Although much progress has been made in the assembly of detailed physical maps of several high eukaryotic genomes, centromere regions typically appear as """"""""holes"""""""" in these maps. One obvious reason for the instability of centromeric DNA during cloning is the existence of long tandem arrays of repetitive DNA which are susceptible to rearrangements and other alterations. Because of the high degree of condensation of centromeric heterochromatin and classical position effect variegation, heterochromatin typically is viewed as devoid of coding sequences. This clearly is an oversimplification since ribosomal precursor genes are located in the centromeres of many organisms, including rodents and man and are often transcribed at very high rates. Thus, the low resolution view of centromeres as refractile to transcription is not completely accurate. In addition, nearly 40 protein-coding genes have been identified in Drosophila which are referred to as heterochromatin genes due to their cytological locations, and at least some of these genes require a heterochromatic environment for normal expression. Although heterochromatin genes may be unique to flies, the investigators propose a series of experiments to attempt to identify genes embedded in centromeric heterochromatin using mouse chromosomes as a model. Two approaches are presented. Centromere regions of mitotic chromosomes on coverslips will be microdissected using a laser """"""""scissors""""""""; unwanted material and surrounding chromosomes will be destroyed by the laser; centromeres will be collected with a microneedle; and the DNA amplified by PCR using several different primer pairs. PCR products will be used to screen cDNA libraries and positive clones will be characterized further and used to select genomic clones. The genomic clones will be used for in situ hybridization studies to document their centromeric origin. cDNAs of centromere-localized clones will be sequenced and expression patterns determined. The second approach involves screening a Bacterial Artificial Chromosome (BAC) library with the major satellite.
