Yeast Artificial Chromosome Insert Research Articles

Recovery and potential utility of YACs as circular YACs/BACs.

A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.

Localization on a Physical Map of the NKC-Linked Cmv1 Locus Between Ly49b and the Prp Gene Cluster on Mouse Chromosome 6

Abstract The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV. Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6. We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters. This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice. Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus. To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples. These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of ∼390 kb. This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.

Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage.

RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site. We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs). Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size. Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA. The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion. The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.

Rapid mapping of markers applying vectorette technology to YAC fragmentation allows easy assembly of a high-density STS bacterial clone contig spanning the markers D6S1260-D6S1918.

We have generated a detailed physical map of the 6p21.3/p22.1 boundary, using a combination of yeast artificial chromosome (YAC) fragmentation and high-resolution sequence tagged site (STS) content mapping. YACs from the CEPH, St. Louis, and ICRF libraries have been used to construct a 4.5-Mb contig spanning the markers D6S306 to D6S1571. YAC insert sizes were determined by pulsed field gel electrophoresis (PFGE). Chimerism of YACs was determined by fluorescent in situ hybridization (FISH), and their integrity was determined by fingerprinting with Alu-PCR. We have identified 10 new CA repeat loci in this region as well as over 50 novel STSs, several tRNA genes, a new histone H2B gene and the phospholipase D gene. Using these new markers, we have rapidly generated a bacterial clone contig of over 250 kb, spanning the markers D6S1260 to D6S1918 (WI-3111) with STSs spaced on average every 6 kb.

Identification of an amplified gene cluster in glioma including two novel amplified genes isolated by exon trapping.

Gene amplification, which occurs in more than 50% of malignant gliomas, is considered to play a pivotal role in tumorigenesis. There are, however, few studies aimed toward the isolation of novel genes from amplified sequences. Previously, we reported amplification of the protooncogene MET (hepatocyte growth factor receptor; 7q31) in more than 20% of glioblastomas. For an approximate size estimation of the amplification unit we analyzed three glioblastomas all of which carried an amplified MET gene, by Southern blot analysis and/or competitive polymerase chain reaction using eight DNA markers. Although the extent of the amplified domain varied, the close vicinity of the MET gene was the only region consistently amplified in these glioblastomas. A yeast artificial chromosome (YAC) contig of 900 kb was refined spanning the amplified region flanking the MET gene. The YAC inserts were subcloned into 59 cosmids, which were used for exon trapping. Eight sequences were identical to parts of the genes MET and CAPZA2 (human actin capping protein alpha-subunit). Two newly identified exons and the CAPZA2 exons were amplified in tumor TX3095, which retains an amplified MET gene. The new exons were localized close to MET and CAPZA2. Characterization of the clones, which were termed glioma-amplified sequence (GAS)7-1 and GAS7-2, showed an open reading frame and a different expression pattern in multiple human tissues. This study reports the identification of a cluster of amplified genes including two novel genes in a region amplified in more than 20% of glioblastomas.

Two vectors for the insertion of mammalian selectable genes into yeast artificial chromosome cloned DNA

The introduction of cloned DNA into mammalian cells allows functional testing of genes contained on the fragments. In many cases, the exogenous DNA introduced into mammalian cells requires selectable genes that mark the presence of input DNA. Two new vectors, carrying mammalian selectable markers encoding for either neomycin-resistance (neo R) or histidinol-resistance (hol R), have been constructed for targeted integration to specific single-copy sites within yeast artificial chromosome (YAC) insert DNA. The integration cassettes comprise a single selectable yeast gene adjacent to a mammalian selectable gene, either LEU2 with neo R or HIS3 with hol R. Modification of the YAC occurs in yeast by transfection with linear DNA containing YAC-specific, unique, recombinogenic ends, thereby ensuring co-integration of the markers. Analysis of modified YACs confirms that both vectors correctly integrate into the targeted unique sites. The precise localization of selectable marker genes in the cloned DNA ensures the integrity of the genomic fragments during functional testing. Placement of mammalian selectable markers within the YAC insert DNA should allow for YAC-based gene targeting experiments in a variety of mammalian cell lines.

Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting.

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.

A physical map of human chromosome 7: an integrated YAC contig map with average STS spacing of 79 kb.

The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project. Our laboratory has focused on developing a physical map of human chromosome 7, a approximately 170-Mb segment of DNA that corresponds to an estimated 5% of the human genome. Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA. The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes. The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries. For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers orders by radiation hybrid (RH) mapping on our YAC contigs. Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are "anchored" on the Genethon genetic map, the RH map, and/or the cytogenetic map. These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain > 98% of the mapped STSs. The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs. An additional 14 contigs, accounting for approximately 1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map. Therefore, these 14 "orphan" contigs are not ordered relative to other contigs. In our contig maps, adjacent STSs are connected by two or more YACs in > 95% of cases. With 2150 mapped STSs, our map provides an average STS spacing of approximately 79 kb. The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome.

A Yeast Artificial Chromosome (YAC) Contig Encompassing the Critical Region of the X-Linked Lymphoproliferative Disease (XLP) Locus

X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein–Barr virus (EBV) infection. Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma. Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence. From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated. Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients. Three more families with interstitial deletions were found. Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescencein situhybridization and which consists of 15 overlapping YAC clones, has been constructed. A detailed restriction enzyme map of the region has been constructed. YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction enzymes. The STSs and lambda subclones were used in Southern hybridization and PCR analyses. The work presented here substantially refines the critical region for XLP. The YAC contig with the overlapping interstitial deletions constitutes the basis for the construction of a transcriptional map of the critical region and facilitates the identification of the XLP gene.

Construction of High-Resolution Physical Maps from Yeast Artificial Chromosomes Using Restriction Landmark Genomic Scanning (RLGS)

We have established a new system for chromosome-specific yeast artificial chromosome (YAC) contig construction using restriction landmark genomic scanning (RLGS-based YAC contig mapper). RLGS is a powerful tool for detecting more than 1000 restriction landmarks distributed on an entire genome in one procedure. In this system, RLGS is applied to sorted chromosomes to cover the target chromosome. Using these landmarks as guideposts, chromosome-specific YAC clones are then ordered. In this paper, we report the construction of a map for a human chromosome 21 YAC contig spanning q22.1 using this new approach. Applying RLGS to sorted chromosomes 21 enables detection of approximately 1400 spots (equivalent of 1050PacI landmarks), covering the entire region of this chromosome. We constructed the 2.5-Mb YAC contig encompassing 21q22.1 with 66 spots (equivalent of 50PacI landmarks). With this contig map, we could detect two deleted regions and chimerism in the YAC insert DNA. Our results demonstrated the usefulness of this approach for finding DNA alterations of YACs, such as deletions and chimerism.

Sequence analysis of the Bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome.

The standard strategies of genome sequencing based on lambda-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli. One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis, established by using yeast artificial chromosomes (YACs). This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it. The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs. The complete 104,109 bp insert sequence of this YAC clone was thus established. The strategy used allowed us to avoid resequencing the two largest, previously sequenced, contigs (13,695 and 20,303 bp) of the YAC insert. We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning, as well as for larger inserts of eukaryotic origin cloned in YACs. Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg. The organization of the newly determined region is presented. Of the 138 ORFs identified in the spollA-kdg region, 57 have no clear putative function from their homology to proteins in the databases.

A Yeast Artificial Chromosome Contig andNotI Restriction Map That Spans the Tumor Suppressor Gene(s) Locus, 11q22.2–q23.3

Human chromosome 11q22–q23 is a pathologically important region in which a high level of loss of heterozygosity has been reported for breast, ovary, cervical, colon, and lung carcinomas, malignant melanomas, and hematologic malignancies. This strongly indicates that one or more tumor suppressor genes reside within the deleted region. In this report, we report the development of a contig map that covers most of the deleted regions found in these malignancies. The map comprises a contig of 66 overlapping yeast artificial chromosomes (YACs) and spans a region of 17 Mb from thePGRgene at 11q22.2 to theMLLgene at q23.3. In the process of screening the YACs, 50 new sequence-tagged site markers were developed from the termini of the YAC inserts. These markers were used for chromosome walking, and the data were then integrated into the contig map.NotI restriction mapping of these YACs revealed the presence of at least 26NotI sites in the region. Using 22 of them, aNotI restriction map of the region fromPGRto D11S939 was developed. This YAC contig will provide efficient tools for identification of the putative tumor suppressor gene(s).

A 2-Megabase Physical Contig Incorporating 43 DNA Markers on the Human X Chromosome at p11.23–p11.22 from ZNF21 to DXS255

A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23–p11.22. As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids. The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internalAlu-PCR product. The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb. The relative order of previously known genes and expressed sequence tags in this region is predicted to be Xpter–ZNF21–DXS7465E (MG66)–DXS7927E (MG81)–WASP, DXS1011E, DXS7467E (MG21)–DXS- 7466E (MG44)–GATA1–DXS7469E (Xp664)–TFE3–SYP (DXS1007E)–Xcen. This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates.

Yeast artificial chromosome (YAC) -based genome mapping of Schistosoma mansoni

Schistosoma mansoni has 7 pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7 × 10 8 bp. We initiated the molecular genetic approach for the detailed characterization and understanding of the evolutionary biology of schistosomes. We have constructed a yeast artificial chromosome (YAC)-library with partially digested parasite genomic DNA, and the chromosome location of each insert was detected by fluorescent in situ hybridization (FISH). The library contains > 2283 clones with an average insert size of 358 kb, which represents a 2.6-fold coverage of the genome (> 7.2 × 10 8 bp). 100 randomly selected YAC clones were localized by FISH and found to be distributed widely among all chromosomes. The assembly of 14 YACs distributed almost the whole region of chromosome 3. Generated expressed sequenced tags (ESTs) derived from a unidirectional cDNA library were also used for further characterization of the YAC inserts. These results indicate that an extensive contig assembly of the entire chromosomes and a reasonably detailed gene map should be feasible in the near future.

Genome manipulation in recalcitrant species: construction and characterization of a yeast artificial chromosome (YAC) library from Erysiphe graminis f. sp. hordei, an obligate fungal pathogen of barley.

Extraction of DNA from organisms where spores are the only source of pure material is a major problem. Methods are described which allow the isolation of high-M(r) DNA, from small quantities of Erysiphe graminis f. sp. hordei (Egh) conidia, suitable for cloning in yeast artificial chromosomes (YACs). A YAC library of 1500 clones was constructed in the vectors, pYAC4 and pYACRC. The average size of YAC inserts is 220 kb and range from 70 to 500 kb, providing ten haploid genome equivalents. Multicopy RFLP markers and an Egh-specific repetitive SINE element were used to characterize the library. The SINE element is effective in fingerprint analysis and contig assembly. Four out of five representative clones containing more than one YAC were mitotically unstable.

Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.

A physical map of the human APP gene in YACs

Several point mutations within exons 16 and 17 of the amyloid precursor protein (APP) gene have been reported that are associated with Alzheimer's disease in a small number of familial cases. To determine the size of the APP gene and the organization of the exons within human genomic DNA, we have characterized 11 Yeast Artificial Chromosome (YAC) recombinants containing human APP gene sequences. The smallest YAC insert was 125 kb, and the largest was 1.4 Mb. The YACs were screened by polymerase chain reaction amplification of APP exons to determine which of the 18 exons coding for APP770 were present. Four of the YACs (D110G1, D110G6, D110E9, and B142F9) contain all 18 exons and at least part of the promoter. Construction of an overlapping map of the gene with all of the YACs demonstrated that 3 of the 11 YACs were chimeric. The orientation and position of the coding sequence on the map was determined by probing digests of the YAC DNA with exon PCR products and the vector arms. The coding region of the APP gene spans approximately 400 kb of genomic DNA.

Genetic transfer and expression of reconstructed yeast artificial chromosomes containing normal and translocated BCL2 proto-oncogenes.

The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection. The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus. Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles. Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line. Fifty-eight percent of the clones contained a copy of the entire YAC insert. Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein. These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S. cerevisiae.

Genetic transfer and expression of reconstructed yeast artificial chromosomes containing normal and translocated BCL2 proto-oncogenes.

The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection. The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus. Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles. Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line. Fifty-eight percent of the clones contained a copy of the entire YAC insert. Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein. These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S. cerevisiae.

Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human beta-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the beta-globin gene. Three cell lines were analyzed by RecA-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.