After the restriction enzyme mapping of the MDV genome, we and others found an amplification of a 132 bp repeat in IRL and TRL, which caused loss of tumorgenicity of MDV. In our recent study, the loss of tumorgenicity was found to be due to the disruption of a 1.8 kb RNA gene as well as a 3.8 kb RNA gene. Exon-I is mapped to the 5'-end of the amplification and exon-II is mapped to the 3'-downstream region of the amplification. In this study we propose to further investigate the nature of the 1.8 kb RNA gene as well as the 3.8 kb RNA gene. We plan to make cDNA libraries of oncogenic and non-oncogenic MDV infected cell RNA and precisely map the 1.8 kb and 3.8 kb RNA be raising monospecific antibody to these genes. Once we determine the open reading frames of these genes and raise monospecific antibodies to these gene products, we will reconstitute oncogenic MDV by insertion of these genes back into the non-oncogenic MDV genome at the region of the TK gene or at the truncated RNA region. This will give direct evidence that these genes of MDV (1.8 kb and/or 3.8 kb RNA) are indispensable for tumor induction in chicken. Furthermore, eukaryotic expression vectors made by inserting the cDNA or the open reding frame of these genes into pZIPneo or 3'-downstream of RSV promoter will be used to determine whether H gene can transform NIH 3T3 cells or CEF. These proposed studies will further clarify the role of these genes in inducing tumors in chicken.
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