Physical mapping of yeast artificial chromosomes containing sequences from the human β-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human β-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult β-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire β-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the β-globin gene cluster.     

Characterization of yeast artificial chromosomes containing interleukin genes on human chromosome 5.

To understand better the organization and linkage of the interleukin genes, IL4 and IL5, we prepared long-range restriction maps of five yeast artificial chromosomes (YACs) containing IL5. We determined that IL4 and IL5 are within 100-170 kb, and that the regions surrounding these genes contain several GC-rich areas. Fluorescence in situ chromosomal analysis demonstrated that three of the five YAC clones contain non-contiguous genomic sequences originating from multiple human chromosomes.     

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.     

Yeast artificial chromosomes containing human Xq24-Xq28 DNA: library construction and representation of probe sequences

A library of yeast artificial chromosomes (YACs) with human DNA inserts has been assembled from a human/hamster somatic cell hybrid containing Xq24-Xqter human DNA. Screening of the agar-embedded transformants for human DNA used a manifold of 3000 stainless-steel pins to transfer colonies onto the surface of media. This facilitated the recovery of the 1 in 300 clones that contained a human DNA insert (the remainder had hamster DNA and were discarded). The library described here consists of about two genomic equivalents (102 Mb) of human DNA in 467 clones: 167 were generated by EcoRI partial digestion and contain 25.5 Mb of human DNA; 252 used partial digestion with TaqI and cover 64.2 Mb; and 48 were from sheared DNA inserts and cover 11.7 Mb. Clones were screened by hybridization with 70 probes previously assigned to Xq24-Xq28. Eleven probes did not hybridize to any YACs in the library, and 16 probes hybridized to one YAC each, 23 to two, 13 to three, and 7 to four. Also, individual YACs large enough to detect features like the clustering of polymorphic sequences in subregions of Xq24-Xqter have been obtained. For example, XY58 contained five probe sequences previously independently isolated. The overall yield of YACs containing probe sequences was indistinguishable from Poisson statistical expectations for random cloning (P = 0.9). Thus, YAC libraries such as the one described here can include most, if not all, of the sequences in the source DNA from which the library is derived. These results support the possibility that YACs may provide a reliable bridge between linkage studies and conventional recombinant DNA analyses in mapping of the human genome.     

Mitotic recombination of yeast artificial chromosomes.

Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.     

Distribution of moderately repetitive sequences pTR5 and LF1 in Xq24-q28 human DNA and their use in assembling YAC contigs.

Xq24-q28 DNA, from a hamster/human hybrid cell containing only that portion of the human X chromosome, was found to contain 56 TaqI restriction fragments that hybridized to the moderately repetitive sequence pTR5. Using the pTR5 sequence as a probe in colony hybridization, 136 cognate yeast artificial chromosome (YAC) clones were detected among a collection of 820 containing about three genomic equivalents of the Xq24-q28 DNA. The YACs were then grouped into 48 contigs and single clones containing one or more of the TaqI fragments. Overlaps were confirmed both by fingerprinting YACs with AluI and L1 probes and by additional information. A less complete analysis was also carried out with a second moderately repetitive sequence, LF1, and some smaller contigs were merged into larger ones. Moderately repetitive sequences can thus be used as probes for multiple loci in single hybridization experiments and can help to organize and confirm YAC overlaps during the development of maps with long-range contiguity.     

利用酵母人工染色体（YAC）减数分裂同源重组构建人免疫球蛋白κ链基因组大片段

具有同源重叠区的酵母人工染色体(YAC)可以利用酵母细胞减数分裂进行同源重组,从而构建更大的人工染色体基因组,这对生命科学基础研究和生物技术应用研究有着非常重要的意义。本实验以两个含人免疫球蛋白κ链基因簇片段的YAC克隆为材料,通过酵母改型、异型接合、二倍体发孢、单孢子筛选和分子生物学鉴定等技术和方法,利用酵母菌减数分裂同源重组机制,构建了一条包含人的免疫球蛋白κ轻链32个Vκ基因、5个Jκ基因、Cκ基因、Eκ基因和κde基因的YAC重组体,长度约400kb。同时,本实验利用溶壁酶消化法获取单孢子重组体,代替了传统的显微分孢操作。使得利用酵母人工染色体减数分裂同源重组的技术更加简便可行。     

Human Xq24-Xq28: approaches to mapping with yeast artificial chromosomes.

One hundred twenty-seven yeast strains with artificial chromosomes containing Xq24-Xqter human DNA were obtained starting from a human/hamster somatic cell hybrid. The clones were characterized with respect to their insert size, stability, and representation of a set of Xq24-Xqter DNA probes. The inserts of the clones add up to 19.3 megabase (Mb) content, or about 0.4 genomic equivalents of that portion of the X chromosome, with a range of 40-650 kb in individual YACs. Eleven clones contained more than one YAC, the additional ones usually having hamster DNA inserts; the individual YACs could be separated by extracting the total DNA from such strains and using it to retransform yeast cells. One of the YACs, containing the probe for the DXS49 locus, was grossly unstable, throwing off smaller versions of an initial 300-kb YAC during subculture; the other YACs appeared to breed true on subculture. Of 52 probes tested, 12 found cognate YACs; the YACs included one with the glucose-6-phosphate dehydrogense gene and another containing four anonymous probe sequences (DX13, St14, cpx67, and cpx6). Xq location of YACs is being verified by in situ hybridization to metaphase chromosomes, and fingerprinting and hybridization methods are being used to detect YACs that overlap.     

Yeast artificial chromosomes: tools for mapping and analysis of complex genomes

Libraries of yeast artificial chromosomes (YACs) are representative of complex genomes, and typical YACs contain single fragments of DNA that are up to a megabase or more in size, are stable during growth, and are faithful to genomic DNA. Such YACs may permit (1) the use of complete gene units for a number of research and medical purposes; and (2) the assembly of chromosome-sized contigs in a single map that unifies genetic and physical data.     

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.     

Chromosomal in situ hybridization using yeast artificial chromosomes

Large DNA fragment cloning methods using yeast artificial chromosomes (YACs) have vastly improved the strategies for constructing physical maps of regions of complex genomes, as well as for isolating and cloning genes important for human disease. We present here a simple and rapid method for carrying out in situ hybridization to metaphase chromosomes using isolated YAC clones by labeling DNA directly in agarose gel slices. Nonisotopic labeling and chromosomal in situ hybridization can be used to determine the chromosomal localization of individual YAC clones on human metaphase chromosomes. This method can also be used to characterize YAC clones consisting of single fragments from those that contain concatamerized, and thus artifactual, inserts. This technique also offers a valuable tool to study consistent translocations in neoplastic diseases by identifying YACs that span a specific chromosomal breakpoint.     

Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes.

Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.     

Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene.

We have isolated and characterized two independent clones containing the chicken adult beta-globin gene. Each clone contains a 6.2-kilobase-pair Eco RI restriction fragment of chicken erythrocyte DNA inserted into the vector, lambda gtWES . lambda B. The orientation of the inserted fragment is opposite in the two clones. Characterization of the clones by electron microscopic R-loop studies, by restriction enzyme mapping, and by filter hybridization shows that the adult beta-globin gene is interrupted by at least one small and one large intervening sequence. In addition to the complete adult beta-globin gene, at least part of a second beta-globin-like gene was identified about 2.7 kilobase pairs from the 3'-end of the adult gene. The two independent clones, while very similar, do differ at two Msp I restriction endonuclease sites in regions flanking the adult beta-globin gene.     

Saturation mapping with subclones of YACs: DNA marker production targeting the rice blast disease resistance gene, Pi-b

Saturation mapping of a very small genomic region is indispensable for map-based cloning. We applied a method based on sub-cloning and the Southern-hybridization technique for generating RFLP markers directly from yeast artificial chromosomes (YACs). Two YACs overlapping each other and covering the locus of the rice blast resistance gene, Pi-b, were used to construct a plasmid sub-library. Rice-specific and single-copy clones suitable as probes for RFLP analysis were selected from this sub-library by hybridization to the blots of digested DNAs of rice, YACs, and yeast. As a result, 22 markers were produced within a small chromosomal region including Pi-b. This case study shows that overlapping YACs known to cover the gene of interest are very useful in fine-scale physical mapping leading to map-based cloning of the target gene. Received: 2 May 1996 / Accepted: 2 August 1996     

A Chlamydomonas Genomic Library in Yeast Artificial Chromosomes

We have constructed and characterized a Chlamydomonas reinhardtii total genomic library in yeast artificial chromosomes (YACs). The library contains 7500 clones with inserts ranging in size from 100-200 kb. The representation of the library was assessed by screening one-third of it with a probe derived from the dispersed repeat, Gulliver, which occurs ~13 times in the genome. At least 10 of these Gulliver loci were isolated within 15 independent YACs. Two of these YACs encompass the Gulliver element designated G, which was reported to map to the uni linkage group (ULG). The end clones of these two YACs have been genetically mapped by RFLP analysis in an interspecific cross and thereby shown to be closely linked to the APM locus on the ULG. A third uni-specific YAC has also been isolated and its ends have been mapped by RFLP analysis. Genetic and RFLP analysis of these and other YACs indicates that the frequency of chimeric YACs in the library is very low. The library was constructed in a second generation vector that enables plasmid rescue of YAC end clones as well as copy number amplification of artificial chromosomes. We provide evidence that amplification of intact YACs requires a rad1:rad52 yeast strain.     

Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR.

Interspersed repetitive element (IRE)-PCR is a useful method for identification of novel human or mouse sequence tagged sites (STSs) from contigs of genomic clones. We describe the use of IRE-PCR with mouse B1 repetitive element primers to generate novel, PCR amplifiable, simple sequence length polymorphisms (SSLPs) from yeast artificial chromosome (YAC) clones containing regions of mouse chromosomes 13 and 14. Forty-two IRE-PCR products were cloned and sequenced from eight YACs. Of these, 29 clones contained multiple simple sequence repeat units. PCR analysis with primers derived from unique sequences flanking the simple sequence repeat units in seven clones showed all to be polymorphic between various mouse strains. This novel approach to SSLP identification represents an efficient method for saturating a genomic interval with polymorphic genetic markers that may expedite the positional cloning of genes for traits and diseases.     

Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination

We have developed a pair of vectors for exchanging yeast artificial chromosome (YAC) arms by targeted homologous recombination. These conversion vectors allow the introduction of copy-number control elements into YACs constructed with pYAC4 or related vectors. YACs modified in this way provide an enriched source of DNA for genetic or biochemical studies. A LYS2 gene on the conversion vector provides a genetic selection for the modified YACs after transformation with appropriately prepared vector. A background of Lys⁺ clones that do not contain modified YACs is also present. However, clones with converted YACs can be distinguished from this background by counter-screening for loss of the original p YAC4 TRP1 arm (Trp^- phenotype). The elimination of yeast replication origins (ARS elements) from the conversion vectors increased the frequency of Lys⁺ Trp^- clones, but resulted in weaker amplification. Several YACs have been converted with these vectors, and the fate of the transformed DNA and of the resident YAC DNA has been systematically investigated.     

Yeast artificial chromosome mapping of the cystinosis locus on chromosome 17p by fluorescence in situ hybridization

The gene locus for cystinosis has been mapped between markers D17S1583 and D17S1584 on the short arm of chromosome 17. Using markers encompassing the cystinosis region, we assigned different yeast artificial chromosome (YAC) clones previously identified by sequence tagged site (STS) screening to 17p13.3. Three of the clones hybridized to the target 17p gene region; one of these was chimeric, hybridizing both to chromosomes 3p and 5q; two of the YACs did not contain sequences of 17p13.3. Our physical mapping has identified candidate YACs as a first step towards a positional cloning approach. Received: 28 February 1996 / Revised: 3 May 1996     

Use of interspersed repetitive sequences-PCR products for cDNA selection

In order to increase the efficiency of cDNA selection approaches, we describe the use of interspersed repetitive sequences-PCR (IRS-PCR) products to isolate genes from large-insert genomic clones. IRS-PCR is conducted on total yeast DNA containing a YAC of interest so that there is no need to purify the starting genomic clone. This enables the production of large amounts of genomic substrate for cDNA selection and allows the use of unstable YAC clones. Moreover, the hybridization of the IRS-PCR product to the cDNA clones after selection introduces a positive selection step. We tested these PCR products from YACs for the presence of exons, using cDNAs originating from seven different genes. In each case, at least one exon was present in the IRS-PCR product. We have applied this strategy to four YAC clones originating from the human X Chromosome (Chr). All the selected cDNAs, strongly positive with the IRS-PCR product, did indeed originate from a gene in the region covered by the YAC. In all cases, the previously known genes contained in the genomic clones have been isolated. In addition, we have isolated human genes that have already been described but not assigned to any chromosomal region.