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PubMed2002

Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17.

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.

Animals↗
PubMed1993

Evaluation of a rapid readout biological indicator for flash sterilization with three biological indicators and three chemical indicators.

OBJECTIVE: Flash sterilization is most commonly used for emergency sterilization of unwrapped items in a gravity displacement sterilizer for three minutes. Sterilization quality assurance is monitored by biological indicators that require a 24-hour incubation prior to reading. In this study, we compared a new biological indicator that provides results within 60 minutes with three conventional, 24-hour biological indicators for monitoring flash sterilization and three chemical indicators. DESIGN: Conventional biological indicators tested included the conventional Attest 1261, Proof Flash and Assert, while the rapid readout indicator tested was Attest 1291. Attest Rapid Readout detects the presence of a Bacillus stearothermophilus enzyme by reading a fluorescent product that is produced by the enzymatic break-down of a nonfluorescent substrate. Chemical indicators tested included Comply, Incheque, and Thermalog S. Survival at 132 degrees C in a gravity displacement sterilizer was measured by media color change after incubation for 24 hours at 56 degrees C for the three conventional biological indicators, fluorescence at 60 minutes for the Attest Rapid Readout biological indicator, and color change for the chemical indicators. Each exposure time was replicated four times with 10 of each biological and chemical indicator per run. RESULTS: The conventional biological indicators (Attest, Proof Flash, and Assert) had 90%, 48%, and 40% spore survival at two minutes exposure; 23%, 3%, and 0% at three minutes exposure; and 3%, 0%, and 0% at four minutes exposure respectively. The Attest Rapid Readout biological indicator had 88%, 33%, and 0% enzyme activity detectable at 2, 3, and 4 minutes exposure. The chemical indicators Comply, Incheque, and Thermalog S revealed sterilization failure rates of 100%, 100%, and 100% at 0 minutes exposure; 100%, 100%, and 45% at one minute; 0%, 0%, and 28% at two minutes exposure; 0%, 0%, and 18% at three minutes exposure; and 0%, 0%, and 0% at four minutes exposure, respectively. CONCLUSION: The sensitivity of the Attest Rapid Readout parallels the conventional biological indicators. These data suggest that a 60-minute rapid readout biological indicator is equivalent to the 24-hour biological indicators. If further studies demonstrate that a four-minute flash sterilization cycle provides a needed safety margin to ensure sterilization, then consideration should be given to requiring a four-minute flash sterilization cycle. Chemical indicators were too sensitive to the processing conditions (eg, steam) and are inadequate to ensure adequate sterilization.

Evaluation Studies as Topic↗
PubMed2025

CDACHIE: chromatin domain annotation by integrating chromatin interaction and epigenomic data with contrastive learning.

MOTIVATION: Chromatin domain annotation identifies functional genomic regions, such as active and inactive zones, based on epigenomic features like histone modifications, DNA methylation, and chromatin accessibility. While recent methods have utilized both chromatin interaction data (e.g. Hi-C) and epigenomic data, they often overlook the direct relationship between these data types. RESULTS: In this study, we introduce Chromatin Domain Annotation using Contrastive Learning for Hi-C and Epigenomic Data (CDACHIE), a method for identifying chromatin domains from Hi-C and epigenomic data. Our approach leverages contrastive learning to generate aligned representative vectors for both data types at each genomic bin. The concatenated vectors are then clustered using K-means to classify distinct chromatin domain types. CDACHIE achieves superior performance in Variance Explained, evaluated across gene expression, replication timing, and ChIA-PET data. This highlights its robust ability to integrate semantic associations between Hi-C and epigenomic features within the embedding space. AVAILABILITY AND IMPLEMENTATION: The source code is available at GitHub: https://github.com/maruyama-lab-design/CDACHIE. An archival snapshot of the code used in this study is available on Zenodo: https://doi.org/10.5281/zenodo.15751780.

Chromatin↗
PubMed2002

A full-coverage, high-resolution human chromosome 22 genomic microarray for clinical and research applications.

We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.

Chromosome Mapping↗
PubMed1996

Genomic imprinting: significance in development and diseases and the molecular mechanisms.

Certain mammalian genes are expressed exclusively from either the paternal or the maternal chromosome because of a differential marking process that occurs during gametogenesis. This epigenetic marking is called genomic imprinting. Monoallelic expression of imprinted genes is responsible for the inability of uniparental mammalian embryos to develop normally and for the abnormal phenotypes observed with particular chromosomal disomies. Many of the imprinted genes identified to date are involved in the regulation of cell proliferation and differentiation and, together with other pieces of evidence, they are suggested to play a role in tumorigenesis. Here we discuss how imprinted genes cause diseases and tumors and summarize the recent advances of studies on the molecular basis of this epigenetic phenomenon. In particular, we focus on two well-characterized imprinted chromosomal regions, namely the human Prader-Willi/Angelman syndrome region and the mouse INS2/IGF2/H19 region. The correlations between the differential gene activity and the changes in DNA methylation, higher order chromatin structure and replication timing, will shed light on gene regulation at the level of the chromosomal domain.

Animals↗
PubMed2006

An isochore map of human chromosomes.

Isochores are large DNA segments (>>300 kb on average) that are characterized by an internal variation in GC well below the full variation seen in the mammalian genome. Precisely defining in terms of size and composition as well as mapping the isochores on human chromosomes have, however, remained largely unsolved problems. Here we used a very simple approach to segment the human chromosomes de novo, based on assessments of GC and its variation within and between adjacent regions. We obtain a complete coverage of the human genome (neglecting the remaining gaps) by approximately 3200 isochores, which may be visualized as the ultimate chromosomal bands. Isochores visibly belong to five families characterized by different GC levels, as expected from previous investigations. Since we previously showed that isochores are tightly linked to basic biological properties such as gene density, replication timing, and recombination, the new level of detail provided by the isochore map will help the understanding of genome structure, function, and evolution.

Chromosome Banding↗
PubMed1997

Earlier finishing of Xp21.2 subband replication of the inactive X chromosome in Rett syndrome girl but not in her 47,XXX mother.

X-inactivation mosaicism has been proposed to explain the origin of Rett syndrome. We present the results of the cytogenetic analysis, including RBG dynamic replication pattern, in a girl with Rett syndrome. The late replicating X chromosome (LRX) showed the earlier replication of subband Xp21.2 in 36% of analysed cells. Unexpectedly the maternal karyotype 47,XXX was found. Replication timing of both maternal LRX chromosomes was normal. The critical region of Xp essential for RS is proposed.

Adult↗
PubMed2001

Targeting silence: the use of site-specific recombination to introduce in vitro methylated DNA into the genome.

DNA methylation has emerged as an important component of transcriptional regulation. However, our understanding of how DNA methylation influences transcription, chromatin structure, replication timing, and imprinting has been limited by the lack of experimental systems that permit control of the methylation state of genes in a chromosomal context. Here, we describe a novel technique that allows for efficient introduction of methylated and unmethylated DNA into defined sites in the mammalian genome. This protocol utilizes bacterial CpG methyltransferases to methylate the DNA of interest in vitro, followed by site-specific targeting using Cre recombinase. Long-term maintenance of the methylation state in vivo allows analysis of the biological consequences of methylation by direct comparison of the methylated and unmethylated state in the same genomic position.

Animals↗
PubMed1996

Parental imprinting and human disease.

Parental imprinting is a process that results in allele-specific differences in transcription, DNA methylation, and DNA replication timing. Imprinting plays an important role in development, and its deregulation can cause certain defined disease states. Absence of a paternal contribution to chromosome 15q11-q13, due to hemizygous deletion or uniparental disomy, results in the Prader-Willi syndrome. The absence of a normal maternal copy of the same region causes Angelman syndrome. The Beckwith-Wiedemann syndrome is associated with the failure of normal biparental inheritance of chromosome 11p15, and loss of imprinting is observed in several cancers including Wilms' tumor. The study of the molecular basis of abnormal imprinting in these disorders will facilitate the identification and characterization of other imprinted human disease loci.

Angelman Syndrome↗
PubMed2001

Genome organization, function, and imprinting in Prader-Willi and Angelman syndromes.

The chromosomal region, 15q11-q13, involved in Prader-Willi and Angelman syndromes (PWS and AS) represents a paradigm for understanding the relationships between genome structure, epigenetics, evolution, and function. The PWS/AS region is conserved in organization and function with the homologous mouse chromosome 7C region. However, the primate 4 Mb PWS/AS region is bounded by duplicons derived from an ancestral HERC2 gene and other sequences that may predispose to chromosome rearrangements. Within a 2 Mb imprinted domain, gene function depends on parental origin. Genetic evidence suggests that PWS arises from functional loss of several paternally expressed genes, including those that function as RNAs, and that AS results from loss of maternal UBE3A brain-specific expression. Imprinted expression is coordinately controlled in cis by an imprinting center (IC), a genetic element functional in germline and/or early postzygotic development that regulates the establishment of parental specific allelic differences in replication timing, DNA methylation, and chromatin structure.

Angelman Syndrome↗
PubMed2002

ICF syndrome cells as a model system for studying X chromosome inactivation.

Mutations in the DNMT3B DNA methyltransferase gene cause the ICF immunodeficiency syndrome. The targets of this DNA methyltransferase are CpG-rich heterochromatic regions, including pericentromeric satellites and the inactive X chromosome. The abnormal hypomethylation in ICF cells provides an important model system for determining the relationships between replication time, CpG island methylation, chromatin structure, and gene silencing in X chromosome inactivation.

Abnormalities, Multiple↗
PubMed2002

Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells.

Inactivation of the X chromosome during early female development and the subsequent maintenance of this transcriptionally inert state through countless cell divisions remain a paradigm for epigenetic regulation in mammals. Nevertheless, the exact mechanisms underlying this chromosome-wide silencing process remain unclear. Using differentiating female embryonic stem (ES) cells as a model system, we recently found that histone H3 tail modifications are among the earliest known chromatin changes in the X inactivation process, appearing as soon as Xist RNA accumulates on the X chromosome, but prior to transcriptional silencing of X-linked genes (Heard et al., 2001). In this report we present an integrated analysis of the sequence of early events and chromatin modifications underlying X inactivation in differentiating female ES cells. We have extended our previous analysis concerning changes in histone tail modification states. We find that the hypomethylation of Arg-17 and that of Lys-36 on histone H3 also characterize the inactive X chromosome, and that these profiles show a similarly early onset during the initiation of X inactivation. In addition, we have investigated the kinetics of the shift in replication timing of the X chromosome undergoing inactivation. This event occurs slightly later than Xist RNA coating and the chromatin modifications. Finally, from an early stage in the X inactivation process, characteristic histone modification patterns can be found on the X chromosome at mitosis, suggesting that they represent true epigenetic marks of the inactive state.

Acetylation↗
PubMed2004

Strand-specific fluorescence in situ hybridization: the CO-FISH family.

The ability to prepare single-stranded chromosomal target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methodologies require functionally single-stranded DNAs in order to facilitate hybridization between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probes to induce temporary separation of the DNA strands. Strand-specific FISH (CO-FISH; Chromosome Orientation-FISH) involves selective removal of newly replicated strands from DNA of metaphase chromosomes which results in single-stranded target DNA. When single-stranded probes are then hybridized to such targets, the resulting strand-specific hybridization is capable of revealing a level of information previously unattainable at the cytogenetic level. Mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5'-->3' towards the termini of all vertebrate chromosomes. Based on this conserved structural organization, CO-FISH with a telomere probe reveals the absolute 5'-->3' orientation of DNA sequences with respect to the pter-->qter direction of chromosomes. Development and various applications of CO-FISH will be discussed: detection of cryptic inversions, discrimination between telomeres produced by leading- versus lagging-strand synthesis, and replication timing of mammalian telomeres.

Animals↗
PubMed1987

A rapid method for the evaluation of new antituberculous agents.

Fifty-one new synthetic compounds belonging to four different series, namely (1a) substituted aryl-4-(substituted phenyl) succinimide; (1b) N-(substituted methyl)-4-(heterocyclic) succinimide; (2) heterocyclic 4-(5'-nitro-2-furyl) thiazoles; (3) substituted aryl-4-(3', 4'-dihydroxy phenyl) thiazoles, and (4) phenyl-N,N-1,2,3-bis-methoxy carbonyl guanidines were screened for antituberculous activity using a conventional broth dilution test (BDT) and a liquid scintillation radiometric method (LSRM). Eight compounds showed in vitro activity. LSRM showed 100% agreement with BDT. LSRM is completed within 60 h, while BDT requires 8-9 days. Unlike BDT, LSRM allowed one to measure the graded changes in the metabolism and the growth rate of Mycobacterium tuberculosis in response to various concentrations of the drug. It permits the measurement of the relative prolongation of the replication time by the drug or the test compound. With LSRM it was possible to detect the phase of growth during which the test compound shows or begins its antituberculous activity. It improves our understanding of the antituberculous activity of the test compound and hence is more advantageous. It is rapid, reliable, quantitative and more sensitive than BDT. LSRM is thus suitable for the evaluation of new drugs.

Antitubercular Agents↗
PubMed2003

Bridging nonliving and living matter.

Assembling non-biological materials (geomaterials) into a proto-organism constitutes a bridge between nonliving and living matter. In this article we present a simple step-by-step route to assemble a proto-organism. Many pictures have been proposed to describe this transition within the origins-of-life and artificial life communities, and more recently alternative pictures have been emerging from advances in nanoscience and biotechnology. The proposed proto-organism lends itself to both traditions and defines a new picture based on a simple idea: Given a set of required functionalities, minimize the physicochemical structures that support these functionalities, and make sure that all structures self-assemble and mutually enhance each other's existence. The result is the first concrete, rational design of a simple physicochemical system that integrates the key functionalities in a thermodynamically favorable manner as a lipid aggregate integrates proto-genes and a proto-metabolism. Under external pumping of free energy, the metabolic processes produce the required building blocks, and only specific gene sequences enhance the metabolic kinetics sufficiently for the whole system to survive. We propose an experimental implementation of the proto-organism with a discussion of our experimental results, together with relevant results produced by other experimental groups, and we specify what is still missing experimentally. Identifying the missing steps is just as important as providing the road map for the transition. We derive the kinetic and thermodynamic conditions of each of the proto-organism subsystems together with relevant theoretical and computational results about these subsystems. We present and discuss detailed 3D simulations of the lipid aggregation processes. From the reaction kinetics we derive analytical aggregate size distributions, and derive key properties of the metabolic efficiency and stability. Thermodynamics and kinetics of the ligation directed self-replication of the proto-genes is discussed, and we summarize the full life cycle of the proto-organism by comparing size, replication time, and energy with the biomass efficiency of contemporary unicells. Finally, we also compare our proto-organism picture with existing origins-of-life and protocell pictures. By assembling one possible bridge between nonliving and living matter we hope to provide a piece in the ancient puzzle about who we are and where we come from.

Artificial Intelligence↗
PubMed2000

Imprint switching for non-random X-chromosome inactivation during mouse oocyte growth.

In mammals, X-chromosome inactivation occurs in all female cells, leaving only a single active X chromosome. This serves to equalise the dosage of X-linked genes in male and female cells. In the mouse, the paternally derived X chromosome (X(P)) is imprinted and preferentially inactivated in the extraembryonic tissues whereas in the embryonic tissues inactivation is random. To investigate how X(P) is chosen as an inactivated X chromosome in the extraembryonic cells, we have produced experimental embryos by serial nuclear transplantation from non-growing (ng) oocytes and fully grown (fg) oocytes, in which the X chromosomes are marked with (1) an X-linked lacZ reporter gene to assay X-chromosome activity, or (2) the Rb(X.9)6H translocation as a cytogenetic marker for studying replication timing. In the extraembryonic tissues of these ng/fg embryos, the maternal X chromosome (X(M)) derived from the ng oocyte was preferentially inactivated whereas that from the fg oocyte remained active. However, in the embryonic tissues, X inactivation was random. This suggests that (1) a maternal imprint is set on the X(M) during oocyte growth, (2) the maternal imprint serves to render the X(M) resistant to inactivation in the extraembryonic tissues and (3) the X(M) derived from an ng oocyte resembles a normal X(P).

Amnion↗
PubMed2004

Epigenetic aspects of differentiation.

A major challenge in biology is to understand how genetic information is interpreted to direct the formation of specialized tissues within a multicellular organism. During differentiation, changes in chromatin structure and nuclear organization establish heritable patterns of gene expression in response to signals. Epigenetic states can be broadly divided into three categories: euchromatin, constitutive heterochromatin and facultative hetereochromatin. Although the static epigenetic profiles of expressed and silent loci are relatively well characterized, less is known about the transition between active and repressed states. Furthermore, it is important to expand on localized models of chromatin structure at specific genetic addresses to examine the entire nucleus. Changes in nuclear organization, replication timing and global chromatin modifications should be integrated when attempting to describe the epigenetic signature of a given cell type. It is also crucial to examine the temporal aspect of these changes. In this context, the capacity for cellular differentiation reflects both the repertoire of available transcription factors and the accessibility of cis-regulatory elements, which is governed by chromatin structure. Understanding this interplay between epigenetics and transcription will help us to understand differentiation pathways and, ultimately, to manipulate or reverse them.

Animals↗
PubMed2006

Species and rotation frequency influence soil nitrogen in simplified tropical plant communities.

Among the many factors that potentially influence the rate at which nitrogen (N) becomes available to plants in terrestrial ecosystems are the identity and diversity of species composition, frequency of disturbance or stand turnover, and time. Replicated suites of investigator-designed communities afforded an opportunity to examine the effects of those factors on net N mineralization over a 12-year period. The communities consisted of large-stature perennial plants, comprising three tree species (Hyeronima alchorneoides, Cedrela odorata, and Cordia alliodora), a palm (Euterpe oleracea), and a large, perennial herb (Heliconia imbricata). Trees were grown in monoculture and in combination with the other two life-forms; tree monocultures were subjected to rotations of one or four years, or like the three-life-form systems, left uncut. The work was conducted on fertile soil in the humid lowlands of Costa Rica, a site with few abiotic constraints to plant growth. Rates of net N mineralization and nitrification were high, typically in the range of 0.2-0.8 microg x g(1) x d(-1), with net nitrification slightly higher than net mineralization, indicating preferential uptake of ammonium (NH4+) by plants and microbes. Net rates of N mineralization were about 30% lower in stands of one of the three tree species, Hyeronima, than in stands of the other two. Contrary to expectations, short-rotation management (one or four years) resulted in higher net rates of N mineralization than in uncut stands, whether the latter were composed of a single tree species or a combination of life-forms. Neither additional species richness nor replenishment of leached N augmented mineralization rates. The net rate at which N was supplied tended to be lowest in stands where demand for N was highest. Careful choice of species, coupled with low frequency of disturbance, can lead to maintenance of N within biomass and steady rates of within-system circulation, whereas pulses, whether caused by cutting and replanting or by the phenological traits of the species selected or combined, subject N supplies to leaching loss.

Biodiversity↗