YAC DNA Research Articles

Liposome-mediated transfer of YAC DNA to tobacco cells

This article describes a set of protocols—for retrofitting, transformation and purification—that together enable the delivery of full-sized YAC-DNA to plant cells. To be able to equip YACs of interest with plant selectable markers, we have constructed a retrofitting vector that carriesnptII anduidA. Furthermore, we established a transformation protocol for plant protoplasts that is sufficiently efficient to support transfer of high-molecular-weight DNA. In this protocol lipofection is combined with PEG-mediated direct gene transfer. Large amounts of purified DNA are necessary for lipofection. To obtain sufficient quantities of concentrated, purified YAC-DNA, we used an optimized two-step, gel-purification method. Transient expression of a YAC-bornuidA demonstrates that both retrofitting vector and transformation protocol are effective.

Growth and storage of YAC clones in Hogness Freezing Medium.

To date, frozen storage of YAC libraries have relied on the administration of glycerol to the medium subsequent to cell growth. By adding Hogness Freezing Medium prior to inoculation, cultures can be frozen directly after cell growth, with no adverse effect on the stability of the YAC DNA or on the viability of the cells even after repeated freezing and defrosting. Although a relatively simple modification, the procedure notably improves the handling of YAC libraries and significantly reduces the risk of contamination, especially when dealing with large numbers of clones.

Transgenic Animals with Various YAC Constructs: Preparation of 400 kb YAC DNA for Microinjection

Transgenic Animals with Various YAC Constructs: Preparation of 400 kb YAC DNA for Microinjection

Human Cart-1: Structural Organization, Chromosomal Localization, and Functional Analysis of a Cartilage-Specific Homeodomain cDNA

Homeoproteins control cell fates during development, specifying pattern formation and the ontogeny of specific tissues and organs in embryogenesis. Cart-1 cDNA was recently cloned from a rat chondrosarcoma tumor and it encodes a protein containing a paired-like homeodomain that is selectively expressed in cartilage during early chondrocyte differentiation. Here we report the molecular cloning of the human Cart-1 cDNA from a HeLa cervical carcinoma cDNA library. The human Cart-1 cDNA sequence is 88% identical and the deduced amino acid sequence is 95% identical to the rat sequence, indicating that Cart-1 structure is highly conserved. Northern and reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed Cart-1 mRNA expression in HeLa cervical carcinoma cells and human cervical tissue, but Cart-1 mRNA was not detected in GH3 rat pituitary cells and murine 10T1/2 one-half fibroblast cells. The Cart-1 gene was localized to human chromosome 12 and regionally mapped to the 12q21.3-q22 by PCR analysis of rodent-X-human somatic cell hybrid DNA and the CEPH megabase-insert YAC DNA pools, respectively. The Holt-Oram syndrome, characterized by upper limb and atrial septal dysplasias, also maps to the 12q21.3-q22 region. Cotransfection studies show that Cart-1 inhibits the rat prolactin promoter and that this repression is mediated by footprint II, an AT-rich element that functions as an inhibitory site of prolactin gene expression in nonpituitary cells and which was used to clone Cart-1. Taken together, these data indicate that Cart-1 may also influence cervix development, identify a putative DNA binding site for Cart-1, and, begin to define its functional role as modulator of gene expression.

Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA.

Construction of animal models of human inherited diseases is particularly important for testing gene therapy approaches. Towards this end, we constructed a mouse model for Charcot-Marie-Tooth disease type 1A by pronuclear injection of a YAC containing the human PMP22 gene. In one transgenic line, the YAC DNA is integrated in about eight copies and the PMP22 gene is strongly expressed to give a peripheral neuropathy closely resembling the human pathology. The disorder is dominant, causes progressive weakness of the hind legs, and there is severe demyelination in the peripheral nervous system including the presence of onion bulb formations. This approach will be valuable for pathologies produced by over-expression of a gene including trisomy and amplification in cancer. Such models will be particularly useful for testing gene therapy approaches if the transgene is human.

YAC DNA preparation and labelling for high throughput FISH analysis.

Journal Article YAC DNA preparation and labelling for high throughput FISH analysis Get access David Markie, David Markie * Paediatric Research Unit, UMDS, 7th Floor Guy's Tower, Guy's HospitalLondon SE1 9RT, UK * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Angela Davies Angela Davies Paediatric Research Unit, UMDS, 7th Floor Guy's Tower, Guy's HospitalLondon SE1 9RT, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 23, Issue 4, 25 February 1995, Pages 720–721, https://doi.org/10.1093/nar/23.4.720 Published: 25 February 1995 Article history Revision received: 16 January 1994 Accepted: 16 January 1994 Received: 09 December 1994 Published: 25 February 1995

CDNA selection from 10 Mb of chromosome 21 DNA: efficiency in transcriptional mapping and reflections of genome organization.

The technique of cDNA hybridization selection has been applied individually to 16 YAC clones mapping to various regions of the long arm of human chromosome 21. YACs represented approximately 10 Mb of non-overlapping DNA, and cDNA sources included fetal brain, whole fetus, and adult testes, thymus, liver and spleen. Sequencing, Northern analysis, RT-PCR and cDNA library screenings have been used to identify and partially characterize a non-redundant set of novel genes. A preliminary analysis strategy of the selected cDNAs has proven rapid and effective for isolation of the more highly represented genes and is suitable for survey transcriptional mapping efforts. By scaling up to screen > 1000 cDNA fragments per 100 kb of YAC DNA, more rare transcripts are identified and lead to comprehensive gene maps. Strong regional variations in transcriptional activity were observed, with gene densities ranging from < 1/2000 kb to > 1/15 kb. This effort has produced a large number of new genes of potential relevance to Down Syndrome.

Arrayed preparation of YAC DNA for pulsed field gel analysis.

Journal Article Arrayed preparation of YAC DNA for pulsed field gel analysis Get access David Markie David Markie Paediatric Research Unit, Division of Medical and Molecular Genetics, United Medical and Dental Schools of Guy's and St Thomas' hospitals, 7th Floor Guy's Tower, Guy's HospitalLondon SEI 9RT, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 23, Issue 21, 11 November 1995, Pages 4526–4527, https://doi.org/10.1093/nar/23.21.4526 Published: 01 January 1995 Article history Published: 01 January 1995 Received: 22 June 1995 Accepted: 13 October 1995

Molecular Cloning, cDNA Sequence, and Chromosomal Localization of the Human Phosphatidylinositol 3-Kinase p110α (PIK3CA) Gene

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme comprising a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that binds to tyrosine phosphopeptide sites linked directly or indirectly to receptors serving diverse signal functions. Knowledge of the structure and function of PI 3-kinase was greatly advanced by the purification, cDNA cloning, and subsequent expression of the bovine enzyme. Here the cloning of the cDNA for the human p110 alpha subunit of PI 3-kinase (PIK3CA), encoding a protein 99% identical to the bovine p110, and of its gene in YAC is described. The chromosomal localization of the gene for PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the gene in 3q26.3.

Isolation of a human YAC contig encompassing a cluster of UGT2 genes and its regional localization to chromosome 4q13.

Previously we mapped the gene encoding a human bile acid UDP-glucuronosyltransferase (UGT2B4) to chromosome 4. Here we report the mapping of two additional human UGT2B genes to chromosome 4 utilizing the polymerase chain reaction (PCR) and a panel of human/rodent somatic cell hybrid cell lines. A yeast artificial chromosome contig containing the UGT2B4, UGT2B9, and UGT2B15 genes was isolated, and pulsed-field gel electrophoresis and PCR revealed that several members of the human UGT2B gene subfamily are clustered within a 195-kb region of the YAC contig. These data permitted a provisional ordering of the genes as UGT2B9-UGT2B4-UGT2B15. Fluorescence in situ hybridization analysis, using the YAC DNA, permitted the regional localization of this gene cluster to chromosome 4q13.

Regional localization of 725 human chromosome 7-specific yeast artificial chromosome clones.

Toward the construction of a complete physical map of human chromosome 7, we have localized 725 YAC clones to cytogenetically defined regions using fluorescence in situ hybridization (FISH) and by screening with DNA markers of known chromosomal locations. These chromosome 7-specific YAC clones are part of a library constructed with DNA isolated from monochromosomal 7 human-hamster somatic cell hybrid lines. The FISH mapping for 575 clones was accomplished by using "Alu-PCR" amplified YAC DNA against metaphase chromosome spreads made from a monochromosomal 7 human-mouse somatic cell hybrid line. Hybridization- or PCR-based screening of previously mapped DNA markers was performed for the mapping of 221 YAC clones. There was excellent correlation between the map locations obtained for the 71 YACs localized with both methods. All of the regionally localized YAC clones are valuable reagents for mapping and identification of disease genes on human chromosome 7.

Structure of the Human N-Cadherin Gene: YAC Analysis and Fine Chromosomal Mapping to 18q11.2

The cadherins are a large family of cell adhesion molecules involved in calcium-dependent recognition and adhesion events. We have used YAC analysis to determine the structure of the human N-cadherin gene. An 850-kb YAC was isolated and the entire N-cadherin gene mapped to a 250-kb region spanning three putative CpG islands. A PCR and cosmid subcloning strategy was used to define the boundaries for the 16 exons that compose the gene. These were shown to be not only highly conserved between mouse and human N-cadherin genes, but also similar to other cadherins. The first and second introns of the gene are large, a property conserved between the mouse and human genes. In situ hybridization with YAC DNA refined the map position of N-cadherin to human chromosome 18q11.2.

Purification and Characterization of YACs Containing Large Inserts

This unit provides protocols for characterizing DNA segments cloned in YACs and for purifying YACs from yeast chromosomes. The first basic protocol describes Southern blotting and partial-digest restriction analysis of YACs. These methods are useful for determining the size and complexity of the cloned insert DNA, the presence and location of particular restriction sites or sequences, and even the species of origin of the insert DNA (indicated by hybridization to species-specific repetitive elements such as Alu repeats). The second basic protocol describes gel purification of YACs for use in procedures requiring pure YAC DNA, such as mammalian-cell transformation and subcloning into smaller insert vectors. The third basic protocol details characterizing and analyzing YACs: in vivo fragmentation via homologous recombination with specialized fragmentation vectors containing specific probe sequences or repetitive elements, followed by Southern blotting with YAC- and human-derived probes.

Targeted Recombination-Based Cloning and Manipulation of Large DNA Segments in Yeast

YAC cloning technology has contributed significantly to physical mapping of genomes and holds enormous potential for identification and functional analysis of genes on large DNA segments. This potential is greatly enhanced by the ability to use homologous recombination-based methodologies to restructure or manipulate exogenous DNA in the yeast host. Three methods are described for manipulation of YAC DNA. "Recombinationally targeted YAC cloning" involves the capture of large DNA segments by recombination in vivo between the YAC vector arms and a target DNA segment. "Two-step gene replacement" exploits targeted homologous recombination to replace a selected segment within a YAC with mutant or exogenous sequences. "YAC transfer by kar1 mating" allows rapid movement of YACs under study into yeast genetic backgrounds that facilitate homologous recombination-based manipulation. These three methods taken together will aid significantly in the construction of physical genome maps and provide powerful genetic tools for the study of genome function.

Human PCK1 encoding phosphoenolpyruvate carboxykinase is located on chromosome 20q13.2.

Cytoplasmic liver phosphoenolpyruvate carboxykinase (GTP) (PEPCK) catalyzes a rate-limiting step in gluconeogenesis. Primers derived from the rat liver PEPCK sequence were used to amplify a portion of the human liver cDNA and to screen a YAC library of human genomic DNA. The sequences of human and rat PEPCK cDNA differed at 16% of the nucleotides compared (45/291). Analysis of a human/rodent hybrid mapping panel demonstrated concordant segregation of PCK1 with chromosome 20. Fluorescence in situ hybridization with YAC DNA further localized PCK1 to subband 20q13.2.

Physical and transcriptional mapping of DXS56-PGK1 1 Mb region: identification of three new transcripts.

Several new techniques for isolation expressed sequences have been recently described considerably speeding up the identification of unknown genes. Here we present a transcriptional map of the 1 Mb DXS56-PGK1 region in Xq13.3. Rare cutter restriction site mapping, direct cDNA selection on membrane discs and probing of Northern blots with total YAC DNA, were the methods explored in order to achieve this goal. In addition to three known genes from this region which have been recloned, two new cDNA clones corresponding to two new genes were isolated, mapped and characterized. Moreover one more transcript, highly expressed in placenta, has been detected in the region with a total YAC as a probe. In summary there are at least six genes known to reside in the DXS56-PGK1 region. As several human disease gene loci (i.e. SCID, CMTX1, WWS, MRX, XDP, ASB) were tightly linked to the markers from the region (PGK, CA repeats), the three new transcripts may be considered as their potential candidate genes.

Analysis of Isolated YAC Clones

This unit provides a series of protocols describing the analysis and manipulation of an isolated YAC clone. The procedures are based upon the use of the YAC vector pYAC4. Once an isolated YAC clone has been obtained from a core laboratory, the clone can be analyzed as described herein. Methods for analysis involve growing and storing YAC-containing yeast strains and purifying YAC DNA in a form suitable for assessing the size of the artificial chromosome and for conventional Southern blotting. Preparation of yeast chromosomes in agarose plugs for subsequent analysis by pulsed-field gel electrophoresis is also described. Additional protocols are provided for recovering DNA fragments from the ends of a YAC genomic insert to be used as probes for detecting chimerism and for chromosome walking. Finally, preparation of high-molecular-weight YAC DNA is described and a general method for subcloning YAC inserts into cosmid or lambda vectors for higher-resolution analysis is provided.

Genomic organization of the human folate receptor genes on chromosome 11q13

There has been interest in the high affinity folate receptor (FOLR) recently because of its high expression in the majority of ovarian tumors. The FOLR genes are part of a family that includes an adult gene, a fetal gene, and one or more pseudogenes, which have been localized to chromosome 11. As a step toward understanding why the adult FOLR gene product is expressed on tumors, we have determined the organization of all the human FOLR-related genes. YAC clones were isolated using the adult FOLR probe. The organization of the locus was determined by PFGE of YAC DNA and by YAC fragmentation. Four FOLR-related genes were found within 140 kb. The adult and fetal genes are not more than 23 kb apart, with the 3' end of the adult gene facing the 5' of the fetal gene. A physical map of over 900 kb of the surrounding region was also constructed. The chromosomal assignment of the FOLR locus was refined to 11q13.3-q13.5 telomeric of the FGF3 locus using fluorescence in situ hybridization.

Overview of Strategies for Screening YAC Libraries and Analyzing YAC Clones

This unit provides an introduction to the use of yeast artificial chromosome-bearing yeast clones (hereafter referred to as YAC clones) in genome analysis. It describes criteria for designing a polymerase chain reaction (PCR) assay to be used in screening a YAC core library and discusses the rationale for verification and characterization of YAC clones obtained from these core laboratories. Protocols for maintaining YAC clones, analyzing YAC insert structure, preparing YAC DNA, and subcloning YAC inserts into other vectors are presented elsewhere in this volume.

Ligation-mediated PCR of restriction fragments from large DNA molecules.

A general method is described for PCR amplification of single restriction fragments from large DNA molecules. The method involves sequence-specific ligation of synthetic oligonucleotides to ambiguous 4-base 5' overhangs produced by type IIS restriction endonucleases. Such "adapter-tags" provide one target for primer annealing in subsequent PCR reactions. The second target for primer annealing is provided by a universal "bubble-tag" ligated to blunt ends produced with another endonuclease. The key advantage of this approach is that specific fragments can be isolated without any prior knowledge of the nucleotide sequence of the target. Using bacteriophage lambda DNA as a test system, unique PCR products could be generated consistently. Conditions of temperature, ionic strength, and substrate concentration in the adapter-tag ligations--which affect sequence specificity--were found to have a major influence on the purity of PCR-generated fragments. In principle, the method permits the amplification of virtually any sequence from purified cosmid or YAC DNA using a library of only 240 adapter-tags.