The long term goal of the proposed research is to understand the function of centric heterochromatin, and its relationship to chromosome segregation. In the process, it is expected that new insights into the nature of heterochromatin will be obtained. A cloned human DNA sequence that has been recently isolated will be used initially to study centromere hererochromatin. This sequence, 308, is homologous to sequences at the centromere of almost all human chromosomes. The way in which these DNA sequences are organized on various human chromosomes will be determined using cosegregation of specific DNA restriction fragments and specific human chromosomes in mouse-human hybrids. The nature of the polymorphism associated with these sequences will be further explored, by localizing the quantitative and qualitative variants to specific chromosomes using the hybrids as well as in situ hybridization to human metaphase chromosomes, and by linkage to other chromosome specific markers. To determine if these centric heterochromatin sequences replicate simultaneously on each chromosome, the timing of replication of chromosome specific restriction fragments will be compared using Southern blots of DNA from synchronized human fibroblasts that have been pulse labelled with 14C thymidine. To determine if some or all of these sequences are transcribed, RNA from human cultured cells and hybrids containing specific human chromosomes will be analyzed using dot blot and northern methodology. The pattern of DNA methylation will be compared for sequences on X and autosomes using restriction enzymes that are methyl sensitive. p308 will be used to screen a human recombinant library to identify any neighboring single copy sequences that might be important in centromeric function. The possibility that antibodies to 308 sequences are present in the serum of patients with anticentromere antibodies will be explored. With affinity chromatography, these anticentromere antibodies will be used to obtain chromatin enriched for centromere DNA. Elucidation of the nature of DNA associated with human centromere function may provide insights into the mechanism of nondisjunction that is a predominant cause of human chromosome aberrations.
