Our central objectives are to understand why anoxia induces normal rat fibroblasts to express very high levels of retrovirus/retrotransposon related VL30 element RNA, and to understand the relationship of this process to malignant neoplasia. This is of particular interest in that VL30 elements have been incorporated into the genomes of two separate rat-derived murine sarcoma viruses, and VL30 RNA is very often found expressed at elevated levels in rodent cancers. 1. To evaluate if gene products are encoded within anoxia inducible VL30 RNA, we will sequence cDNA clones. Immune precipitation with antibodies to synthetic polypeptides will confirm that open reading frames are actually used. 2. Antipeptide sera to VL30 sequences detects at least one polypeptide of 61 kilodaltons, p61. Like VL30 itself, p61 is also anoxia inducible. p61 will be purified and partially sequenced, to facilitate cloning and to define relatedness to known proteins. Oligonucleotide probes will be used to evaluate if p61 is a non-VL30 gene which was incorporated into the VL30 region of Harvey carcoma virus. 3. With VL30 representing a multigene family with 50-100 copies per cell in rat DNA, we will use restriction mapping of cDNA clones to determine if only one or many of these elements are expressed in response to anoxia. 4. Does the anoxia inducible control site reside within the VL30 element itself, or elsewhere in the rat cell? Anoxia responsive rat VL30 DNA will be introduced into non-rat cells via retroviral pseudotyping or DNA transfection, and anoxia responsiveness elevated. 5. What is the nature of the human homolog of the anoxia inducible rat VL30? Is it a true VL30 element? Does it encode anything? Is it expressed as RNA anomalously in tumor tissues? The human VL30 homolog will be cloned and sequenced, and RNA transcripts quantitated in normal and tumor tissues. Together, these studies should advance our understanding of the roles played by VL30 elements in normal cells responding to anoxia. Our work should further clarify the role of VL30 elements in cancer.
Showing the most recent 10 out of 11 publications
