EUKARYOTIC GENE ORGANIZATION

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The long term goal of this project is to contribute to the analysis of eukaryotic genome organization at the DNA nucleotide sequence level. The proposed work aims to provide insights into mammalian gene regulation, and should add to understanding of the basis of genetic diseases. Two genetic elements in the laboratory mouse will be studied: 1) The Hbb complex locus in the mouse. This locus encodes the genes for the beta and beta-like chains of hemoglobin. The DNA sequences for a 65 Kb region including the seven known beta- globin genes and pseudogenes from the BALB/c mouse will be completed. The sequence will be extensively analyzed. The mouse sequence will be compared to the human Hbb sequence to identify features of functional and evolutionary significance. Conserved sequence elements will be investigated to determine their genomic copy number, transcriptional activity, and to look for specific protein binding sites. 2) The L1 (LINES-1) retroposon. This is the major long interspersed repetitive element in the mammalian genome. Full length L1 elements are about 7 Kb in length with two open reading frames (ORFs) 1137-bp and 3900-bp in length. These ORF's will be translated at high levels in E. coli and in yeast. The ORF products will be assayed for suspected functions, for example reverse transcriptase activity, nucleic acid binding, and protease activity. A search will be made for L1 RNA packaged in virus-like particles. Antibodies directed against L1 ORF products will be used to probe for L1 coded proteins in mouse tissues and cultured cells. Possible promoter and enhancer activity of 200-bp repeated sequences found at the 5' end of L1 in mouse will be investigated. An experimental system will be developed to detect L1 transposition, with a long term goal of harnessing L1 as an insertional mutagen for mammalian systems. Proposed approaches include L1 overexpression, and detection of L1 insertion into the herpes virus TK gene. A search for likely intermediates in KL1 propagation will be made, for example DNA/RNA hybrids and extrachromosomal L1 DNA.