Assuntos
Proteínas de Ligação a Calmodulina/genética , Transformação Celular Neoplásica/genética , Leucemia Mielomonocítica Aguda/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Animais , Transformação Celular Neoplásica/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mielomonocítica Aguda/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismoRESUMO
DNA was isolated from a circular derivative of chromosome III to prepare a library of recombinant plasmids enriched in chromosome III sequences. An ordered set of recombinant plasmids and bacteriophages carrying the contiguous 210-kilobase region of chromosome III between the HML and MAT loci was identified, and a complete restriction map was prepared with BamHI and EcoRI. Using the high frequency transformation assay and extensive subcloning, 13 ARS elements were mapped in the cloned region. Comparison of the physical maps of chromosome III from three strains revealed that the chromosomes differ in the number and positions of Ty elements and also show restriction site polymorphisms. A comparison of the physical map with the genetic map shows that meiotic recombination rates vary at least tenfold along the length of the chromosome.
Assuntos
Replicação do DNA , DNA Circular/genética , DNA Fúngico/genética , Replicon , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Clonagem Molecular , Elementos de DNA Transponíveis , DNA Circular/biossíntese , DNA Fúngico/biossíntese , Polimorfismo de Fragmento de Restrição , Mapeamento por Restrição , Transformação GenéticaRESUMO
The poliovirus P2/P712 strain is an attenuated virus that is closely related to the type 2 Sabin vaccine strain. By using a mouse model for poliomyelitis, sequences responsible for attenuation of the P2/P712 strain were previously mapped to the 5' noncoding region of the genome and a central region encoding VP1, 2Apro, 2B, and part of 2C. To identify specific determinants that attenuate the P2/P712 strain, recombinants between this virus and the mouse-adapted P2/Lansing were constructed and their neurovirulence in mice was determined. By using this approach, the attenuation determinant in the central region was mapped to capsid protein VP1. Candidate attenuating sequences in VP1 and the 5' noncoding region were identified by comparing the P2/P712 sequence with that of vaccine-associated isolate P2/P117, and the P2/117 sequences were introduced into the P2/Lansing-P2/P712 recombinants by site-directed mutagenesis. Results of neurovirulence assays in mice indicate that an A at nucleotide 481 in the 5' noncoding region and isoleucine (Ile) at position 143 of capsid protein VP1 are the major determinants of attenuation of P2/P712. These determinants also attenuated neurovirulence in transgenic mice expressing human poliovirus receptors, a new model for poliomyelitis in which virulent viruses are not host restricted. These results demonstrate that A-481 and Ile-143 are general determinants of attenuation.
Assuntos
Vacina Antipólio de Vírus Inativado , Poliovirus/genética , Vacinas Atenuadas , Animais , Sequência de Bases , Capsídeo/genética , Capsídeo/imunologia , Proteínas do Capsídeo , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Poliovirus/imunologia , Poliovirus/patogenicidade , Conformação Proteica , RNA Viral/genética , Virulência/genéticaRESUMO
A human poliovirus receptor (PVR) gene was used to generate transgenic mice that express PVR transcripts and poliovirus binding sites in a wide range of tissues. Intracerebral inoculation of PVR transgenic mice with poliovirus type 1, Mahoney strain, resulted in viral replication in the brain and spinal cord and development of paralytic poliomyelitis. P1/Mahoney did not replicate or cause paralysis in nontransgenic mice. PVR transgenic mice failed to develop clinical disease when inoculated intracerebrally with the live attenuated Sabin type 1 vaccine strain. These results demonstrate that the PVR is the major determinant of poliovirus host range in mice. Transgenic mice expressing human PVR should be useful for studying poliovirus neurovirulence, attenuation, and tissue tropism, and for development and testing of poliovirus vaccine strains.
