Heterochromatic features of an 11-megabase transgene in brain cells

Transgenic mice provide a remarkable experimental setting for the study of nuclear architecture. The three-dimensional localization and fine structure of a foreign DNA within the mouse genome can be conveniently followed by using high-resolution in situ hybridization. Foreign DNAs designed with specific characteristics, such as base bias, sequence motif(s), and size can stably integrate into finite positions on host chromosomes. Thus the relative importance of each of these characteristics in determining the three-dimensional nuclear position and the detailed morphology of the transgene can be evaluated in different cell types. The aim of this study was to evaluate a transgene with sequence characteristics that might contribute to the de novo formation of heterochromatin in interphase nuclei. The structure of a phenotypically silent 11-megabase transgene, containing tandem repeats of beta-globin-pBR sequences integrated into the peritelomeric region of both mouse chromosome 3 homologs, was determined in adult brain cells. Neurons that are largely euchromatic were especially informative in three-dimensional studies of transgene position. The two transgenic loci behaved much like centromeric or paracentromeric A + T-rich satellite DNAs of comparable length from a single chromosome; one or both transgene domains localized together with centromeric satellite DNA on the nucleolus. This is an unusual nuclear position for a telomeric or chromosome arm region that does not contain a substantial amount of constitutively heterochromatic satellite DNA. G + C richness did not prevent these regions from assembling as dense heterochromatic bodies of approximately 1 micron3 in volume. Ultrastructurally, transgenic domains were often intimately connected with constitutive heterochromatin and were highly condensed. Labeled supercoils, formed by a discrete approximately 250-nm-wide fiber, were observed in oblique thin sections through the center of the domain. The structural data were consistent with negligible transcriptional activity detected for this locus, as well as the predicted conformation of constitutive heterochromatin. Interestingly, in transgenic but not control mice, a substantial number of large neurons, including approximately 30% of cerebellar Purkinje cells, showed excessive invaginations of the nuclear membrane.