RESUMEN
A 'sandwich' enzyme-linked immunosorbent assay has been developed for measuring humanized anti-Tac (HAT), a humanized antibody to the IL-2 receptor on activated T cells (Tac), in human serum. The working range of this assay is 25-400 ng/ml with an overall precision of 5%. In this assay, the analyte, HAT, is sandwiched between Tac which is bound to a microtiter plate and biotinylated Tac that is conjugated to peroxidase labelled streptavidin. This assay was utilized to determine the pharmacokinetic parameters of HAT in patients with graft-versus-host disease.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Anticuerpos Monoclonales/inmunología , Biotina/química , Trasplante de Médula Ósea/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedad Injerto contra Huésped/inmunología , Humanos , RatonesRESUMEN
A rabbit serum raised against Eimeria tenella merozoites was used to screen a lambda gt11 cDNA library made from merozoite mRNA of E. tenella. The insert of the phage clone lambda Mz 5-7 revealed an open reading frame consisting of 945 nucleotides, encoding a 33-kDa protein. This size is consistent with the size of a protein translated in vitro from merozoite mRNA and immunoprecipitated with monospecific anti-Mzp 5-7 antibodies. A smaller protein of 24 kDa, located on the surface of the parasite, also reacted with the monospecific antiserum and is the potential processed form of the Mzp 5-7. Furthermore, a recombinant vaccinia virus expressing the Mzp 5-7 antigen was constructed and used to immunize chickens.
Asunto(s)
Antígenos de Protozoos/biosíntesis , Eimeria tenella/metabolismo , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Escherichia coli , Biblioteca de Genes , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Plásmidos , Biosíntesis de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Recombinación Genética , Mapeo Restrictivo , Transfección , Virus Vaccinia/genéticaRESUMEN
The cytoplasmic, poly(A)-containing RNA species complementary to all regions of the adenovirus type 2 (Ad2) genome expressed before the onset of viral DNA synthesis have been examined by "Northern" blotting. HeLa cells infected with low multiplicities of Ad2 were harvested at 2-hr intervals until late in the infectious cycle to establish the temporal patterns of expression of each viral gene. Under these conditions, such late mRNA products as those of the L3 and L5 families were not detected until 14 hr after infection. Although individual mRNA species complementary to several genes showed different patterns of expression, the order of appearance in the cytoplasm of substantial concentrations of adenoviral mRNA species was E1A, E3, and E4 (4 to 6 hr), E2A and E2B (8 hr), 3.7- and 4.1-kb L1 mRNA species (10-12 hr), IX and IVa2 mRNAs (12 hr), and those encoded in the major late transcriptional unit, such as members of the L3 and L5 families (14 hr). The mRNA species encoding polypeptides IX and IVa2 were not produced when viral DNA synthesis was blocked, whereas the larger L1 mRNA species was made under these conditions. Two E2B mRNA species, some 5.0 and 7 kb, were observed at low concentrations at 8 hr after infection and their concentration increased until 24 hr after infection, as did that of the E2A mRNA species: the products of the E2 transcription unit appeared to be expressed coordinately and at a constant ratio throughout infection. Few of the early mRNA species decreased in concentration after the onset of the late phase of infection. Examination of the viral mRNA produced when protein synthesis was inhibited by 10 microM anisomycin added 3 hr after infection suggested that processing of certain viral, early transcripts was altered in the presence of the drug.
Asunto(s)
Adenovirus Humanos/genética , Transformación Celular Viral , Genes Virales , ARN Mensajero/genética , Clonación Molecular , ADN/análisis , Replicación del ADN , Enzimas de Restricción del ADN , Electroforesis en Gel de Poliacrilamida , Células HeLa/fisiología , Humanos , Peso Molecular , Hibridación de Ácido Nucleico , Poli A/genética , Poli A/aislamiento & purificación , ARN/genética , ARN/aislamiento & purificaciónRESUMEN
The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.
Asunto(s)
Adenovirus Humanos/genética , Transformación Celular Viral , Desoxirribonucleoproteínas/genética , Genes Virales , Nucleoproteínas/genética , Precursores de Proteínas/genética , Proteínas Virales/genética , Humanos , Hibridación de Ácido Nucleico , ARN Mensajero/genética , ARN Viral/genéticaRESUMEN
The virus-specific RNA sequences synthesized in nuclei isolated from adenovirus type 2-infected HeLa cells comprise a fraction of the total RNA similar to that observed with RNA made in vivo. By 16 h after infection, for example, some 25% of the RNA made in isolated nuclei is transcribed from adenoviral DNA. Only 10 to 15% of the adenoviral RNA sequences synthesized in nuclei isolated during the late phase of infection are transcribed by form III RNA polymerase. This RNA, whose synthesis is resistant to 0.5 microgram/ml, but sensitive to 200 microgram/ml of alpha-amanitin, sediments at about 5 S in denaturing sucrose gradients and must, therefore, represent the virus-associated RNAs. The remainder of the sequences are transcribed by form II RNA polymerase and sediment as several RNA species in denaturing sucrose gradients. The largest of these exhibits the size, 55 to 60 S, expected of a complete transcript of the major, adenoviral transcriptional unit expressed during the late phase. Hybridization of RNA 32P-labeled in nuclei isolated during the late phase of infection to restriction endonuclease fragments of adenoviral DNA immobilized on nitrocellulose filters suggests that sequences of this transcriptional unit are indeed transcribed in vitro. To make a detailed assessment of the fidelity of transcription in isolated nuclei, transcription reactions were performed in the presence of 5-mercuricytidine 5'-triphosphate and the RNA mercurated in vitro separated from endogenous RNA by chromatography on sulfhydryl-agarose columns by a stringent procedure. After demercuration, RNA made in nuclei isolated 20 h following adenovirus type 2 infection was hybridized to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus type 2 DNA. Such RNA is complementary to the r strand of adenoviral DNA from 16.6 units to a point to the right of 98.3 units. These sequences comprise the major transcriptional unit expressed during the late phase (Fig. 1). It is therefore clear that the fidelity of transcription of adenoviral DNA by form II RNA polymerase is preserved in isolated nuclei. Two 1-strand transcriptional units, those of the IVa2 and ts36 genes (see Fig. 1) are also active at 20 h after infection. The results of similar analysis of RNA made in nuclei isolated 4 and 12 h after infection are also presented and discussed in terms of the mapping of individual transcriptional units within the type 2 adenoviral genome and the temporal regulation of adenoviral gene expression during productive infection.
Asunto(s)
Adenovirus Humanos/metabolismo , Núcleo Celular/metabolismo , ARN Viral/biosíntesis , Transcripción Genética , Secuencia de Bases , Enzimas de Restricción del ADN , Células HeLa/metabolismo , Humanos , Cinética , Hibridación de Ácido Nucleico , ARN Neoplásico/biosíntesisRESUMEN
Genetic and biochemical analysis has revealed additional early gene sequences in the group C human adenovirus genome. Physical mapping by an improved marker rescue method showed that a group of "early function" mutations previously found by us to map within the left quarter of the genome do not map within the known early sequence (0-11.2) in that region, but instead lie within the coordinates 18.5-22.0. A search for early expression in this region of the genome using saturation hybridization techniques showed the presence of two discrete sets of viral cytoplasmic RNA sequences, present early in infection at low copy number and complementary to sequences on the I strand of the adenovirus genome lying between 11.2-14.5 and 19.8-23.5 untis.