Heterochromatin forms on artificial chromosomes in the 3-20 Mb size range but is depleted on smaller artificial chromosomes that are approximately 1-3 Mb. Indirect immunofluorescence using an antibody that recognizes modification of histone H3 by trimethylation at lysine 9/lysine 27 (H3TrimK9/K27) (red signal) demonstrated that these heterochromatin markers are not detectable on the smaller D17Z1-based artificial chromosomes (arrowheads) in lines (a) 17-D34 and (b) 17-E29, but are readily detectable on the larger D17Z1- and DXZ1-based artificial chromosomes (arrowheads) as shown in lines (c) 17-B12, (d) 17-C20, (e) X-4 and (f) X-5. Arrows indicate chromosome 17 centromere regions (a-d) or host X centromere regions (e, f). Host D17Z1 sequences typically stained positive for H3TrimK9/K27 in most spreads (arrows in a-d). It was difficult to detect the X centromere signal (for example, arrow in (e)) but in about 30% of spreads there was a clearly positive signal as indicated by the arrow in (f). (g) Variation in H3TrimK9/K27 levels at host centromere regions is shown in a larger area of the spread shown in (c): artificial chromosomes are indicated by arrowheads; arrows point to the consistently strongly positive signals on the long arm of the Y chromosome (Yq). Artificial chromosome size estimates are listed in Table 2. Confirmation of artificial chromosomes and relevant host centromere regions were determined by FISH analyses with appropriate alpha-satellite probes (data not shown).