Chromosomal region of the cystic fibrosis gene in yeast artificial chromosomes: a model for human genome mapping

A general strategy for cloning and mapping large regions of human DNA with yeast artificial chromosomes (YAC's) is described. It relies on the use of the polymerase chain reaction to detect DNA landmarks called sequence-tagged sites (STS's) within YAC clones. The method was applied to the region of human chromosome 7 containing the cystic fibrosis (CF) gene. Thirty YAC clones from this region were analyzed, and a contig map that spans more than 1,500,000 base pairs was assembled. Individual YAC's as large as 790 kilobase pairs and containing the entire CF gene were constructed in vivo by meiotic recombination in yeast between pairs of overlapping YAC's.     

Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors

Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with which clones can be generated is high enough to allow the construction of comprehensive libraries from the genomes of higher organisms. By offering a tenfold increase in the size of the DNA molecules that can be cloned into a microbial host, this system addresses a major gap in existing experimental methods for analyzing complex DNA sources.     

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.     

Complementary DNA sequencing: expressed sequence tags and human genome project

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.     

人类3个多肽基因的人工合成和对人工基因合成意义的探讨

本文报道了胰岛素A,B链基因,人表皮生长因子基因和人α心钠素基因的人工合成。人工合成是在DNA合成仪上完成的,先合成上述基因的平均为60个核苷酸的各寡核苷酸片段,经过对各片段的纯化、磷酸化和连接,得到上述多肽基因。并进行了电泳检测。人工基因合成的重要意义在于可以正确地、高效地合成基因工程所需的基因,是获得在原核生物中表达的多肽基因的主要途径,并可将重组DNA和表达所需的碱基顺序同时组合在合成的基因中。人工基因合成有广泛的应用前途和价值。     

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.     

Mapping the Drosophila genome with yeast artificial chromosomes

The ability to clone large fragments of DNA in yeast artificial chromosomes (YAC's) has created the possibility of obtaining global physical maps of complex genomes. For this application to be feasible, most sequences in complex genomes must be able to be cloned in YAC's, and most clones must be genetically stable and colinear with the genomic sequences from which they originated (that is, not liable to undergo rearrangement). These requirements have been met with a YAC library containing DNA fragments from Drosophila melanogaster ranging in size up to several hundred kilobase pairs. Preliminary characterization of the Drosophila YAC library was carried out by in situ hybridization of random clones and analysis of clones containing known sequences. The results suggest that most euchromatic sequences can be cloned. The library also contains clones in which the inserted DNA is derived from the centromeric heterochromatin. The locations of 58 clones collectively representing about 8 percent of the euchromatic genome are presented.     

瘤胃微生物基因组文库中BAC末端序列分析

基于前期从瘤胃微生物基因组文库中筛选的功能酶克隆菌,利用T7和pIBRP引物,测定功能酶克隆菌的BAC末端序列,并利用NCBI Blast系统对BAC末端序列进行分析.结果表明:共得到26条BAC末端序列,经Blast比对分析,与已知编码基因的匹配度低,而大部分BAC末端序列与阪崎肠杆菌和环境中的微生物匹配度高,说明瘤胃中的微生物与其它环境中的微生物具有一定的相似性,进一步丰富了对瘤胃微生物的认识.     

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.     

南瓜基因组DNA的细菌人工染色体(BAC)文库的构建

[目的]构建南瓜基因组DNA的细菌人工染色体(BAC)文库。[方法]以南瓜幼芽为材料,利用HindⅢ酶切体系,初步构建南瓜基因组DNA的BAC文库。[结果]研究成功构建了南瓜基因组DNA的BAC文库。[结论]该技术为南瓜相关基因的克隆、功能验证、物理图谱的构建和基因组测序等研究工作奠定了基础。     

Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons

Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them. To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs. Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals. Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases.     

棉花晋A细胞质雄性不育系及其保持系线粒体基因组文库的构建

【目的】构建棉花晋A细胞质雄性不育系及其保持系的线粒体BAC文库,为研究棉花线粒体基因组结构以及棉花细胞质雄性不育的形成机理提供基础。【方法】在借鉴其它作物的线粒体基因组BAC文库构建方法的基础上,对棉花线粒体DNA的提取及其BAC文库的构建进行探索和优化,构建了棉花晋A细胞质雄性不育系及其保持系的线粒体BAC文库,并采用同源克隆的方法克隆棉花线粒体的功能基因。【结果】构建的不育系和保持系的线粒体基因组文库分别包括2 600个克隆,插入DNA片段大小在10.3 kb和37.5 kb之间,平均分别为22.29 kb和21.36 kb。重组子的覆盖率分别达到棉花线粒体基因组的79倍和76倍。获得了3个棉花线粒体功能基因序列,通过比较,证明晋A不育系与其保持系的这3个基因序列无差异;以这3个基因为探针筛选文库,均获得阳性克隆。【结论】分别构建了棉花晋A细胞质雄性不育系及其保持系线粒体基因组的BAC文库。获得了晋A不育系与其保持系的orfB,coxⅠ和nad4L 3个基因的全序列,通过比较,证明晋A不育系与其保持系在orfB、coxⅠ和nad4L基因全序列上无差异。     

Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation

A mouse-human somatic cell hybrid clone, deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and containing a structurally normal inactive human X chromosome, was isolated. The hybrid cells were treated with 5-azacytidine and tested for the reactivation and expression of human X-linked genes. The frequency of HPRT-positives clones after 5-azacytidine treatment was 1000-fold greater than that observed in untreated hybrid cells. Fourteen independent HPRT-positive clones were isolated and analyzed for the expression of human X markers. Isoelectric focusing showed that the HPRT expressed in these clones is human. One of the 14 clones expressed human glucose-6-phosphate dehydrogenase and another expressed human phosphoglycerate kinase. Since 5-azacytidine treatment results in hypomethylation of DNA, DNA methylation may be a mechanism of human X chromosome inactivation.     

小麦LMW-GS基因PCR初步研究

用CTAB法提取小麦材料基因组DNA,根据基因库中公布的已知LMW-GS基因序列,设计并合成染色体位点特异的PCR引物1～7;探索出优化的PCR反应体系,即20μL反应体积中,Mg2+浓度为2.5mmol/L,dNTP浓度为200μmol/L,模板DNA30～60ng,每种引物50ng,Taq酶0.5U。利用特殊小麦材料——六倍体普通小麦(染色体组为AABBDD)、四倍体小麦(AABB)及二倍体一粒小麦(AA)和节节麦(DD)等的基因组DNA为模版,在优化的PCR反应体系下进行特异性扩增和引物验证。结果表明,引物3和引物4为小麦谷蛋白Glu-D3位点LMW-GS基因的特异引物,用其进行扩增时,循环反应条件为94℃变性1min,62℃退火1min,72℃延伸2min;扩增产物大小约为1.63kb,包括启动子和整个编码区。引物5和7为小麦谷蛋白Glu-B3位点LMW-GS基因的特异引物,用其进行扩增时,循环反应条件为94℃变性1min,64℃退火1min,72℃延伸2min;扩增产物大小约为1.45kb,包括启动子和整个编码区。     

Isolation of human transcribed sequences from human-rodent somatic cell hybrids

A method was developed for selectively isolating genes from localized regions of the human genome that are contained in interspecific hybrid cells. Complementary human DNA was prepared from a human-rodent somatic cell hybrid that contained less than 1% human DNA, by using consensus 5' intron splice sequences as primers. These primers would select immature, unspliced messenger RNA (still retaining species-specific repeat sequences) as templates. Screening a derived complementary DNA library for human repeat sequences resulted in the isolation of human clones at the anticipated frequency with characteristics expected of exons of transcribed human genes--single copy sequences that hybridized to discrete bands on Northern (RNA) blots.     

酸马奶中酵母菌5.8 S rDNA及ITS的基因序列分析

实验将两株从酸马奶中分离提纯的酵母菌,提取单菌株基因组DNA,用一对真菌通用引物ITS1和ITS4 PCR 扩增它们的5.8S rRNA和ITS基因,连接入pMD19-T载体,克隆鉴定后,测定序列,将测得的序列与GenBank数据库的序列进行同源性分析,并建立系统进化树.结合系统发育树及5.8 S rDNA和ITS的序列分析结果,将菌株J14和S33判定为Kluyveromyces marxianus(马克斯克鲁维酵母).酸马奶中传统的酵母菌分类主要依靠形态学和生理生化等鉴定方法,实验首次利用5.8S rRNA和ITS基因扩增和测序的分子生物学方法对其进行鉴定.