以绵羊MHC区段BAC克隆酶切片段为探针杂交筛选绵羊混合组织cDNA文库

在已知中国美利奴羊MHC(Major histocompatibility complex)区段BAC(Bacterial artificial chromosome)克隆序列信息和预测的基因注释前提下,用位于中国美利奴羊基因组BAC文库MHC区段的6个BAC克隆酶切片段为探针,以噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA文库(库库杂交),对分离到的cDNA阳性克隆进行全序列测定,并与相应的已知序列信息和基因注释的BAC克隆比对以及在NCBI Blastn数据库中序列相似性检索,旨在验证基因注释结果的准确性和对基因(序列)功能的初步分析。实验中,经过两轮杂交共筛选出27个cDNA阳性克隆(序列),并发现这些序列均可定位到相应的BAC克隆上,且25条序列处在注释基因的外显子部分;在NCBI数据库中经Blastn序列相似性检索发现,23条序列与牛基因的序列相似性最高,且与免疫功能密切相关。     

细菌人工染色体基因组文库构建方法的改进

目的:建立一种改进的更简便、易操作的细菌人工染色体(BAC)文库构建方法。方法:在构建猪霍乱沙门氏菌基因组大片段DNA的BAC文库时,对改进的基因组BAC文库构建方法和常规的BAC文库构建方法进行比较。结果:利用改进的方法可简便快速地构建猪霍乱沙门氏菌基因组BAC文库。结论:使用2种方法构建BAC文库,其转化效率,以及在BAC克隆中插入的DNA片段的大小和BAC克隆的稳定性等都相同,从而表明改进的方法更简单、更方便,它能使BAC文库的构建更为高效。     

水稻抗白叶枯病基因Xa4位点跨叠BAC克隆群的构建

水稻白叶枯病抗性基因Xa4已被定位于第11染色体长臂末端的分子标记VG181和L1044之间,并与抗性基因同源序列片段RS13共分离。利用这3个标记筛选IRBB56的BAC文库,共得到128个阳性BAC克隆,其中RS13获得18个阳性克隆,这18个克隆中有4个和6个我隆分别同时为G181和L1044的阳性克隆,选其中的12克隆进行分析,构建了一个从G181到L1044区间的BAC跨叠克隆,全长420kb,并且56M22、106P13和104B153个BAC克隆可覆盖整个跨叠克隆群。这一研究结果为进一步分离Xa4基因打下基础。     

棉花BAC文库快速筛选法

目的:构建棉花细菌人工染色体(Bacterial Artificial Chromosome,BAC)文库的快速筛选法,以期从BAC文库中大量、快速、高效筛选出特定BAC克隆,为从事基因组测序、分离和分析特定基因、构建物理图谱及基因图位克隆等生物学技术研究奠定基础。方法:构建了整板、行、列的三维混合池,以菌液PCR为基础,从BAC文库中筛选出含有特定DNA片段的BAC单克隆。结果:从BAC文库的3 456个克隆中,共筛选出16个阳性单克隆,涉及13条染色体、11个SSR标记。结论:该文构建的棉花BAC文库筛选体系,筛选快速、准确,适合从BAC文库中大量筛选BAC单克隆。结合当前的多种BAC文库筛选方法进行探讨,根据不同的实验目的选择更合适的筛选方法和操作步骤。     

利用染色体区段混合BAC探针鉴定食管癌细胞中的染色体畸变

染色体畸变是恶性肿瘤细胞的重要遗传学特征, 文章旨在应用BAC DNA克隆鉴定食管癌细胞中的染色体臂和染色体区段的畸变。针对染色体各区段选取5~10个1 Mb BAC DNA, 分别混合制备成特定染色体区段的BAC DNA混合克隆, 然后将染色体臂上覆盖所有区段的上述混合克隆进一步混合制备成特定染色体臂BAC DNA混合克隆。利用简并寡核苷酸引物聚合酶链反应(Degenerate oligonucleotide primed PCR, DOP-PCR)标记染色体臂探针, 利用切口平移法(Nick translation)标记染色体区段探针, 并对食管癌细胞中期染色体进行荧光原位杂交(Fluorescence in situ hybridization, FISH)分析。正常人外周血淋巴细胞中期染色体FISH结果显示, 上述方法标记的探针具有较高的特异性。进一步利用染色体臂混合探针, 确定了多个食管癌细胞中的染色体重排所涉及的特定染色体臂; 利用染色体区段混合探针, 鉴定出KYSE140的t(1q;7q)衍生染色体中1q上的断点范围位于1q32-q41。文章成功建立了1 Mb BAC DNA混合克隆探针标记技术, 并鉴定出多个食管癌细胞中的染色体臂和染色体区段畸变, 不仅为利用M-FISH技术鉴定肿瘤细胞中的染色体畸变提供了更为准确的方法, 而且还可能进一步将该法推广应用于恶性血液病的核型分析以及产前诊断。     

以BAC为基础的水痘-带状疱疹病毒的感染性克隆技术及其应用

水痘-带状疱疹病毒（VZV）属于疱疹病毒科α亚科,其原发感染为水痘,潜伏再度激活则引起带状疱疹。目前对其基因功能和疫苗的减毒机制尚不十分清楚。细菌人工染色(BAC)是一种新的用于大分子DNA克隆的载体系统,它具有容量大、遗传稳定、操作简单等优点。将VZV全基因组克隆至BAC系统构建成VZV的感染性克隆,并利用现代基因修饰技术可极大促进对该病毒的研究。就近年来以BAC为基础VZV感染性克隆技术的建立和应用做一综述。     

拟南芥细胞死亡突变体mod1突变座位的精细物理图谱构建

拟南芥细胞死亡突变体mod1突变位点位于第2染色体分子标记IGS1和mi421之间. 以这一区域YAC重叠群中的YAC克隆末端DNA片段CIC9A3R, CIC11C7L, CIC2G5R及RFLP分子标记克隆CDs3为探针筛选TAMU BAC文库, 获得31个阳性BAC克隆. 用BAC克隆的末端DNA片段杂交所有阳性BAC克隆, 确立了由T6P5, T7M23, T12A21, T8L6及T18A18等克隆组成的MOD1基因所在区域的BAC重叠群. 同时在这一区域发展出11个CAPS分子标记和12个STS序标, 为MOD1基因的图位克隆与鉴定分析及这一区域的全序列测定奠定了基础.     

温敏核不育水稻5460S细菌人工染色体文库的构建和鉴定

为了构建温敏核不育 (TGMS)基因区域的精细物理图谱并最终分离TGMS基因 ,以温敏核不育水稻 5 46 0S为材料 ,摸索优化了构建植物细菌人工染色体 (BAC)文库的方法 ,构建了一个高质量的BAC文库 .该文库由 1 95 84个克隆构成 ,插入片段平均长度为 1 1 0kb ,相当于水稻单倍体基因组大小的 5倍 ;以分子量分别为 1 40和2 5 0kb的 2个大BAC克隆进行稳定性传代实验 ,经 1 0 0代传代后其插入的DNA片段仍然稳定存在 ;以线粒体和叶绿体基因为探针筛选BAC文库 ,未检验出叶绿体和线粒体DNA的污染 ;以和tms1基因连锁的 3个分子标记作为探针对BAC文库进行了筛选 ,每个探针至少可获得一个阳性克隆 ,利用热不对称性交错PCR(Tail PCR)法成功分离了阳性克隆的左右末端序列 .     

东方田鼠部分BAC文库的微卫星筛选

目的 从东方田鼠的部分BAC文库中筛选微卫星.方法 应用非放射性的菌落杂交方法和磁珠富集法从东方田鼠的BAC文库中筛选高质量的微卫星标记.结果 以地高辛标记的寡聚核苷酸(CA)20为探针,通过菌落杂交法从136个东方田鼠BAC克隆中筛选出杂交信号最强的20个阳性克隆.再将这20个阳性克隆分别通过链霉亲和素磁珠法构建亚克隆文库,从中选取400个经PCR鉴定为阳性的亚克隆进一步测序分析,共得到220个微卫星序列,阳性率55％.选取重复次数高,侧翼序列完整的微卫星序列设计74对引物,共有35对引物能扩增出清晰的条带,其中16对引物具有多态性.结论 成功且高效地从阳性BAC克隆中筛选出微卫星序列,这些微卫星和阳性BAC克隆可用于后续的定位研究.     

大豆BAC克隆基因注释

根据来源于大豆BAC克隆库中的基因组序列,与其比对的基因或蛋白质序列可以从一些数据库中搜索,如GenBank序列数据库、EMBL数据库、PDB数据库等,然后利用NCBI的Entrz程序在数据库中搜索大豆基因类似物,获取其登录号及核苷酸序列,以及这些基因编码的氨基酸序列,通过GENSCAN等软件分析,得出的是与拟南芥等物种序列比对的结果,根据GENSCAN的预测结果,可以初步得知序列长度、基因数目及具有编码某种功能蛋白的基因存在的可能性,进而对预测出的氨基酸或核苷酸序列利用数据库NCBI中的BLAST进行序列的相似性搜索及比对分析,寻找其保守区域。最后对其功能基因进行注释。     

A comprehensive BAC resource

The Human Genome Project has generated extensive map and sequence data for a large number of Bacterial Artificial Chromosome (BAC) clones. In order to maximize the efficient use of the data and to minimize the redundant work for the research community, The Institute for Genomic Research (TIGR) comprehensive BAC resource (cBACr) (http://www.tigr.org/tdb/BacResource/BAC_resourc e_intro. html) was built as an expansion of the TIGR human BAC ends database. This resource collects, integrates and reports the information on library, maps, sequence, annotation and functions for each human and mouse BAC. The current database contains 635 016 human BACs and 265 617 mouse BACs that were characterized by various approaches, among which 22 705 human clones and 1000 mouse clones have sequence and annotation data.     

MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea

We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.     

GenMapDB: a database of mapped human BAC clones

GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9-22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequences of the clones. GenMapDB is freely available to the public. The main purpose of GenMapDB is to organize the mapping data and to allow the research community to search for mapped BAC clones that can be used in gene mapping studies and chromosomal mutation analysis projects.     

Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.     

FISH physical mapping with barley BAC clones

Fluorescence in situ hybridization (FISH) is a useful technique for physical mapping of genes, markers, and other single- or low-copy sequences. Since clones containing less than 10 kb of single-copy DNA do not reliably produce detectable signals with current FISH techniques in plants, a bacterial artificial chromosome (BAC) partial library of barley was constructed and a FISH protocol for detecting unique sequences in barley BAC clones was developed. The library has a 95 kb average barley insert, representing about 20% of a barley genome. Two BAC clones containing hordein gene sequences were identified and partially characterized. FISH using these two BAC clones as probes showed specific hybridization signals near the end of the short arm of one pair of chromosomes. Restriction digests of these two BAC clones were compared with restriction patterns of genomic DNA; all fragments contained in the BAC clones corresponded to bands present in the genomic DNA, and the two BAC clones were not identical. The barley inserts contained in these two BAC clones were faithful copies of the genomic DNA. FISH with four BAC clones with inserts varying from 20 to 150 kb, showed distinct signals on paired chromatids. Physical mapping of single- or low-copy sequences in BAC clones by FISH will help to correlate the genetic and physical maps. FISH with BAC clones also provide an additional approach for saturating regions of interest with markers and for constructing contigs spanning those regions.     

Mapping of quantitative trait locus related to submergence tolerance in rice with aid of chromosome walking.

The major QTL for submergence tolerance was locate in the 5.9 cM interval between flanking RFLP markers. To narrow down this region, a physical map was constructed using YAC and BAC clones. A 400-kb YAC was identified in this region and later its end fragments were used to screen a rice BAC library. Through chromosome walking, 24 positive BAC clones formed two contigs around linked-RFLP markers, R1164 and RZ698. Using one YAC end, six BAC ends and three RFLP markers, a fine-scale map was constructed of the 6.8-cM interval of S10709-RZ698 on rice chromosome 9. The submergence tolerance and related trait were located in a small, well-defined region around BAC-end marker 180D1R and RFLP marker R1164. The physical-to-map distance ratio in this region is as small as 172.5 kb/cM, showing that this region is a hot spot for recombination in the rice genome.     

Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

Although the centromeres of some plants have been investlgated prevlously, our knowledge of the wheat centromere Is still very llmlted. To understand the structure and functlon of the wheat centromere, we used two centromeric repeats （RCS1 and CCS1-5ab） to obtain some centromere-assoclated bacterial artificial chromosome （BAC） clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat （Triticum boeoticum Boiss.; 2n=2x=14） BAC library. Southern hybridization results indicated that, of the 32 candidates, there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization （FISH） analysis Indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromerlc regions in hexaplold wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or Dgenome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific （or rich） sequences dispersing on chromosome arms in the BAC clone TbBACS. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation In the period before metaphase.     

一种存放大基因组文库的新方法

用野生一粒小麦为材料,以细菌人工染色体（pECBAC1)为载体构建了细菌人工染色体克隆混合池（Bacterial artificial chromosome pool）,每池100个克隆。经初步验证,池中靶克隆经12h的培养后仍稳定存活。克隆之间的竞争实验仍在进行当中。