoire of mouse variable regions was cloned from mouse spleens.
Asunto(s)
Clonación Molecular , Biblioteca de Genes , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Antineoplásicos/genética , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Secuencia de Bases , Sitios de Unión de Anticuerpos/genética , Línea Celular , ADN de Cadena Simple , Expresión Génica , Vectores Genéticos/genética , Humanos , Regiones Constantes de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
Mouse monoclonal antibody (mAb) 0.5 beta binds to the envelope protein gp120 of human immunodeficiency virus (HIV) and neutralizes infection by HIV in vitro. Mouse mAb 0.5 beta, therefore, has potential as a therapeutic agent for the prevention and treatment of acquired immunodeficiency syndrome (AIDS). Since mouse mAbs are highly immunogenic in humans, efforts are being made to humanize mouse mAbs that are being considered for use in humans. This report describes the design, construction, and expression of reshaped human 0.5 beta antibodies. In these antibodies, the entire constant (C) regions were derived from human sequences. The variable (V) regions were derived from human framework regions (FRs) and mouse 0.5 beta complementarity determining regions (CDRs). One version of reshaped human 0.5 beta light (L) chain and six versions of reshaped human 0.5 beta heavy (H) chain were made and tested. Following transient expression in cos cells, all of the constructions were capable of producing humanlike antibody. Three of the H chain constructions (RHc, RHe, and RHf), when co-expressed with the L chain construction (RL), produced reshaped human antibody capable of binding to the epitope on gp120 recognized by mouse 0.5 beta mAb. The best version (RL + RHe) of reshaped human 0.5 beta antibody had both binding affinity and neutralizing activity that were within twofold that of the mouse or chimeric 0.5 beta antibody.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Proteína gp120 de Envoltorio del VIH/inmunología , VIH/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Secuencia de Bases , Unión Competitiva , Humanos , Datos de Secuencia Molecular , Pruebas de NeutralizaciónRESUMEN
Expression systems for producing foreign proteins in mammalian cells are built from two components. One component is the DNA expression vector and the other is the mammalian host-cell line. Functional elements of DNA are better understood and generally easier to manipulate than complex mammalian cells. The standard approach, therefore, has been to manipulate the expression vectors to work well in convenient host cell lines. A wide variety of DNA regulatory signals for efficient transcription and translation have been tested in mammalian-cell-expression vectors. Many of the most successful and widely used regulatory signals are derived from eukaryotic viral DNAs. In addition to optimizing the vectors to give efficient transcription and translation of the foreign protein, higher expression levels can be achieved by increasing the number of foreign gene copies per cell. High gene copy number is usually attained by including an amplifiable gene, such as dhfr, in the expression vector, introducing the vector DNA into the host-cell lines, and then using a toxic agent to select for resistant cell lines containing high copy numbers of the amplifiable gene. Cells with amplified copy numbers of the selected gene generally also contain high copy numbers of the foreign gene and thus produce elevated levels of the foreign protein product. Although gene amplification has been most successful in creating cell lines producing high levels of foreign proteins, there are inherent instability problems with cell lines forced to carry extremely high copy numbers of foreign genes. One means of avoiding instability problems due to continuous high gene copy and continuous high foreign protein production, is to develop regulatable expression systems. The regulatable expression systems being developed are based either on regulating the gene copy number by regulating DNA replication or on regulating the level of transcription by using a regulatable promoter to transcribe the foreign protein coding cDNA. In addition to designing a good expression vector, it is important to consider the mammalian host cell. Although most potential mammalian host-cell lines are capable of post-translational processing and secretion, certain processing steps, such as gamma-carboxylation, may be done efficiently only in specialized cell types. It is also important to estimate how much of the foreign protein will be needed and to decide whether the proposed host-cell line can be easily and economically grown to produce that amount. Regulatory considerations are also important in choosing a host-cell line for commercial production. Many of the potential host-cell lines are tumorigenic and carry retroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Proteínas Recombinantes/biosíntesis , Animales , Células Cultivadas , Expresión Génica , Regulación de la Expresión Génica , Ingeniería Genética , Vectores Genéticos , Humanos , Biosíntesis de Proteínas , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
The promoters from Drosophila and human 70,000-dalton heat shock protein (hsp70) genes were linked to human tissue plasminogen activator (tPA) cDNA. Mouse C127 cells were transformed with bovine papilloma virus (BPV) vectors carrying the hybrid hsp70/tPA genes. Stable BPV-transformed cell lines were selected and analyzed for tPA expression before and after heat shock. In most cell lines, there was a low level of tPA production even in the absence of heat shock or other obvious stress. After heat shock (42 degrees C, 2 hr), there was up to a 40-fold increase in tPA production. Production of tPA protein occurred within the first 5 h after the heat shock and was due to a burst of hsp70/tPA transcription during the heat shock. The hsp70/tPA transcripts appeared to have a short half-life. Thus, stable mouse cell lines carrying hsp70/tPA hybrid genes can be induced by a short heat shock to transcribe high levels of hsp70/tPA mRNAs and, subsequently, to produce elevated levels of tPA protein.
Asunto(s)
Proteínas de Choque Térmico/genética , Regiones Promotoras Genéticas , Activador de Tejido Plasminógeno/genética , Animales , Papillomavirus Bovino 1 , Línea Celular , Transformación Celular Viral , ADN Recombinante , Regulación de la Expresión Génica , Calor , Ratones , Plásmidos , Proteínas/genética , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
Xenopus oocytes are versatile cells capable of carrying out many post-translational processes. Although previously reported not to be capable of C-terminal amidation, this report demonstrates that Xenopus oocytes do indeed have an amidating enzyme. The amidating activity from Xenopus ovaries is compared to the known amidating activity found in porcine pituitaries. The demonstration of C-terminal amidation by Xenopus oocytes extends their usefulness in studying post-translational events.
Asunto(s)
Oocitos/enzimología , Procesamiento Proteico-Postraduccional , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Ovario/enzimología , Hipófisis/enzimología , Porcinos , Xenopus laevisRESUMEN
We have adapted the fibrin overlay assay for plasminogen activators (Jones et al., 1975) into a gene transfer expression assay which has the advantage of being very sensitive and nondestructive. In this assay plasminogen activators convert plasminogen to plasmin, which then degrades fibrin, resulting in clearings in a fibrin overlay. Furthermore, the assay can be used as a signal indicating the efficiency of gene transfer or the loss of introduced genetic elements in unstable cell lines.
Asunto(s)
Genes , Activadores Plasminogénicos/genética , Transfección , Acetiltransferasas/genética , Virus del Sarcoma Aviar/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa , ADN/metabolismo , Fibrina/metabolismo , Humanos , Plásmidos , Activadores Plasminogénicos/metabolismo , Regiones Promotoras Genéticas , Virus 40 de los Simios/genéticaRESUMEN
We have isolated a polyomavirus with 134 base pairs of foreign DNA between the origin of replication and the early promoter. The insertion reduces the infectivity of the virus by interfering with events required for the initiation of infection. mRNA transcripts from the early region exhibit a marked heterogeneity of 5' termini.
Asunto(s)
ADN Viral/genética , Poliomavirus/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Mapeo Cromosómico , Mutación , ARN Viral/genética , Transcripción Genética , Replicación ViralRESUMEN
The Xenopus laevis alpha 1- and beta 1-globin genes were injected into oocytes and unfertilized eggs of X. laevis. In oocytes, the injected globin genes were actively transcribed, but the majority of the transcripts were incorrectly initiated. In unfertilized eggs, the injected genes were transcribed at a low level but only from the correct start sites. In oocytes, the injected circular plasmid DNA containing the cloned globin genes persisted but did not replicate. In contrast, DNA injected into unfertilized eggs replicated up to 15-fold within a 22-h period. We suggest that the ability of the egg to selectively transcribe the injected X. laevis globin genes from the correct promoter sites may be related to differences in chromatin structure between the oocyte and the unfertilized egg.
Asunto(s)
Globinas/genética , Oocitos/metabolismo , Transcripción Genética , Amanitinas/farmacología , Animales , Afidicolina , ADN/metabolismo , Replicación del ADN , Diterpenos/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Microinyecciones , Óvulo/metabolismo , Factores de Tiempo , Xenopus laevisRESUMEN
Using previously cloned Xenopus nucleosomal core histone genes as hybridization probes, a genomic DNA library of Xenopus laevis was screened for histone gene clusters. From over 200 histone-gene containing clones identified, 36 were selected as possibly containing H1 histone genes by hybridization to a probe derived from a sea urchin H1 histone gene. These 36 clones were further analyzed by hybrid-selected translation for the definitive presence of H1 histone genes. The genes for three different H1 histone variants were found: H1A , H1B and H1C . Mapping of the histone genes within each clone showed that at least three different gene arrangements can occur within a cluster and that the type of H1 histone variant present in a cluster may be related to the cluster type. S1-mapping experiments indicated that histone genes found in different cluster-types can be expressed in oocytes. Also, the H1 gene found in one cluster-type was expressed in at least three different cell-types: oocytes, gastrula-stage embryos, and erythroblasts.
Asunto(s)
Genes , Variación Genética , Histonas/genética , Transcripción Genética , Animales , Clonación Molecular , Enzimas de Restricción del ADN , Vectores Genéticos , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genética , XenopusRESUMEN
Xenopus laevis tadpole and adult beta-globin genes were injected into fertilized X. laevis eggs. Both injected genes replicated and were retained in the developing embryos with equal efficiency. Transcripts of the injected adult gene were detectable at gastrulation and reached a maximum level shortly thereafter. In contrast, transcripts of the injected tadpole gene were not detected until much later stages of development. The level of expression of both the injected genes was low compared with the level of expression of the chromosomal genes during erythropoiesis.