RESUMO
Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for D-amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824-8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3'-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1-151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal any significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.
Assuntos
D-Aminoácido Oxidase/genética , DNA/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Dados de Sequência Molecular , SuínosRESUMO
A colorimetric microplate hybridization assay was developed previously to simplify detection procedures of DNA fragments resulting from polymerase chain reactions (PCR). This format has now been adapted for the simultaneous detection and identification of three human papillomavirus (HPV), types 16, 18 and 33, associated frequently with cervical cancer. This post-PCR detection system uses three type-specific capture oligonucleotides linked covalently to a single microplate well and three type-specific multibiotinylated oligonucleotidic probes for detection. It therefore offers a double specificity; the first is conferred by pairs of primers, specific of each type of virus tested, and the second, by the sets of capture and detection probes which are complementary to internal regions of the amplified DNA fragments. The detection format outperformed agarose gel electrophoresis of amplified DNA products in sensitivity and specificity. The rapidity and simplicity of this hybridisation system would justify its use in routine diagnostic examination of cervical specimens (smears and biopsies).
Assuntos
Colorimetria/métodos , DNA Viral/análise , Proteínas de Ligação a DNA , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/métodos , Infecções Tumorais por Vírus/virologia , Estudos de Avaliação como Assunto , Feminino , Humanos , Hibridização de Ácido Nucleico , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/patologia , Sensibilidade e Especificidade , Infecções Tumorais por Vírus/patologiaRESUMO
Human haptoglobin (Hp alpha 2 beta) was synthesized in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) as an expression vector. Viruses carrying the proHp alpha 2 beta cDNA, either fused or non fused to viral polyhedrin DNA sequences, expressed intracellularly low levels of unglycosylated and non maturated haptoglobin. On the contrary, recombinant viruses containing the preproHp alpha 2 beta cDNA directed the expression of high levels of prohaptoglobin. To a large extent, the uncleaved product was found in the culture medium as a glycosylated molecule. Despite the lack of maturation into subunits, the secreted recombinant prohaptoglobin was able to bind hemoglobin in vitro, although less efficiently than plasma-derived haptoglobin.
Assuntos
Haptoglobinas/genética , Vírus de Insetos/genética , Insetos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Glicosilação , Haptoglobinas/metabolismo , Humanos , Técnicas In Vitro , Vírus de Insetos/isolamento & purificação , Insetos/microbiologia , Dados de Sequência Molecular , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
The DNA coding for the circumsporozoite protein (CPS) of Plasmodium falciparum has been cloned into the baculovirus expression vector pAcYM1 and expressed in Spodoptera frugiperda (Sf9) insect cells. Three DNA constructs have been made: the first one directs the synthesis of the complete CSP (aa 1-412), the second leads to the production of a species devoid of the anchor domain (aa 1-391) and the third one to a molecule lacking both signal and membrane anchor sequences (aa 18-391). All three recombinant CPS were produced at about 3 micrograms per 10(6) infected cells and were characterized in terms of immunoreactivity and apparent molecular weight. Analytical purification of the recombinant proteins was achieved by a combination of heat treatment, acidification, isoelectric focusing and ion exchange chromatography. The purified material, when injected into mice, generated only modest antibody responses, although antisera from immunized mice reacted with control CSP antigens carrying or not the major immunodominant repeat region.