Chromosomes In Interphase Nuclei Research Articles

Plant Cytogenetics: From Chromosomes to Cytogenomics.

Chromosomes have been studied since the late nineteenth century in the disciplines of cytology and cytogenetics. Analyzing their numbers, features, and dynamics has been tightly linked to the technical development of preparation methods, microscopes, and chemicals to stain them, with latest continuing developments described in this volume. At the end of the twentieth and beginning of the twenty-first centuries, DNA technology, genome sequencing, and bioinformatics have revolutionized how we see, use, and analyze chromosomes. The advent of in situ hybridization has shaped our understanding of genome organization and behavior by linking molecular sequence information with the physical location along chromosomes and genomes. Microscopy is the best technique to accurately determine chromosome number. Many features of chromosomes in interphase nuclei or pairing and disjunction at meiosis, involving physical movement of chromosomes, can only be studied by microscopy. In situ hybridization is the method of choice to characterize the abundance and chromosomal distribution of repetitive sequences that make up the majority of most plant genomes. These most variable components of a genome are found to be species- and occasionally chromosome-specific and give information about evolution and phylogeny. Multicolor fluorescence hybridization and large pools of BAC or synthetic probes can paint chromosomes and we can follow them through evolution involving hybridization, polyploidization, and rearrangements, important at a time when structural variations in the genome are being increasingly recognized. This volume discusses many of the most recent developments in the field of plant cytogenetics and gives carefully compiled protocols and useful resources.

Molecular conflicts disrupting centromere maintenance contribute to Xenopus hybrid inviability

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when the eggs of the frog X.tropicalis are fertilized with either X.laevis or X.borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. Invitro reactions combining X.tropicalis egg extract with either X.laevis or X.borealis sperm chromosomes revealed that paternally matched or overexpressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, although the X.laevis chromosomes maintained centromeric CENP-A in metaphase, X.borealis chromosomes did not and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X.borealis mitotic chromosomes could be rescued by inhibiting RNA polymerase I or preventing the collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to the disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids.

Celebrating Mendel, McClintock, and Darlington: On end-to-end chromosome fusions and nested chromosome fusions.

The evolution of eukaryotic genomes is accompanied by fluctuations in chromosome number, reflecting cycles of chromosome number increase (polyploidy and centric fissions) and decrease (chromosome fusions). Although all chromosome fusions result from DNA recombination between two or more nonhomologous chromosomes, several mechanisms of descending dysploidy are exploited by eukaryotes to reduce their chromosome number. Genome sequencing and comparative genomics have accelerated the identification of inter-genome chromosome collinearity and gross chromosomal rearrangements and have shown that end-to-end chromosome fusions (EEFs) and nested chromosome fusions (NCFs) may have played a more important role in the evolution of eukaryotic karyotypes than previously thought. The present review aims to summarize the limited knowledge on the origin, frequency, and evolutionary implications of EEF and NCF events in eukaryotes and especially in land plants. The interactions between nonhomologous chromosomes in interphase nuclei and chromosome (mis)pairing during meiosis are examined for their potential importance in the origin of EEFs and NCFs. The remaining open questions that need to be addressed are discussed.

The evolution of preimplantation genetic testing for aneuploidy

Abnormal chromosome number or aneuploidy has been recognised as a major contributor to implantation and pregnancy failure and miscarriage since the earliest years of IVF following the birth of Louise Brown forty years ago. Already in 1986, for example, Angell and colleagues, reported Evidence was found of nondisjunction, resulting in trisomy, monosomy and nullisomy; structural abnormalities, haploidy and triploidy. Despite the lethality of their chromosome complements, these embryos could not be distinguished morphologically from those with normal (Angell et al., 1986). These early observations were made using conventional cytogenetic methods to karyotype cells arrested in metaphase, which is technically difficult in human eggs and embryos. This led to the use of molecular approaches, initially using dual colour fluorescent in situ hybridisation (FISH) to identify X and Y chromosomes in interphase nuclei to identify the gender of the embryo in couples at risk of X-linked diseases (Griffin et al., 1992). Multicolour FISH methods with expanded numbers of chromosome specific probes and two or more hybridisations were then developed for polar body analysis of maternal meiotic errors by Verlinsky, Kuliev and colleagues (Verlinsky et al., 1996) and Munne and colleagues for preimplantation genetic screening (Munne et al., 1995), now termed preimplantation genetic testing for aneuploidy (PGT-A). Coincidentally, these studies and the analysis of aneuploid embryos confirmed and extended all of the abnormalities observed earlier by conventional cytogenetics. In particular, chromosomal mosaicism was commonly observed among cells in cleavage stage embryos. The discovery of efficient methods for whole genome amplification allowing micrograms of DNA to be amplified from single cells in the mid 2000's, heralded the prospect of using genomic approaches to analyse copy number for all 24 chromosomes. Recent developments over the last ten years from comparative genomic hybridisation to next generation sequencing will be reviewed.

Morphometric and Densitometric Analysis of Heterochromatin during Cell Differentiation Using the Leukaemic Granulocytic Lineage as a Convenient Model

Granulocytic early progenitors and terminally differentiated – mature granulocytes with segmented nuclei were studied using computer-assisted diameter and heterochromatin optical image densitometry to provide more information on the nuclear size and heterochromatin condensation state. Bone marrow smears of patients suffering from chronic myeloid leukaemia untreated as well as treated with “specific” anti-leukaemic therapy with imatinib mesylate are a convenient model for such study because they possess a satisfactory number of cells for diameter and optical density measurements. In addition, the identification of developmental stages of granulocytes is very easy and the morphology is not different from that in not-leukaemic persons. As it was expected, the mean diameter of nuclear segments in fully differentiated and mature granulocytes was much smaller than that in non-segmented nuclei of early granulocytic precursors. Therefore, no wonder that the heterochromatin condensation state in nuclear segments of mature granulocytes was much larger than in non-segmented nuclei of granulocytic progenitors. On the other hand, the sum of mean diameters of all nuclear segments per cell was close to the mean nuclear diameter of early granulocytic progenitors. The heterochromatin condensation state in granulocytic progenitors or fully differentiated mature granulocytes exhibited marked stability and did not change after the anti-leukaemic therapy. In addition, Barr bodies of characteristic drumstick appearance bearing inactive X chromosome in interphase nuclei of mature granulocytes in fertile female patients exhibited a heterochromatin condensation state similar to nuclear segments. This heterochromatin condensation state was also stable and constant, and was not apparently influenced by the anti-leukaemic therapy.

The arrangement of Brachypodium distachyon chromosomes in interphase nuclei.

The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biological processes, such as DNA replication, transcription, and repair. However, detailed data about the CT arrangement in monocotyledonous plants are scarce. In this study, chromosome painting was used to analyse the distribution and associations of individual chromosomes in the 3-D preserved nuclei of Brachypodium distachyon root cells in order to determine the factors that may have an impact on the homologous CT arrangement. It was shown that the frequency of CT association is linked to the steric constraints imposed by the limited space within the nucleus and may depend on chromosome size and morphology as well as on the nuclear shape. Furthermore, in order to assess whether the distribution of interphase chromosomes is random or is subject to certain patterns, a comparison between the experimental data and the results of a computer simulation (ChroTeMo), which was based on a fully probabilistic distribution of the CTs, was performed. This comparison revealed that homologous chromosome arm CTs associate more often than if they were randomly arranged inside the interphase nucleus.

3D nuclear organization and genomic instability in cancer.

To understand the genetic changes that occur at tumor initiation and during tumor progression, we focus on changes in nuclear architecture that promote the onset of genomic instability [1,2]. To determine changes in nuclear organization, we measure the 3D nuclear organization of telomeres, the ends of chromosomes. The measurements of telomeres allow one to trace the positions of chromosomes in interphase nuclei and, by using a fluorescent telomere-specific probe, all telomeres can be visualized in a single image. During the past years, we have defined the organization of telomeres in nuclei of normal, immortalized and tumor cells. We have developed quantitative software that enables us to measure the three-dimensional (3D) organization of telomeres [3,4]. The parameters we measure include: number of telomeres, sizes of telomeres, nuclear distribution of telomeres, and the presence of telomeric aggregates. The latter are clusters of telomeres that are absent from normal cells. More recently, we started to automate the 3D image acquisition and analysis and we are able to scan 15 000 cells per hour [5]. We observed changes in the nuclear organization of telomeres as a result of conditional c-Myc deregulation dependent on a functional myc box II [6,7]. We found Epstein-Barr Virus-induced changes in 3D telomere organization and resulting genomic instability [8]. Encouraged by these findings, we focused on tumor initiation and progression in patient samples and observed cancer-associated 3D nuclear telomere changes in lymphoid and solid tumors [9-12]. Moreover, the use of 3D nuclear telomere profiling permitted, for the first time, the identification of patient (and tumor) subpopulations that were not detectable up to that point. For example, we blindly defined three distinct subpopulations that correlated with short-term, intermediate and long-term survival in glioblastoma [11]. Using the same 3D imaging approach, we defined subpopulations in myelodysplastic syndromes and acute myeloid leukemias [12]. Additional studies are currently ongoing. We provided evidence that genomic instability is a result of these nuclear changes. Dynamic nuclear alterations directly result from 3D telomere aberrations [1,2,6,13-15]. These genomic changes include aneuploidy, Robertsonian fusions, breakage-bridge fusion (BBF) cycles with resulting terminal deletions and unbalanced translocations and continued rounds of BBF cycles. While these changes can be followed during cancer progression in patients, their true origin can only be examined in conditional expression studies or longitudinally in mouse models. Using conditional c-Myc deregulation, we demonstrated that changes in 3D telomere profiles precede the onset and propagation of genomic instability [6,13]. In Hodgkin’s lymphoma, in collaboration with Dr. Hans Knecht, we showed that mono-nucleated H cells become multinucleated Reed Sternberg (RS) cells through telomere dysfunction as measured by 3D nuclear profiles [10,16]. These changes coincide with localized shelterin dysfunction, aberrant centrosome duplication as well as spindle formation and significantly elevated levels of DNA damage foci [10,16]. Spectral karyotyping (SKY) and super resolution 3D imaging confirmed the dynamics and complexity of these genetic changes in which RS cells are the end-stage cells generated through multiple defects traced back to the H cells and propagated from there on [15]. Most recently, we have reported that Hodgkin’s patients with recurrent/non-recurrent disease display distinct 3D telomeric profiles [17]. Ongoing studies focus on a variety of cancers where clinicians currently lack the ability to define the risk of an individual patient to progress, the ability of early detection or of monitoring of disease progression. In conclusion, 3D telomere profiling provides a platform technology able to determine normal and aberrant nuclear organization that is a measure of genomic instability and cancer.

To see an interphase chromosome or: How a disease can be associated with specific nuclear genome organization

The last hierarchical level of cellular genome organization is the spatial arrangement of chromosomes within the nuclear space. Despite of high regulatory potential and functional implications, issues concerning nuclear organization at chromosomal level are rarely addressed because of limitations in visualizing interphase chromosomes. The problem is especially seen when an attempt to associate specific patterns of nuclear genome organization with a pathological condition is made. Fortunately, advances in molecular cytogenetics have provided for a solution to visualize chromosomes in interphase nuclei at molecular resolution. A study in this issue of <em>BioDiscovery</em> shows the way of how to identify interphase chromosome architecture at molecular resolutions and demonstrates the involvement of specific nuclear genome organization in generating a cancer-causing chromosomal aberration (translocation between chromosomes 8 and 21 in acute myelogenous leukemia). Authors’ findings suggest interphase molecular cytogenetic techniques (i.e. interphase chromosome-specific multicolor banding or ICS-MCB) to be required to perform studies regarding nuclear genome organization at chromosomal level and its role in disease pathogenesis.

Chromobility: the rapid movement of chromosomes in interphase nuclei

There are an increasing number of studies reporting the movement of gene loci and whole chromosomes to new compartments within interphase nuclei. Some of the movements can be rapid, with relocation of parts of the genome within less than 15 min over a number of microns. Some of these studies have also revealed that the activity of motor proteins such as actin and myosin are responsible for these long-range movements of chromatin. Within the nuclear biology field, there remains some controversy over the presence of an active nuclear acto-myosin motor in interphase nuclei. However, both actin and myosin isoforms are localized to the nucleus, and there is a requirement for rapid and directed movements of genes and whole chromosomes and evidence for the involvement of motor proteins in this relocation. The presence of nuclear motors for chromatin movement is thus an important and timely debate to have.

Location of Chromosomes in the Nucleus of Human Mesenchymal Stem Cells

For evaluation of the spatial structure of chromatin in nuclei of mesenchymal SC we determined the position of centromeres and individual chromosomes in interphase nucleus of mesenchymal SC. More than 300 nuclei in 7 cultures of mesenchymal SC were analyzed. Centromeres of chromosomes 6, 8, and 11 lie at a longer (0.68, 0.67, 0.7), while centromere of chromosome 18 at a shorter radial distance (0.49). Homologues of each chromosome had different radial distances. No differences in radial distances of centromeres were detected between mesenchymal SC from the adipose tissue and BM. After passage 8, distal displacement of chromosome 6 centromere (from 0.66 to 0.72) was observed, which probably indicates aging or spontaneous differentiation of cells.

Comprehensive embryo analysis of advanced maternal age–related aneuploidies and mosaicism by short comparative genomic hybridization

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

Oligonucleotide (ODN) fluorescence in situ hybridization (Oligo-FISH) and conventional FISH allow enumeration of 24 chromosomes in 6 successive hybridizations performed in a single day

OBJECTIVE: Enumerate 24 human chromosomes on a single cell in less than 24 h by FISH. DESIGN: Using conventional FISH experiments, no more than 3 hybridizations can be performed sequentially. Oligo-FISH probes are currently available in 4 visible fluors permitting enumeration of 4 chromosomes per hybridization. Because of low stringency conditions and short hybridization time (5 min), up to 6 successive FISH experiments can be performed on the same slide without signal loss. Performing 5 successive Oligo-FISH with a combination of 4 chromosomes (FISH 1: 3, 7, 12, 16; FISH 2: 2, 13-21, 18, 20; FISH 3: 6, 8, 9q12, 11; FISH 4: X, Yq12, 15, 17; FISH 5: 1q12, 4, 10, 14-22) and 1 hybridization with 4 chromosome speceific BAC probes (FISH 6: 5, 19, 21, 22), 24 chromosomes in the same cell were enumerated in less than 24 h. All cocktail probes were evaluated on peripheral blood and on discarded human blastomeres. MATERIALS AND METHODS: Cytogenetic Slides from 5 chromosomally normal males were prepared as previously described. Blastomere Slides from 20 discarded embryos were prepared as previously described. ODN sequence Design: ODN probes have been designed to detect 20 human chromosomes that harbor specific repeats. BAC probes were selected from the RP11 library (Empire Genomics, LLC, NY) to detect chromosomes 5, 19, 21 and 22. FISH was performed as previously described. Image Acquisition was performed using a Nikon microscope equipped with a CCD camera. Images were captured using NIS elements software. Analytical Specificity, Sensitivity and S/N were calculated as previously described according to the Standards and Guidelines for Clinical Genetic Laboratories of the American College of Medical Genetics. RESULTS: We successfully detected 24 chromosomes in 6 successive hybridizations in a single day. CONCLUSIONS: ODN probes' short length allows rapid hybridization and reduces cell damage, which are significant advantages for time critical procedures such as enumeration of chromosomes in interphase nuclei for PGD.

Rapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvae

AbstractTwo different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female‐specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W‐specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2‐EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2‐EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co‐amplified the CpW2‐EcoRI sequence to identify the W chromosome and the Z‐linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth.

Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions

The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.

Oligo fluorescence in situ hybridization (oligo-fish), a new strategy for enumerating chromosomes in interphase nuclei

Oligo fluorescence in situ hybridization (oligo-fish), a new strategy for enumerating chromosomes in interphase nuclei

In Situ Hybridization to Metaphase Chromosomes and Interphase Nuclei

In situ hybridization is used to determine the chromosomal map location and the relative order of genes and DNA sequences within a chromosomal band. It can also be used to detect aneuploidy, gene amplification, and subtle chromosomal rearrangements. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in the first basic protocol. Two support protocols are provided to amplify weak fluorescent signals obtained in FISH. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described in alternate protocols. Nonisotopic labeling of DNA probes by nick translation is described in a support protocol. The order of closely spaced FISH probes along chromosomes in interphase nuclei can be determined. A basic protocol for isotopic in situ hybridization (IISH) with (3)H is provided followed by a support protocol for preparation of autoradiographic emulsion.

Comparison of the rates of aneuploidy occurrence in quiescent and dividing cells of humans exposed to adverse environmental factors

Fluorescence in situ hybridization (FISH) was used to compare aneuploidy rates in four autosomes and two sex chromosomes in interphase nuclei of noncultivated (quiescent) and cultivated (induced to divide with phytohemagglutinin (PHA)) leukocytes in people engaged in nuclear-chemical industry and in a control group of people not exposed to mutagenic factors occupationally or at home. The overall rates of numerical chromosome aberrations for all of the six chromosomes studied showed little difference, although a higher rate of loss of the X- and Y-chromosomes was observed in the exposed group. In individuals exposed to several adverse environmental factors, the overall rate of numerical chromosome aberrations in cultivated cells after at least one DNA replication cycle exceeded that in noncultivated cells by 52% (P = 0.01), whereas only a trend for its increase was observed in the control group (23%, P = 0.25). Thus, the effect of adverse environmental factors in humans caused more than a twofold increase in the difference between the rates of aneuploid cells in cultivated and non-cultivated leukocytes in the exposed group as compared to control. It is conjectured that cell division is accompanied by the expression of potential damage of mitotic chromatid segregation apparatus accumulated in vivo. These defects, realized during cell division, bring about numerical chromosome aberrations.

An Approach for Quantitative Assessment of Fluorescence In Situ Hybridization (FISH) Signals for Applied Human Molecular Cytogenetics

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.

The DNA-Based Structure of Human Chromosome 5 in Interphase

In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.

Chromosome dynamics in the yeast interphase nucleus.

Little is known about the dynamics of chromosomes in interphase nuclei. By tagging four chromosomal regions with a green fluorescent protein fusion to lac repressor, we monitored the movement and subnuclear position of specific sites in the yeast genome, sampling at short time intervals. We found that early and late origins of replication are highly mobile in G1 phase, frequently moving at or faster than 0.5 micrometers/10 seconds, in an energy-dependent fashion. The rapid diffusive movement of chromatin detected in G1 becomes constrained in S phase through a mechanism dependent on active DNA replication. In contrast, telomeres and centromeres provide replication-independent constraint on chromatin movement in both G1 and S phases.