Construction of a general human chromosome jumping library, with application to cystic fibrosis

In many genetic disorders, the responsible gene and its protein product are unknown. The technique known as "reverse genetics," in which chromosomal map positions and genetically linked DNA markers are used to identify and clone such genes, is complicated by the fact that the molecular distances from the closest DNA markers to the gene itself are often too large to traverse by standard cloning techniques. To address this situation, a general human chromosome jumping library was constructed that allows the cloning of DNA sequences approximately 100 kilobases away from any starting point in genomic DNA. As an illustration of its usefulness, this library was searched for a jumping clone, starting at the met oncogene, which is a marker tightly linked to the cystic fibrosis gene that is located on human chromosome 7. Mapping of the new genomic fragment by pulsed field gel electrophoresis confirmed that it resides on chromosome 7 within 240 kilobases downstream of the met gene. The use of chromosome jumping should now be applicable to any genetic locus for which a closely linked DNA marker is available.     

A genetic map and recombination parameters of the human malaria parasite Plasmodium falciparum

Genetic investigations of malaria require a genome-wide, high-resolution linkage map of Plasmodium falciparum. A genetic cross was used to construct such a map from 901 markers that fall into 14 inferred linkage groups corresponding to the 14 nuclear chromosomes. Meiotic crossover activity in the genome proved high (17 kilobases per centimorgan) and notably uniform over chromosome length. Gene conversion events and spontaneous microsatellite length changes were evident in the inheritance data. The markers, map, and recombination parameters are facilitating genome sequence assembly, localization of determinants for such traits as virulence and drug resistance, and genetic studies of parasite field populations.     

Sequence-directed DNA translocation by purified FtsK

DNA translocases are molecular motors that move rapidly along DNA using adenosine triphosphate as the source of energy. We directly observed the movement of purified FtsK, an Escherichia coli translocase, on single DNA molecules. The protein moves at 5 kilobases per second and against forces up to 60 piconewtons, and locally reverses direction without dissociation. On three natural substrates, independent of its initial binding position, FtsK efficiently translocates over long distances to the terminal region of the E. coli chromosome, as it does in vivo. Our results imply that FtsK is a bidirectional motor that changes direction in response to short, asymmetric directing DNA sequences.     

Paired-end mapping reveals extensive structural variation in the human genome

Structural variation of the genome involves kilobase- to megabase-sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements. We introduce high-throughput and massive paired-end mapping (PEM), a large-scale genome-sequencing method to identify structural variants (SVs) approximately 3 kilobases (kb) or larger that combines the rescue and capture of paired ends of 3-kb fragments, massive 454 sequencing, and a computational approach to map DNA reads onto a reference genome. PEM was used to map SVs in an African and in a putatively European individual and identified shared and divergent SVs relative to the reference genome. Overall, we fine-mapped more than 1000 SVs and documented that the number of SVs among humans is much larger than initially hypothesized; many of the SVs potentially affect gene function. The breakpoint junction sequences of more than 200 SVs were determined with a novel pooling strategy and computational analysis. Our analysis provided insights into the mechanisms of SV formation in humans.     

鸡致病性大肠杆菌的质粒DNA指纹图谱分析

从山东省诸城地区6个集约化养鸡场的大肠杆菌感染鸡群的死鸡、毛蛋的心血中分离到32株细菌,经形态、培养特性及生化反应鉴定为大肠埃希氏杆菌(Escherichia.coli)简称大肠杆菌(E.coli)。对其作动物回归试验结果表明,全部为致病性大肠杆菌。从而证明从死鸡(毛蛋)心血中分离到的大肠杆菌与其致病性密切相关。将分离到的鸡大肠杆菌的14株优势血清型菌株进行了质粒DNA分析,通过碱裂解法小量提取质粒,然后对质粒进行了琼脂糖凝胶电泳分析,用hindⅢ限制性内切酶对质粒进行了酶切分析,结果表明:质粒的得率为100%,来源相同的菌株具有相同或相似的质粒图谱,来源不同的菌株的质粒图谱一般不同。将血清型和质粒图谱比较发现:同一血清型可以有不同的质粒图谱,不同血清型可以有相似的质粒图谱,血清型与质粒图谱没有直接的联系。本试验同一来源菌株的质粒指纹图谱相同、但它们的血清型不一致的现象可作为自然条件下同一质粒在不同血清型大肠杆菌中扩散转化的一个分子生物学依据。     

AFLP分子标记与作物改良

建立了于ＤＮＡ基础之上的分子标记对于作物改良具有重要作用，ＡＦＬＰ（Amplified fragment length polymorphism，简称ＡＦＬＰ）国内译为扩增片段长度多态性，是一种ＤＮＡ分子标记技术。利用 一方法，在不需要预先知道ＤＮＡ序列住处的情况下，可以同时进行多数ＤＮＡ酶切片断的ＰＣＲ扩增，目前该技术不仅在小麦、玉米、棉花和大豆等主要农作物上得以应用，而且在蔬菜（番茄、马铃薯、鹰嘴豆等）以植物基因组研究的模式植物拟南芥上广泛应用。讨论ＡＦＬＰ在主要作物的品种鉴定、遗传多样性分析，遗传作图，基因定位以及辅助选择等方面的应用进展。     

Location of the c-yes gene on the human chromosome and its expression in various tissues

Analysis of DNA from human embryo fibroblasts showed that ten Eco RI fragments were hybridizable with the Yamaguchi sarcoma virus oncogene (v-yes). Four of the Eco RI fragments were assigned to chromosome 18 and one to chromosome 6. There was evidence for multiple copies of yes-related genes in the human genome; however, only a single RNA species, 4.8 kilobases in length, was related to yes in various cells.     

用RFLP技术检测水稻染色体片段的来源

利用已定位的28个RFLP标记为探针,测定了IR8及其亲本在9种限制性内切酶酶切条件下的RFLP表现,以此来判断IR8品种中这些标记所在的染色体片段来源。结果,有18个标记揭示了品种间的RFLP存在。根据RFLP图谱,IR8品种中9个标记的图谱相同于Peta,5个标记的图谱相同于低脚乌尖（DGWG）,这表明这些标记所在的染色体片段分别来自Peta和DGWG。另外3个标记显示了IR8特有的RFLP图谱,表明IR8中这些标记所在的染色体片段的DNA顺序已经发生了重组。在IR8不同个体的测定中,发现了一个不与RG365标记DNA杂交的个体,该个体可能是缺少RG365标记同源顺序的突变体。RG237标记的EcoRV酶切条件下,也揭示了个体间的RFLP存在。     

P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster

We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage PhiC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. PhiC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.     

链霉菌高拷贝质粒pIJ101DNA的研究 I．在大肠杆菌中的启动子活性

用大肠杆菌座子与Tn5个去除了自身启动子区域的卡那霉素抗性基因（neo)作为指标标记来探测广寄主、高拷贝的链霉菌质粒pIJ101上DNA的启动子活性所做的基因融合试验揭示，pIJ101上至少有两个可大大肠杆菌中行使启动子功能的DNA片段。基因融合后所产生的杂合质粒能够赋予大肠杆菌ED8767较高的卡那霉素抗性。对含质粒菌株在液体培养基中的生长动力学分析揭示，这种菌株在从无药物向有药物的生物环境中过渡时，生长并不受到暂时的抑制。说明这种启动子活性不是pIJ101DNA上DNA的突变所引起。用大肠杆菌的启动子探针载体pKK232-8所做的体外亚克隆试验更进一步证实了这项观察，并把两个启动子功能区分别缩小到0．54kb和1．18kb的范围内。这项试验成功地组建了质粒pIJ101亚克隆库，便于对这个重要的链霉菌质粒进行精细的分子生物学研究。     

DNA ligase: structure, mechanism, and function

DNA ligase of E. coli is a polypeptide of molecular weight 75,000. The comparable T4-induced enzyme is somewhat smaller (63,000 to 68,000). Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA, coupled to the cleavage of the pyrophosphate bond of DPN (E. coli) or ATP (T4). Phosphodiester bond synthesis catalyzed by both enzymes occurs in a series of these discrete steps and involves the participation of two covalent intermediates (Fig. 1). A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and further demonstrates that enzyme-adenylate and DNA-adenylate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis. A strain of E. coli with a mutation in the structural gene for DNA ligase which results in the synthesis of an abnormally thermolabile enzyme is inviable at 42 degrees C. Although able to grow at 30 degrees C, the mutant is still defective at this temperature in its ability to repair damage to its DNA caused by ultraviolet irradiation and by alkylating agents. At 42 degrees C, all the newly replicated DNA is in the form of short 10S "Okazaki fragments," an indication that the reason for the mutant's failure to survive under these conditions is its inability to sustain the ligation step that is essential for the discontinuous synthesis of the E. coli chromosome. DNA ligase is therefore an essential enzyme required for normal DNA replication and repair in E. coli. Purified DNA ligases have proved to be useful reagents in the construction in vitro of recombinant DNA molecules.     

五种麻黄亲缘关系的RAPD分析

应用RAPD技术对5种麻黄亲缘关系进行了分析.从100条引物中筛选出18条重复性好,多态性高的随机引物.18条引物共扩增出103条带,其中多态性带为89条,占总扩增带的87.5%,平均每条引物扩增出4.9条多态性带.对这103条带进行聚类分析,结果表明:5个麻黄种间遗传差异明显,具有丰富的遗传多样性;膜果麻黄与其他4种药用麻黄的差异较大;木贼麻黄和斑子麻黄相对独立;草麻黄与中麻黄遗传距离较近,具有较为相似的遗传关系,这与传统的植物分类学结果一致,说明RAPD标记适用于麻黄植物的遗传多样性研究.     

一株高产脂肪酶产生菌16S rDNA的序列分析

[目的]对分离获得的高产脂肪酶菌株进行鉴定,为其改造和更好利用奠定基础.[方法]对从食堂下水道中分离获得的一株高效产脂肪酶细菌(JLΠ-4)进行培养,提取其基因组DNA.设计16S rDNA通用引物,扩增16S rDNA基因片段,并连接到pUC19-T载体上,转化大肠杆菌DH5X,经PCR鉴定的阳性克隆摇菌培养后测序.[结果]提取获得较高质量的基因组DNA,扩增获得新分离菌株16S rDNA基因片段,长度为1528 bp,BLAST相似性比对分析结果表明,其与伯克霍尔德氏菌16S rDNA序列相似性达97%,是一株与伯克霍尔德氏菌最近的革兰氏阴性菌.[结论]初步将高产脂肪酶细菌JTΠ-4鉴定为唐菖蒲伯克霍尔德菌.     

The mosaic genome of warm-blooded vertebrates

Most of the nuclear genome of warm-blooded vertebrates is a mosaic of very long (much greater than 200 kilobases) DNA segments, the isochores; these isochores are fairly homogeneous in base composition and belong to a small number of major classes distinguished by differences in guanine-cytosine (GC) content. The families of DNA molecules derived from such classes can be separated and used to study the genome distribution of any sequence which can be probed. This approach has revealed (i) that the distribution of genes, integrated viral sequences, and interspersed repeats is highly nonuniform in the genome, and (ii) that the base composition and ratio of CpG to GpC in both coding and noncoding sequences, as well as codon usage, mainly depend on the GC content of the isochores harboring the sequences. The compositional compartmentalization of the genome of warm-blooded vertebrates is discussed with respect to its evolutionary origin, its causes, and its effects on chromosome structure and function.     

Selective cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage

Current methods for sequence-specific cleavage of large segments of DNA are severely limited because of the paucity of possible cleavage sites. A method is described whereby any Eco RI site can be targeted for specific cleavage. The technique is based on the ability of RecA protein from Escherichia coli to pair an oligonucleotide to its homologous sequence in duplex DNA and to form a three-stranded complex. This complex is protected from Eco RI methylase; after methylation and RecA protein removal, Eco RI restriction enzyme cleavage was limited to the site previously protected from methylation. When pairs of oligonucleotides are used, a specific fragment can be cleaved out of genomes. The method was tested on lambda phage, Escherichia coli, and human DNA. Fragments exceeding 500 kilobases in length and yields exceeding 80 percent could be obtained.     

Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasmic male-sterile maize

Mitochondrial DNA from normal and cytoplasmic male-sterile maize was digested with restriction endonucleases RI from Escherichia coli or dIII from Hemophilus influenzae. Electrophoresis of resulting fragments revealed distinctions between the two cytoplasmic types. These distinctions suggest that factors responsible or cytoplasmic male sterility are located in the mitochondrial DNA, and that the mitochondrial genome is not inherited paternally.     

Integrating the physical and genetic map of bread wheat facilitates the detection of chromosomal rearrangements

The bread wheat genome harbors a high content of repetitive DNA, which is amenable to detection and characterization using fluorescence in situ hybridization(FISH) karyotyping. An integrated genetic map was derived from a recombinant inbred population bred from a cross between a synthetic hexaploid wheat and a commercial Chinese bread wheat cultivar, based on 28 variable FISH sites and 150 000 single nucleotide polymorphism(SNP) loci. The majority(20/28) of the variable FISH sites were physically located within a chromosomal region consistent with the genetic location inferred from that of their co-segregating SNP loci. The eight exceptions reflected the presence of either a translocation(1 R/1 B, 1 A/7 A) or a presumptive intra-chromosomal inversion(4 A). For eight out of the nine FISH sites detected on the Chinese Spring(CS) karyotype, there was a good match with the reference genome sequence, indicating that the most recent assembly has dealt well with the problem of placing tandem repeats. The integrated genetic map produced for wheat is informative as to the location of blocks of tandemly repeated DNA and can aid in improving the quality of the genome sequence assembly in regions surrounding these blocks.     

梨遗传连锁图谱的构建与比较分析

【目的】利用已公开发表的梨和苹果的SSR（Simple Sequence Repeat）引物以及从梨转录组开发的SSR引物构建本研究作图群体的遗传连锁图谱,为后期梨重要性状QTL定位和分子标记辅助选择等奠定基础。【方法】以西洋梨品种‘红茄’（Red Clapp Favorite）为母本,东方梨品种‘晚秀’（Mansoo）为父本,构建F1代作图群体。将所选用的SSR引物在亲本和4个子代个体进行PCR扩增,初步筛选出扩增结果符合JoinMap 4.0软件中“CP”作图模式要求的引物,随后在F1群体中检测,选用JoinMap 4.0软件对分离数据进行连锁分析,分别构建亲本的连锁图谱。以双亲图谱在各连锁群上的同源标记作为锚定位点,对双亲图谱进行整合。【结果】利用PCR技术对不同来源的共909对SSR引物（526对梨和283对苹果公开发表的SSR引物,从梨转录组开发的100对SSR引物）进行初步筛选后,发现来自苹果的SSR引物有效扩增片段的比例和多态性均较低,而来自梨和梨转录组开发的SSR引物相对较高。筛选出207对符合作图要求的SSR引物在群体中扩增,构建亲本的连锁图谱。母本图谱中的141个标记分布在17个连锁群上,总长度757.34 cM,标记间平均5.37 cM;父本图谱中的153个标记分布在19个连锁群上,总长度1 149.43 cM,标记间平均7.51 cM。【结论】对不同来源的SSR引物构建的双亲连锁图谱进行整合,最终得到一张由186个SSR标记,覆盖基因组长度1 125.33 cM的整合图谱。     

Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome

We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.