The long-term objectives of this research are to gain insights into chromatin structure and gene expression.
The specific aims described in this application are: (1) To gain new information concerning the molecular architecture of active genes in metaphase chromosomes. Metaphase chromosomes and interphase nuclei will be isolated from synchronized chicken cells. The organizations of active genes, including the glyceradehyde-3-phosphate dehydrogenase, thymidine kinase and Beta-tubulin genes, in chromosomes/nuclei will be investigated using nucleases and recombinant DNA molecules containing these genes as probes. The longtitudinal distribution of active genes in metaphase chromosomes will be studied using a chromosome nick-translation method. (2) To investigate the nuclear skeletal structure (synaptonemal complex) in prophase stage of meiotic cells. Monoclonal antibodies against synaptonemal complex proteins will be prepared and used as probes to investigate the structural and physiological roles of the synaptonemal complex inside the nuclei. (3) The chromatin structure in the abnormal chromosomes such as homogeneously staining regions (HSR) and double minutes (dm), which are most often observed in drug-resistant mutants and neoplastic cells, will be studied similarly using recombinant DNA and nucleases as probes. The possibilities that gene amplification may take place in the vincristine-resistant mutants which contain HSR and dm will be investigated using a differential screening method of a cDNA library or genomic DNA library constructed from the mutants. The results obtained from these described projects may provide useful information in molecular genetics and enhance fundamental knowledge in human diseases.
