Centromeric chromatin: what makes it unique?

Centromeres represent the final frontier of eukaryotic genomes. Although they are defining features of chromosomes--the points at which spindle microtubules attach--the fundamental features that distinguish them from other parts of the chromosome remain mysterious. The function of centromeres is conserved throughout eukaryotic biology, but their DNA sequences are not. Rather, accumulating evidence favors chromatin-based centromeric identification. To understand how centromeric identity is maintained, researchers have studied DNA-protein interactions at native centromeres and ectopic "neocentromeres". Other studies have taken a comparative approach focusing on centromere-specific proteins, of which mammalian CENP-A and CENP-C are the prototypes. Elucidating the assembly and structure of chromatin at centromeres remain key challenges.     

Centromeres are specialized replication domains in heterochromatin

The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable from surrounding noncentromeric sequences. However, centromeric chromatin contains variant H3-like histones that may specify centromeric regions. Nucleosomes are normally assembled during DNA replication; therefore, we examined replication and chromatin assembly at centromeres in Drosophila cells. DNA in pericentric heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast to expectation, we show that centromeres replicate as isolated domains early in S phase. These domains do not appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like variant proceeds by a replication-independent pathway. We suggest that late-replicating pericentric heterochromatin helps to maintain embedded centromeres by blocking conventional nucleosome assembly early in S phase, thereby allowing the deposition of centromeric histones.     

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

Somatic Instability of a Drosophila Chromosome

A mitotically unstable chromosome, detectable because of mosaic expression of marker genes, was generated by X-ray mutagenesis in Drosophila. Nondisjunction of this chromosome is evident in mitotic chromosome preparations, and premature sister chromatid separation is frequent. The mosaic phenotype is modified by genetic elements that are thought to alter chromatin structure. We hypothesize that the mitotic defects result from a breakpoint deep in the pericentric heterochromatin, within or very near to the DNA sequences essential for centromere function. This unique chromosome may provide a tool for the genetic and molecular dissection of a higher eukaryotic centromere.     

Release of growth hormone from ox pituitary slices after pronase treatment

Proteolytic enzymes have been used both to modify properties of the cell membrane and to dissociate cells from many tissues including pituitary (4, 5, 12). Exposure of secretory tissues to pronase can alter their secretory response. Thus incubation of pancreatic islets of Langerhans in the presence of low concentrations of pronase increased the subsequent release of insulin in the presence of stimulatory and nonstimulatory glucose concentrations (7). The purpose of the present investigation was to determine whether low concentrations of pronase have the same stimulatory effect on the release of a pituitary hormone, growth hormone. Such an effect on hormone release could be of some importance in view of the development of dissociated cell systems as models for the study of the control of hormone release (4, 5).