Pathogenesis and insertional mutagenesis by akv and akv-derived vectors
Author
Martín Hernández, JavierEntity
UAM. Departamento de Biología Molecular; Universidad de Aarhus (Dinamarca)Date
2005-02-17Subjects
Leucemia murina - Tesis doctorales; Enfermedades por virus - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-02-2005Abstract
My Ph. D. project focuses on retroviral pathogenesis and insertional mutagenesis of murine
leukemia viruses, and occasionally on the genes that are target of retroWal insertion by Akv and
Akvl-99 vinises. The sites of integration are located in the genome of tumor cells that have
undergone a long selection process during tumorigenesis and the identified proviruses single out
genes that may be involved in cancer development. PCR amplification of flanking sequences fiom
DNA isolated from 21 independent lymphornas induced by the Akv and Akvl-99 viruses, revealed
56 retrovirus integration sites (RISs). The analyses of the RISs showed a significant difference
between the integration site preferences of the two vinises with respect to integrations within genes
and in regions near CpG islands. Moreover, 21 of the RISs presented here have previously not
been identified and represent new candidate genes that might be involved in B-cell lymphoma
development. The N-ras gene was also shown to be involved in ihis type of disease since three
integrations were found at the N-ras/unr locus in three independent Akvl-99 tumors. Analyses of
the effects induced by the proviruses between N-rus and unr genes showed that the insertions
altered the expression of N-rus only. Moreover, no point mutations were detected in the codon
sequences of N-rus analyzed. Altogether, the íirsi part of the project describes that despite of the
similar pathogenic properties of Akv and Akvl-99 MLVs, the two viruses show different
integration site patterns. Besides, new genes targeted by proviral insertions ihat might contribute in
the carcinogenesis process are also reported.
The second part of my project consists of the analyses of three different Akv-based replicationcompetent
vectors, containing activated N-ras or Ha-rus genes or the non-mutated sequence of N-rus,
respectively. Newbom mice were infected and monitored for disease development during up to twoyear
observation periods. The mice succumbed to the pathogenic effects of the vectors cmybg the
activated ras genes and showed no symptoms of disease in the case of the proto-oncogene. The
animals presented with enlarged spleens, and occasionally tumors elsewhere in the corpse. Single cell
suspension samples fiom the organs were analyzed by flow cytometry but failed to diagnose the
nature of the disease. Fnrthermore, retrovirai tagging of DNA ftom infected mice isolated a small
number of proviral integration sites of rus maxiviruses.
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