The telomeric Cdc13-Stn1-Ten1 complex regulates RNA polymerase II transcription.

Specialized telomeric proteins have an essential role in maintaining genome stability through chromosome end protection and telomere length regulation. In the yeast Saccharomyces cerevisiae, the evolutionary conserved CST complex, composed of the Cdc13, Stn1 and Ten1 proteins, largely contributes to these functions. Here, we report genetic interactions between TEN1 and several genes coding for transcription regulators. Molecular assays confirmed this novel function of Ten1 and further established that it regulates the occupancies of RNA polymerase II and the Spt5 elongation factor within transcribed genes. Since Ten1, but also Cdc13 and Stn1, were found to physically associate with Spt5, we propose that Spt5 represents the target of CST in transcription regulation. Moreover, CST physically associates with Hmo1, previously shown to mediate the architecture of S-phase transcribed genes. The fact that, genome-wide, the promoters of genes down-regulated in the ten1-31 mutant are prefentially bound by Hmo1, leads us to propose a potential role for CST in synchronizing transcription with replication fork progression following head-on collisions.

Detection of the alternative lengthening of telomeres pathway in malignant gliomas for improved molecular diagnosis.

Human malignant gliomas exhibit acquisition of either one of two telomere maintenance mechanisms, resulting from either reactivation of telomerase expression or activation of an alternative lengthening of telomeres (ALT) mechanism. In the present study, we analyzed 63 human malignant gliomas for the presence of ALT-specific extrachromosomal circles of telomeric DNA (C-circles) and measured telomerase expression, telomeric DNA content (Telo/Alu method), and telomeric repeat-containing RNAs (TERRA) levels. We also assessed histomolecular markers routinely used in clinical practice. The presence of C-circles significantly correlated with IDH1/2 mutation, MGMT exon 1 methylation, low Ki-67 immunostaining, increased telomeric DNA content, absence of functional ATRX protein and level of HTERT gene expression. In multivariate analysis, we observed a trend to a correlation between elevated TERRA levels and increased survival. Interestingly, the C-circles assay allowed to detect ALT activation in glioblastomas exhibiting wild-type IDH1/2 and ATRX expression. These results suggest that, after the correlations uncovered here have been confirmed on larger numbers of tumors, telomeric markers might be useful in improving diagnosis. They also point out to the utility of using the specific, sensitive and quantitative C-circle and Telo/Alu assays that can work with as few as 30 ng of tumor DNA.

Measurement of Telomere Length in Colorectal Cancers for Improved Molecular Diagnosis.

All tumors have in common to reactivate a telomere maintenance mechanism to allow for unlimited proliferation. On the other hand, genetic instability found in some tumors can result from the loss of telomeres. Here, we measured telomere length in colorectal cancers (CRCs) using TRF (Telomere Restriction Fragment) analysis. Telomeric DNA content was also quantified as the ratio of total telomeric (TTAGGG) sequences over that of the invariable Alu sequences. In most of the 125 CRCs analyzed, there was a significant diminution in telomere length compared with that in control healthy tissue. Only 34 tumors exhibited no telomere erosion and, in some cases, a slight telomere lengthening. Telomere length did not correlate with age, gender, tumor stage, tumor localization or stage of tumor differentiation. In addition, while telomere length did not correlate with the presence of a mutation in BRAF (V-raf murine sarcoma viral oncogene homolog B), PIK3CA (phosphatidylinositol 3-kinase catalytic subunit), or MSI status, it was significantly associated with the occurrence of a mutation in KRAS. Interestingly, we found that the shorter the telomeres in healthy tissue of a patient, the larger an increase in telomere length in the tumor. Our study points to the existence of two types of CRCs based on telomere length and reveals that telomere length in healthy tissue might influence telomere maintenance mechanisms in the tumor.

Rat pulmonary responses to inhaled nano-TiO2: effect of primary particle size and agglomeration state.

BACKGROUND: The exact role of primary nanoparticle (NP) size and their degree of agglomeration in aerosols on the determination of pulmonary effects is still poorly understood. Smaller NP are thought to have greater biological reactivity, but their level of agglomeration in an aerosol may also have an impact on pulmonary response. The aim of this study was to investigate the role of primary NP size and the agglomeration state in aerosols, using well-characterized TiO2 NP, on their relative pulmonary toxicity, through inflammatory, cytotoxic and oxidative stress effects in Fisher 344 male rats. METHODS: Three different sizes of TiO2 NP, i.e., 5, 10-30 or 50 nm, were inhaled as small (SA) (< 100 nm) or large agglomerates (LA) (> 100 nm) at 20 mg/m³ for 6 hours. RESULTS: Compared to the controls, bronchoalveolar lavage fluids (BALF) showed that LA aerosols induced an acute inflammatory response, characterized by a significant increase in the number of neutrophils, while SA aerosols produced significant oxidative stress damages and cytotoxicity. Data also demonstrate that for an agglomeration state smaller than 100 nm, the 5 nm particles caused a significant increase in cytotoxic effects compared to controls (assessed by an increase in LDH activity), while oxidative damage measured by 8-isoprostane concentration was less when compared to 10-30 and 50 nm particles. In both SA and LA aerosols, the 10-30 nm TiO2 NP size induced the most pronounced pro-inflammatory effects compared to controls. CONCLUSIONS: Overall, this study showed that initial NP size and agglomeration state are key determinants of nano-TiO2 lung inflammatory reaction, cytotoxic and oxidative stress induced effects.

Oral p-tert-octylphenol exposures induce minimal toxic or estrogenic effects in adult female Sprague-Dawley rats.

Contamination of the environment with endocrine-disrupting chemicals (EDC) has raised concerns about potential health hazards for humans and wildlife. Human and wildlife exposure to one such ubiquitous chemical, p-tert-octylphenol (OP), are likely, due to its persistence in the environment and its presence in food, water, and items of daily use. OP is reported to bind to the estrogen receptor (ER) and alter expression of estrogen-responsive genes. Detrimental effects of OP exposures on the reproductive system have been observed in most, but not all, in vivo experiments. This study examined estrogenic effects of oral exposures of adult female rats to OP. In vitro, OP bound weakly to human ER and a co-activator protein, and accelerated proliferation of MCF-7 cells. Adult Sprague-Dawley rats were given OP by gavage daily for 35 d (25, 50, or 125 mg/kg/d). Body and organ weights and ovarian follicle populations were not significantly altered in OP-exposed adult rats, despite detectable levels of OP in reproductive organs. The estrous cycle of rats was slightly altered, but there were no significant estrogen-like changes in histomorphology or gene expression of the uterus. Prepubertal rats given 125 or 250 mg/kg OP by gavage for 3 d had reduced body weight compared to vehicle-exposed rats but failed to show any uterotrophic response, although 17alpha-ethinyl estradiol (EE, 10 microg/kg/d, ip) induced a threefold increase in uterine weight. Overall, results suggest that toxicity will occur before estrogenic effects with oral exposures to OP. Relevant environmental exposures likely pose little risk for estrogenic effects.

Inhibition of the alternative lengthening of telomeres pathway by subtelomeric sequences in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, telomerase is constitutively active and is essential for chromosome end protection and illimited proliferation of cell populations. However, upon inactivation of telomerase, alternative mechanims of telomere maintenance allow proliferation of only extremely rare survivors. S. cerevisiae type I and type II survivors differ by the nature of the donor sequences used for repair by homologous recombination of the uncapped terminal TG1-3 telomeric sequences. Type I amplifies the subtelomeric Y' sequences and is more efficient than type II, which amplifies the terminal TG1-3 repeats. However, type II survivors grow faster than type I survivors and can easily outgrow them in liquid cultures. The mechanistic interest of studying S. cerevisiae telomeric recombination is reinforced by the fact that type II recombination is the equivalent of the alternative lengthening of telomeres (ALT) pathway that is used by 5-15 % of cancer types as an alternative to telomerase reactivation. In budding yeast, only around half of the 32 telomeres harbor Y' subtelomeric elements. We report here that in strains harboring Y' elements on all telomeres, type II survivors are not observed, most likely due to an increase in the efficiency of type I recombination. However, in a temperature-sensitive cdc13-1 mutant grown at semi-permissive temperature, the increased amount of telomeric TG1-3 repeats could overcome type II inhibition by the subtelomeric Y' sequences. Strikingly, in the 100 % Y' strain the replicative senescence crisis normally provoked by inactivation of telomerase completely disappeared and the severity of the crisis was proportional to the percentage of chromosome-ends lacking Y' subtelomeric sequences. The present study highlights the fact that the nature of subtelomeric elements can influence the selection of the pathway of telomere maintenance by recombination, as well as the response of the cell to telomeric damage caused by telomerase inactivation.

Effects of chronic exposure to octylphenol on the male rat reproductive system.

p-tert-Octylphenol (OP) is a degradation product of alkylphenol ethoxylates. OP is an endocrine disruptor known to bind to the estrogen receptor; however, effects on males are controversial. The objective of this study was to evaluate the effects of chronic exposure to OP on male reproduction. Adult Sprague-Dawley rats were administered OP for 60 d, representing 1.5 cycles of spermatogenesis. Experimental groups included a vehicle control, and three doses of OP (25, 50, or 125 mg/kg body weight [bw]) administered daily by gavage. There was a significant decrease in body weight in the 125-mg/kg group after 60 d of treatment. Both testicular and epididymal weights and histology were not altered by treatment with OP at any of the doses administered. There were no marked differences in cauda epididymal sperm counts at any doses; however, total percent sperm motility was significantly lower in rats exposed to the intermediate dose (50 mg/kg bw). There was an increase in percent static sperm cells in all OP-treated groups, with the intermediate dose (50 mg/kg) displaying a significantly higher proportion of static cells relative to untreated controls. Caput epididymal sperm motility was unaltered by OP treatment. Gene expression profiles of testes from control and high-dose-exposed rats indicate that 14 genes were modulated by at least twofold, although these changes were not statistically significant. Taken together, results from this study indicate that OP treatment of adult rats does not appear to exert major effects on male reproductive endpoints at relevant environmental exposure doses.

Control of the yeast telomeric senescence survival pathways of recombination by the Mec1 and Mec3 DNA damage sensors and RPA.

Saccharomyces cerevisiae telomerase-negative cells undergo homologous recombination on subtelomeric or TG(1-3) telomeric sequences, thus allowing Type I or Type II post-senescence survival, respectively. Here, we find that the DNA damage sensors, Mec1, Mec3 and Rad24 control Type II recombination, while the Rad9 adaptor protein and the Rad53 and Chk1 effector kinases have no effect on survivor type selection. Therefore, the Mec1 and Mec3 checkpoint complexes control telomeric recombination independently of their roles in generating and amplifying the Mec1-Rad53-Chk1 kinase cascade. rfa1-t11 mutant cells, bearing a mutation in Replication Protein A (RPA) conferring a defect in recruiting Mec1-Ddc2, were also deficient in both types of telomeric recombination. Importantly, expression of an Rfa1-t11-Ddc2 hybrid fusion protein restored checkpoint-dependent arrest, but did not rescue defective telomeric recombination. Therefore, the Rfa1-t11-associated defect in telomeric recombination is not solely due to its failure to recruit Mec1. We have also isolated novel alleles of RFA1 that were deficient in Type I but not in Type II recombination and proficient in checkpoint control. Therefore, the checkpoint and recombination functions of RPA can be genetically separated, as can the RPA-mediated control of the two types of telomeric recombination.

The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas.

All cancer cells need to maintain functional telomeres to sustain continuous cell division and proliferation. In human diffuse gliomas, functional telomeres are maintained due either to reactivation of telomerase expression, the main pathway in most cancer types, or to activation of a mechanism called the alternative lengthening of telomeres (ALT). The presence of IDH1/2 mutations (IDH-mutant) together with loss of ATRX expression (ATRX-lost) are frequently associated with ALT in diffuse gliomas. However, detection of ALT, and a fortiori its quantification, are rarely, if ever, measured in neuropathology laboratories. We measured the level of ALT activity using the previously described quantitative "C-circle" assay and analyzed it in a well characterized cohort of 104 IDH-mutant and ATRX-lost adult diffuse gliomas. We report that in IDH-mutant ATRX-lost anaplastic astrocytomas, the intensity of ALT was inversely correlated with age (p < 0.001), the younger the patient, the higher the intensity of ALT. Strikingly, glioblastomas having progressed from anaplastic astrocytomas did not exhibit this correlation. ALT activity level in the tumor did not depend on telomere length in healthy tissue cells from the same patient. In summary, we have uncovered the existence, in anaplastic astrocytomas but not in glioblastomas with the same IDH and ATRX mutations, of a correlation between patient age and the level of activity of ALT, a telomerase-independent pathway of telomere maintenance.

Protection against chromosome degradation at the telomeres.

Telomeres, the ends of linear chromosomes, contain repeated TG-rich sequences which, in dividing cells, must be constantly replenished in order to avoid chromosome erosion and, hence, genomic instability. Moreover, unprotected telomeres are prone to end-to-end fusions. Telomerase, a specialized reverse transcriptase with a built-in RNA template, or, in the absence of telomerase, alternative pathways of telomere maintenance are required for continuous cell proliferation in actively dividing cells as well as in cancerous cells emerging in deregulated somatic tissues. The challenge is to keep these free DNA ends masked from the nucleolytic attacks that will readily operate on any DNA double-strand break in the cell, while also allowing the recruitment of telomerase at intervals. Specialized telomeric proteins, as well as DNA repair and checkpoint proteins with a dual role in telomere maintenance and DNA damage signaling/repair, protect the telomere ends from degradation and some of them also function in telomerase recruitment or other aspects of telomere length homeostasis. Phosphorylation of some telomeric proteins by checkpoint protein kinases appears to represent a mode of regulation of telomeric mechanisms. Finally, recent studies have allowed starting to understand the coupling between progression of the replication forks through telomeric regions and the subsequent telomere replication by telomerase, as well as retroaction of telomerase in cis on the firing of nearby replication origins.

Physiologically based pharmacokinetic modeling of persistent organic pollutants for lifetime exposure assessment: a new tool in breast cancer epidemiologic studies.

BACKGROUND: Despite experimental evidence, most epidemiologic studies to date have not supported an association between exposure to persistent organic pollutants (POP) and breast cancer incidence in humans. This may be attributable to difficulties in estimating blood/tissue POP concentration at critical time periods of carcinogenesis. OBJECTIVES: In this work we aimed to develop a tool to estimate lifetime POP blood/tissue exposure and levels during any hypothesized time window of susceptibility in breast cancer development. METHODS: We developed a physiologically based pharmacokinetic (PBPK) model that can account for any given physiologic lifetime history. Using data on pregnancies, height, weight, and age, the model estimates the values of physiologic parameters (e.g., organ volume, composition, and blood flow) throughout a woman's entire life. We assessed the lifetime toxicokinetic profile (LTP) for various exposure scenarios and physiologic factors (i.e., breast-feeding, growth, pregnancy, lactation, and weight changes). RESULTS: Simulations for three POPs [hexachlorobenzene, polychlorinated biphenyl (PCB)-153, PCB-180] using different lifetime physiologic profiles showed that the same blood concentration at 55 years of age can be reached despite totally different LTP. Aside from exposure levels, lactation periods and weight profile history were shown to be the factors that had the greatest impact on the LTP. CONCLUSIONS: This new lifetime PBPK model, which showed the limitations of using a single sample value obtained around the time of diagnosis for lifetime exposure assessment, will enable researchers conducting environmental epidemiology studies to reduce uncertainty linked to past POP exposure estimation and to consider exposure during time windows that are hypothesized to be mechanistically critical in carcinogenesis.

Synergistic effect of 5-Aza-2'-deoxycytidine and genistein in combination against leukemia.

5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation, is an effective agent for the treatment of leukemia. The aim of this study was to investigate the antileukemic activity of this epigenetic agent in combination with genistein, a nontoxic isoflavone with chemopreventive activity. The combined treatment produced a synergistic loss of clonogenicity in human myeloid (HL-60) and lymphoid (MOLT-3) leukemic cell lines. Genistein alone showed a significant antileukemic activity against murine 5-AZA-CdR-resistant cells, and this effect was enhanced when used in combination with 5-AZA-CdR. The combined treatment also produced a synergistic increase in life span of mice with L1210 leukemia. These results suggest that genistein may have the potential to increase the clinical efficacy of 5-AZA-CdR for the treatment of leukemia.

Sexual dimorphism in the regulation of liver connexin32 transcription in hexachlorobenzene-treated rats.

Hexachlorobenzene (HCB), an epigenetic carcinogen, causes female rats to be more susceptible to liver tumor formation than males. HCB exposure in females downregulates the expression of Cx32, a gap junction protein, through the activation of Akt. The objectives of this study were to determine (1) the implication of different regions of the hepatic Cx32 promoter in the observed sexual dimorphism in the expression of Cx32, (2) the implication of different regions of the hepatic Cx32 promoter in the HCB-induced downregulation of Cx32 in female rat liver, and (3) if HCB exposure modulates the binding of transcription factors on the Cx32 promoter through Akt activation. Male and female rats were exposed to HCB during 5 consecutive days and sampled 45 days later. Electrophoresis mobility shift assays showed that the intensity of only one nuclear protein-DNA complex differed between males and females. The formation of this complex requires two binding sites to be intact in a fragment of the basal promoter (Fr26). Following HCB exposure, the intensity of two complexes (Fr26 and Fr110) was decreased in females, but not in males, consistent with the decrease in Cx32 expression observed only in HCB-treated females. In vitro studies using a rat hepatocyte cell line (MH1C1) showed that the formation of the Fr110 protein-DNA complex appears to be controlled by Akt and requires the integrity of a Myb site. Overall, results suggest that both the sexual dimorphism and the downregulation of Cx32 in HCB-treated female rats are mediated by a reduction in the binding of activating transcription factors on the Cx32 promoter.

Mitotic cyclins regulate telomeric recombination in telomerase-deficient yeast cells.

Telomerase-deficient mutants of Saccharomyces cerevisiae can survive death by senescence by using one of two homologous recombination pathways. The Rad51 pathway amplifies the subtelomeric Y' sequences, while the Rad50 pathway amplifies the telomeric TG(1-3) repeats. Here we show that telomerase-negative cells require Clb2 (the major B-type cyclin in this organism), in association with Cdc28 (Cdk1), to generate postsenescence survivors at a normal rate. The Rad50 pathway was more sensitive to the absence of Clb2 than the Rad51 pathway. We also report that telomerase RAD50 RAD51 triple mutants still generated postsenescence survivors. This novel Rad50- and Rad51-independent pathway of telomeric recombination also appeared to be controlled by Clb2. In telomerase-positive cells, a synthetic growth defect between mutations in CLB2 and RAD50 or in its partners in the conserved MRX complex, MRE11 and XRS2, was observed. This genetic interaction was independent of Mre11 nuclease activity but was dependent on a DNA repair function. The present data reveal an unexpected role of Cdc28/Clb2 in telomeric recombination during telomerase-independent maintenance of telomeres. They also uncover a functional interaction between Cdc28/Clb2 and MRX during the control of the mitotic cell cycle.

The Rad51 pathway of telomerase-independent maintenance of telomeres can amplify TG1-3 sequences in yku and cdc13 mutants of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, Cdc13, Yku, and telomerase define three parallel pathways for telomere end protection that prevent chromosome instability and death by senescence. We report here that cdc13-1 yku70delta mutants generated telomere deprotection-resistant cells that, in contrast with telomerase-negative senescent cells, did not display classical crisis events. cdc13-1 yku70delta cells survived telomere deprotection by exclusively amplifying TG(1-3) repeats (type II recombination). In a background lacking telomerase (tlc1delta), this process predominated over type I recombination (amplification of subtelomeric Y' sequences). Strikingly, inactivation of the Rad50/Rad59 pathway (which is normally required for type II recombination) in cdc13-1 yku70delta or yku70delta tlc1delta mutants, but also in cdc13-1 YKU70(+) tlc1delta mutants, still permitted type II recombination, but this process was now entirely dependent on the Rad51 pathway. In addition, delayed senescence was observed in cdc13-1 yku70delta rad51delta and cdc13-1 tlc1delta rad51delta cells. These results demonstrate that in wild-type cells, masking by Cdc13 and Yku prevents the Rad51 pathway from amplifying telomeric TG(1-3) sequences. They also suggest that Rad51 is more efficient than Rad50 in amplifying the sequences left uncovered by the absence of Cdc13 or Yku70.

Involvement of the integrin-linked kinase pathway in hexachlorobenzene-induced gender-specific rat hepatocarcinogenesis.

Overexpression of the integrin-linked kinase (ILK) pathway disrupts cell-cell interactions, an epigenetic event leading to epithelial cell transformation. Female rats exposed to hexachlorobenzene (HCB) for 5 consecutive days and sampled 45 days later show a decrease in liver gap junctional intercellular communication. We hypothesized that HCB also alters E-cadherin expression and that this alteration is mediated by the ILK pathway. Hepatic ILK levels were markedly increased in HCB-treated female rats. Cytoplasmic/membrane levels of protein kinase B (Akt), a target of ILK, and its phosphorylated active form were decreased in treated female rats. Flow cytometric analysis showed a concomitant increase in nuclear Akt levels. Both ILK and Akt can phosphorylate glycogen synthetase kinase-3beta (GSK3beta), rendering it inactive. Phosphorylated-GSK3beta levels were higher in treated females and resulted in a twofold decrease in the activity of GSK3beta. The inactivation of GSK3beta in HCB-treated female rats resulted in the nuclear translocation of beta-catenin, as demonstrated by both immunocytochemistry and flow cytometric analyses. Western blot analysis showed an 84% decrease in E-cadherin levels in HCB-treated rats as compared to controls, and this decrease was not mediated by Snail activation. Mimicking the activation of ILK with specific GSK3beta inhibitors resulted in downregulation of E-cadherin levels but had no effect on Cx32 expression in the MH(1)C(1) cells. Overall, these results indicate that hepatic E-cadherin is downregulated as a result of an overexpression of the ILK pathway. The concomitant HCB-induced downregulation of intercellular communication does not occur as a result of either E-cadherin downregulation or GSK3beta inactivation.

Physiologically based modeling of the accumulation in plasma and tissue lipids of a mixture of PCB congeners in female Sprague-Dawley rats.

This study aimed to develop a physiologically based model for simulating the concentrations of polychlorinated biphenyls (PCBs) in tissue and plasma lipids of rats exposed to PCB mixtures. The model was based on the assumption that the neutral lipid fraction is the only critical determinant of the tissue distribution of PCBs, and that the solubility/retention in other tissue components is negligible. The volumes of the model compartments reflected the volumes of neutral lipids, whereas the flow rates corresponded to those of the neutral lipids in blood. Since the equilibrium ratio of PCB concentrations in neutral lipids of tissues and plasma equals 1, the present modeling approach does not require the use of tissue:blood partition coefficients. Metabolism rates were derived from the best visual fit of the model to the PCB concentrations in hepatic lipids determined on d 41 and 90 in rats exposed to a mixture containing 5, 50, or 500 microg PCBs (118, 138, 153, 170, 180 and 187) per kilogram body weight according to various protocols: (a) every-day dosing, (b) once-a-week dosing, (c) consecutive dosing for 13 d with no further treatment, and (d) 13 irregularly spaced doses. The resulting model consistently simulated the concentrations of PCBs in adipose tissue and plasma lipids of rats exposed according to the four described protocols. The physiologically based pharmacokinetic (PBPK) model developed in this study should be useful as a basis for interpretating blood or plasma lipid concentration data on PCBs collected during biomonitoring studies.

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells.

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX.

Polysaccharide Complexes in Full-Grown Oocytes of the Newt, Pleurodeles, and Changes in their Distribution During Progesterone-Induced Maturation: (glycogen granules/oocyte maturation/polysaccharide complexes/urodele amphibian).

Immature full-grown oocytes of Pleurodeles waltlii contain large amounts of small electron-dense polysaccharidic granules. These granules lack a limiting membrane, and have a dense but heterogeneous matrix and an apparent diameter of 24-36 nm. Their structure, organization and distribution strongly suggest that they are glycogen granules. On the other hand, mature oocytes both after oviposition or 22-24 hr after in vitro progesterone stimulation contain no polysaccharide granules or complexes. During the first 9-10 hr after hormonal stimulation, granules were abundant and present both individually and as large strands occupying most of the space between the organelles. Granules were frequently found packed together and arranged in regularly arrayed stacks within large subcortical ant cortical vacuoles. Between 4 and 6 hr after progesterone addition, oocytes released the contents of vacuoles to the outside. Between about 11 and 14 hr after progesterone addition, oocytes still contained large amounts of polysaccharide complexes, but the vacuoles were empty. From about 15 hr after progesterone treatment until the end of maturation, the complexes progressively disappeared from the cytoplasm, coincident with the detachment of the follicle cell layer from the oocytes and a reduction in the number and size of microvilli.

A Freeze-Fracture Study of the Cortex of Xenopus laevis Eggs: (amphibian egg/cortical endoplasmic reticulum/cortical granules/Freeze-fracture/membrane junctions).

The organization of the cortex of Xenopus laevis eggs was investigated by freeze-fracture electron microscopy. The cortical endoplasmic reticulum (CER) formed a network surrounding and interconnecting the cortical granules. It formed junctions with the plasma membrane and was confluent with the ER in subcortical regions. Intramembranous particles (IMP1 ) were only present in the P face of the CER, the E face being apparently devoid of pits and particles. Arrays of densely packed IMP1 , having a mean diameter of 17 nm, were restricted to the microvillar region of the plasma membrane. The cortical granule membrane also contained IMP1 (mean diameter, 21 nm) that were sparsely and randomly distributed. Several types of cortical granule seemed to exist based on an analysis of the distribution of the different IMP sizes.