YAC克隆的筛选及鉴定分析技术

人工酵母染色体（ＹＡＣ）技术是人类基因组分极及疾病相关基因的分离、克隆中的关键技术。在基因组ＹＡＣ文库基础上特定目的基因的分离克隆涉及ＹＡＣ克隆的筛选,嵌合体、缺失体和共转染克隆的检测与处理,插入片段的分离及及其结构特征的分析,亚克隆的快速构建等等。近年来,有关技术取得了重要进展,已趋于成熟,并正得到了广泛应用。     

YAC克隆的筛选及鉴定分析技术

人工酵母染色体（ＹＡＣ）技术是人类基因组分析及疾病相关基因的分离、克隆中的关键技术。在基因组ＹＡＣ文库基础上特定目的基因的分离克隆涉及ＹＡＣ克隆的筛选,嵌合体、缺失体和共转染克隆的检测与处理,插入片段的分离及其结构特征的分析,亚克隆的快速构建等等。近年来,有关技术取得了重要进展,已趋于成熟,并正得到广泛应用 。     

桃果实采后生理特性初探

桃果实采后生理特性初探杨映根,张立军,李钰（中国科学院植物研究所,北京１０００４４）ＳＴＵＤＩＥＳＯＮＴＨＥＰＯＳＴＨＡＲＶＥＳＴＰＨＹＳＩＯＬＯＧＹＰＫＯＰＥＲＴＩＥＳＯＦＰＥＡＣＨＥＲＵＩＴＹａｎｇＹｉｎｇ－ｇｅｎ;ＺｈａｎｇＬｉ－ｊｕｎ;ＬｉＹ...     

人X染色体35cMYAC重叠群的构建及物理图谱分析

人Ｘ染色体Ｘｐ１１．２－ｐ２１．３区域具有重要的基础遗传学和医学遗传学意义。为了对该区段编码的基因,尤其是疾病基因进行克隆与变异研究,对该区段染色体ＤＮＡ进行了ＹＡＣ克隆并将其依染色体排序,采用了一系列ＤＮＡ位标,尤其是多肽性微卫星序列位标筛选了３个ＹＡＣ方库,得到了１５１个ＹＡＣ克隆,对这些ＹＡＣ克隆进行了物理图谱分析,构建了这一区域的一系列ＹＡＣ重叠群,这些ＹＡＣ重叠群的总跨度约３５摩,基本覆     

铜对马铃薯块茎产量与生理生化特性的影响

铜对马铃薯块茎产量与生理生化特性的影响白嵩吕芳芝＊白宝璋李秀坤刘志清＊＊陈文荣（吉林农业大学,长春１３０１１８）ＥＦＦＥＣＴＳＯＦＣＯＰＰＥＲＯＮＴＵＢＥＲＹＩＥＬＤＡＮＤＰＨＹＳＩＯ┐ＬＯＧＩＣＡＬＡＮＤＢＩＯＣＨＥＭＩＣＡＬＣＨＡＲＡＣ┐ＴＥＲ...     

荧光标记的染色体原位杂交及其在人类基因组研究中的…

ＦＩＳＨ技术是８０年代开始发展起来的一种新的定位技术,在人类基因组研究中得到了广泛的应用,通过中期染色体的ＦＩＳＨ可以进行ＳＣＰ,Ｃｏｓｍｉｄ和ＹＡＣ的染色体定位,嵌合克隆的鉴别。通过间期核的ＦＩＳＨ可能在５０ｋｂ的分辨率下进行基因作图;最新的研究进展已可以进行伸展的染色质丝的ＦＩＳＨ,直接测量基因的长度,从而达到高精度基因作图的目的。总之,随着ＦＩＳＨ技术本身的发展,它将在人类基因组研究中发挥更     

茉莉酸类物质与脱落酸生理作用的比较研究

茉莉酸类物质与脱落酸生理作用的比较研究江月玲郑燕玲（广州师范学院生物系,广州５１０４００）潘瑞炽（华南师范大学生物系,广州５１０６３１）ＣＯＭＰＡＲＡＴＩＶＥＳＴＵＤＹＯＦＰＨＹＳＩＯＬＯＧＩＣＡＬＥＦＦＥＣＴＳＯＦＡＢＡ,ＪＡＳＭＯＮＩＣＡＣＩＤ...     

庚型肝炎病毒包膜糖蛋白E2基因在昆虫细胞中的表达

用ＰＣＲ扩增出ＨＧＶＥ２全基因,克隆进杆状病毒表达载体ｐＦＡＳＴＢＡＣＨＴａ中,构建成重组转座载体ｐＦＡＳＴＢＡＣＥ２,转化ＤＨ１０ＢＡＣ大肠杆菌感受态细胞,筛选阳性菌落,抽提大分子质粒ＤＮＡ,获得含ＨＧＶＥ２基因的重组杆状病毒穿梭载体,转染昆虫草地夜蛾Ｓｆ９细胞,出现细胞病变后,收集含有重组病毒颗粒的培养上清,重新感染草地夜蛾Ｓｆ９单层细胞及甜菜夜蛾幼虫,分别收集Ｓｆ９细胞和甜菜夜蛾幼虫体内的血淋巴细胞,进行１２％ＳＤＳ聚丙烯酰胺凝胶电泳,可见表达的融合蛋白带,经亲和层析进行蛋白纯化,用ＥＬＩＳＡ方法检测各类血清标本,初步研究ＨＧＶＥ２糖蛋白的抗原性     

猕猴桃果实的生理生化特征

猕猴桃果实的生理生化特征魏玉凝,李曜东（中国科学院植物所,北京１０００４４）ＴＨＥＰＨＹＳＩＯＬＯＧＩＣＡＬＡＮＤＢＩＬＣＨＥＭＩＣＡＬＣＨＡＲＩＣＴＥＲＩＳＴＩＣＳＯＦＫＩＷＩＦＲＵＩＴ￥ＷｅｉＹｕ－ｎｉｎｇ;ＬｉＹａｏ－ｄｏｎｇ（Ｉｎｓｔｉｔｕｔ...     

人分裂细胞核抗原基因片段的筛选、测序及对启动子的分析

经６．６×１０５个克隆筛选,从装在λ噬菌体载体Ｃｈａｒｏｎ３０中的人基因库中筛选到了一个含人分裂细胞核抗原（ＰＣＮＡ）基因的克隆。经Ｓｏｕｔｈｅｒｎ杂交分析插入基因长约１４ｋｂ,有较长的５＇上游区,但３＇端缺少一部分。经亚克隆和测序已确定从５＇上游１２６３ｂｐ到３＇端与λ载体接点共４９６９ｂｐＰＣＮＡ基因片段的核苷酸序列。将ＰＣＮＡ基因启动子核苷酸序列与ＤＮＡ聚合酶α,拓扑异构酶Ⅱα,胸苷酸激酶基因的启动子进行比较有３０％以上同源性,具有“看家基因”特征。在转录起始点的５＇上游几百ｂｐ的范围内都有与ＣＡＴ,ＳＰ１,Ｅ２Ｆ,ＮＦＨＢ,Ｏｃｔ１和ＡＴＦ等转录因子的结合位点相似的核苷酸序列。     

Construction and Characterization of a 10-Genome Equivalent Yeast Artificial Chromosome Library for the Laboratory Rat,Rattus norvegicus

Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescencein situhybridization were found to be chimeric, indicating a proportion of about 20–30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species.     

Physical Mapping of Rice Chromosomes 8 and 9 with YAC Clones

First efforts for physical mapping of rice chromosomes 8 and9 were carried out by ordering YAC clones of a rice genomicDNA library covering six genome equivalents with mapped DNAmarkers. A total of 79 and 74 markers from chromosomes 8 and9, respectively, were analyzed by YAC colony and Southern hybridizationusing RFLP markers of cDNA and genomic clones, and by polymerasechain reaction (PCR) screening using PCR-derived and sequence-taggedsite (STS) markers. As a result, 252 YAC clones were confirmedto contain the mapped DNA fragments on both chromosomes. A contigmap was constructed by ordering these YAC clones and about 53%and 43% genome coverage was obtained for chromosomes 8 and 9,respectively, assuming a YAC clone size of 350 kb and overlapbetween neighboring YACs of 50%. A continuous array of YAC cloneswith minimum overlap gave a total size of 18.9 Mb for chromosome8 and 15.6 Mb for chromosome 9, which are close to previousestimates. These contig maps may provide valuable informationthat can be useful in understanding chromosome structure andisolating specific genes by map-based cloning.     

A 2-Mb YAC Contig and Physical Map Covering the Chromosome 8q12 Breakpoint Cluster Region in Pleomorphic Adenomas of the Salivary Glands

Pleomorphic adenomas are benign epithelial tumors originating from the major and minor salivary glands. Extensive cytogenetic studies have demonstrated that they frequently show chromosome abnormalities involving chromosome 8, with consistent breakpoints at 8q12. In previous studies, we have shown that these breakpoints are located in a 9-cM interval betweenMOS/D8S285 and D8S260. Here, we describe directional chromosome walking studies starting from D8S260 as well as D8S285. Using the CEPH and ICRF YAC libraries, these studies resulted in the construction of two nonoverlapping YAC contigs of about 2 and 5 Mb, respectively. Initial fluorescencein situhybridization (FISH) analysis suggested that the majority of 8q12 breakpoints clustered within the 2-Mb contig, which was mapped to the centromeric part of chromosome band 8q12. This contig has at least double coverage and consists of 34 overlapping YAC clones. The localization of the YACs was confirmed by FISH analysis. On the basis of mapping data of landmarks with an average spacing of 65 kb as well as restriction enzyme analysis, a long-range physical map was established for the chromosome region spanned by the 2-Mb contig. The relative positions of various known genes and expressed sequence tags within this contig were also determined. Subsequent FISH analyses of pleomorphic adenomas using YACs as well as cosmids revealed that all but two of the 8q12 breakpoints in the primary tumors tested mapped within a 300-kb interval between theMOSproto-oncogene and STS EM156. The target gene affected by the chromosome aberrations mapping within this interval was recently shown to be thePLAG1gene, which encodes a novel zinc finger protein.     

Detection and characterization of chimeric yeast artificial-chromosome clones.

Methods for the construction of yeast artificial-chromosome (YAC) clones have been designed to isolate single, large (100-1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such "chimeric" YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in yeast, between two Alu sequences located on unrelated segments of human DNA.     

A Chlamydomonas Genomic Library in Yeast Artificial Chromosomes

We have constructed and characterized a Chlamydomonas reinhardtii total genomic library in yeast artificial chromosomes (YACs). The library contains 7500 clones with inserts ranging in size from 100-200 kb. The representation of the library was assessed by screening one-third of it with a probe derived from the dispersed repeat, Gulliver, which occurs ~13 times in the genome. At least 10 of these Gulliver loci were isolated within 15 independent YACs. Two of these YACs encompass the Gulliver element designated G, which was reported to map to the uni linkage group (ULG). The end clones of these two YACs have been genetically mapped by RFLP analysis in an interspecific cross and thereby shown to be closely linked to the APM locus on the ULG. A third uni-specific YAC has also been isolated and its ends have been mapped by RFLP analysis. Genetic and RFLP analysis of these and other YACs indicates that the frequency of chimeric YACs in the library is very low. The library was constructed in a second generation vector that enables plasmid rescue of YAC end clones as well as copy number amplification of artificial chromosomes. We provide evidence that amplification of intact YACs requires a rad1:rad52 yeast strain.     

The CIC library: a large insert YAC library for genome mapping in Arabidopsis thaliana

A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial Eco RI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively. Only one clone was found to carry the 160 bp paracentromeric repeated element. Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy out of 76 pairs of primers identified from one to seven YAC clones. Thus at least 92% of the genome is represented in the CIC library. The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.     

Theoretical analysis of library screening using a N-dimensional pooling strategy.

A solution to the problem of library screening is analysed. We examine how to retrieve those clones that are positive for a single copy landmark from a whole library while performing only a minimum number of laboratory tests: the clones are arranged on a matrix (i.e in 2 dimensions) and pooled according to the rows and columns. A fingerprint is determined for each pool and an analysis allows selection of a list containing all the positive clones, plus a few false positives. These false positives are eliminated by using another (or several other) matrix which has to be reconfigured in a way as different as possible from the previous one. We examine the use of cubes (3 dimensions) or hypercubes of any dimension instead of matrices and analyse how to reconfigure them in order to eliminate the false positives as efficiently as possible. The advantage of the method proposed is the low number of tests required and the low number of pools that require to be prepared [only 258 pools and 282 tests (258 + 24 verifications) are needed to screen the 72,000 clones of the CEPH YAC library (1) with a sequence-tagged site]. Furthermore, this method allows easy and systematic screenings and can be applied to a large physical mapping project, which will lead to an interesting map with a low, precisely known, rate of error: when fingerprinting a 150 Mb chromosome with the CEPH YAC library and 1750 sequence-tagged sites, 903,000 tests would be necessary to obtain about 20 contigs of an average length of 6.7 Mb, while only about one false positive would be expected in the resultant map. Finally, STSs can be ordered by dividing a clone library into sublibraries (corresponding to groups of microplates for example) and testing each STS on pooled clones from each sublibrary. This allows to dedicate to each STSs a fingerprint that consists in the list of the positive pools. In many cases these fingerprints will be enough to order the STSs. Indeed if large YACs (greater than 1 Mb) can be obtained, the combined screening of DNA families and YAC DNA pools would allow an integrated construction of both genetic and physical maps of the human genome, that will also reduce the optimal number of meioses needed for a 1 centimorgan linkage map.     

Genes and genomes: Towards construction of an overlapping YAC library of the Arabidopsis thaliana genome

Arabidopsis thaliana (Thale cress, Arabidopsis) is an ideal model organism for the molecular genetic analysis of many plant processes. The availability of a complete physical map would greatly facilitate the gene cloning steps in these studies. The small genome size of Arabidopsis makes the construction of such a map a feasible goal. One of the approaches to construct an overlapping library of the Arabidopsis genome takes advantage of the many mapped markers and the availability of Arabidopsis yeast artificial chromosome (YAC) libraries. Mapped molecular markers are used to identify corresponding YAC clones and thereby place them on the genetic map. Subsequently, these YAC clones provide the framework for directed walking experiments aimed at closing the gaps between the YAC contigs. Adopting this strategy, YAC clones comprising about 10% of the genome have been assigned to the top halves of Arabidopsis chromosomes 4 and 5. Extensive walking experiments in a 10 cM interval of chromosome 4 have resulted in two contiguous regions in the megabase size range.     

Construction of a swine YAC library allowing an efficient recovery of unique and centromeric repeated sequences

A swine DNA genomic library was constructed in yeast artificial chromosome (YAC) using the pYAC4 vector and the AB1380 strain. The DNA prepared from two Large White males was partially digested with EcoRI and size selected after both digestion and ligation. The YAC library contained 33792 arrayed clones with an average size of 280 kb as estimated by analysis of 2% of the clones, thus representing a threefold coverage of the swine haploid genome. The library was organized in pools to facilitate the PCR screening. The complexity of the library was tested both for unique and centromeric repeated sequences. In all, 20 out of 22 primer sets allowed the characterization of one to six clones containing specific unique sequences. These sequences are known to be on Chromosomes (Chrs) 1, 2, 5, 6, 7, 8, 13, 14, 15, 17, and X. Eight additional clones carrying centromeric repeat units were also isolated with a single primer set. The sequencing of 37 distinct repeat units of about 340 bp subcloned from these eight YACs revealed high sequence diversity indicating the existence of numerous centromeric repeat unit subfamilies in swine. Furthermore, the analysis of the restriction patterns with selected enzymes suggested a higher order organization of the repeat units. According to preliminary FISH experiments on a small number of randomly chosen YACs and YACs carrying specific sequences, the chimerism appeared to be low. In addition, primed in situ labeling experiments favored the idea that the YACs with centromeric repeat sequences were derived from a subset of metacentric and submetacentric chromosomes. Received: 14 July 1996 / Accepted: 24 October 1996     

A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage.

The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described. The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome. The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential. To date we have used 14 single copy probes to screen this library by colony hybridisation as well as PCR and have isolated between 1 and 5 YAC clones for every probe.