Dieter Schweizer

Reverse fluorescent chromosome banding with chromomycin and DAPI.

Two DNA binding guanine-specific antibiotics, chromomycin A3 (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.

Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin

Post-translational modifications of histone amino termini are an important regulatory mechanism that induce transitions in chromatin structure, thereby contributing to epigenetic gene control and the assembly of specialized chromosomal subdomains. Methylation of histone H3 at lysine 9 (H3–Lys9) by site-specific histone methyltransferases (Suv39h HMTases) marks constitutive heterochromatin. Here, we show that H3–Lys9 methylation also occurs in facultative heterochromatin of the inactive X chromosome (Xi) in female mammals. H3–Lys9 methylation is retained through mitosis, indicating that it might provide an epigenetic imprint for the maintenance of the inactive state. Disruption of the two mouse Suv39h HMTases abolishes H3-Lys9 methylation of constitutive heterochromatin but not that of the Xi. In addition, HP1 proteins, which normally associate with heterochromatin, do not accumulate with the Xi. These observations suggest the existence of an Suv39h-HP1-independent pathway regulating H3-Lys9 methylation of facultative heterochromatin.

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

Abstract A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.

Isolation and Characterization of Suv39h2, a Second Histone H3 Methyltransferase Gene That Displays Testis-Specific Expression

ABSTRACT Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. We have recently discovered mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) and shown that they modulate chromatin dynamics in somatic cells. We describe here the isolation, chromosomal assignment, and characterization of a second murine gene, Suv39h2. Like Suv39h1,Suv39h2 encodes an H3 HMTase that shares 59% identity with Suv39h1 but which differs by the presence of a highly basic N terminus. Using fluorescent in situ hybridization and haplotype analysis, theSuv39h2 locus was mapped to the subcentromeric region of mouse chromosome 2, whereas the Suv39h1 locus resides at the tip of the mouse X chromosome. Notably, although bothSuv39h loci display overlapping expression profiles during mouse embryogenesis, Suv39h2 transcripts remain specifically expressed in adult testes. Immunolocalization of Suv39h2 protein during spermatogenesis indicates enriched distribution at the heterochromatin from the leptotene to the round spermatid stage. Moreover, Suv39h2 specifically accumulates with chromatin of the sex chromosomes (XY body) which undergo transcriptional silencing during the first meiotic prophase. These data are consistent with redundant enzymatic roles for Suv39h1 and Suv39h2 during mouse development and suggest an additional function of the Suv39h2 HMTase in organizing meiotic heterochromatin that may even impart an epigenetic imprint to the male germ line.

Application of Giemsa banding to orchid karyotype analysis

A method for obtaining orchid chromosome squash preparations from ovular tissues and a Giemsa C-band technique are described. Jointly applied, they result in well-defined chromosome banding patterns. Preliminary tests with two species of the genusCephalanthera show that Giemsa banding is also well suited for orchids. Besides aiding in chromosome identification and karyotype analysis, it should prove valuable in studies of chromosomal variation and karyotype evolution of this large family.

Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution

The extent of 5S and 45S ribosomal DNA (rDNA) variation was investigated in wild and domesticated common beans (Phaseolus vulgaris) chosen to represent the known genetic diversity of the species. 5S and 45S rDNA probes were localized on mitotic chromosomes of 37 accessions by fluorescent in situ hybridization (FISH). The two 5S rDNA loci were largely conserved within the species, whereas a high variation in the number of 45S rDNA loci and changes in position of loci and number of repeats per locus were observed. Domesticated accessions from the Mesoamerican gene pool frequently had three 45S rDNA loci per haploid genome, and rarely four. Domesticated accessions from Andean gene pool, particularly from the race Peru, showed six, seven, eight or nine loci, but seven loci were found in all three races of this gene pool. Between three and eight loci were observed in accessions resulting from crosses between Andean and Mesoamerican genotypes. The presence of two to eight 45S rDNA loci in wild common beans from different geographic locations indicates that the 45S rDNA amplification observed in the Andean lineage took place before domestication. Our data suggest that ectopic recombination between terminal chromosomal regions might be the mechanism responsible for this variation.

Meiotic chromosome condensation and pairing in Saccharomyces cerevisiae studied by chromosome painting.

Non-isotopic high resolution in sity hybridization was applied to cytological preparations of sporulating yeast cells. Ribosomal DNA (rDNA) and chromosome V-specific recombinant lambda clones were used to tag individual chromosomes and chromosome subregions. This allowed the study of chromosome behaviour during early meiotic prophase. It was found that chromatin becomes condensed and homologous DNA sequences then appear to become aligned prior to synaptonemal complex formation.

Equilocality of heterochromatin distribution and heterochromatin heterogeneity in acridid grasshoppers

Comparative fluorescence studies on the chromosome of ten species of acridid grasshoppers, with varying amounts and locations of C-band positive heterochromatin, indicate that the only regions to fluoresce differentially are those that C-band. Within a given species there is a marked tendency for groups of chromosomes to accumulate heterochromatin with similar fluorescence behaviour at similar sites. This applies to all three major categories of heterochromatin — centric, interstitial and telomeric. Different sites within the same complement, however, tend to have different fluorescence properties. In particular, centric C-bands within a given species are regularly distinguishable in their behaviour from telomeric C-bands. Different species, on the other hand, may show distinct forms of differential fluorescence at equilocal sites. These varying patterns of heterochromatin heterogeneity, both within and between species, indicate that whatever determines the differential response to fluorochromes has tended to operate both on an equilocal basis and in a concerted fashion. This is reinforced by the fact that structural rearrangements that lead to the relocation of centric C-bands, either within or between species, may also be accompanied by a change in fluorescence behaviour.

Selective Interactions between Vertebrate Polycomb Homologs and the SUV39H1 Histone Lysine Methyltransferase Suggest that Histone H3-K9 Methylation Contributes to Chromosomal Targeting of Polycomb Group Proteins

ABSTRACT Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.

Giemsa banded karyotypes, systematics, and evolution inAnacyclus (Asteraceae-Anthemideae)

Giemsa C-banding allows the differentiation of six, otherwise very similar karyotypes from the small genusAnacyclus. “Banding style”—with stable centromeric and nucleolar bands, and diverse specific banding patterns in distal chromosome segments—contributes significantly to generic demarcation and systematic grouping. The amount of banding corresponds to heterochromatic chromocentres and increases from perennials to annuals. Relationships with other nucleotype parameters and evolutionary mechanisms are discussed.