Construction of a MluI-YAC library from barley (Hordeum vulgare L.) and analysis of YAC insert terminal regions.

We describe the construction of a specific yeast artificial chromosome (YAC) library from barley (Hordeum vulgare L.) using the vector pYAC-RC. The library was generated by total digestion of high molecular weight DNA with the infrequently cutting restriction enzyme MluI. Only 10-30% of the colonies were recombinant, as visualized by red-white selection and subsequent pulsed-field gel electrophoresis analysis. About 17 000 individual recombinant YAC clones with insert sizes ranging from 50 to 700 kb, with a mean of 170 kb, were selected. No chloroplast sequences were detected and the proportion of YAC clones containing BARE-1 copia-like retroelements is about 5%. Screening of the library with a single-copy RFLP marker closely linked to the Mla locus yielded three identical clones of the same size. Insert termini of randomly chosen YAC clones were investigated with respect to their redundancy in the barley genome and compared with termini of YAC clones from an EcoRI-based YAC library, resulting in a fourfold enrichment of single-copy sequences at the MluI vector-insert junctions.     

Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation

To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies.     

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 β-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 β-globin gene. Three cell lines were analyzed by Rec A-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.     

YAC cloning of DNA embedded in an agarose matrix

Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions. It minimizes any shearing that may result from handling of high molecular weight DNA and can be done with nanogram to microgram amounts of material, which facilitates construction of YACs from sources of DNA other than genomic DNA isolated from cells. The average size of the YACs recovered (200-1000 kb) and efficiency of transformation of ligation products (200-1000 cfu/micrograms) are similar to those reported using aqueous protocols. This method has been used to construct chromosome specific YACs, and it should be possible to apply the technique to the construction of chromosome specific libraries using flow sorted chromosomes as source material, and the cloning of restriction fragments isolated by preparative pulsed field gel electrophoresis.     

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。     

Isolation of the human sex determining region from a Y-enriched yeast artificial chromosome library

We describe the isolation and characterization of yeast artificial chromosome (YAC) clones spanning the male sex determining region on the short arm of the human Y chromosome. The clones were isolated by hybridizing probes in the interval between the genes MIC2 and ZFY to a Y chromosome-enriched YAC library. The YAC clones were consistent with the order of probes established for this interval and may be useful for functional studies of the region in male sex determination. However, many of the YAC clones from this library carried only one arm of the vector ("half-YACs"), deleted sequences from one end, and contained much smaller inserts (148 kb average) than the size of ligated fragments selected by pulsed-field gel electrophoresis (greater than 440 kb). These problems were overcome by protecting DNA with polyamines during YAC library construction and a second Y-enriched YAC library was constructed with an average insert size of 627 kb.     

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.     

Construction and characterization of a rice YAC library for physical mapping

Genomic libraries of rice,Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of theEco RI- andNot I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transformSaccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs withEco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.     

Application of cDNA Selection Techniques to Regions of the Human MHC

Identification of transcribed sequences by cDNA selection is a potentially rapid and efficient way of scanning large genomic DNA fragments for the presence of genes. To evaluate this approach further, we have applied it to three yeast artificial chromosomes (YACs) and examined the products obtained from a total of about 1100 kb from two regions of the human major histocompatibility complex (MHC). One YAC was derived from an extensively studied portion of the Class II region of the MHC. The cDNAs recovered from this YAC included representatives of the previously described genes as well as one or more cDNA clones not described in the databases. A second YAC spanned about 330 kb of DNA surrounding the Class I gene HLA-A. In addition to Class I clones, 10 distinct cDNA products were identified from this YAC. A third YAC contained about 700 kb of human DNA, including 260 kb of overlap with the second YAC, and recovered an additional cDNA complementary to YAC B30 H3 DNA. Overall, the method is shown to be able to detect very scarce cDNAs and to detect a large fraction of coding sequences in YAC clones. Advantages and limitations of the approach are discussed.     

Analysis of the occurrence and nature of repeated DNA in an 850 kb region of Arabidopsis thaliana chromosome 4

The occurrence and nature of repeated DNA sequences has been analysed within an 850 kb YAC contig on Arabidopsis thaliana chromosome 4. Hybridization analysis with seven RFLP markers, six cosmid contigs, 29 YAC end probes and eight YAC clones showed that a least 585 kb of the 850 kb contained only low-copy sequences. One YAC end probe, EG15C8LE, hybridized to multiple genomic fragments and contained a sequence with predicted protein homology to cytochrome P450 monooxygenases. Another one, EG11B7RE, was found to be non-contiguous with the other YAC clones and contained a dispersed repetitive sequence associated with centromeric regions     

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.     

Direct Complementation ofChlamydomonasMutants with Amplified YAC DNA

We previously described the construction and characterization of aChlamydomonasgenomic library in yeast artificial chromosomes (YACs). Here we describe the isolation and genetic mapping of YACs at the FLA10 locus on theunichromosome as well as isolation of a YAC spanning the PF14 locus on chromosome VI. Genetic mapping of YAC end clones by RFLP analyses in interspecific crosses reveals that YACs with a physical size of 150 kb commonly span genetic intervals defined by one or two recombination events in crosses of approximately 20 tetrads. This promises to make chromosomal walking inChlamydomonasa relatively efficient enterprise. We also describe our development of a method for direct complementation of mutant genes by transformation with amplified wildtype YAC DNA. The use of positional cloning using YACs and this direct functional assay for the presence of a gene in a YAC represent powerful molecular genetic tools enabling the cloning of most anyChlamydomonasgene.     

A test case for physical mapping of human genome by repetitive sequence fingerprints: construction of a physical map of a 420 kb YAC subcloned into cosmids.

A rapid and safe method of Yeast Artificial Chromosome (YAC) physical mapping by cosmid 'fingerprinting' is presented. YACs are subcloned into cosmids which are prepared without previous separation of cloned DNA from host DNA. Groups of overlapping clones are detected according to their restriction fragments size and intensity after hybridization with total human DNA. To test this approach, a cosmid library was constructed from total DNA of a yeast strain containing a 420 kb YAC. A single contig of 84 clones was obtained with a minimal detectable overlap of 60% i.e. a 9.2 fold representative library. Large scale physical mapping of YACs would take full advantage of the DNA preparation procedure employed in this work and allows to take into account restriction fragment intensities.     

Construction of a yeast artificial chromosome library of pepper (Capsicum annuum L.) and identification of clones from the Bs2 resistance locus

A yeast artificial chromosome (YAC) library was constructed using high-molecular-weight DNA isolated from pepper (Capsicum annuum L.) leaf protoplasts. Insert DNA was prepared by partial digestion using EcoRI and subjected to electrophoretic fractionation before in-gel ligation to the pJS97/98 YAC vector. Prior to transformation of yeast spheroplasts, ligation products were subjected to a second electrophoretic size selection. The library consists of about 19 000 clones with an average insert size of 500 kb, thus representing approximately three haploid genome equivalents. Three PCR-based markers tightly linked to the pepper Bs2 resistance gene were used to assess the utility of this library for positional cloning. Three YAC clones containing pepper genomic DNA from the Bs2 resistance locus were isolated from the library. The clones ranged in size from 270 kb to 1.2 Mb and should prove useful for the cloning of the Bs2 gene. Received: 15 January 1999 / Accepted: 11 May 1999     

A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes

A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%. The YACs show segregative and replicative stability.     

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.     

Construction and characterization of a yeast artificial chromosome library containing 1.5 equivalents of human chromosome 21.

A library of yeast artificial chromosomes (YACs) was constructed from a human/hamster somatic cell hybrid containing human chromosome 21 (q11-qter). Cells were embedded in agarose, and the DNA was partially digested with EcoRI, released into solution by agarase treatment of the agarose plugs, ligated into pYAC4, and transferred into yeast. Double screening of the yeast transformants with human and hamster genomic DNA allowed the selection of clones hybridizing only with human DNA. The library consists of 321 clones, amounting to 1.5 equivalents (61 Mb) of chromosome 21. The mean YAC size calculated from 178 clones is 190 +/- 100 kb. Screening of the library with eight sequence-tagged sites gave six positives. Among 21 YACs tested by in situ hybridization, 17 mapped to chromosome 21.     

Construction and characterization of a soybean yeast artificial chromosome library and identification of clones for the<Emphasis Type="Italic"> Rps6</Emphasis> region

We report the construction and characterization of the first soybean yeast artificial chromosome (YAC) library using high-molecular weight DNA isolated from leaf nuclei of the cultivar Conrad 94 that carries Phytophthora resistance genes Rps1-k and Rps6. The quality of this library has been evaluated through analysis of 393 randomly selected YAC clones. The library consists of 36,864 clones, of which 19,956 carry single soybean YACs with an average size of about 285 kb. The library represents approximately five soybean genome equivalents. The probability of finding any soybean sequences from this library is about 0.99. The library was screened for 43 SSR markers representing the whole soybean genome. We were able to identify positive YAC pools for 95% of the SSR markers. Two YAC clones carrying molecular markers linked to the Rps6 gene were identified. The YAC library reported here would be a useful resource for map-based cloning of agronomically important soybean genes and also to complement the effort towards construction of the physical map for the soybean genome.     

水稻核基因组DNA的YAC克隆和鉴定

将EcoRI部分消化的水稻(Oryza sativa L.)细胞核高分子量DNA电泳分部,回收大于200kb的片段,与内切酶消化过的酵母人工染色体(YAC)双质粒载体pJs97。pJS98DNA连接,转化酵母YPH252感受态原生质球,用ura^-,TrP^-双选择培养基直接筛选转化子,已获得2 000多个克隆。转化子DNA的southern杂交显示插入片段在200～820kb范围。     

Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones

As part of the worldwide efforts at molecular analysis of Arabidopsis thaliana as a model plant the complete structure of the mitochondrial genome has been determined. The mitochondrial DNA molecules were mapped by restriction fragment analysis of more than 300 cosmid clones and purified mitochondrial DNA. The entire genome of 372 kb is contained in three different configurations of circular molecules and is split into two additional subgenomic molecules of 234 kb and 138 kb, respectively. These arrangements result from recombinations of the two sets of repeats present in combinations of inverted and/or direct orientation. Alignment of YAC clones confirms the in vivo presence of continuous DNA molecules of more than 300 kb in A. thaliana mitochondria. The presence of this comparatively large mitochondrial genome in a plant with one of the smallest nuclear genomes shows that different size constraints act upon the different genomes in plant cells.