Yeast Artificial Chromosome DNA Research Articles

Purification of Yeast Artificial Chromosome DNA for Microinjection Using a Two-Gel Electrophoresis Procedure.

This protocol describes a method to purify yeast artificial chromosome (YAC) DNA for microinjection. YAC DNA solutions cannot be concentrated by standard DNA precipitation methods and resuspended into smaller volumes. Attempts to precipitate YAC DNA solutions will, unavoidably, break and destroy the DNA molecules. Therefore YAC DNA must always be kept in a solution that maintains the integrity of the YAC DNA molecules. In the method described here, YAC DNA is extracted from pulsed-field gel electrophoresis (PFGE) agarose plugs by using two gel electrophoresis steps. The first gel electrophoresis step is the PFGE itself. The region of agarose containing the YAC DNA of interest is excised and run on a standard electrophoresis gel setup at a 90° angle to the PFGE run. The YAC DNA migrates out of the agarose slice and enters into a thicker low-melting agarose gel, thereby promoting compaction of YAC DNA molecules. The aim of this method is to convert a slice of agarose into a cube of agarose of smaller volume. The volume of this cube will determine the final concentration of the YAC DNA precipitation.

Purification of Yeast Artificial Chromosome DNA for Microinjection Using Pulsed-Field Gel Electrophoresis and Ultrafiltration.

This protocol describes a method for concentrating yeast artificial chromosome (YAC) DNA preparations following separation of chromosomes by pulsed-field gel electrophoresis (PFGE). The agarose slice containing the YAC DNA of interest is melted and digested. The resulting YAC DNA-containing solution is concentrated using ultrafiltration units with a 30,000-mwt cutoff. The DNA is retained in the upper container, where it concentrates sixfold to sevenfold. Ideally, YAC DNA preparations at 10-60 ng/µL can be obtained. However, great care must be taken to recover the YAC DNA molecules that sometimes irreversibly attach to the filter membrane. This procedure can also be used to concentrate BAC DNA from PFGE gel slices of BAC DNA-containing agarose exactly as described for YACs.

Large-Scale Preparation of Yeast Agarose Plugs to Isolate Yeast Artificial Chromosome DNA.

The microinjection of DNA directly into the pronuclei of fertilized zygotes is the most extensively used method of gene transfer in the mouse. The injection of very large pieces of DNA, including bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), has become increasingly popular because their size normally accommodates all of the regulatory elements that are needed for a given expression domain to function adequately in an ectopic genomic location. This protocol describes how to prepare large-scale yeast agarose plugs to isolate YAC DNA. The quality of the YAC DNA preparation is the most important consideration in optimizing transgenic mouse production.

Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast–embryonic stem cell fusion

Introducing human genes into mice offers the opportunity to analyze their in vivo function or to obtain therapeutic molecules. For proper gene regulation, or in case of multigene families, megabase (Mb)-sized DNA fragments often have to be used. Yeast artificial chromosome (YAC)-mediated transgenesis is irreplaceable for this purpose, because alternative methods such as the use of bacterial artificial chromosomes (BACs) cannot introduce DNA fragments larger than 500 kb into the mouse germ line. However, YAC libraries often contain only partial gene loci. Time-consuming reconstruction of YACs, genetic instability and the difficulty in obtaining intact YAC DNA above a certain size impede the generation of humanized mice. Here we describe how to reconstruct YACs containing Mb-sized human DNA, such as the T cell receptor-α (TRA) gene locus, thus facilitating the introduction of large DNA fragments into the mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids the need for YAC DNA purification. These ES cells are then used to stably introduce the functional TRA gene locus into the mouse germ line. The protocol takes ∼1 year to complete, from reconstruction of the entire TRA gene locus from YACs containing partial but overlapping TRA regions to germline transmission of the YAC.

Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies

Circular yeast artificial chromosomes (YACs) provide significant advantages for cloning and manipulating large segments of genomic DNA in Saccharomyces cerevisiae. However, it has been difficult to exploit these advantages, because circular YACs are difficult to isolate and purify. Here we describe a method for purification of large circular YACs that is more reliable compared with previously described protocols. This method has been used to purify YACs up to 600 kb in size. The purified YAC DNA is suitable for restriction enzyme digestion, DNA sequencing and functional studies. For example, YACs carrying full-size genes can be purified from yeast and used for transfection into mammalian cells or for the construction of a synthetic genome that can be used to produce a synthetic cell. This method for isolating high-quality YAC DNA in microgram quantities should be valuable for functional and synthetic genomic studies. The entire protocol takes ∼3 d to complete.

Purification of circular YACs from yeast cells for DNA sequencing

We describe a method for the purification of circular yeast artificial chromosome (YAC) DNA 120-150 kilobases (kb) in size that is of sufficient quantity and quality for restriction enzyme analysis and DNA sequencing. This method preferentially enriches for circular YAC DNA and avoids the time-consuming step of centrifugation in CsCl--ethidium bromide (EtBr) gradients. We applied this method to the purification of circular YACs carrying DNA segments that are extremely unstable in E. coli, including those that correspond to GAP2 and GAP3 on human chromosome 19. We showed that YAC DNA (GAP2 and GAP3) purified using this new method is clearly resolved in EtBr-stained gels. The sequence of YAC-GAP3 was obtained, representing the first GAP clone sequenced in YAC form. At present, it is estimated that there are more than 1000 gaps in the human genome that cannot be cloned using bacterial vectors. Thus, our new method may be very useful for completing the last stage of the human genome project.

Preparation of intact yeast artificial chromosome DNA for transgenesis of mice

Transgenesis with large DNA molecules such as yeast artificial chromosomes (YACs) has an advantage over smaller constructs in that an entire locus and all its flanking cis-regulatory elements are included. The key to obtaining animals bearing full-length transgenes is to avoid physical shearing of the DNA during purification and microinjection. This protocol details how to prepare intact YAC DNA for transgenesis of mice and involves separation of YAC DNA from yeast chromosomal DNA by pulsed field gel electrophoresis, concentration to a range suitable for microinjection by second dimension electrophoresis and enzymatic digestion of matrix-embedded YAC DNA to produce a solution that can be injected. The YAC is maintained in an agarose gel matrix to avoid damage until the final steps before microinjection. Special precautions are also taken during the microinjection protocol. Transgenesis efficiency is approximately 15%; most animals carry 1-5 copies of the desired locus. This method takes 6 d for completion.

A porcine BAC library with tenfold genome coverage: a resource for physical and genetic map integration.

A porcine BAC library with tenfold genome coverage: a resource for physical and genetic map integration.

Overexpression of the human VPAC2 receptor in the suprachiasmatic nucleus alters the circadian phenotype of mice.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to a superfamily of structurally related peptide hormones that includes glucagon, glucagon-like peptides, secretin, and growth hormone-releasing hormone. Microinjection of VIP or PACAP into the rodent suprachiasmatic nucleus (SCN) phase shifts the circadian pacemaker and VIP antagonists, and antisense oligodeoxynucleotides have been shown to disrupt circadian function. VIP and PACAP have equal potency as agonists of the VPAC(2) receptor (VPAC(2)R), which is expressed abundantly in the SCN, in a circadian manner. To determine whether manipulating the level of expression of the VPAC(2)R can influence the control of the circadian clock, we have created transgenic mice overexpressing the human VPAC(2)R gene from a yeast artificial chromosome (YAC) construct. The YAC was modified by a strategy using homologous recombination to introduce (i) the HA epitope tag sequence (from influenza virus hemagglutinin) at the carboxyl terminus of the VPAC(2)R protein, (ii) the lacZ reporter gene, and (iii) a conditional centromere, enabling YAC DNA to be amplified in culture in the presence of galactose. High levels of lacZ expression were detected in the SCN, habenula, pancreas, and testis of the transgenic mice, with lower levels in the olfactory bulb and various hypothalamic areas. Transgenic mice resynchronized more quickly than wild-type controls to an advance of 8 h in the light-dark (LD) cycle and exhibited a significantly shorter circadian period in constant darkness (DD). These data suggest that the VPAC(2)R can influence the rhythmicity and photic entrainment of the circadian clock.

Deletion mapping at 12p12-13 in metastatic prostate cancer

The identification of homozygous deletions in malignant tissue is a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the polymerase chain reaction (PCR) to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes, such as BRCA2 and PTEN. We have recently identified a 1-5-cM homozygous deletion on 12p12-13 in a prostate cancer xenograft and found that 47% of patients who died of prostate carcinoma demonstrate focal loss of heterozygosity (LOH) in this region in metastatic deposits. We have now characterized the region of interest by assembling a yeast artificial chromosome (YAC) contig spanning the homozygous deletion and identifying which known genes and expressed sequence tags (EST) lie within the homozygous deletion. A rib metastasis was harvested at autopsy and placed subcutaneously in a male SCID mouse. Genomic DNA from this xenograft and from the patient's normal renal tissue was extracted. Multiplex PCR, with the xenograft and normal DNA used as template, was performed using primers for loci on the Whitehead contig 12.1 believed to be near our region of interest. We found that our deletion lay in a 1-2-Mb interval between WI-664 and D12S358. We then used the same primers to construct a YAC contig across the homozygous deletion. PCR amplification of YAC DNA, using primers for the genomic sequences of known genes and ESTs reported to lie on 12p12-13, was used to identify candidate genes that lay within the deletion. Duplex PCR, with control primers known not to be deleted in the xenograft, was used to confirm that both the CDKN1B and ETV6 genes were homozygously deleted in the xenograft. Mutations in either or both of these genes may play an important role in metastatic prostate carcinoma.

Detailed transcript map of a 810-kb region at 11p14 involving identification of 10 novel human 3' exons.

A limited number of genes, including the human brain-derived neutrotrophic factor (BDNF) gene, have been identified in the human chromosome 11p14 region. Since this area is involved in a genetic disorder (WAGR syndrome) and because of interest in studying the regulation of the human BDNF gene, we have established a detailed transcript map of a 810-kb region clone in a yeast artificial chromosome (YAC), corresponding to a portion of this genomic locus. A set of nested deletion mutants has been generated to map genes at a mean resolution of 75kb. Four genic markers from available mapping databases have been mapped on the YAC. Ten potential novel human exons have been isolated by a 3' terminal exon trapping procedure directly applied to purified YAC DNA. Most of these exons display polyadenylation signals and they all yield positive signals in RT-PCR experiments, confirming their status of transcribed sequences. The BDNF gene is now co-localised with three other genes on a 120 kb DNA fragment.

Identification of a ζ-Crystallin (Quinone Reductase)-like 1 Gene (CRYZL1) Mapped to Human Chromosome 21q22.1

To identify a new gene(s) located on the yeast artificial chromosome (YAC) clone D142H8 that was mapped to human chromosome 21q22.1, purified YAC DNA from the clone was utilized directly as a probe to screen a human brain cDNA library after the suppression of human repetitive DNA. One cDNA clone hybridizing specifically to the YAC D142H8 DNA was identified. The clone has an insert of 1341 bp and the longest open reading frame of 349 amino acids. A search of GenBank revealed that the clone has a high degree of homology to ζ-crystallin (quinone reductase) at the amino acid level, and its nucleotide sequence represents the expressed sequence from the 50-kb segment of the human chromosome 21q11.1. Thus a new gene was namedCRYZL1(ζ-crystalline-like 1). Genomic Southern blot with total human and yeast DNAs suggests thatCRYZL1might be a single-copy gene. The fluorescencein situhybridization procedure was applied, and the results showed that the gene mapped to the human chromosome 21q22.1 subband. TheCRYZL1mRNA was expressed in heart, brain, skeletal muscle, kidney, pancreas, liver, and lungs but at different levels in different tissues.

Regional insertional mutagenesis of genes on Arabidopsis thaliana chromosome V using the Ac/Ds transposon in combination with a cDNA scanning method.

For regional insertional mutagenesis of Arabidopsis thaliana genes, we combined a cDNA scanning method (Hayashida et al. Gene 1995; 165:155-161) and an Ac/Ds transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on A. thaliana chromosome 5. We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub-libraries in which cDNAs for genes on each YAC DNA were concentrated. From each sub-library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs). In total, 113 non-redundant groups of cDNAs were obtained. Forty-four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms. In parallel, we transposed Ds from a donor Ds-GUS-T-DNA line, Ds4391-20, already mapped to the CIC7E11/8B11 region. We obtained Ds-transposed lines and recovered their Ds-flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR). Dot-blot analysis indicated that 20% of the lines contained transposed Ds in the CIC7E11/8B11 region, suggesting that this Ac/Ds transposon system is effective for regional insertional mutagenesis. To isolate Ds insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800 Ds-containing lines using Ds-specific and gene-specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method. We found that 49 lines contain Ds insertion mutations, and that five lines contain Ds mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region. These results indicate that combining the cDNA scanning method and the Ac/Ds transposon gives a powerful tool for regional insertional mutagenesis not only in Arabidopsis but also in other plants or crops whose genomes are not sequenced.

Assay of centromere function using a human artificial chromosome.

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.

Arabidopsis YAC restriction mapping.

The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (approximately 27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region. The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used. This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterized genes involved in male gametogenesis (e.g., Ms1 and Ms37). The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterized floral development genes which lie in this region. This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.

Modification of bacterial artificial chromosomes through chi-stimulated homologous recombination and its application in zebrafish transgenesis.

The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish GATA-2 genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that GATA-2 expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable.

The effect of neoagaro-oligosaccharides on lipofectAMINE-mediated transfection

▼Yeast artificial chromosomes (YACs) are widely used as vectors for the study of gene expression in mammalian cells (Ref. 1). The genomic environment of the YAC DNA provides a more representative way to study gene expression and, in particular, tissue-specific gene expression. Normally, the YAC DNA is isolated using the methods described by Maule et al. (Ref. 2) and Schedl et al. (Ref. 3) in which the YAC DNA from a preparative pulsed-field gel slice is concentrated into a 4% low-melting-temperature agarose gel and the intact DNA subsequently recovered by melting the agarose and digesting it with β-agarase (New England Biolabs). The purified and concentrated YAC DNA is then used to transfect mammalian cells. We have been using this method to purify and concentrate YAC DNA in order to transfect myoblasts. We observed that the presence of neoagaro-oligosaccharides, the end products of β-agarase-digested agarose, inhibits the transfection efficiency when lipofectAMINE (Pharmacia) is used. The number of transformants obtained in the presence 4% and 1% neoagaro-oligosaccharides could differ between 20 and 200 times (data not shown). In order to establish the effect of neoagaro-oligosaccharides on transfection efficiency, we used a standard amount (2 μg/transfection) of reporter construct [pSVβ-gal (Promega)] DNA and varied the concentration of neoagaro-oligosaccharides between 4 and 1% (w/v) in our transfection experiment. The results show that neoagaro-oligosaccharides will inhibit the liposome-

YAC transgenesis: a study of conditions to protect YAC DNA from breakage and a protocol for transfection

Yeast artificial chromosomes (YACs) are providing a great boon to transgene technology by allowing the easy mutagenesis and study of very large DNAs. The large insert sizes of these vectors permit more accurate analysis of the regulation of transgene expression than smaller, more artificially assembled constructs. Transfection of mammalian cells by YACs can be accomplished by a number of methods; the most prevalent, using gel-purified DNA, is dependent upon compaction by salts to protect the large YAC DNA from breakage. We show that the common reliance on NaCl to compact YAC DNA sufficiently to protect it from breakage is not well-founded. Even the use of mixtures of polyamines and NaCl allows substantial damage to purified YACs. The use of polyamines alone in low salt buffers to compact YAC DNA provides the best protection from breakage and allows very effective transfection of murine embryonic stem (ES) cells. We provide a detailed method for ES cell transfection by YACs utilizing the DOTAP lipofection reagent that optimizes transfection efficiency and recovery of intact YACs.

Transient gene expression from yeast artificial chromosome DNA in mammalian cells is enhanced by adenovirus.

The introduction of high molecular weight DNA into mammalian cells is useful for gene expression studies. However, current transfection strategies are inefficient, necessitating propagation of stable DNA transformants prior to analysis of gene expression. Here we demonstrate that transient lipid-mediated DNA transfection can be used to assess gene expression from yeast artificial chromosomes (YACs) containing the 230 kb cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and Escherichia coli lacZ . We also show that psoralen-UV inactivated adenovirus significantly enhances transfection efficiency. The ability to deliver high molecular weight DNA using lipid-mediated transfection should expedite the analysis of large human genes contained within artificial chromosome vectors.

Generation of novel human MHC class II mutant B-cell lines by integrating YAC DNA into a cell line homozygously deleted for the MHC class II region.

The human B lymphoblastoid cell line (LCL) 721.174 sustains a large homozygous deletion in the major histocompatibility complex (MHC) class II region that results in an absence of DQ and DR molecules as well as a deficiency in the assembly and transport of class I molecules to the cell surface. The deleted genes include the transporters associated with antigen processing TAP1 and TAP2, DMA and DMB which are involved in editing class II bound peptides, and four genes whose roles in antigen processing are unclear; the low mass polypeptide genes LMP2 and LMP7, and DNA and DOB. To study this region we have integrated into 721.174 two overlapping yeast artificial chromosome (YAC) clones which cover the interval LMP2-DRA inclusive. Three clones (11.2A1.1, 4D1D10.1 and 4D1D10.2), containing complete copies of the transfected YAC, produced varying levels of mRNA from the LMP, TAP, DQ and DR genes and corresponding levels of LMP and TAP protein. Class I cell surface expression was restored in 11.2A1.1 and 4D1D10.1, as was DR expression in both 4D1D10 transfectants. These studies demonstrate the feasibility of introducing large groups of functional genes back into human lymphoblastoid cells sustaining deletions, with full restoration of biological function. The procedure could be exploited in order to restore all but one gene covered by the deletion, effectively producing a single gene defect. This could be used to introduce copies of genes engineered to contain mutations and to study cis regulatory elements at some distance from the chosen loci.