RESUMEN
Cladograms of iridoviruses were inferred from bootstrap analysis of molecular data sets comprising all published protein and DNA sequences of the major capsid protein, ATPase and DNA polymerase genes of members of the Iridoviridae family Iridovirus. All data sets yielded cladograms supporting the separation of the Iridovirus, Ranavirus and Lymphocystivirus genera, and the cladogram based on data derived from major capsid proteins further divided both the Iridovirus and Ranavirus genera into two groups. Tests of alternative hypotheses of topological constraints were also performed to further investigate relationships between infectious spleen and kidney necrosis virus (ISKNV), an unclassified fish iridovirus for which the complete genome sequence data is available, and other iridoviruses. Cladograms inferred and results of Shimodaira-Hasegawa tests indicated that ISKNV is more closely related to the Ranavirus genus than it is to the other genera of the family.
Asunto(s)
Proteínas de la Cápside/química , ADN Viral/química , Iridovirus/clasificación , Filogenia , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de la Cápside/genética , Iridovirus/genéticaRESUMEN
The nucleotide sequence of the infectious spleen and kidney necrosis virus (ISKNV) genome was determined and found to comprise 111,362 bp with a G+C content of 54.78%. It contained 124 potential open reading frames (ORFs) with coding capacities ranging from 40 to 1208 amino acids. The analysis of the amino acid sequences deduced from the individual ORFs revealed that 35 of the 124 potential gene products of ISKNV show significant homology to functionally characterized proteins of other species. Some of the putative gene products of ISKNV showed significant homologies to proteins in the GenBank/EMBL/DDBJ databases including enzymes and structural proteins involved in virus replication, transcription, protein modification, and virus-host interaction. In addition, one major repeated sequence showing significant homology to the Red Sea bream iridovirus (RSIV) genome was identified. Based on the information obtained from biological properties (including histopathology, tissue tropisms, natural host range, and geographic distribution), physiochemical and physical properties, and genome analysis, we suggest that ISKNV, RSIV, sea bass iridovirus, grouper iridovirus, and African lampeye iridovirus may belong to a new genus of the Iridoviridae family and are tentatively referred to as cell hypertrophy iridoviruses.
Asunto(s)
Genoma Viral , Iridovirus/genética , Perciformes/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Replicación del ADN , ADN Viral , Bases de Datos de Proteínas , Iridovirus/clasificación , Iridovirus/fisiología , Riñón , Datos de Secuencia Molecular , Nucleótidos , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Bazo , Transcripción Genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/fisiología , Proteínas Estructurales Virales/genéticaRESUMEN
The porcine somatoropin gene was inserted into the Pichia pastoris expression vector of pPICZ alpha A which contains AOX I promoter and alpha-factor signal sequence. The recombinant plasmid of pPICZ alpha A-pST was linearnized by Sac I and transformed into X-33 by electroporation. The multi-copy insert transformants were selected and cultivated in flasks. SDS-PAGE and Western blot analysis showed that PST gene products were observed in the supernants with a little larger molecular weight size than the natural PST's, however, the molecular weight size of the PST gene products in the soluble cellular proteins were identical to the natural PST's. Retransformation of the linearnized pPICZ alpha-pST showed the expression level was improved greatly and rPST has the same antigenicity as natural one. The expressed rPST accumulated up to about 956 mg/L. The N-glycosylation analysis showed rPST had no N-glycosylation.
Asunto(s)
Hormona del Crecimiento/metabolismo , Pichia/genética , Animales , Western Blotting , Medios de Cultivo Condicionados/química , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Glicosilación , Hormona del Crecimiento/genética , Pichia/metabolismo , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Porcinos , Transformación GenéticaRESUMEN
Endothelin(ET) is the most potent mammalian vasoconstrictor identified to data. As a pathogenic factor, ET is involved in the genesis of many diseases. In this study, a pair of primers was designed and synthesized according to the human ETB receptor gene (hETBR) sequence. A 394 bp of DNA fragment was amplified by polymerase chain reaction(PCR) and labeled with alpha-32P-CTP using Random Primer-Labeling method. With this probe, rabbit lung cDNA library was screened by in situ hybridization and 11 positive clones were identified. Sequencing result showed that a complete reading frame of rabbit ETB receptor(rETBR) cDNA could be produced from three positive clones of eleven. By a series of subcloning, a recombinant plasmid including the 1326 bp of rETBR coding sequences, named pBlu Script-rETBR, was constructed. The deduced amino acid sequence indicated that the rETBR is 441 residues in length, with an expected molecular mass of approximately 49.44 kD. N-terminal 18 residues is the potential signal peptide (Score = 11.11) and therefore the molecular mass of mature rETBR is 47.65 kD with 423 amino acid residues. Analysis of the rETBR hydropathy profile indicates the presence of seven hydrophobic regions, putative transmembrane domains. Potential N-glycosylation sites are the 60th and the 118th. The structure exhibits a significant sequence and topographical similarity with G protein-coupled receptors.
Asunto(s)
ADN Complementario/química , Receptores de Endotelina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Hibridación in Situ , Datos de Secuencia Molecular , Conejos , Receptor de Endotelina B , Receptores de Endotelina/químicaRESUMEN
Following mutagenesis of cultured lepidopteran cells (Spodoptera frugiperda) by ethylmethanesulfonate, three variants resistant to 5-bromodeoxyuridine (BrdUrd) were isolated. These clones were 100- to 200-fold more resistant to BrdUrd than the parental cells and were shown to be deficient in thymidine kinase (TK). The drug-resistant phenotype was stable for up to three years of culture under nonselective conditions. It was also found that the S. frugiperda cell line was highly resistant to aminopterin. A selective medium was formulated and used to select herpes simplex virus thymidine kinase (HSV-TK) transfectants from the TK-deficient cells.
Asunto(s)
Bromodesoxiuridina/farmacología , Línea Celular , Simplexvirus/genética , Timidina Quinasa/genética , Transfección , Animales , Línea Celular/efectos de los fármacos , ADN Recombinante , Resistencia a Medicamentos , Genes Virales , Mariposas Nocturnas/citología , Simplexvirus/enzimologíaRESUMEN
The gene coding for the hepatitis B virus surface antigen (HBsAg) under the control of Autographa californicanuclear polyhedrosis virus polyhedrin promoter was successfully inserted into the genome of the Trichoplusia ni nuclear polyhdrosis virus. Infection of Spodoptera frugiperda cells with this recombinant virus produced a significant amount of HBsAg protein and secreted 22 nm particles containing the HBsAg. The expression of HBsAg gene was also obtained both in Trichoplusia ni larvae and in Philosamia cynthia ricini prepupae when infected with the recombinant virus. The HBsAg proteins expressed by baculovirus vector systems have morphological and antigenic properties identical to the 22 nm particles secreted by human cells.