Higher-Order Structure of Mammalian Chromatin Deduced from Viscoelastometry Data

Abstract

The results of viscoelastometry (VE) for mammalian DNA have been puzzling because they have two orders of magnitude smaller measured viscoelastic relaxation times for mammalian chromosomes than that expected for DNA linear coils of chromosomal size. In an attempt to resolve this discrepancy, we have applied a recent model of Gl chromosome structure (J.Y. Ostashevsky, Mol Biol. Cell 9, 3031–3040, 1998) in which the 30 nm chromatin fiber of each chromosome forms a string of loop clusters (micelles). This model has two parameters: the number of loops per micelle (f) and the average loop size (M_f), which can be estimated independently from VE data. Using our VE data for plateau phase V79 Chinese hamster cells (unirradiated and X-irradiated with doses up to 40 Gy) we show that f-13, which is close to other estimates made using the model (f ranges from 10–20), and M_f～ 2 Mbp, which is similar to estimates made from our nucleoid data (1.3 Mbp) and to estimates made in the literature using a variety of techniques (1–3 Mbp).     

Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.     

The fraction of DNA released on pulsed-field gel electrophoresis gels may differ significantly between genomes at low levels of double-strand breaks

A common way to use pulsed-field gel electrophoresis (PFGE) for measuring the induction and repair of DNA double-strand breaks (DSBs) in mammalian cells is by using the fraction of total DNA released, FR, from the plug. We have analyzed the general relationship between initial chromosome sizes and FR. It is shown that, because of the difference in initial chromosomal size, the discrepancy in FR values between human and rodent cells may become significant at doses of radiation producing approximately 5 DSBs/100 Mbp or less.     

Nuclear DNA content of the whitefly Bemisia tabaci (Aleyrodidae: Hemiptera) estimated by flow cytometry

The nuclear DNA content of the whitefly Bemisia tabaci (Gennnadius) was estimated using flow cytometry. Male and female nuclei were stained with propidium iodide and their DNA content was estimated using chicken red blood cells and Arabidopsis thaliana L. (Brassicaceae) as external standards. The estimated nuclear DNA content of male and female B. tabaci was 1.04 and 2.06 pg, respectively. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid. Conversion between DNA content and genome size (1 pg DNA=980 Mbp) indicate that the haploid genome size of B. tabaci is 1020 Mbp, which is approximately five times the size of the genome of the fruitfly Drosophila melanogaster Meigen. These results provide an important baseline that will facilitate genomics-based research for the B. tabaci complex.     

Construction and characterization of a BAC library for the molecular dissection of a single wild beet centromere and sugar beet (Beta vulgaris) genome analysis.

We have constructed a sugar beet bacterial artificial chromosome (BAC) library of the chromosome mutant PRO1. This Beta vulgaris mutant carries a single chromosome fragment of 6-9 Mbp that is derived from the wild beet Beta procumbens and is transmitted efficiently in meiosis and mitosis. The library consists of 50,304 clones, with an average insert size of 125 kb. Filter hybridizations revealed that approximately 3.1% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents eight genome equivalents. Thus, there is a greater than 99.96% probability that any sequence of the PROI genome can be found in the library. Approximately 0.2% of the clones hybridized with centromeric sequences of the PRO1 minichromosome. Using the identified BAC clones in fluorescence in situ hybridization experiments with PRO1 and B. procumbens chromosome spreads, their wild-beet origin and centromeric localization were demonstrated. Comparative Southern hybridization of pulsed-field separated PROI DNA and BAC inserts indicate that the centromeric region of the minichromosome is represented by overlapping clones in the library. Therefore, the PRO1 BAC library provides a useful tool for the characterization of a single plant centromere and is a valuable resource for sugar beet genome analysis.     

A survey of Penstemon's genome size

Penstemon is the largest genus in North America with more than 270 reported species. However, little is known about its genome size. This information may be useful in developing hybrids for landscape use and for gaining insight into its current taxonomy. Using flow cytometry, we estimated the genome size of approximately 40% of the genus (115 accessions from 105 different species). Genome sizes for both reported and probable diploids range from P. dissectus 2C = 0.94 pg (1C = 462 Mbp) to P. pachyphyllus var. mucronatus 2C = 1.88 pg (1C = 919 Mbp), and the polyploids range from P. attenuatus var. attenuatus 2C = 2.35 pg (1C = 1148 Mbp) to P. digitalis 2C = 6.45 pg (1C = 3152 Mbp). Chromosome counts were done for ten previously published and four previously unreported Penstemon species (P. dissectus, P. navajoa, P. caespitosus var. desertipicti, and P. ramaleyi). These counts were compiled with all previously published chromosome data and compared with the flow cytometry results. Ploidy within this study ranged from diploid to dodecaploid. These data were compared and contrasted with the current taxonomy of Penstemon and previously published internal transcribed spacer and chloroplast DNA phylogenetic work. Based on genome size and previous studies, reassigning P. montanus to the subgenus Penstemon and P. personatus to the subgenus Dasanthera, would better reflect the phylogeny of the genus. Furthermore, our data concur with previous studies suggesting that the subgenus Habroanthus be included in the subgenus Penstemon. The DNA content of subgenus Penstemon exhibits high plasticity and spans a sixfold increase from the smallest to the largest genome (P. linarioides subsp. sileri and P. digitalis, respectively). Our study found flow cytometry to be useful in species identification and verification.     

Physical maps and recombination frequency of six rice chromosomes

We constructed physical maps of rice chromosomes 1, 2, and 6-9 with P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones. These maps, with only 20 gaps, cover more than 97% of the predicted length of the six chromosomes. We submitted a total of 193 Mbp of non-overlapping sequences to public databases. We analyzed the DNA sequences of 1316 genetic markers and six centromere-specific repeats to facilitate characterization of chromosomal recombination frequency and of the genomic composition and structure of the centromeric regions. We found marked changes in the relative recombination rate along the length of each chromosome. Chromosomal recombination at the centromere core and surrounding regions on the six chromosomes was completely suppressed. These regions have a total physical length of about 23 Mbp, corresponding to 11.4% of the entire size of the six chromosomes. Chromosome 6 has the longest quiescent region, with about 5.6 Mbp, followed by chromosome 8, with quiescent region about half this size. Repetitive sequences accounted for at least 40% of the total genomic sequence on the partly sequenced centromeric region of chromosome 1. Rice CentO satellite DNA is arrayed in clusters and is closely associated with the presence of Centromeric Retrotransposon of Rice (CRR)- and RIce RetroElement 7 (RIRE7)-like retroelement sequences. We also detected relatively small coldspot regions outside the centromeric region; their repetitive content and gene density were similar to those of regions with normal recombination rates. Sequence analysis of these regions suggests that either the amount or the organization patterns of repetitive sequences may play a role in the inactivation of recombination.     

Separation of chromosomal DNA molecules from C.albicans by pulsed field gel electrophoresis.

Modifications have been made to standard pulse field gel electrophoresis (PFGE) systems to enable very large DNA molecules to be resolved. The single most important modification was to elevate the temperature of electrophoresis to 35 degrees C. This enabled the largest Saccharomyces cerevisiae chromosome to be reproducibly resolved. More impressively, it enabled the DNA of Candida albicans to be clearly resolved into six bands, a feat which was very difficult at lower temperatures. Even so, optimal resolution could only be obtained by carefully adjusting field voltages and switching times. The DNA from the two largest C. albicans chromosomes, which was estimated to be at least 5-10Mbp in size, ran somewhat anomalously, giving fuzzy bands which did not migrate in the direction of the average electric field. That the highest molecular weight band was a distinct chromosome was demonstrated by specific hybridisation to the C. albicans ADE2 gene probe. With further fine tuning, the PFGE system described here should be capable of resolving DNA from the smallest human chromosomes.     

Inheritance of chromosome heteromorphisms analyzed by high-resolution bivariate flow karyotyping.

The DNA content of the mitotic chromosomes from 10 children and their parents in four families were quantified by bivariate flow karyotyping. In all cases, each chromosome peak in the flow karyotype of the child could be traced to one of the two parents. The measured absolute difference in homologue DNA content between children and their parents averaged 0.8%, or approximately 1 Mbp over all chromosome types. This study demonstrates that flow karyotypes of a proband's parents can be an aid in detecting and quantifying the size of de novo deletions that involve heteromorphic chromosome types.     

Generation of high-density DNA markers from yeast artificial chromosome DNA by single unique primer-polymerase chain reaction

We have developed a method for the whole sequence amplification of yeast artificial chromosome (YAC) DNA excised from preparative pulsed-field gel electrophoresis using single unique primer-polymerase chain reaction procedures. We used seven contiguous YAC clones, which span 2 Mbp of the Huntington disease gene region on 4p16.3, to amplify the YAC DNAs. The average size of the amplified DNA was ∼300 bp long, and 12 DNA markers located on the YAC clones positively hybridized with these amplified products, implying that the sequences of the YAC clones were comprehensively amplified by our procedures. These amplified YAC DNAs greatly facilitate the characterization of YAC clones, leading to the detailed analysis of the defined chromosomal region.     

Flow cytometric analysis of genome size variation in some Passiflora species

Nuclear genome size variation was studied in eight taxa of Passiflora. Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. 2C DNA content ranged from 3.16-5.36 pg for diploids and 1.83 pg for tetraploid. Differences in nuclear genome size were observed among Passiflora species (pg): P. suberosa 1.83, P. edulis f. edulis 3.16, P. edulis f. flavicarpa (Brazil) 3.19, P. edulis f. flavicarpa (Mexico) 3.21, P. mucronata 3.40, Passiflora edmundoi 3.43, P. laurifolia 3.88, P. giberti 3.92, P. quadrangularis 5.36, the largest value being up to 192% greater than the smallest. The means of 2C DNA content were compared by the Tukey test, and the differences in genome size permitted the recognition of five taxa groups. The result was the same for the means 2C genome size (Mbp) values. The genetic parameters were studied with their respective estimators, phenotypic variance (sigma2F), genotypic variability (PhiG), and the genotypic determination index (H2). The genotypic determination index presented high magnitude estimates (greater than 99%) emphasizing the reliability of the results and demonstrating the efficiency of determining the DNA content in the species using only one leaf per plant. Passiflora species show great phenotypic variability and have different geographic distribution that might implicate in genetic diversity.     

Unique multiple sex chromosomes of the tree mouse Vandeleuria o. oleracea: identification of X1 and X2.

The tree mouse Vandeleuria o. oleracea has an odd diploid chromosome complement (2n = 29 female/male) accompanied by a unique multiple sex-chromosome mechanism (X1X2Ymale/X1X1X2female). In the present paper the sex chromosomes have been identified unequivocally with the help of G-banding and DNA replication patterns which confirm our earlier suggestion that the X1 is smaller in size than the simple mammalian X, and also that the combined sizes of the X1 and X2 (3-8 and 1-7 per cent respectively) approximate to the size of the conservative mammalian X (5 per cent). It is proposed that the mammalian X has attained a minimum functional size so that further reduction is not tolerated.     

Estimation of hominoid ancestral population sizes under bayesian coalescent models incorporating mutation rate variation and sequencing errors

Estimation of population parameters for the common ancestors of humans and the great apes is important in understanding our evolutionary history. In particular, inference of population size for the human-chimpanzee common ancestor may shed light on the process by which the 2 species separated and on whether the human population experienced a severe size reduction in its early evolutionary history. In this study, the Bayesian method of ancestral inference of Rannala and Yang (2003. Bayes estimation of species divergence times and ancestral population sizes using DNA sequences from multiple loci. Genetics. 164:1645-1656) was extended to accommodate variable mutation rates among loci and random species-specific sequencing errors. The model was applied to analyze a genome-wide data set of approximately 15,000 neutral loci (7.4 Mb) aligned for human, chimpanzee, gorilla, orangutan, and macaque. We obtained robust and precise estimates for effective population sizes along the hominoid lineage extending back approximately 30 Myr to the cercopithecoid divergence. The results showed that ancestral populations were 5-10 times larger than modern humans along the entire hominoid lineage. The estimates were robust to the priors used and to model assumptions about recombination. The unusually low X chromosome divergence between human and chimpanzee could not be explained by variation in the male mutation bias or by current models of hybridization and introgression. Instead, our parameter estimates were consistent with a simple instantaneous process for human-chimpanzee speciation but showed a major reduction in X chromosome effective population size peculiar to the human-chimpanzee common ancestor, possibly due to selective sweeps on the X prior to separation of the 2 species.     

利用同源重组对酵母人工染色体左右臂进行多基因修饰

构建携带哺乳动物细胞筛选基因和酵母人工染色体(YAC)同源序列的载体,利用酵母中能够发生高频率同源重组的特点对YAC分别进行左、右臂修饰,依次将NEO、EGFP及PURO基因定点整合到YAC左右臂上。用营养缺陷筛选的方法排除酵母发生突变或随机整合等情况后,用PCR及Southern杂交方法证实各筛选基因定点整合于YAC两臂上,从而获得携带3个哺乳动物细胞筛选基因的YAC克隆。并且由此建立了通过同源重组将哺乳动物标记基因定点引入YAC左右臂的多基因修饰平台。     

Stretching the rules: monocentric chromosomes with multiple centromere domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel "meta-polycentric" functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.     

Construction of BAC libraries from two apomictic grasses to study the microcolinearity of their apospory-specific genomic regions

We have constructed bacterial artificial chromosome (BAC) libraries from two grass species that reproduce by apospory, a form of gametophytic apomixis. The library of an apomictic polyhaploid genotype (line MS228-20, with a 2C genome size of approximately 4,500 Mbp) derived from a cross between the obligate apomict, Pennisetum squamulatum, and pearl millet (P. glaucum) comprises 118,272 clones with an average insert size of 82 kb. The library of buffelgrass (Cenchrus ciliaris, apomictic line B-12-9, with a 2C genome size of approximately 3,000 Mbp) contains 68,736 clones with an average insert size of 109 kb. Based on the genome sizes of these two lines and correcting for the number for false-positive and organellar clones, library coverages were found to be 3.7 and 4.8 haploid genome equivalents for MS 228-20 and B12-9, respectively. Both libraries were screened by hybridization with six SCARs (sequence-characterized amplified regions), whose tight linkage in a single apospory-specific genomic region had been previously demonstrated in both species. Analysis of these BAC clones indicated that some of the SCAR markers are actually amplifying duplicated regions linked in coupling in both genomes and that restriction enzyme mapping will be necessary to sort out the duplications.     

Genome sizes and chromosomes in the basal metazoan Hydra

Hydras belong to one of the earliest eumetazoan animal groups, but to date very little is known about their genome sizes, gene numbers, and chromosomes. Here we provide genome size estimates and corresponding karyotypes for five Hydra species. Nuclear DNA contents were assessed by slide-based Feulgen microphotometry. Hydra oligactis possesses the largest genome of 1450 Mbp, followed by similar 1 C capacities in H. carnea (1350 Mbp), H. vulgaris (1250 Mpb) and H. circumcincta (1150 Mbp). The smallest genome of 380 Mbp was determined in H. viridissima. While the number of chromosomes is identical in all five Hydra species (2n = 30), the size of the chromosomes is strictly correlated to the size of the genome, with H. viridissima having conspicuously small chromosomes. The taxonomic and evolutionary significance of the C-value and chromosomal size variation in this ancient group of metazoans as well as its impact on genomic organization and forthcoming genome projects are discussed.     

Cytological and electrophoretic karyotyping of the chestnut blight fungus Cryphonectria parasitica

The karyotypes of nine strains including three transformants of the chestnut blight fungus Cryphonectria parasitica were analyzed by pulsed-field gel electrophoresis (PFGE) and cytology using a fluorescence microscope. Cytology of the mitotic metaphase showed n = 9 for both standard strain EP155 and field strain GH2 infected by Cryphonectria hypovirus 3. Chromosomes were morphologically characterized by size, heterochromatic segment, and constriction. PFGE resolved 5 or 6 chromosomal DNA bands ranging from 3.3 Mbp to 9.7 Mbp, but accurate determination of the chromosome number was hampered by clumping of some bands. Banding profiles in PFGE were similar among the strains except for GH2, in which a chromosome translocation was detected by Southern blot analysis. By integrating the data from cytology and PFGE, the genome size of C. parasitica was estimated to be ca. 50 Mbp. This is the first report of a cytological karyotype in the order Diaporthales.