Heterochromatin of the Indian muntjac: Replication, condensation, DNA ultracentrifugation, fluorescent and heterochromatin staining

Abstract The following features of the Indian muntjac (miniature Asian deer) make it ideal for cytogenetic studies: 1. 1. A low chromosome number (female 6, male 7) with each chromosome individually identifiable; 2. 2. Large chromosomes; 3. 3. The presence of a very long centromere region on a chromosome formed by fusion of the X-chromosome with an autosomal telocentric chromosome; 4. 4. A prominent secondary constriction on this autosomal telocentric chromosome. DNA replication studies demonstrate that the centromere regions and the Y chromosome undergo relatively intense replication during a short period occurring 5 to 7 h into the 8-h S period. Other portions of the chromosome complement continued replication after replication had ceased in these areas. These replicative characteristics indicated that all the centromere regions and the Y chromosome were heterochromatic. This was confirmed by staining for constitutive heterochromatin. The rest of the chromosomes showed a diffuse replication pattern during all periods of the S phase. It is suggested that any segmental labeling that may have been present was obscured by the high packing ratio of chromatin fibers into these large chromosomes. Measurements were made on the chromosomes with and without exposure to Colcemid. These indicated that the heterochromatic centromere region of the X-autosome chromosome remained long and narrow following Colcemid treatment while the non-heterochromatic regions became short and thick. These studies parallel the observations that following exposure to cold or Colcemid the heterochromatic areas in plant chromosomes tend to condense less than the euchromatic regions. These measurements also indicated that there was a gradient of chromosome contraction with the least contraction occurring in chromatin nearest the heterochromatin (or centromere) and the greatest contraction occurring in those segments furthest removed from the heterochromatin. Analytical Ultracentrifugation of muntjac DNA showed a curve that was moderately non-gaussian because of a heavy shoulder component constituting approx. 13% of the total DNA. When the DNA was denatured, renatured for 5 h, and centrifuged a small satellite component constituting about 4% of the total DNA became apparent. Similar studies of main band and heavy shoulder DNA isolated by preparative Ultracentrifugation showed that the satellite was buried primarily within the main band DNA, indicating that its GC content was essentially the same as that of the main band. The heterochromatin staining reaction suggested that this rapidly renaturing DNA was probably localized to the centromeric heterochromatin. Centrifugation after denaturation and partial renaturation indicated the heavy shoulder DNA did not contain a disproportionate amount of rapidly renaturing DNA. After treatment with quinacrine mustard the fluorescent patterns showed a marked decrease in staining over the centromeric heterochromatin, in the region of the secondary constriction, and at numerous other bands in the chromosome arms. The fact that there was decreased staining in the centromeric heterochromatin, which was probably enriched in the rapidly renaturing fraction whose base composition was the same as that of the main band DNA, suggests that the base composition of DNA may play only a secondary or minor role in determining the fluorescent patterns. Consideration of many facets of fluorescent staining, including the results following treatment of the chromosomes with numerous chemicals and enzymes, suggests that the fluorochromes may be sensitive to variations in the amount and type of non-histone protein bound to certain segments of the genome. It is suggested that on the basis of location, content of rapidly renaturing DNA, ability to stain with the Arrighi-Hsu technique, late replication patterns, and AT content, there are probably at least two types or modes of constitutive heterochromatin.