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.     

Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs

Almost two decades after CFTR was identified as the gene responsible for cystic fibrosis (CF), we still lack answers to many questions about the pathogenesis of the disease, and it remains incurable. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but the mutant mice do not develop the characteristic manifestations of human CF, including abnormalities of the pancreas, lung, intestine, liver, and other organs. Because pigs share many anatomical and physiological features with humans, we generated pigs with a targeted disruption of both CFTR alleles. Newborn pigs lacking CFTR exhibited defective chloride transport and developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF. The pig model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of strategies for prevention and treatment.     

Identification of the cystic fibrosis gene: genetic analysis

Approximately 70 percent of the mutations in cystic fibrosis patients correspond to a specific deletion of three base pairs, which results in the loss of a phenylalanine residue at amino acid position 508 of the putative product of the cystic fibrosis gene. Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations. A small set of these latter mutant alleles (about 8 percent) may confer residual pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient. The ability to detect mutations in the cystic fibrosis gene at the DNA level has important implications for genetic diagnosis.     

Identification of the cystic fibrosis gene: chromosome walking and jumping

An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences, encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.     

Very low gene duplication rate in the yeast genome

The gene duplication rate in the yeast genome is estimated without assuming the molecular clock model to be approximately 0.01 to 0.06 per gene per billion years; this rate is two orders of magnitude lower than a previous estimate based on the molecular clock model. This difference is explained by extensive concerted evolution via gene conversion between duplicated genes, which violates the assumption of the molecular clock in the analyses of duplicated genes. The average length of the period of concerted evolution and the gene conversion rate are estimated to be approximately 25 million years and approximately 28 times the mutation rate, respectively.     

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.     

The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome

We have sequenced and annotated the genome of the filamentous ascomycete Ashbya gossypii. With a size of only 9.2 megabases, encoding 4718 protein-coding genes, it is the smallest genome of a free-living eukaryote yet characterized. More than 90% of A. gossypii genes show both homology and a particular pattern of synteny with Saccharomyces cerevisiae. Analysis of this pattern revealed 300 inversions and translocations that have occurred since divergence of these two species. It also provided compelling evidence that the evolution of S. cerevisiae included a whole genome duplication or fusion of two related species and showed, through inferred ancient gene orders, which of the duplicated genes lost one copy and which retained both copies.     

Isolation of single-copy human genes from a library of yeast artificial chromosome clones

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.     

卷烟真伪的鉴别

\t\t\t\t\t目的\t\t\t\t\t利用顶空—离子分子反应质谱(HS-IMR-MS)对真品卷烟和冒牌卷烟的挥发性成分进行分析,以期为卷烟真伪鉴别提供一种新方法,进一步提高卷烟真伪鉴别的准确性和客观性。\t\t\t\t\t\t\t\t\t\t\t\t\t方法\t\t\t\t\t以云烟(软珍品)和云烟(紫)两个牌号各20批次卷烟及各5种冒牌卷烟为试验材料,研究不同电离源、顶空平衡温度及平衡时间对分析效果的影响,通过选取群体特征离子,利用平均法构建两个牌号卷烟的特征质谱指纹图谱,并利用以相关系数法确定的合格阈值及主成分分析,对不同批次真品卷烟及冒牌卷烟进行评价。\t\t\t\t\t\t\t\t\t\t\t\t\t结果\t\t\t\t\t(1)以Xe做电离源,顶空平衡温度140 ℃,顶空平衡时间15 min时,卷烟分析效果最佳;(2)两个牌号不同批次卷烟与其特征质谱指纹图谱的相关系数均在0.99以上,而冒牌卷烟分别为0.914 7~0.934 3和0.884 7~0.908 8,且在主成分分析投影图上,同一牌号卷烟表现为相互聚集。\t\t\t\t\t\t\t\t\t\t\t\t\t结论\t\t\t\t\tHS-IMR-MS法能有效区分真品卷烟和冒牌卷烟,可为卷烟真伪鉴别提供新的参考依据。\t\t\t\t     

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.     

Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes

Radiation hybrid (RH) mapping, a somatic cell genetic technique, was developed as a general approach for constructing long-range maps of mammalian chromosomes. This statistical method depends on x-ray breakage of chromosomes to determine the distances between DNA markers, as well as their order on the chromosome. In addition, the method allows the relative likelihoods of alternative marker orders to be determined. The RH procedure was used to map 14 DNA probes from a region of human chromosome 21 spanning 20 megabase pairs. The map was confirmed by pulsed-field gel electrophoretic analysis. The results demonstrate the effectiveness of RH mapping for constructing high-resolution, contiguous maps of mammalian chromosomes.     

Antigens encoded by the 3'-terminal region of human T-cell leukemia virus: evidence for a functional gene

Antibodies in sera from patients with adult T-cell leukemia-lymphoma or from healthy carriers of type I human T-cell leukemia virus (HTLV) recognize an antigen of approximately 42 kilodaltons (p42) in cell lines infected with HTLV-I. Radiolabel sequence analysis of cyanogen bromide fragments of p42 led to the conclusion that this antigen is encoded in part by LOR, a conserved portion of the "X" region that is flanked by the envelope gene and the 3' long terminal repeat of HTLV-I. It is possible that this novel product mediates the unique transformation properties of the HTLV family.     

OrchidBase 3.0:一个兰花基因功能和基因组进化研究的数据库

兰科为被子植物的最大科,兰科植物是经济价值非常高的世界性观赏植物,其基因组学和转录组学数据可为进一步推动兰花育种和研究提供帮助.本研究团队在建立的OrchidBase 1.0、2.0数据库的基础上,利用小兰屿蝴蝶兰的全基因组数据建立了基于网页版的OrchidBase 3.0数据库.OrchidBase 3.0数据库包含了小兰屿蝴蝶兰基因组拼接支架序列的信息以及基因注释、基因定位、基因结构、KEGG途径和BLAST搜索等内容;此外,该数据库也提供了小兰屿蝴蝶兰的基因序列和每千碱基读数每百万映射读数(RPKM).OrchidBase 3.0数据库的在线资源可以通过网页浏览、文本和BLAST搜索获取,搜索结果会显示在网站上并可供下载,用户可通过该数据库下载小兰屿蝴蝶兰的基因组拼接支架序列及预测的基因和蛋白质序列.http://orchidbase.itps.ncku.edu.tw为OrchidBase3.0数据库的免费网站链接.     

A region of the Herpesvirus saimiri genome required for oncogenicity

Herpesvirus saimiri naturally infects squirrel monkeys (Saimiri sciureus) without producing signs of disease; infection of other New World primates, however, results in a rapidly progressing, malignant, T-cell lymphoma. Results described in this report identify a region of the viral genome that is required for oncogenicity in owl monkeys (Aotus trivirgatus); this region is not required for replication of the virus. This is believed to be the first such genomic region identified in a herpesvirus system.     

Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III

The retrovirus involved in acquired immune deficiency syndrome (HTLV-III/LAV) contains a region that is necessary for stimulation of gene expression directed by the viral long terminal repeat. This region is located between nucleotides 5365 and 5607, immediately 5' to the envelope gene. A doubly-spliced message containing this region could encode an 86-amino acid protein with structural features similar to those of nucleic acid-binding proteins.