Topological domains in mammalian genomes identified by analysis of chromatin interactions

The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term 'topological domains', as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.     

Similar level of polyteny in bands and interbands of Drosophila giant chromosomes

The giant polytene chromosomes of Drosophila melanogaster have long been of interest to the geneticist because of the visible map of the genome provided by their characteristic banding patterns. An issue in the understanding of the molecular basis of chromosome banding has been whether the chromosomal DNA is replicated to the same extent in bands and interbands. Although various suggestions have been put forward the point has remained controversial. We have isolated 315 kilobases (kb) of contiguous Drosophila genomic DNA which spans an interval of approximately 13 bands and interbands of the polytene chromosomes. We report here the measurement of the relative level of DNA replication in polytene chromosomes of 84 adjacent restriction fragments of our cloned DNA. We conclude that there are no significant differences in the level of polyteny within the large band and between bands and interbands of this region. This result supports the 'folded fibre' model of polytene chromosome organization, rather than models involving disproportionate replication along the banding pattern.     

Integration of cytogenetic landmarks into the draft sequence of the human genome

We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.     

Integration of telomere sequences with the draft human genome sequence

Telomeres are the ends of linear eukaryotic chromosomes. To ensure that no large stretches of uncharacterized DNA remain between the ends of the human working draft sequence and the ends of each chromosome, we would need to connect the sequences of the telomeres to the working draft sequence. But telomeres have an unusual DNA sequence composition and organization that makes them particularly difficult to isolate and analyse. Here we use specialized linear yeast artificial chromosome clones, each carrying a large telomere-terminal fragment of human DNA, to integrate most human telomeres with the working draft sequence. Subtelomeric sequence structure appears to vary widely, mainly as a result of large differences in subtelomeric repeat sequence abundance and organization at individual telomeres. Many subtelomeric regions appear to be gene-rich, matching both known and unknown expressed genes. This indicates that human subtelomeric regions are not simply buffers of nonfunctional 'junk DNA' next to the molecular telomere, but are instead functional parts of the expressed genome.     

Genome linking with yeast artificial chromosomes

The haploid genome of Caenorhabditis elegans consists of some 80 x 10(6) base pairs of DNA contained in six chromosomes. The large number of interesting loci that have been recognized by mutation, and the accuracy of the genetic map, mean that a physical map of the genome is highly desirable, because it will facilitate the molecular cloning of chosen loci. The first steps towards such a map used a fingerprinting method to link cosmid clones together. This approach reached its practical limit last year, when 90-95% of the genome had been cloned into 17,500 cosmids assembled into some 700 clusters (contigs), but the linking clones needed were either non-existent or extremely rare. Anticipating this, we had planned to link by physical means--probably by hybridization to NotI fragments separated by pulse field gel electrophoresis. NotI recognizes an eight base sequence of GC pairs; thus the fragments should be large enough to bridge regions that clone poorly in cosmids, and, with no selective step involved, would necessarily be fully representative. However, with the availability of a yeast artificial chromosome (YAC) vector, we decided to use this alternative source of large DNA fragments to obtain linkage. The technique involves the ligation of large (50-1,000 kilobase) genomic fragments into a vector that provides centromeric, telomeric and selective functions; the constructs are then introduced into Saccharomyces cerevisiae, and replicate in the same manner as the host chromosomes.     

Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans

Chromosomes are divided into domains of open chromatin, where genes have the potential to be expressed, and domains of closed chromatin, where genes are not expressed. Classic examples of open chromatin domains include 'puffs' on polytene chromosomes in Drosophila and extended loops from lampbrush chromosomes. If multiple genes were typically expressed together from a single open chromatin domain, the position of co-expressed genes along the chromosomes would appear clustered. To investigate whether co-expressed genes are clustered, we examined the chromosomal positions of the genes expressed in the muscle of Caenorhabditis elegans at the first larval stage. Here we show that co-expressed genes in C. elegans are clustered in groups of 2-5 along the chromosomes, suggesting that expression from a chromatin domain can extend over several genes. These observations reveal a higher-order organization of the structure of the genome, in which the order of the genes along the chromosome id correlated with their expression in specific tissues.     

Chromosome sorting and its applications in common wheat (Triticum aestivum) genome sequencing

The large genome size (～17000 Mb) and complicated DNA structures of common wheat (Triticum aestivum) hamper its genome sequencing.By means of flow cytometry,systematic investigations on individual chromosome sorting have been carried out to construct chromosome-specific bacterial artificial chromosome (BAC) libraries since the 1980s.Several wheat chromosome-specific BAC libraries,such as chromosome 3B,three D genome chromosomes (1D,4D and 6D),and the short arm of chromosome 1B,have been developed,and the ph...     

Genetic analysis of autoimmune type 1 diabetes mellitus in mice.

Two genes, Idd-3 and Idd-4, that influence the onset of autoimmune type 1 diabetes in the nonobese diabetic mouse have been located on chromosomes 3 and 11, outside the chromosome 17 major histocompatibility complex. A genetic map of the mouse genome, analysed using the polymerase chain reaction, has been assembled specifically for the study. On the basis of comparative maps of the mouse and human genomes, the homologue of Idd-3 may reside on human chromosomes 1 or 4 and Idd-4 on chromosome 17.     

Determinants of nucleosome organization in primary human cells

Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.     

Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.     

Chromatin organization is a major influence on regional mutation rates in human cancer cells

Cancer genome sequencing provides the first direct information on how mutation rates vary across the human genome in somatic cells. Testing diverse genetic and epigenetic features, here we show that mutation rates in cancer genomes are strikingly related to chromatin organization. Indeed, at the megabase scale, a single feature—levels of the heterochromatin-associated histone modification H3K9me3—can account for more than 40% of mutation-rate variation, and a combination of features can account for more than 55%. The strong association between mutation rates and chromatin organization is upheld in samples from different tissues and for different mutation types. This suggests that the arrangement of the genome into heterochromatin- and euchromatin-like domains is a dominant influence on regional mutation-rate variation in human somatic cells.     

Functional genetic analysis of mouse chromosome 11

Now that the mouse and human genome sequences are complete, biologists need systematic approaches to determine the function of each gene. A powerful way to discover gene function is to determine the consequence of mutations in living organisms. Large-scale production of mouse mutations with the point mutagen N-ethyl-N-nitrosourea (ENU) is a key strategy for analysing the human genome because mouse mutants will reveal functions unique to mammals, and many may model human diseases. To examine genes conserved between human and mouse, we performed a recessive ENU mutagenesis screen that uses a balancer chromosome, inversion chromosome 11 (refs 4, 5). Initially identified in the fruitfly, balancer chromosomes are valuable genetic tools that allow the easy isolation of mutations on selected chromosomes. Here we show the isolation of 230 new recessive mouse mutations, 88 of which are on chromosome 11. This genetic strategy efficiently generates and maps mutations on a single chromosome, even as mutations throughout the genome are discovered. The mutations reveal new defects in haematopoiesis, craniofacial and cardiovascular development, and fertility.     

黄雀(Carduelis spinus)上橄榄核的传入性联系—HRP顺逆行追踪法研究

分别将HRP微电泳入黄雀的上橄榄核(SO)、角状核、层状核和巨细胞核,观察其顺、逆行标记物的分布.结果表明,SO接受同侧层状标和双侧角状核的传入,双侧SO之间有交互投射,SO的纤维投射至外侧丘系核腹侧部、外侧丘系腹核和中脑背外侧核.因此,它属于听觉传入通路中的第二级中继站.此外,SO向巨细胞核投射并形成巨细胞核→层状核→SO→巨细胞核的闭合环路.此环路可能参与听觉信息的反馈及声源定向等的初步整合机能.     

Mitochondrial DNA repairs double-strand breaks in yeast chromosomes

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.     

A physical map of the mouse genome

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.     

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.     

An SNP map of human chromosome 22

The human genome sequence will provide a reference for measuring DNA sequence variation in human populations. Sequence variants are responsible for the genetic component of individuality, including complex characteristics such as disease susceptibility and drug response. Most sequence variants are single nucleotide polymorphisms (SNPs), where two alternate bases occur at one position. Comparison of any two genomes reveals around 1 SNP per kilobase. A sufficiently dense map of SNPs would allow the detection of sequence variants responsible for particular characteristics on the basis that they are associated with a specific SNP allele. Here we have evaluated large-scale sequencing approaches to obtaining SNPs, and have constructed a map of 2,730 SNPs on human chromosome 22. Most of the SNPs are within 25 kilobases of a transcribed exon, and are valuable for association studies. We have scaled up the process, detecting over 65,000 SNPs in the genome as part of The SNP Consortium programme, which is on target to build a map of 1 SNP every 5 kilobases that is integrated with the human genome sequence and that is freely available in the public domain.     

Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii

Species of malaria parasite that infect rodents have long been used as models for malaria disease research. Here we report the whole-genome shotgun sequence of one species, Plasmodium yoelii yoelii, and comparative studies with the genome of the human malaria parasite Plasmodium falciparum clone 3D7. A synteny map of 2,212 P. y. yoelii contiguous DNA sequences (contigs) aligned to 14 P. falciparum chromosomes reveals marked conservation of gene synteny within the body of each chromosome. Of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. This is the first genome sequence of a model eukaryotic parasite, and it provides insight into the use of such systems in the modelling of Plasmodium biology and disease.