Analysis of clones carrying repeated DNA sequences in two YAC libraries of Arabidopsis thaliana DNA

YAC clones carrying repeated DNA sequences from the Arabidopsis thaliana genome have been characterized in two widely used Arabidopsis YAC libraries, the EG library and the EW library. Ribosomal, chloroplast and the paracentromeric repeat sequences are differentially represented in the two libraries. The coordinates of YAC clones hybridizing to these sequences are given. A high proportion of EG YAC clones were classified as containing chimaeric inserts because individual clones carried unique sequences and repetitive sequences originating from different locations in the genome. None of the EW YAC clones analysed were chimaeric in this way. YAC clones carrying tandemly repeated sequences, such as the paracentromeric or rDNA sequences, exhibited a high degree of instability. These observations need to be taken into account when using these libraries in the development of a physical map of the Arabidopsis genome and in chromosome walking experiments.     

Preparation of Pooled Arabidopsis YAC DNAs for PCR-Based Mapping

Physical mapping of an arabidopsis DNA sequence of interest can easily be performed by PCR. This is done by using specific primers and pooled DNA templates isolated from publicly available YAC or BAC libraries. We present simple protocols for preparing pooled YAC DNAs and PCR-based detection of sequences in them by which several sequences can be mapped in a short time.     

基因组YAC文库构建方法的几点改进

以pJS97、pJS98为载体, 对中国人基因组YAC文库的构建进行了尝试.建立了一种对小片段DNA“原位电泳筛除”的方案, 采用多胺溶液处理以减少大片段DNA的降解, 使整个建库工作更加简便、有效. 此外, 改进了转化子的挑取和保存的方法, 既简化了操作, 又减少了杂菌污染和交叉污染. 这些改进的方案, 可以推广到其他高等生物基因组YAC文库的构建和应用工作.     

Growth and storage of YAC clones in Hogness Freezing Medium.

To date, frozen storage of YAC libraries have relied on the administration of glycerol to the medium subsequent to cell growth. By adding Hogness Freezing Medium prior to inoculation, cultures can be frozen directly after cell growth, with no adverse effect on the stability of the YAC DNA or on the viability of the cells even after repeated freezing and defrosting. Although a relatively simple modification, the procedure notably improves the handling of YAC libraries and significantly reduces the risk of contamination, especially when dealing with large numbers of clones.     

An improved method of partially digesting plant megabase DNA suitable for YAC cloning: application to the construction of a 5.5 genome equivalent YAC library of tomato

An improved method for preparing partially digested tomato DNA has been developed, that is suitable for YAC cloning. It involves (i) isolation of high molecular-weight DNA from agarose-embedded leaf protoplasts, (ii) controlled partial digestion in situ using Eco RI endonuclease in the presence of Eco RI methylase (M. Eco RI), and (iii) fractionation of the partial digest on a Clamped Homogeneous Electric Fields (CHEF) gel. Unlike methods commonly used for generating partial digests, the present method allows one to produce digests in which the bulk of restriction fragments are of the desired size. Use of these partial digests in constructing YAC libraries of the tomato lines Moneymaker- Cf4 and VFNT Cherry resulted in libraries (total 21 060 clones, 5.5 genome equivalents) in which 80% of the YACs have inserts between 200 and 600 kb. Both libraries have been screened with selected RFLP markers linked to the Cladosporium fulvum Cf4 locus on chromosome 1, using a three-dimensional PCR-based screening technique. To this end, the RFLP markers have been sequenced to allow for the synthesis of specific primers. Thus, for each marker tested several YAC clones have been isolated, including a family of clones that carry leucine-rich repeat sequences located around the Cf4/ Cf9 locus.     

Construction and characterization of a YAC library with a lowfrequency of chimeric clones from flow-sorted human chromosome 9

Human chromosome 9 DNA, flow-sorted from somaticcell hybrid PK-87-9, has been used to construct two complete digest YAC libraries. The combined representation of chromosome 9 in these libraries, estimated by hybridization of chromosome 9-specific sequences to YAC colony grids, is 95%. The frequency of chimeric clones, analyzed by fluorescence in situ hybridization of chromosome 9 YACs to human metaphase chromosomes, was estimated to be 4%. These libraries provide a resource for physical mapping and for moving from genetic markers to disease loci on chromosome 9.     

Application of Interspersed Repetitive Sequence Polymerase Chain Reaction for Construction of Yeast Artificial Chromosome Contigs

Construction of physical maps across candidate regions is one of the rate-limiting steps of positional cloning projects. To date, most physical maps have been constructed by polymerase chain reaction (PCR)-based sequence-tagged site (STS) content mapping. While effective, this technique has a number of disadvantages including problems with yeast artificial chromosome (YAC) chimerism, the time and effort required to generate new STSs from YAC ends, the cost of primer synthesis for large contiging projects, and the time, effort, and expense necessary for screening each STS in the two-tiered hierarchical YAC library screening format. An alternative strategy, interspersed repetitive sequence (IRS) PCR genomics, alleviates many of these constraints. Clonal overlap is detected by hybridization of individual IRS-PCR products to IRS-PCR product pools of the three-dimensional coordinate pools of YAC libraries in dot-blot format. Entire libraries can be screened in a single step, and multiple libraries can be screened simultaneously. Cloning YAC fragments, sequencing, and primer generation are eliminated, increasing the efficiency of contig construction and reducing the expense. In addition, the genomic location of the individual IRS-PCR products can also be simultaneously determined by screening either interspecific backcrosses or radiation hybrid panels, in dot-blot format, confirming contig extension in the region of interest.     

Identification and map position of YAC clones comprising one-third of the Arabidopsis genome.

YAC clones corresponding to 125 Arabidopsis thaliana RFLP markers have been identified. At least one YAC clone has been isolated for each of the RFLP markers tested. Based on CHEF gel analysis of 196 clones, the mean insert size of the available Arabidopsis YAC libraries is approximately 160 kb. The YACs of known genetic map location encompass about 30% of the Arabidopsis genome. The results presented here represent a first step towards assembly of an overlapping YAC library of the A. thaliana genome.     

Retrofitting YACs for direct DNA transfer into plant cells

The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.     

Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation.

Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized. In each collection, 80-85% of YAC strains contained a single X YAC. Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences. Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning. In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents. Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure.     

Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis.

A method for constructing yeast artificial chromosome (YAC) libraries with large insert sizes is reported. High molecular weight human DNA was partially digested with EcoRI and cloned in the vector pYAC4. When unfractionated DNA was used, the mean YAC size was 120kb. Fractionation by pulsed-field gel electrophoresis using a 'waltzer' apparatus to remove small DNA fragments increased the mean YAC size to congruent to 220kb or congruent to 370kb depending on the fractionation conditions. Ligated DNA prepared by this method was stable at 4 degrees C and routinely yielded transformation efficiencies of greater than 700 colonies/micrograms. It should be possible to extend the method to produce even larger inserts and to use high molecular weight DNA from any source.     

Introduction of YACs into intact yeast cells by a procedure which shows low levels of recombinagenicity and co-transformation.

Yeast artificial chromosomes (YACs) enable the cloning and analysis of large segments of genomic DNA and permit the isolation of sequences which are impossible to maintain in Escherichia coli. However, the construction of genome libraries in YAC vectors is beset by a number of technical problems, not least of which is the creation of cloned fragments which are not true representatives of the donor genome. These artefactual clones arise mainly due to intra-fragment rearrangements or inter-fragment chimaera formation, both phenomena resulting from the activity of the host yeast's mitotic recombination system. We demonstrate that this system is significantly stimulated by the spheroplasting step of the standard YAC transformation system. In contrast, the transformation of intact yeast cells by either the lithium method or a new lithium-free protocol is much less recombinagenic. It is not possible to introduce high molecular weight YACs into yeast using the lithium protocol, but we find that such molecules may be introduced into pde2-mutants using the lithium-free approach. Since intact cells are transformed by this method, automation of post-transformation steps in the construction of YAC libraries is facilitated. Moreover, the frequency of cotransformation (and, therefore, chimera formation) is significantly reduced. However, these advantages do incur a penalty. Yields of YAC transformants by this simplified intact cell approach are reduced some 25- to 30-fold compared to those obtained by the spheroplast transformation route. Nevertheless, the considerable advantages of the new system recommend it for a number of applications.     

Yeast artificial chromosomes: an alternative approach to the molecular analysis of mouse developmental mutations

Mammalian genetics now allows a molecular study of genomic regions previously analysed by genetic and embryological techniques. To simplify such an analysis, we have established a number of libraries of mouse DNA in Yeast Artificial Chromosome (YAC) vectors, constructed either by partial digestion with EcoRI, or by complete digestion with enzymes which cut rarely in the mammalian genome. In this paper we report the construction of complete digest libraries prepared from mouse genomic DNA using the rare cutter enzymes NotI and BssHII, and the detection of gene loci from the H-2 complex, the t-complex, and other loci from the mouse genome. Due to their large insert size, YAC clones simplify the cloning of extended regions of the mouse genome surrounding known developmental mutations and should, after introduction into the germ line, offer a high probability of correct expression of the genes contained within the cloned region. We hope that this will allow the use of YAC clones to scan regions of interest such as the t-complex for specific genes by testing DNA introduced into transgenic mice for the ability to complement mutations localised to this region.     

人基因组YAC分子克隆库的构建及DMD基因YAC克隆的筛选

以pYAC4为载体,以正常人白细胞和含4条X染色体的细胞株GM1414为DNA源构建成人基因组YAC(Yeast Artificial Chromosome,酵母人工染色体)分子克隆库,已得到原始克隆近2万个,插入DNA片段长度在400—1000kb,从其中选出一组YAC克隆,它们含有DMD基因全部DNA顺序。     

Rapid screening of a YAC library by pulsed-field gel Southern blot analysis of pooled YAC clones

A new method for screening of YAC libraries is described. Individual YACs were pooled into groups of 384 clones and prepared as samples suitable for pulsed-field gel electrophoresis. A five hit human YAC library (Brownstein et al., 1989) containing approximately 60,000 clones was condensed into 150 such pools and chromosomal DNAs in each sample were separated on three pulsed field gels containing 50 samples each. Southern blots prepared from these gels were hybridized with probes of interest to identify pools containing homologous YACs. Further purification was performed using standard colony hybridization procedures. Twenty-one probes used thus far have identified 47 positive pools and corresponding YACs have been purified from 28 of these. Some significant advantages of this method include avoidance of DNA sequence analysis and primer generation prior to YAC screening and the ability to handle the entire library on three filters. The screening approach described here permits rapid isolation of YACs corresponding to unsequenced loci and will accelerate establishment of YAC contigs for large chromosomal segments.     

Characterization of a YAC Contig Spanning the Pseudoautosomal Region

Due to its unique biology of partial sex linkage and high recombination rates, the pseudoautosomal region (PAR1) on both X and Y chromosomes has attracted considerable interest. In addition, an extremely high level of YAC instability has been observed in this region. We have derived 82 YAC clones from six different YAC libraries mapping to this 2.6-Mb region. Of these a subset of 22 YACs was analyzed in detail. YAC contigs were assembled using 67 pseudoautosomal probes, of which 64 were unambiguously ordered. All markers are well distributed over the entire region, including the middle part of the region, which has previously been found difficult to contig. Two gaps of less than 50 kb within the genomic locus of CSF2RA and around XE7 remain, which could not be covered with YACs, cosmids, or phages. This YAC contig anchored on the physical map of PAR1 represents one of the best characterized large regions of the human genome with a map completion greater than 90% at 100-kb resolution and has permitted the accurate localization of all known genes within this region.     

Automatable screening of yeast artificial-chromosome libraries based on the oligonucleotide-ligation assay.

The systematic screening of yeast artificial-chromosome (YAC) libraries is the limiting step in many physical mapping projects. To improve the screening throughput for a human YAC library, we designed an automatable strategy to identify YAC clones containing a specific segment of DNA. Our approach combines amplification of the target sequence from pooled YAC DNA by the polymerase chain reaction (PCR) with detection of the sequence by an ELISA-based oligonucleotide-ligation assay (OLA). The PCR-OLA approach eliminates the use of radioactive isotopes and gel electrophoresis, two of the major obstacles to automated YAC screening. Furthermore, the use of the OLA to test for the presence of sequences internal to PCR primers provides an additional level of sensitivity and specificity in comparison to methods that rely solely on the PCR.     

The construction and analysis of M13 libraries prepared from YAC DNA.

Yeast artificial chromosomes (YACs) provide a powerful way to isolate and map large regions of genomic DNA and their use in genome analysis is now extensive. We modified a series of procedures to produce high quality shotgun libraries from small amounts of YAC DNA. Clones from several different libraries have been sequenced and analyzed for distribution, sequence integrity and degree of contamination from yeast DNA. We describe these procedures and analyses and show that sequencing at about 1-fold coverage, followed by database comparison (survey sequencing) offers a relatively quick method to determine the nature of previously uncharacterized cosmid or YAC clones.     

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.