Heterochromatin Protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster localized principally in the pericentric heterochromatin, telomeres, and fourth chromosome, all regions associated with HP1. Mutations in HP2 can suppress position effect variegation, indicating a role in gene silencing and heterochromatin formation [Shaffer, C.D. et al. (2002) Proc. Natl. Acad. Sci. 99, 14332-14337]. In vitro coimmunoprecipitation experiments with various peptides from HP2 have identified a single HP1 binding domain. Conserved domains in HP2, including those within the HP1 binding region, have been identified by recovering and sequencing Su(var)2-HP2 from D. willistoni and D. virilis, as well as examining available sequence data from D. pseudoobscura. A PxVxL motif, shown to be an HP1 binding domain in many HP1-interacting proteins, is observed but is not well conserved in location and sequence, and does not mediate HP2 binding to HP1. The sole HP1 binding domain is composed of two conserved regions of 12 and 16 amino acids separated by 19 amino acids. Site-directed mutagenesis within the two conserved regions has shown that the 16 amino acid domain is critical for HP1 binding. This constitutes a novel domain for HP1 interaction, providing a critical link for heterochromatin formation in Drosophila.Regions of every euchromatic genome, prominently the centromeres and the telomeres, are packaged as constitutive heterochromatin. These regions contain relatively few genes, and are made up primarily of repetitious sequences, such as satellite DNA and transposable elements. Heterochromatic domains are characterized by a condensed appearance of the interphase chromatin, a low level of meiotic recombination, and late replication in S phase. In Drosophila, a normally euchromatic gene that is mislocalized to heterochromatin by tranposition or rearrangment will be silenced in some cells but not others, resulting in a variegating phenotype. This phenomenon is known as PEV 1 , or position effect variegation (1). Mutations that suppress PEV (resulting in a loss of silencing) have identified many genes whose products are essential for heterochromatin formation (2,3).Heterochromatin Protein 1 (HP1), one of the best-characterized nonhistone chromosomal proteins, has been implicated in both heterochromatin formation and gene silencing. HP1 is conserved from yeast (Schizosaccharomyces pombe) to humans and is consistently associated with pericentric heterochromatin (4). HP1 has been found to be part of multiprotein complexes that are necessary for the induction of heterochromatin formation. Proteins in the HP1 family contain an amino-terminal chromodomain and a carboxy-terminal chromoshadow domain separated by a linker region of variable length. The chromodomain has been found to bind to histone H3 methylated at lysine 9 (H3-mK9) (5,6); the chromoshadow domain is a proteinCorrespondence to be sent to: Gena Eve Stephens, protein interaction domain that forms a homodimer (7) and binds to many other proteins, in...

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Drosophila dot chromosomes

Sequencing and analysis of fosmid hybridization to the dot chromosomes of Drosophila virilis and D. melanogaster suggest that repetitive elements and density are important in determining higher-order chromatin packaging.

Drosophila melanogaster heterochromatin protein 2 (HP2) interacts with heterochromatin protein 1 (HP1). In polytene chromosomes, HP2 and HP1 colocalize at the chromocenter, telomeres, and the small fourth chromosome. We show here that HP2 is present in the arms as well as the centromeric regions of mitotic chromosomes. We also demonstrate that Su(var)2-HP2 exhibits a dosage-dependent modification of variegation of a yellow reporter transgene, indicating a structural role in heterochromatin formation. We have isolated and characterized 14 new mutations in the Su(var)2-HP2 gene. Using w m4h , many (but not all) mutant alleles show dominant Su(var) activity. Su(var)2-HP2 mutant larvae show a wide variety of mitotic abnormalities, but not the telomere fusion seen in larvae deficient for HP1. The Su(var)2-HP2 gene codes for two isoforms: HP2-L (365 kDa) and HP2-S (175 kDa), lacking exons 5 and 6. In general, mutations that affect only the larger isoform result in more pronounced defects than do mutations common to both isoforms. This suggests that an imbalance between large and small isoforms is particularly deleterious. These results indicate a role for HP2 in the structural organization of chromosomes and in heterochromatin-induced gene silencing and show that the larger isoform plays a critical role in these processes. T HE DNA found inside a eukaryotic nucleus does not exist as such, but is packaged with proteins to form chromatin. By weight, chromatin is approximately one-third DNA, one-third histones, and one-third nonhistone chromosomal proteins, plus a small RNA component. The histones play a major role in packaging and organizing the very long DNA molecules of each chromosome. Analysis of post-transcriptional modifications of histones shows that these basic proteins also play a significant role in determining specific modes of packaging and concomitant gene regulation, interacting with both enzymes and structural proteins that define alternative chromatin states (for reviews, see Richards and Elgin 2002;Khorasanizadeh 2004). One level of packaging, evident by cytological examination of interphase nuclei, is the partitioning of chromatin into euchromatin and heterochromatin. While euchromatin decondenses during interphase, heterochromatin remains relatively more condensed, showing intense staining.In Drosophila melanogaster, the entire Y chromosome, most of the fourth chromosome, the proximal 40% of the X chromosome, and the pericentric 20% of the major autosomes are heterochromatic. Euchromatic DNA has a high proportion of genes and unique sequences and tends to replicate throughout S phase. Heterochromatin has relatively few genes, is rich in repetitive sequences, and tends to replicate late in S phase; it is often found to be underreplicated (compared to euchromatin) in the polytene chromosomes of insects such as D. melanogaster. Heterochromatin is also characterized by a very low rate of meiotic recombination (Weiler and Wakimoto 1995;Zhimulev et al. 2004).Euchromatin and heterochromatin appear t...

Background: Diplomonads are common free-living inhabitants of anoxic aquatic environments and are also found as intestinal commensals or parasites of a wide variety of animals. Spironucleus vortens is a putatively commensal diplomonad of angelfish that grows to high cell densities in axenic culture. Genomic sequencing of S. vortens is in progress, yet little information is available regarding molecular and cellular aspects of S. vortens biology beyond descriptive ultrastructural studies. To facilitate the development of S. vortens as an additional diplomonad experimental model, we have constructed and stably transformed an episomal plasmid containing an enhanced green fluorescent protein (GFP) tag, an AU1 epitope tag, and a tandem affinity purification (TAP) tag. This construct also contains selectable antibiotic resistance markers for both S. vortens and E. coli.