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The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Objective: Improving antimicrobial stewardship in the livestock sector requires an understanding of the motivations for antimicrobial use and the quantities consumed. However,detailed information on antimicrobial use in livestock sectors is lacking. This cross‐sectional study aimed to better understand antimicrobial use in the beef feedlot sector in Australia. Design: A self‐administered questionnaire asking about antimicrobial use and reasons for use was designed and mailed to beef feedlot operators in Australia. Respondents were asked to report the percentage of animals treated, purpose of use, and disease conditions targeted for 26antimicrobial agents. Results: In total, 83 of 517 (16.1%) beef feedlot operators completed the survey. Monensin (61.0%of respondents) and virginiamycin (19.5%of respondents) were the most commonly reported in‐feed antimicrobials. In‐feed antimicrobial agents were most frequently used by respondents for treatment of gastrointestinal diseases (52.8%). Antimicrobials were used for growth promotion by 42.1% of respondents, with most (85.7%) reporting the use of ionophores(a group of compounds not used in human medicine). Short‐acting penicillin(69.1%), short‐acting oxytetracycline, and tulathromycin (both 57.3%) werethe most common injectable antimicrobial agents used. Injectable antimicrobials were most frequently used to treat respiratory (72.3%) and musculoskeletal (67.5%) conditions. Conclusion: Overall,the use of antimicrobials was appropriate for the purpose indicated, and there was a strong preference for drugs of low‐importance in human medicine. The data described here stand to be a strong influence on the implementation of an antimicrobial stewardship program in the sector. [ABSTRACT FROM AUTHOR]

Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this "centromere effect" is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the satellite-less centromere of chromosome 11 causes crossover suppression, similarly to satellite-based centromeres. These results suggest that the centromere effect does not depend on satellite DNA. During this analysis, we observed a peculiar phenomenon: while, as expected, the centromere of the majority of meiotic bivalent chromosomes was labelled with a single immunofluorescence centromeric signal, double-spotted or extended signals were also detected. Their number varied from 0 to 7 in different cells. This observation can be explained by positional variation of the centromeric domain on the two homologs and/or misalignment of pericentromeric satellite DNA arrays during homolog pairing confirming the great plasticity of equine centromeres.

Feedlot cattle consuming large amounts of feed and gaining weight rapidly generate significant amounts of metabolic heat. In summer, failure to dissipate this heat leads to heat accumulation and heat stress. Respiratory rates, panting scores, and behavioral changes are useful indicators of heat stress in cattle. Ceasing cattle movement, providing supplementary water tanks in the pens, cooling the pen surface, and manipulation of nutrition and feeding management should be considered to mitigate the risk and manage a heat stress crisis. Removing manure from the pens and provisions of shade has been found to be beneficial for cattle exposed to hot climates., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries., (© 2018 Nergadze et al.; Published by Cold Spring Harbor Laboratory Press.)

Centromeres are highly distinctive genetic loci whose function is specified largely by epigenetic mechanisms. Understanding the role of DNA sequences in centromere function has been a daunting task due to the highly repetitive nature of centromeres in animal chromosomes. The discovery of a centromere devoid of satellite DNA in the domestic horse consolidated observations on the epigenetic nature of centromere identity, showing that entirely natural chromosomes could function without satellite DNA cues. Horses belong to the genus Equus which exhibits a very high degree of evolutionary plasticity in centromere position and DNA sequence composition. Examination of horses has revealed that the position of the satellite-free centromere is variable among individuals. Analysis of centromere location and composition in other Equus species, including domestic donkey and zebras, confirms that the satellite-less configuration of centromeres is common in this group which has undergone particularly rapid karyotype evolution. These features have established the equids as a new mammalian system in which to investigate the molecular organization, dynamics and evolutionary behaviour of centromeres.

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres., (© 2016 Prendergast et al.; Published by Cold Spring Harbor Laboratory Press.)

The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.

The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. 'Spindle free' nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability.

The functional identity of centromeres arises from a set of specific nucleoprotein particle subunits of the centromeric chromatin fibre. These include CENP-A and histone H3 nucleosomes and a novel nucleosome-like complex of CENPs -T, -W, -S and -X. Fluorescence cross-correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that human CENP-S and -X exist principally in complex in soluble form and retain proximity when assembled at centromeres. Conditional labelling experiments show that they both assemble de novo during S phase and G2, increasing approximately three- to fourfold in abundance at centromeres. Fluorescence recovery after photobleaching (FRAP) measurements documented steady-state exchange between soluble and assembled pools, with CENP-X exchanging approximately 10 times faster than CENP-S (t1/2 ∼ 10 min versus 120 min). CENP-S binding to sites of DNA damage was quite distinct, with a FRAP half-time of approximately 160 s. Fluorescent two-hybrid analysis identified CENP-T as a uniquely strong CENP-S binding protein and this association was confirmed by FRET, revealing a centromere-bound complex containing CENP-S, CENP-X and CENP-T in proximity to histone H3 but not CENP-A. We propose that deposition of the CENP-T/W/S/X particle reveals a kinetochore-specific chromatin assembly pathway that functions to switch centromeric chromatin to a mitosis-competent state after DNA replication. Centromeres shuttle between CENP-A-rich, replication-competent and H3-CENP-T/W/S/X-rich mitosis-competent compositions in the cell cycle.

Purpose: We created a shorter version of the American Urological Association symptom score, called UWIN (urgency, weak stream, incomplete emptying and nocturia)., Materials and Methods: Participants in Prostate Cancer Awareness Week from 2006 and 2007 were administered the regular American Urological Association symptom score and UWIN. A total of 278 participants completed each questionnaire. Total scores of each participant for the American Urological Association symptom score (range 0 to 35) and UWIN (range 0 to 12) were evaluated using Spearman's correlation coefficients and Bland-Altman plots to determine the level of agreement between the 2 questionnaires., Results: The correlation between the total American Urological Association symptom score (range 0 to 35) and the total UWIN score (range 0 to 12) was 0.913 (p<0.0001). The correlation between the quality of life question on the American Urological Association symptom score and UWIN was 0.821 using the Spearman correlation coefficient (p<0.0001). A second analysis performed using Bland-Altman plots showed good agreement between the American Urological Association symptom score and UWIN. Overall, respondents tended to have slightly higher UWIN total scores than their American Urological Association symptom scores., Conclusions: This study validates that the UWIN questionnaire can be used in place of the American Urological Association symptom score. The UWIN questionnaire will lessen the burden on the respondent, broaden the applicability of the instrument and make collecting data as efficient and effective as possible., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: The American Urological Association symptom score instrument is widely used to assess lower urinary tract symptom severity in men. We describe the methods used to develop a shorter form of the American Urological Association symptom score that may provide symptom score assessment with minimal compromise in accuracy., Materials and Methods: Complete American Urological Association symptom score data were collected on 8,731 men who attended Prostate Cancer Awareness Week in 2003 or 2004. Correlation analysis and area under the ROCs were used to determine the best reduced index and cutoff points in scores for the severity categories of mild, moderate and severe., Results: The number of responses in the original 7 American Urological Association symptom score items was lowered from 6 to 4 and for the bothersome index it was lowered from 7 to 3. Four of the original 7 items were retained. Cronbach's α was 0.851 for the symptom score items in our data. The combination of items with the best joint correlation to the American Urological Association symptom score and bothersome score was UWIN (urgency, weak stream, incomplete emptying and nocturia). The correlation of UWIN with the American Urological Association symptom score was 0.938. The correlation of UWIN bother to the American Urological Association bothersome score was 0.638. The ROC for the mild, moderate and severe UWIN categories compared to the categorized American Urological Association symptom score was 0.96, 0.97 and 0.99, respectively., Conclusions: The UWIN instrument may potentially be a valuable tool to assess American Urological Association symptom score severity and bother. Clinical validation of this instrument is indicated in a prospective comparative study., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin., Competing Interests: The authors have declared that no competing interests exist.

Prostate cancer (CaP) is the second most common cause of cancer deaths in the United States and the incidence of CaP has remained constant at 165 cases per 100,000 men. Since 1990, the age-adjusted death rate has decreased by 31%. In this article, the authors review the current literature on the experimental therapy for HIFU. The HIFU technique, its mechanism of action, patient selection, current efficacy studies, complications, follow-up after HIFU treatment, and future developments are discussed., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The faithful replication of DNA and the accurate segregation of genomic material from one generation to the next is critical in the maintenance of genomic stability. This chapter will describe the structure and assembly of an epigenetically inherited locus, the centromere, and its role in the processes by which sister chromatids are evenly segregated to daughter cells. During the G2 phase of the cell cycle kinetochores are assembled upon the chromatids. During mitosis, kinetochores attach chromosome(s) to the mitotic spindle. The kinetochore structure serves as the interface between the mitotic spindle and the chromatids and it is at the kinetochore where the forces that drive chromatid separation are generated. Unattached chromosomes fail to satisfy the spindle assembly checkpoint (SAC), resulting in cell cycle arrest. The centromere is the locus upon which the kinetochore assembles, and centromeres themselves are determined by their unique protein composition. Apart from budding yeast, centromeres are not specified simply by DNA sequence, but rather through chromatin composition and architecture and are thus epigenetically determined. Centromeres are built on a specific nucleosome not found elsewhere in the genome, in which histone H3 is replaced with a homologue - CENP-A or CenH3. This domain is flanked by heterochromatin and is folded to provide a 3-dimensional cylinder-like structure at metaphase that establishes the kinetochore on the surface of the mitotic chromosomes. A large family of CENtromere Proteins (CENPs) associates with centromeric chromatin throughout the cell cycle and are required for kinetochore function. Unlike the bulk of histones, CENP-A is not assembled concurrently with DNA synthesis in S-phase but rather assembles into the centromere in the subsequent G1 phase. The assembly of CENP-A chromatin following DNA replication and the re-establishment of this network of constitutive proteins have emerged as critical mechanisms for understanding how the centromere is replicated during the cell cycle.

Improvements in prostate cancer diagnosis and treatment have resulted in a decreasing age-adjusted death rate. But improved diagnostic tools have not delivered a proportionate decrease in mortality, primarily because physicians now are diagnosing - and treating - more clinically insignificant tumors. Targeted focal therapy (TFT) uses three dimensional (3D) mapping biopsies to guide cryotherapy so that it targets lesions themselves while sparing surrounding healthy tissues, thereby avoiding side effects associated with more invasive treatments. As such, TFT can provide a bridge between active surveillance and more aggressive treatments for patients with low-risk tumors. It is appropriate for men who either do not want or are not good candidates for more aggressive therapies. Identifying patients who are appropriate candidates for TFT is challenging, but the mapping biopsy technique helps identify individuals who have localized disease and could benefit from this treatment. In recent years, improvements in cryotherapy have increased its efficacy while decreasing complications. At the present time using cryotherapy to target and destroy the cancer(s) is appealing. Other approaches to less aggressive therapies are discussed including gland hemi ablation, high intensity ultrasound (HIFU) and others. Going forward, patients will benefit further from development of better imaging technologies and completion of long-term survival studies.

Purpose of Review: Prostate cancer screening has shifted the diagnosis of prostate cancer to lower grade, organ confined disease. Radical prostatectomy or radiation therapy has been shown to be overtreatment in 30% of patients. In this review, we will discuss targeted focal therapy (TFT) using the modalities of cryotherapy or high intensity focused ultrasound as an alternate treatment for low-risk prostate cancers., Recent Findings: TFT uses 3-D mapping biopsies to guide treatment so lesions of interest are ablated whereas sparing surrounding healthy tissues, avoiding the side-effects associated with more invasive treatments. In recent years, improvements in cryotherapy and HIFU have increased efficacy whereas decreasing complications. Prostate cancer control reported following TFT is promising., Summary: With data demonstrating effective treatment in select patients, physicians can better inform their patients of the options available to eradicate their prostate cancer. TFT provides an alternative to active surveillance and more aggressive treatments for patients with low-risk tumors. As studies mature, more information regarding long-term survival and benefit will become more evident.

Autofluorescent proteins (AFPs) have revolutionized molecular cell biology, and applications continue to harness the power of these genetically encoded fluorescent tags. Here, we review the discovery and physical properties of AFPs as well as their development through mutational optimization for several functional parameters. A practical guide to selection and use of major AFPs is provided as well as an overview of techniques for experimental applications.

Confinement of enzymatic reactions to nuclear and chromosomal subdomains regulates functional organization of the nucleus. Aurora-B kinase regulates cell-cycle-dependent phosphorylation of chromosomal substrates through sequential localization to a series of sites on chromosomes and the mitotic spindle. In G2 nuclei, Aurora-B recruitment to heterochromatin restricts histone H3S10 phosphorylation to a domain around centromeres (pericentromeres). However, no intrinsic chromosomal determinants have been implicated in Aurora-B recruitment to interphase pericentromeres. Using cyclin B1 as a cell-cycle marker, we found that the great majority of nuclei exhibiting H3S10 phosphorylated foci were positive for cyclin B1, thus revealing that H3S10 phosphorylation arises at pericentromeres during late S phase and persists in G2. By immunofluorescent in situ hybridization, Aurora-B and H3S10 phosphorylated foci were found more frequently at larger pericentromeres than at smaller ones, revealing a preferential phosphorylation of pericentromeres, exhibiting a high density of methyl cytosines. Disruption of DNA methylation inhibited pericentromeric Aurora-B targeting and H3S10 phosphorylation in G2 nuclei, thus demonstrating the role of DNA methylation in Aurora-B targeting to pericentromeres. These results favour the idea that DNA methylation maintains a local environment essential for regulating the functional properties of sub-chromosomal domains during S-G2 progression.

Pathways and dilution of a point source ocean discharge in the farfield (approximately to 10-66 km) were measured using the deliberate tracer sulfur hexafluoride (SF6). The injection of SF6 was performed by bubbling the gas over a period of 6 days into an ocean outfall pipe discharging into the southeast Florida coastal ocean. The surface SF6 concentrations show that the discharged water flowed northward parallel to the coast with a broadening of the width of the plume to about 3 km at the farthest point sampled, 66 km from the outfall. The discharge was fully mixed throughout the water column within 13 km of the outfall terminus. In the first 20 km from the outfall, SF6 surface concentrations were highly variable, while beyond this the SF6 concentrations decreased monotonically going northward. The currents were measured during the study with a bottom-mounted acoustic Doppler current profiler (ADCP) located 5.5 km from the outfall. Velocities were variable in magnitude and direction but showed a net northward flow during the 6-day study. Maximum concentrations decreased by about 200-fold per kilometer from the outfall to the northern end of the study area. The study shows that SF6 is an effective method to trace point source releases far from their origin.

We have previously described 2D array ultrasound transducers operating up to 10 MHz for applications including real time 3D transthoracic imaging, real time volumetric intracardiac echocardiography (ICE), real time 3D intravascular ultrasound (IVUS) imaging, and real time 3D transesophageal echocardiography (TEE). We have recently built a pair of 2D array transducers for real time 3D laparoscopic ultrasonography (3D LUS). These transducers are intended to be placed down a trocar during minimally invasive surgery. The first is a forward viewing 5 MHz, 11 x 19 array with 198 operating elements. It was built on an 8 layer multilayer flex circuit. The interelement spacing is 0.20 mm yielding an aperture that is 2.2 mm x 3.8 mm. The O.D. of the completed transducer is 10.2 mm and includes a 2 mm tool port. The average measured center frequency is 4.5 MHz, and the -6 dB bandwidth ranges from 15% to 30%. The 50 omega insertion loss, including Gore MicroFlat cabling, is -81.2 dB. The second transducer is a 7 MHz, 36 x 36 array with 504 operating elements. It was built upon a 10 layer multilayer flex circuit. This transducer is in the forward viewing configuration and the interelement spacing is 0.18 mm. The total aperture size is 6.48 mm x 6.48 mm. The O.D. of the completed transducer is 11.4 mm. The average measured center frequency is 7.2 MHz, and the -6 dB bandwidth ranges from 18% to 33%. The 50 omega insertion loss is -79.5 dB, including Gore MicroFlat cable. Real-time in vivo 3D images of canine hearts have been made including an apical 4-chamber view from a substernal access with the first transducer to monitor cardiac function. In addition, we produced real time 3D rendered images of the right pulmonary veins from a right parastemal access with the second transducer, which would be valuable in the guidance of cardiac ablation catheters for treatment of atrial fibrillation.

The availability of iron is known to exert a controlling influence on biological productivity in surface waters over large areas of the ocean and may have been an important factor in the variation of the concentration of atmospheric carbon dioxide over glacial cycles. The effect of iron in the Southern Ocean is particularly important because of its large area and abundant nitrate, yet iron-enhanced growth of phytoplankton may be differentially expressed between waters with high silicic acid in the south and low silicic acid in the north, where diatom growth may be limited by both silicic acid and iron. Two mesoscale experiments, designed to investigate the effects of iron enrichment in regions with high and low concentrations of silicic acid, were performed in the Southern Ocean. These experiments demonstrate iron's pivotal role in controlling carbon uptake and regulating atmospheric partial pressure of carbon dioxide.

White pine blister rust caused by Cronartium ribicola was introduced into North America in the early 20th century and is spreading throughout the range of five-needle pines. In northern Colorado, this pathogen was first observed in 1998 on limber pine (Pinus flexilis) (1). It has not been reported on Rocky Mountain or Great Basin bristlecone pine (Pinus aristata and P. longaeva, respectively) in nature. However, Rocky Mountain bristlecone pine is susceptible to the disease when artificially inoculated (2). In October 2003, a Rocky Mountain bristlecone pine was found infected with C. ribicola in the Great Sand Dunes National Monument, Alamosa County, Colorado. Seven branch cankers were observed on the tree. Cankers ranged in length from 15 to 41 cm and were estimated to be approximately 5 to 7 years old. Distinct C. ribicola branch symptoms were observed, including flagging, spindle-shaped swellings, and 6 mm long aecial scars. A branch was deposited at the Colorado State Herbarium. Microscopic examination of spores within remnant aecial blisters revealed aeciospores characteristic of C. ribicola (yellow-orange, ellipsoid, verrucose, and 19 × 25 μm). Cankers were only observed on one bristlecone pine. However, most limber pines in the area were infected with C. ribicola, including a limber pine less than 1 m from the infected bristlecone pine. To our knowledge, this is the first report that shows Rocky Mountain bristlecone pine can become infected naturally, and the pathogen is further south in Colorado on limber pine than previously reported. These observations suggest the need for a more complete investigation of this disease on bristlecone pines. References: (1) D. W. Johnson and W. R. Jacobi. Plant Dis. 84:595, 2000. (2) B. R. Stephan, Allg. Forst Z. 28:695, 1985.

The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined. The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy).

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

Recent studies have shed new light on how the physical association between sister cells is severed at the end of cytokinesis while the membrane is resealed. Comparisons with yeast suggest that daughter cell shape may feed back to regulate cytokinesis through the Bub2 checkpoint system.

Centromeres provide a distinctive mechanical function for the chromosomes as the site of kinetochore assembly and force generation in mitosis and meiosis. Recent studies show that a unique form of chromatin, based on the histone-H3-like protein CENP-A and homologues, provides a conserved foundation for this mechanical chromatin domain. CENP-A plays a role in templating kinetochore assembly and may be a central element in the epigenetic maintenance of centromere identity. Cohesion at the centromere, intimately linked to kinetochore assembly, is required for integrating spindle forces exerted across the centromere and for establishing the bipolar geometry of sister kinetochores.

We investigated the chromatin organization of living cells with a combination of recently developed approaches for histone and DNA labeling. Nucleosomal DNA was labeled with a histone H2B-GFP (green fluorescent protein) fusion protein and the chromatin organization of living HeLa cells was analyzed by high resolution confocal microscopy. Within the perinuclear and perinucleolar regions chromatin was organized into large-scale fibers of 2 to 8 microm in length and 300 to 500 nm in diameter. Within the nuclear interior we observed similar large-scale fibers, but in addition focal as well as diffuse forms of organization. Comparison with standard labeling and detection procedures revealed major differences in the chromatin organization observed. Chromatin organization revealed by the distribution of histone H2B-GFP was directly compared with the functional organization of chromatin by Cy3-dUTP labeling of DNA replicating at a specific time. DNA regions replicating at a specific time display characteristic physical and functional properties. Analysis of Cy3-labeled foci revealed that they are associated with all three forms of chromatin organization (fibrillar, focal and diffuse). In particular, Cy3-labeled foci appeared as discontinuous regions of large-scale fibers. These results demonstrate that large-scale chromatin fibers have discontinuous functional characteristics.

Cells infected by herpes simplex virus type 1 in the G2 phase of the cell cycle become stalled at an unusual stage of mitosis defined as pseudoprometaphase. This block correlates with the viral immediate-early protein ICP0-induced degradation of the centromere protein CENP-C. However, the observed pseudoprometaphase phenotype of infected mitotic cells suggests that the stability of other centromere proteins may also be affected. Here, we demonstrate that ICP0 also induces the proteasome-dependent degradation of the centromere protein CENP-A. By a series of Western blot and immunofluorescence experiments we show that the endogenous 17-kDa CENP-A and an exogenous tagged version of CENP-A are lost from centromeres and degraded in infected and transfected cells as a result of ICP0 expression. CENP-A is a histone H3-like protein associated with nucleosome structures in the inner plate of the kinetochore. Unlike fully transcribed lytic viral DNA, the transcriptionally repressed latent herpes simplex virus type 1 genome has been reported to have a nucleosomal structure similar to that of cellular chromatin. Because ICP0 plays an essential part in controlling the balance between the lytic and latent outcomes of infection, the ICP0-induced degradation of CENP-A is an intriguing feature connecting different aspects of viral and/or cellular genome regulation.

After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that around H3 Ser10, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, we generated peptide antibodies against the putative phosphorylation site of CENP-A. ELISA, western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression.

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.

The nucleus is known to be compartmentalized into units of function, but the processes leading to the spatial organization of chromosomes and nuclear compartments are not yet well defined. Here we report direct quantitative analysis of the global structural perturbations of interphase chromosome and interchromosome domain distribution caused by infection with herpes simplex virus-1 (HSV-1). Our results show that the peripheral displacement of host chromosomes that correlates with expansion of the viral replication compartment (VRC) is coupled to a twofold increase in nuclear volume. Live cell dynamic measurements suggest that viral compartment formation is driven by the functional activity of viral components and underscore the significance of spatial regulation of nuclear activities.

Here we show that suppression of synthesis of the microtubule motor CENP-E (centromere-associated protein E), a component of the kinetochore corona fibres of mammalian centromeres, yields chromosomes that are chronically mono-orientated, with spindles that are flattened along the plane of the substrate. Despite apparently normal microtubule numbers and the continued presence at kinetochores of other microtubule motors, spindle poles fragment in the absence of CENP-E, which implicates this protein in delivery of components from kinetochores to poles. CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signalling cascade, as depletion of this motor leads to profound checkpoint activation, whereas immunoprecipitation reveals a nearly stoichiometric association of CENP-E with the checkpoint kinase BubR1 during mitosis.

White pine blister rust, caused by Cronartium ribicola Fisch., was found in 1997 infecting white pines (genus Pinus, subgenus Strobus) at two locations in the Carson Range of western Nevada. Rust incidence, infection age, damage to trees, rust phenology, and host distribution were evaluated at one of these locations and a nearby location in California in 1998. At the first location (39.3°N, 119.9°W), C. ribicola was found infecting 24 of 49 whitebark pines (P. albicaulis Engelm.) near Mt. Rose Summit on Highway 27 at 2,710 m elevation, ≈6 km northeast of Incline Village, Washoe County, NV. Among infected trees, 33% had only branch cankers, 54% had live stem cankers, and 12% had stem cankers that had killed portions of trees distal to cankers. No trees had died from infection. At the second location (39.1°N, 119.9°W), we found only 6 of 50 (12%) infected western white pines (P. monticola Dougl.) near Genoa Peak (≈2,750 m elevation), 3 km east of Lake Tahoe, Douglas County, NV; however, stem cankers occurred on 4 of the 6 infected trees. In September 1998, whitebark pines at Mt. Rose and Tahoe Meadows (2,550 m elevation, 1.5 km southwest of Mt. Rose Summit) were examined, and the following was observed: (i) aeciospore production was at its peak, indicating that sporulation can occur exceptionally late in the season in this region; (ii) signs of blister rust infection were absent on the telial hosts of C. ribicola (Ribes cereum, R. montigenum, and R. nevadense) at both locations; (iii) ≈80% of the cankers occurred on host wood produced in 1978 and 1979; and (iv) the oldest cankers originated on wood produced in 1968 and the youngest on wood produced in 1980. In October 1998, infected western white pines were examined at a location (2,650 m elevation) ≈30 km north of Lake Tahoe on Babbitt Peak, Sierra County, CA (39.6°N, 120.1°W). At this location, no trees had died from infection, fresh aeciospores were abundant on live cankers, R. montigenum and R. cereum were present but did not show signs of infection, and 19 of 20 cankers examined were on wood produced between 1978 and 1980. White pine blister rust was not found at any of 10 other locations examined throughout Nevada from 1995 to 1997. This is believed to be the first documented report of C. ribicola infecting white pines in Nevada and the easternmost extension of blister rust in the Sierra Nevada Region. These observations suggest that our understanding of blister rust spread and infection dynamics east of the Sierra Nevada crest is incomplete and that future surveys and research in this region must address, among other issues, timing of aeciospore production on pine, and the possibility of blister rust spread into the Great Basin.

The centromere-kinetochore complex can be divided into distinct domains based on structure and function. Previous work has used CREST auto-antibodies with various microscopic techniques to map the locations of proteins within the centromere-kinetochore complex and to analyze the maturation of prekinetochores before mitosis. Here we have focused on the centromere-specific histone Centromere Protein (CENP)-A and its spatial relationship to other histones and histone modifications found in condensed chromatin. We demonstrate that the phosphorylation of histone H3 is essentially excluded from a specific region of centromeric chromatin, defined by the presence of CENP-A. Interspersion of CENP-B with phosphorylated H3 in the inner centromere indicates that the exclusion of H3 modification is not a general property of alpha-satellite DNA. We also demonstrate that these regions are functionally distinct by fragmenting mitotic chromatin into motile centromere-kinetochore fragments that contain CENP-A with little or no phosphorylated H3 and nonmotile fragments that contain exclusively phosphorylated H3. The sequence of CENP-A diverges from H3 in a number of key residues involved in chromosome condensation and in transcription, potentially allowing a more specialized chromatin structure within centromeric heterochromatin, on which kinetochore plates may nucleate and mature. This specialized centromere subdomain would be predicted to have a very tight and static nucleosome structure as a result of the absence of H3 phosphorylation and acetylation.

The centromere/kinetochore complex is a chromosomal assembly that mediates chromosome motility and mitotic regulation by interacting with microtubules of the mitotic spindle apparatus. Centromere protein A (CENP-A) is a histone H3 homolog that is concentrated in the chromatin of the inner kinetochore plate of human chromosomes. To identify DNA sequences associated with the inner kinetochore plate, we used anticentromere autoantibodies to immunoprecipitate CENP-A associated chromatin selectively from Indian muntjac fibroblasts. DNA was cloned from immunoprecipitated CENP-A- associated chromatin and characterized by DNA sequence and hybridization analyses. A novel centromeric satellite DNA sequence was identified and shown by fluorescence in situ hybridization analysis to be present at all centromeres of the Indian muntjac. This satellite DNA constitutes a 972 bp monomer repeat and shows partial homology with satellite II DNA of the white-tailed deer. Southern blot analysis of muntjac genomic DNA suggests that this satellite DNA is present in repetitive tandem arrays and contains complex internal arrangements. In conjunction with previous work showing the association of CENP-A with human alpha-satellite DNA, we conclude that the mammalian inner kinetochore plate contains a unique form of chromatin that contains CENP-A in association with complex satellite DNA.

Using these methods we have shown that the alpha-satellite domain of the human centromere behaves as an elastic element, stretching in response to spindle forces applied during prometaphase and metaphase (Shelby et al., 1996). These data complement previous observations of centromere stretching during mitosis (e.g., Skibbens et al., 1993) by demonstrating a specific molecular compartment within the centromere, the satellite heterochromatin domain, that supports this strain. Centromere stretching reports on the net force applied across the centromere during mitosis and the availability of a fluorescence-based assay system in human cells provides a robust assay system to complement the elegant DIC-based methods that have been perfected using marsupial and newt cell cultures (Cassimeris et al., 1990; Skibbens et al., 1993, 1995; Rieder et al., 1994). Current applications of this method are directed toward examining the relationship between centromere tension and microtubule dynamics using pharmacological approaches and the behavior of kinetochore-associated regulatory proteins, such as Mad2 and Bub1 (Li and Benezra, 1996; Chen et al., 1996; Taylor and McKeon, 1997), as a function of centromere distortion. In addition, GFP-labeled centromeres can be observed during interphase, providing a novel window into chromosome organization within the nucleus. Our observations show that centromeres distribute into the newly forming nucleus at telophase by what is apparently a uniform isometric expansion, with little evidence for directed motion of individual centromeres contributing to the formation of the G1 nucleus. During interphase, centromeres show very little movement in general, behaving as though embedded in a rigid matrix. Sustained movements of individual centromeres or groups of centromeres are occasionally observed, however, suggesting that chromosome position is subject to change during interphase (Shelby et al., 1996). These experiments complement those described by Belmont and colleagues, who have developed a method to mark specific chromosomal sites with GFP for analysis in vivo (see Chapter 13; Robinett et al., 1996; Straight et al., 1996). These new GFP-based techniques for direct observation of defined DNA sequence domains in vivo carry the logic of in situ hybridization analysis into living cells and, coupled with new methods for observing global chromatin architecture as well as functional nuclear protein domains (Huang et al., 1997; Misteli et al., 1997), promise significant progress toward understanding the dynamic organization of the genome within the living nucleus.

Anticentromere antibodies (ACA) are associated with systemic sclerosis (scleroderma) patients exhibiting the more benign or so called limited manifestation of the disease (lSSc). ACA reactivity is directed against multiple polypeptide targets, the smallest of which is designated CENP-A. CENP-A is not an abundant cellular constituent; therefore to maximize recovery, we developed a protocol with a minimum of steps to isolate CENP-A from a human cell line. The trace cellular amount of this protein clearly dictated the production of its recombinant counterpart to facilitate determination of the role of the CENP-A antigen in scleroderma pathogenesis. Here we describe the eukaryotic expression of CENP-A cDNA using baculovirus-mediated infection of insect cells. The non-fusion recombinant protein spans the natural residues of the human CENP-A protein and rCENP-A followed the same chromotographic sequence for purification as did the natural source. The availability of the bona fide antigen provided a critical standard upon which to document authenticity of the recombinant polypeptide. The two forms of this antigen have been compared and shown to exhibit similar physical and antigenic properties., (Copyright 1998 Academic Press)

Background: The amplification of oncogenes in cancer cells is often mediated by paired acentric chromatin bodies called double minute chromosomes (DMs), which can accumulate to a high copy number because of their autonomous replication during the DNA synthesis phase of the cell cycle and their subsequent uneven distribution to daughter cells during mitosis. The mechanisms that control DM segregation have been difficult to investigate, however, as the direct visualization of DMs in living cells has been precluded because they are far smaller than normal chromosomes. We have visualized DMs by developing a highly sensitive method for observing chromosome dynamics in living cells., Results: The human histone H2B gene was fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria and transfected into human HeLa cells to generate a stable line constitutively expressing H2B-GFP. The H2B-GFP fusion protein was incorporated into nucleosomes without affecting cell cycle progression. Using confocal microscopy, H2B-GFP allowed high-resolution imaging of both mitotic chromosomes and interphase chromatin, and the latter revealed various chromatin condensation states in live cells. Using H2B-GFP, we could directly observe DMs in living cancer cells; DMs often clustered during anaphase, and could form chromosomal 'bridges' between segregating daughter chromosomes. Cytokinesis severed DM bridges, resulting in the uneven distribution of DMs to daughter cells., Conclusions: The H2B-GFP system allows the high-resolution imaging of chromosomes, including DMs, without compromising nuclear and chromosomal structures and has revealed the distinctive clustering behavior of DMs in mitotic cells which contributes to their asymmetric distribution to daughter cells.

The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood [1]. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly [2], but whose molecular role in kinetochore structure and function is unknown. Here we have raised for the first time monospecific antisera to CENP-A [3], a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 [4,5] and that resembles the yeast centromeric component CSE4 [6]. We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A was previously shown to co-purify with nucleosomes [7], our data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA [8]. However, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.

The pathway of molecular interactions leading to kinetochore assembly on mammalian chromosomes is unknown. Kinetochores could be specified by structural features of centromeric satellite DNA [1-3] or by specific DNA sequences, analogous to budding yeast centromeres, interspersed in centromeric satellite DNA arrays [4,5]. Alternatively, kinetochores could be epigenetic structures that replicate without strict dependence on DNA sequence [6-8]. We purified kinetochore-associated chromatin from human chromosomes by immunoprecipitation of CENP-A, a centromere-specific histone H3 homologue located in the inner plate of the kinetochore [6,9,10]. Hybridization and DNA sequence analyses of cloned kinetochore DNA fragments revealed alpha-satellite as the predominant sequence associated with CENP-A. A major site of micrococcal nuclease digestion was identified by mapping the termini of alpha-satellite clones, suggesting that the inner kinetochore plate contains phased arrays of CENP-A-alpha-satellite nucleosomes. These experiments demonstrate for the first time that complex satellite DNA is a structural component of the kinetochore. Further, because complex satellite DNA is evolutionarily unconserved, these results suggest that molecular recognition events necessary for kinetochore formation take place at the level of DNA conformation or epigenetic mechanisms rather than DNA sequence per se.