Assuntos
Escherichia coli/genética , Genes de Plantas , Nucleopoliedrovírus/genética , Proteínas de Plantas/genética , Animais , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Expressão Gênica , Técnicas Genéticas , Vetores Genéticos , Corpos de Inclusão/química , Proteínas de Plantas/isolamento & purificação , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Spodoptera , TransfecçãoRESUMO
The 3.5 kb long mRNA of the maize transposable element Ac contains an open reading frame (ORFa) which encodes a polypeptide of 807 amino acids, the putative transposase of Ac. The Ac mRNA is a rare transcript: we now estimate the fraction of Ac mRNA in wx-m7::Ac seedlings to be 2-13 x 10(-5) of the polyA RNA. Assuming that maize cells contain similar amounts of polyA RNA as another monocot (0.16 pg/cell), this is equivalent to 1.5-10 transcripts in each cell. A protein with an apparent molecular weight of 112 kDa is detected, by five antisera directed against different segments of ORFa, exclusively in nuclear extracts from Ac-containing maize. This protein is most likely the full-length Ac ORFa protein. We estimate its concentration to be in the range of 3 x 10(-7) of the nuclear proteins, or about 1000 molecules per triploid endosperm cell containing one Ac element.
Assuntos
Elementos de DNA Transponíveis , Nucleotidiltransferases/genética , RNA Mensageiro/genética , Zea mays/genética , Northern Blotting , Western Blotting , Peso Molecular , Nucleotidiltransferases/metabolismo , Fases de Leitura Aberta , RNA Mensageiro/análise , Mapeamento por Restrição , Transcrição Gênica , Transposases , Zea mays/enzimologiaRESUMO
The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%-2% of the newly synthesized protein.
