RESUMO
A four-kilobase (kb) region (HindIII-N, map units 7.0-11.3) of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was found to contain sequences that conferred upon plasmids the ability to undergo infection-dependent replication. The plasmid DNA appeared to be replicated to form high molecular weight multimers. Plasmids with deletions of up to 1.8 kb from either end of the HindIII-N region were replication competent. However, a discrete sequence, contained within the region bracketed by the deletions, capable of specifying replication was not identified. No evidence for sequence homology was found between the OpMNPV HindIII-N region and regions elsewhere in the OpMNPV genome or to putative Autographa californica MNPV (AcMNPV) replication origins. Origin-dependent plasmid replication was shown to require the presence of the OpMNPV DNA polymerase gene. The OpMNPV origin replicated poorly in AcMNPV-infected Spodoptera frugiperda cells and, conversely, a putative AcMNPV origin (hr2) replicated at low levels in OpMNPV-infected Lymantria dispar cells.
Assuntos
Replicação do DNA/genética , DNA Viral/genética , Nucleopoliedrovírus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Cosmídeos , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/genética , Genoma Viral , Insetos/citologia , Insetos/microbiologia , Dados de Sequência Molecular , Nucleopoliedrovírus/crescimento & desenvolvimento , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Análise de Sequência de DNA , Deleção de Sequência , TransfecçãoRESUMO
The DNA polymerase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was cloned and sequenced. The predicted DNA polymerase protein (1113 amino acids, 115.9K) was found to have an amino acid identity of 48% with the corresponding gene of the Autographa californica MNPV (AcMNPV). It contains five domains associated with substrate binding, primase interaction, and pyrophosphate hydrolysis and three domains associated with 3'-->5' exonuclease activity common to other DNA polymerases. A region with a conserved TATA promoter and a CAGT mRNA start site sequence motif was identified and shown to be transcribed by RNA polymerase II, indicating that the LdMNPV DNA polymerase gene is expressed as an early gene. An open reading frame possibly expressed as a late gene, oriented in the opposite direction and overlapping the N-terminal coding region of the DNA polymerase gene was found in the LdMNPV sequence and was shown to be conserved in the same position in AcMNPV.
Assuntos
Baculoviridae/genética , DNA Polimerase Dirigida por DNA/genética , Genes Virais , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Baculoviridae/enzimologia , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Mapeamento por Restrição , Alinhamento de Sequência , Transcrição GênicaRESUMO
We have generated transgenic flies expressing R7 cell-specific opsins in the major class of photoreceptor cells of the Drosophila retina and characterized their spectral properties using high-resolution microspectrophotometry and sensitivity recordings. We show that the Rh3 and Rh4 opsin genes encode UV-sensitive opsins with similar spectral properties (lambda max = 345 nm and 375 nm), and that Rh3 corresponds to the R7p and R7marg class of visual pigments. We have also generated Rh3 and Rh4 isoform-specific antibodies and present an R7 cell map of the Drosophila retina. In a related set of experiments, we show that it is possible to coexpress two different visual pigments functionally in the same cell and produce photoreceptors that display the summed spectral response of the individual pigments. These findings open up the possibility of tuning an animal's visual behavior by targeted expression of combinations of opsin genes to selective types of photoreceptors.
Assuntos
Percepção de Cores , Células Fotorreceptoras/metabolismo , Rodopsina/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Células Fotorreceptoras/química , Células Fotorreceptoras/fisiologia , Regiões Promotoras Genéticas , Rodopsina/análise , Opsinas de Bastonetes/análise , Opsinas de Bastonetes/metabolismoRESUMO
The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV), which is used for the overexpression of eukaryotic genes and is being engineered for possible use as a viral insecticide, has a circular, supercoiled genome of approximately 128 kilobases. Despite its widespread use, little is known about the mechanism by which AcMNPV replicates. Evidence is presented in this report that AcMNPV origins of DNA replication are repeated sequences each containing several closely related imperfect palindromes that are present in six regions distributed around the genome. Although AcMNPV infection-dependent plasmid replication was initiated by a single complete palindrome, the amount of replication was substantially increased in plasmids containing six or eight palindromes.
Assuntos
Baculoviridae/genética , Replicação do DNA , DNA Super-Helicoidal/genética , DNA Viral/genética , Genes Virais/genética , Replicação Viral , Sequência de Bases , DNA Super-Helicoidal/química , DNA Viral/química , Dados de Sequência Molecular , Mutagênese , Hibridização de Ácido Nucleico , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , TransfecçãoRESUMO
A 6.4 kb region from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome was sequenced and found to contain open reading frames (ORFs) homologous to the polyhedron envelope (PE) protein coding sequence, and the C-terminal half of ORF 1, which is a gene located upstream of the PE protein gene in other baculoviruses. The proteins predicted from the LdMNPV genes encoding the PE protein, and ORF 1 demonstrated 27 and 34% amino acid sequence identity, respectively, with the corresponding genes in the Autographa californica multicapsid nuclear polyhedrosis virus.
Assuntos
Baculoviridae/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Viral , Dados de Sequência Molecular , Mariposas/microbiologia , Proteínas de Matriz de Corpos de Inclusão , Fases de Leitura Aberta , Mapeamento por Restrição , Alinhamento de Sequência , Proteínas Estruturais ViraisRESUMO
A 1.85 kb region, containing an open reading frame (ORF) homologous to the baculovirus p39-capsid gene, was sequenced from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome. Analysis of the p39-capsid gene demonstrated that it was 39% and 47% identical in amino acid sequence with the homologous genes in the Autographa californica and Orgyia pseudotsugata MNPVs, respectively. Two late promoter elements located upstream of the p39 gene in the LdMNPV genome are conserved with two other baculoviruses, whereas an ORF located downstream is not conserved.
