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.     

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.     

Simplified construction and characterization of yeast artificial chromosome libraries.

Three yeast artificial chromosome (YAC) libraries were constructed using two human cell lines and the pYAC-RC vector. The main differences from the previously described methods were: i) genomic DNA was digested in low melting point (LMP) agarose blocks with the rare cutting enzyme ClaI; ii) DNA was ligated in melted LMP agarose after agarase treatment; iii) spheroplast regeneration plating was done in calcium alginate thin layer. In addition, a panel of PCR primers was used to identify quickly the presence in the libraries of repetitive and single copy human DNA sequences.     

Rescue of a single yeast artificial chromosome from a cotransformation event utilizing segregation at meiosis

During the construction of yeast artificial chromosome (YAC) libraries to facilitate mapping of the human genome, two YACs may be cotransformed into the same yeast cell, making further analysis very difficult. We present a simple method to rescue the required YAC that utilizes the segregation of chromosomes at meiosis. In brief, we crossed the cotransformed yeast cell with a non-YAC-containing strain and induced the resulting diploid to sporulate and undergo meiosis. The new haploid generation included some yeast cells that contained only the desired YAC. These YACs were analyzed by conventional methods. To exclude the possibility that major rearrangement occurred during the procedure, we analyzed the YACs with restriction enzymes that cut only rarely. We conclude that this is a useful technique to rescue cotransformed YACs.     

Construction of yeast artificial chromosome libraries by pulsed-field gel electrophoresis

Yeast artificial chromosome (YAC) libraries have been constructed from a variety of organisms using different approaches. This protocol outlines in detail the construction of YAC libraries with large inserts using size fractionation of partially digested DNA by pulsed-field gel electrophoresis.     

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.     

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.     

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.     

Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability.

In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.     

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.     

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.     

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.     

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.     

Description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5

In order to generate a physical map of Arabidopsis thaliana chromosome 5, 142 molecular markers mapping to chromosome 5 have been used in colony hybridization experiments with four Arabidopsis, ecotype Columbia, yeast artificial chromosome (YAC) libraries. This resulted in 634 YAC clones being anchored on chromosome 5. Southern blot analysis confirmed their positioning and provided data, which along with knowledge of the sizes of all the YAC clones, enabled the clones to be arranged into 31 contigs. Genetic mapping of markers located within 29 of these contigs on the Landsberg erecta/Columbia recombinant inbred lines allowed positioning of the contigs along the chromosome. A high proportion of the YAC clones were found to contain chimaeric inserts. The availability of this YAC contig map will accelerate chromosome-walking experiments, provide substrates for large-scale genomic sequencing projects and facilitate the mapping of new probes to this chromosome.     

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.     

Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization.

"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.     

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma.

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.     

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.     

Molecular cloning of lin-29, a heterochronic gene required for the differentiation of hypodermal cells and the cessation of molting in C.elegans.

The lin-29 gene product of C.elegans activates a temporal developmental switch for hypodermal cells. Loss-of-function lin-29 mutations result in worms that fail to execute a stage-specific pattern of hypodermal differentiation that includes exist from the cell cycle, repression of larval cuticle genes, activation of adult cuticle genes, and the cessation of molting. Combined genetic and physical mapping of restriction fragment length polymorphisms (RFLPs) was used to identify the lin-29 locus. A probe from the insertion site of a Tc1 (maP1), closely linked and to the left of lin-29 on the genetic map, was used to identify a large set of overlapping cosmid, lambda and yeast artificial chromosome (YAC) clones assembled as part of the C.elegans physical mapping project. Radiolabeled DNA from one YAC clone identified two distinct allele-specific alterations that cosegregated with the lin-29 mutant phenotype in lin-29 intragenic recombinants. lin-29 sequences were severely under-represented in all cosmid and lambda libraries tested, but were readily cloned in a YAC vector, suggesting that the lin-29 region contains sequences incompatible with standard prokaryotic cloning techniques.     

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.