Identification of a zeta-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 zeta-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 named CRYZL1 (zeta-crystalline-like 1). Genomic Southern blot with total human and yeast DNAs suggests that CRYZL1 might be a single-copy gene. The fluorescence in situ hybridization procedure was applied, and the results showed that the gene mapped to the human chromosome 21q22.1 subband. The CRYZL1 mRNA was expressed in heart, brain, skeletal muscle, kidney, pancreas, liver, and lungs but at different levels in different tissues.     

A yeast artificial chromosome telomere clone spanning a possible location of the Huntington disease gene

The Huntington disease (HD) gene has been mapped to the most distal subband of chromosome 4p. Analysis of recombination events has not provided an unequivocal location of the HD gene, but it indicates a position very close to the telomere as one possibility. We have constructed a yeast artificial chromosome (YAC) vector (containing a rare-cutter polylinker) for the cloning of mammalian telomeres, used it to prepare a BssHII-telomere library with DNA from an individual homozygous for HD, and have identified a 115-kb clone containing the telomere of 4p. One probable recombinant would confine the telomeric candidate location for the gene to the region covered by the YAC, which makes it possible that the clone described here contains the HD locus in its mutant form.     

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.     

Molecular cloning of the human DNA excision repair gene ERCC-6.

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.     

The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion

A 680-kb yeast artificial chromosome (YAC) that contains a functional copy of the human hypoxanthine phosphoribosyltransferase (HPRT) gene has been isolated. This YAC, yHPRT, and another YAC, yXY837, which contains the 3' end of the HPRT gene, have been mapped with restriction enzymes that cleave human DNA infrequently. The HPRT gene lies near the center of yHPRT. Fusion of yHPRT-containing yeast spheroplasts with mouse L A-9 cells, which are HPRT-negative, gives rise to HPRT-positive colonies. These colonies contain the human HPRT gene and express human HPRT mRNA. Fusion of yeast with mammalian cells is an efficient way of testing the integrity and functionality of human DNA contained in YACs.     

A yeast artificial chromosome contig encompassing the cystic fibrosis locus.

The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.     

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.     

Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.     

A shuttle system for transfer of YACs between yeast and mammalian cells.

The development of a system for shuttling DNA cloned as yeast artificial chromosomes (YACs) between yeast and mammalian cells requires that the DNA is maintained as extrachromosomal elements in both cell types. We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (oriP) are maintained as stable, episomal elements in a human kidney cell line constitutively expressing the viral transactivator protein EBNA-1. Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple alkaline lysis procedure and shuttled back into Saccharomyces cerevisiae by spheroplast transformation. In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells. The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.     

Construction and characterization of a partial library of yeast artificial chromosomes from human chromosome 21

We report a protocol for cloning large DNA fragments in yeast artificial chromosomes (YAC). A partial library has been constructed from a somatic hybrid containing chromosome 21 as the single source of human DNA. About 4.0 Mb of human DNA was recovered in 17 YAC clones. Three clones were analyzed by in situ hybridization and mapped on chromosome 21. One clone hybridized with the chromosome 21 centromeric region and may provide new insight both on the molecular structure of centromere and on the localization of Alzheimer disease gene.     

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。     

Cloning and expression in Escherichia coli of a yeast mannosyltransferase from the asparagine-linked glycosylation pathway

The yeast Saccharomyces cerevisiae temperature-sensitive lethal mutant alg1-1, has been previously shown to lack the activity necessary for the addition of the first mannose residue in the synthesis of lipid-linked precursor oligosaccharide. The gene ALG1 has been cloned by complementation of the temperature-sensitive mutation alg1-1 with a total genomic DNA library. The original DNA fragment isolated was 11,300 base pairs and has been subcloned to a 1,500-base pair fragment which is still capable of complementing alg1-1. The gene ALG1 has been mapped on chromosome II at a distance of 2.1 map units from LYS2. The ALG1 gene product has been shown to catalyze the transfer of a mannosyl residue from GDP-mannose to the lipid-linked acceptor GlcNAc2, yielding Man beta 1-4GlcNAc2-lipid, in lysates from Escherichia coli transformants. This result proves that ALG1 is the structural gene for the first mannosyltransferase in lipid-linked oligosaccharide assembly.     

Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain.

The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.     

A versatile and general splitting technology for generating targeted YAC subclones

Yeast artificial chromosomes (YAC) splitting technology was developed as a means to subclone any desired region of eukaryotic chromosomes from one YAC into new YACs. In the present study, the conventional YAC splitting technology was improved by incorporating PCR-mediated chromosome splitting technique and by adding autonomously replicating sequence (ARS) to the system. To demonstrate the performance of the improved method, a 60-kb region from within a 590-kb YAC (clone CIC9e2 from Arabidopsis thaliana chromosome 5) that could not be subcloned using the original method was split to convert into a replicating YAC. Two template plasmids, pSK-KCA and pSKCLY, were used to generate two splitting fragments by PCR. Two splitting fragments consisted of telomeric (C₄A₂)₆ repeats, 400-bp target region, CEN4, H4ARS and Km^r (selective marker for plant transformants), or CgLEU2. These splitting fragments were introduced into Saccharomyces cerevisiae harboring the 100-kb split YAC generated by splitting of the 590-kb YAC and containing the 60-kb region. Among 12 Leu⁺ transformants, four exhibited the expected karyotype in which two newly split 40- and 60-kb chromosomes were generated. These results demonstrate that the improved method can convert a targeted region of a eukaryotic chromosome within a YAC into a replicating YAC.     

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.     

High-Resolution Physical Map of the Immunoglobulin λ Variant Gene Cluster Assembled by Quantitative DNA Fiber Mapping

Quantitative DNA fiber mapping (QDFM) allows rapid construction of near-kilobase-resolution physical maps by hybridizing specific probes to individual stretched DNA molecules. We evaluated the utility of QDFM for the large-scale physical mapping of a rather unstable, repeat-rich 850-kb region encompassing the immunoglobulin λ variant (IGLV) gene segments. We mapped a minimal tiling path composed of 32 cosmid clones to three partially overlapping yeast artificial chromosome (YAC) clones and determined the physical size of each clone, the extent of overlap between clones, and contig orientation, as well as the sizes of gaps between adjacent contigs. Regions of germline DNA for which we had no YAC coverage were characterized by cosmid to cosmid hybridizations. Compared to other methods commonly used for physical map assembly, QDFM is a rapid, versatile technique delivering unambiguous data necessary for map closure and preparation of sequence-ready minimal tiling paths.     

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.