A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discusse

Expression of Telomerase and Telomere Length Are Unaffected by either Age or Limb Regeneration in Danio rerio

BACKGROUND:The zebrafish is an increasingly popular model for studying many aspects of biology. Recently, ztert, the zebrafish homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintains telomerase activity for much of its adult life and has remarkable regenerative capacity. To date, there has been no longitudinal study to assess whether this retention of telomerase activity equates to the retention of chromosome telomere length through adulthood. METHODOLOGY/PRINCIPAL FINDINGS:We have systematically analyzed individual organs of zebrafish with regard to both telomere length and telomerase activity at various time points in its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years of age by Southern blot analysis. We found that telomeres do not appreciably shorten throughout the lifespan of the zebrafish in any organ. In addition, there was little difference in telomere lengths between organs. Even when cells were under the highest pressure to divide after fin-clipping experiments, telomere length was unaffected. All aged (2 year old) tissues examined also expressed active amounts of telomerase activity as assessed by TRAP assay. CONCLUSIONS/SIGNIFICANCE:In contrast to several other species including humans, the retention of lifelong telomerase and telomeres, as we have reported here, would be necessary in the zebrafish to maintain its tremendous regenerative capacity. The ongoing study of the zebrafish's ability to maintain telomerase activity may be helpful in unraveling the complexity involved in the maintenance (or lack thereof) of telomeres in other species such the mouse or human

Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Background: Histone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression. Results: Using immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo. Conclusions: These results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Characterization of phycobilisome glycoproteins in the cyanobacterium Anacystis nidulans R2.

Concanavalin A-reactive linker and anchor subunits of phycobilisomes from Anacystis nidulans R2 (H. C. Riethman, T. P. Mawhinney, and L. A. Sherman, FEBS Lett. 215:209-214, 1987) were purified electrophoretically and analyzed for carbohydrate composition and quantity. Different quantities of glucose and N-acetylgalactosamine were found on the concanavalin A-reactive subunits analyzed. Proteolytic analysis of the purified subunits suggested that small regions of the 33- and 27-kilodalton linker polypeptides previously shown to be important for in vitro phycobilisome assembly contained the concanavalin A-reactive carbohydrates present on these subunits. The linker and anchor subunits from the morphologically different phycobilisome of Synechocystis sp. strain PCC6714 were also shown to be concanavalin A reactive. Membranes from iron-starved Anacystis nidulans, which lack assembled phycobilisomes and are associated with glycogen deposits, were shown to be depleted of linker and anchor proteins and to accumulate very large quantities of a concanavalin A-reactive, extrinsic membrane glycoprotein. We suggest that this iron stress-induced glycoprotein is associated with the glycogen deposits on the thylakoid surface and that the glycosylation of phycobilisome linker and anchor subunits is involved in the physiological regulation of phycobilisome assembly and degradation