Cloning of the essential myotonic dystrophy region and mapping of the putative defect.

Myotonic dystrophy is a common dominant disorder (global incidence of 1:8,000) with variable onset and a protean nature of symptoms mainly involving progressive muscle wasting, myotonia and cataracts. To define the molecular defect, we have cloned the essential region of chromosome 19q13.3, including proximal and distal markers in a 700-kilobase contig formed by overlapping cosmids and yeast artificial chromosomes (YACs). The central part of the contig bridges an area of about 350 kilobases between two new flanking crossover borders. This segment has been extensively characterized through the isolation of five YAC clones and the subsequent subcloning in cosmids from which a detailed EcoRI, HindIII, MluI and NotI restriction map has been derived. Two genomic probes and two homologous complementary DNA probes were isolated using the cosmids. These probes are all situated within approximately 10 kilobases of genomic DNA and detect an unstable genomic segment in myotonic dystrophy patients. The length variation in this segment shows similarities to the instability seen at the fragile X locus. The physical map location and the genetic characteristics of the length polymorphism is compatible with a direct role in the pathogenesis of myotonic dystrophy.     

Construction and use of human chromosome jumping libraries from NotI-digested DNA

A basic difficulty in the molecular analysis of genes identified by mutations in the mammalian genome is the need to cover genetic distances corresponding to several hundred kilobases or more by molecular techniques like chromosome walking. In chromosome jumping, this limitation is overcome by the deletion of all but the extreme ends of large DNA molecules before cloning. We describe here the construction and characterization of a NotI 'jumping library' from human DNA. To characterize this library, random clones were analysed by restriction mapping. Clones carrying unique end fragments were characterized further by hybridization to Southern blots of NotI-cleaved human DNA separated on pulsed field gradient (PFG) gels. As a first step in a directional walk, the library was screened with a clone containing a NotI site cleaved in genomic DNA ('NotI linking clone') localized to the distal third of the short arm of human chromosome 4 (A.-M.F. & T.P., unpublished data). Starting and end points of two identified clones were positioned within a restriction map covering 850 kilobases.     

Construction of single-chromosome DNA library fromLilium regale Wilson

A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA fromLilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0.5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300–2500 base pairs (bp) with predominant fragments at about 1000 bp. Southern blot analysis confirmed that the PCR products originated from the genome ofLilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1800 bp, with an average of780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).     

Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation.

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the generalizability of the recognition motif, and the cleavage yield. Oligonucleotide-directed triple helix formation is a generalizable motif for specific binding to sequences longer than 12 base pairs within DNA of high complexity. Studies with plasmid DNA show that triple helix formation can limit the operational specificity of restriction enzymes to endonuclease recognition sequences that overlap oligonucleotide-binding sites. Triple helix formation, followed by methylase protection, triple helix-disruption, and restriction endonuclease digestion produces near quantitative cleavage at the single overlapping triple helix-endonuclease site. As a demonstration that this technique may be applicable to the orchestrated cleavage of large genomic DNA, we report the near quantitative single-site enzymatic cleavage of the Saccharomyces cerevisiae genome mediated by triple helix formation. The 340-kilobase yeast chromosome III was cut uniquely at an overlapping homopurine-EcoRI target site 27 base pairs long to produce two expected cleavage products of 110 and 230 kilobases. No cleavage of any other chromosome was detected. The potential generalizability of this technique, which is capable of near quantitative cleavage at a single site in at least 14 megabase pairs of DNA, could enable selected regions of chromosomal DNA to be isolated without extensive screening of genomic libraries.     

禾本科同源染色体对趋同进化的比较基因组学

多个禾本科物种全基因组测序的相继完成为禾本科植物基因组物理和遗传结构进化历史的研究提供了前所未有的良好机遇。以五个禾本科物种为研究对象,利用基因同源共线性方法对其基因组进行了比对分析,获得了物种的同源信息,并根据同源信息结合基因组同源结构分析,确定了物种基因组内和基因组间的同源染色体片段。比较分析同源染色体对上重复DNA片段之间的分子距离,初步揭示了禾本科植物同源染色体对间趋同进化规律,研究结果有助于理解染色体结构受非正常遗传重组影响的进化机制。     

基于松材线虫全基因组序列的SSR标记开发

为了研究松材线虫在我国的群体遗传关系及传播路径,获得更为稳定的松材线虫分子标记,使用MISA软件对松材线虫全基因组10 432条DNA片段进行搜索,共获得95个gSSR位点。其中,二核苷酸重复出现频率最高,占全部SSR位点的66.3%。依据所有gSSR位点共设计出36对引物,以1份松材线虫DNA pooling(包含我国46个不同地理来源的松材线虫虫株DNA)为模板进行PCR,产物由QIAxcel全自动凝胶电泳分析系统检测,获得17对可能具有多态性的gSSR引物。进一步对这17对引物的PCR产物进行单克隆试验并测序,BioEdit软件拼接比对结果表明,其中9对确实具有多态性。     

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...     

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.     

A five-fold pig bacterial artificial chromosome library: a resource for positional cloning and physical mapping

A pig BAC library was constructed with genomic DNA from a male Erhualian pig. After partial digestion with Hind III or BamH I the fragments obtained were cloned into the pBeloBAC11 vector. The library consists of 184320 clones which stored in 480 pieces 384-well plates (20 plates per superpool). A two-step 4-dimension PCR screening system was established to screen the positive clones. An average insert size of 128 kb was estimated from 105 randomly isolated clones, which indicates that the library is more than five times of genomic coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 10 single-copy genes were screened out and the positive clones were yielded between 1 and 8 with an average of 3.6. Positive superpools were obtained for 32 microsatellite markers selected from different regions of pig genome. The number of positive superpools for each marker varies from 1 to 9 with an average of 4.78. This BAC library provides an additional resource for pig physical mapping and gene identification.     

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.     

A physical map of the human Y chromosome

The non-recombining region of the human Y chromosome (NRY), which comprises 95% of the chromosome, does not undergo sexual recombination and is present only in males. An understanding of its biological functions has begun to emerge from DNA studies of individuals with partial Y chromosomes, coupled with molecular characterization of genes implicated in gonadal sex reversal, Turner syndrome, graft rejection and spermatogenic failure. But mapping strategies applied successfully elsewhere in the genome have faltered in the NRY, where there is no meiotic recombination map and intrachromosomal repetitive sequences are abundant. Here we report a high-resolution physical map of the euchromatic, centromeric and heterochromatic regions of the NRY and its construction by unusual methods, including genomic clone subtraction and dissection of sequence family variants. Of the map's 758 DNA markers, 136 have multiple locations in the NRY, reflecting its unusually repetitive sequence composition. The markers anchor 1,038 bacterial artificial chromosome clones, 199 of which form a tiling path for sequencing.     

草鱼基因组随机扩增多态性引物及多态性位点的筛选

为了建立草鱼基因组DNA多态性分析的指标体系,应用RAPD技术,从180条10碱基随机引物中筛选出了31条能扩增出多态性DNA片段的引物。用这31条多态性引物共扩增出了327条重复性好、带型清晰、分辨率高的谱带。扩增产物的片段大小范围在400—2000bp之间。单一引物扩增条带为5—14条。用这些多态性引物在草鱼基因组DNA中检测到了93个多态性位点,并对这些多态性位点上的等位基因频率进行了统计分析。这31条多态性引物和所检测到的93个多态性位点初步为草鱼基因组的多态性分析提供了可靠的分析指标体系。     

The DNA sequence and analysis of human chromosome 14

Chromosome 14 is one of five acrocentric chromosomes in the human genome. These chromosomes are characterized by a heterochromatic short arm that contains essentially ribosomal RNA genes, and a euchromatic long arm in which most, if not all, of the protein-coding genes are located. The finished sequence of human chromosome 14 comprises 87,410,661 base pairs, representing 100% of its euchromatic portion, in a single continuous segment covering the entire long arm with no gaps. Two loci of crucial importance for the immune system, as well as more than 60 disease genes, have been localized so far on chromosome 14. We identified 1,050 genes and gene fragments, and 393 pseudogenes. On the basis of comparisons with other vertebrate genomes, we estimate that more than 96% of the chromosome 14 genes have been annotated. From an analysis of the CpG island occurrences, we estimate that 70% of these annotated genes are complete at their 5' end.     

Comparison of human genetic and sequence-based physical maps

Recombination is the exchange of information between two homologous chromosomes during meiosis. The rate of recombination per nucleotide, which profoundly affects the evolution of chromosomal segments, is calculated by comparing genetic and physical maps. Human physical maps have been constructed using cytogenetics, overlapping DNA clones and radiation hybrids; but the ultimate and by far the most accurate physical map is the actual nucleotide sequence. The completion of the draft human genomic sequence provides us with the best opportunity yet to compare the genetic and physical maps. Here we describe our estimates of female, male and sex-average recombination rates for about 60% of the genome. Recombination rates varied greatly along each chromosome, from 0 to at least 9 centiMorgans per megabase (cM Mb(-1)). Among several sequence and marker parameters tested, only relative marker position along the metacentric chromosomes in males correlated strongly with recombination rate. We identified several chromosomal regions up to 6 Mb in length with particularly low (deserts) or high (jungles) recombination rates. Linkage disequilibrium was much more common and extended for greater distances in the deserts than in the jungles.     

Reagents for the site-specific cleavage of megabase DNA.

The physical mapping of chromosomes will be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the number of different sequences that can be targeted and the cleavage yield.     

Construction of a BAC library from cucumber （Cucumis sativus L.） and identification of linkage group specific clones

A bacterial artificial chromosome （BAC） library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber （Cucumis sativus L.） inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions （SCAR） and seven simple sequence repeats （SSR） markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction （PCR）. Fifteen markers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.     

A physical map of the chicken genome

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones. In an accompanying paper we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes.     

Construction of an electronic physical map of Magnaporthe oryzae using genomic position-ready SSR markers

Magnaporthe oryzae is a model for plant pathogenic filamentous fungi. We have assembled a simple sequence repeat (SSR)-based physical map of the species, using in silico sequence data. A set of 120 SSR markers was developed from the genomic sequence of the reference isolate 70-15. These markers were readily amplified from the genomic DNA of other isolates, and high levels of allelic variation characterised the parental isolates of the two crosses tested. All the markers were locatable to one of the seven M. oryzae chromosomes. An SSR-based physical in silico map was constructed, and pre-existing SSR and RFLP loci were integrated into the map, along with 23 Avr (avirulence) genes and two other genes of importance to the plant/pathogen interaction. This map provides a platform for population genetics and functional genomics studies in the model pathogen, and even in other evolu- tionally related pathogens.     

Reassembly of shattered chromosomes in Deinococcus radiodurans

Dehydration or desiccation is one of the most frequent and severe challenges to living cells. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called 'extended synthesis-dependent strand annealing' (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become 'sticky ends' that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.