De novo formation of several features of a centromere following introduction of a Y alphoid YAC into mammalian cells

The DNA sequence requirements for mammalian centromere functionhave been Investigated by re-Introducing human YAC clones containingeither centromeric or non-centromeric sequences Into hamsterand human cells. All YACs integrated into the host chromosomes.In most cell lines produced by spheroplast fusion into hamstercells, intact copies of the YAC and a large amount of yeastDNA were found. Cell lines produced by lipofection Into humancells usually contained simple structures without yeast DNA.YACs containing Y alphoid DNA reformed several of the propertiesof a centromere, Including a cytogenetlcally visible constriction,CREST antiserum binding and disruption of anaphase chromosomemovement. In contrast, YACs containing non-centromeric sequencesproduced none of these results. This work suggests that a fewhundred kb of alphoid DNA is sufficient to reconstitute severalImportant features of a centromere.     

New vector for transfer of yeast artificial chromosomes to mammalian cells

A modification vector has been constructed to facilitate the transfer of yeast artificial chromosomes (YACs) to mammalian cells in culture by targeting a dominant selectable marker (G418 resistance) to the right arm of pYAC4 clones. The ADE2 gene is used for yeast selection with consequent disruption of the URA3 gene, allowing direct modification of YACs within the common host strain AB1380, and providing a simple test for correct targeting. This vector has been tested by modification of a 550-kb YAC containing part of the human MHC class II region and transfer to CHO cells by protoplast fusion. Analysis of 15 independent G418-resistant CHO lines obtained following fusion suggests the majority contain a complete YAC with moderate amplification in some lines.     

The proopiocortin (adrenocorticotropin/β-lipotropin) gene is located on chromosome 2 in humans

The proopiocortin gene is located on chromosome 2 in humans. A 13-kb DNA fragment containing proopiocortin gene sequences was identified in human cells while proopiocortin-related gene sequences of 9.8 and 6.2 kb were present in mouse cells. In human-mouse cell hybrids which contained reduced numbers of human chromosomes and a complete set of mouse chromosomes, the 9.8- and 6.2-kb fragments were always present while the 13-kb fragment segregated with human chromosome 2 and the chromosome 2 enzyme markers acid phosphatase-1 (ACP1), malate dehydrogenase-1 (MDH1), and isocitrate dehydrogenase-1 (IDH1). Analysis of a single cell hybrid with a broken chromosome 2 indicates that the proopiocortin andACP1 genes are closely linked and in the distal region of the short arm of chromosome 2.     

A large inverted duplicated DNA region associated with an amplified oncogene is stably maintained in a YAC

In the polyoma virus (Py) transformed 3B rat cell line the Pyoncogene and adjacent cellular DNA are amplified in arrays ofvery large inverted duplications. A region of the 3B amplifiedDNA was cloned as a 550 kb insert in a Yeast Artificial Chromosome(YAC) vector, designated y3B01. Analysis of the y3B01 clonedinsert revealed it contained a large inverted duplicated DNAregion which was approximately 400 kb in size (two palindromicarms of about 200 kb). At least 420 kb of the 550 kb YAC inserthas been identified as being derived from the 3B amplified DNAand the amplicon in 3B cells is at least 220 kb in size. NoDNA instability of the y3B01 YAC clone was detected. The y3B01DNA replicated as efficiently as yeast chromosomes and was structurallystable in yeast cells during more than 30 cell divisions. Comparisonof the restriction endonuclease maps of the inverted duplicatedregion of the y3B01 DNA insert and the amplified 3B genomicDNA did not reveal any gross differences suggesting that norearrangements had occurred during or after the cloning intothe YAC vector. These results suggest that large inverted duplications,which can show instability in prokaryotic cloning systems, canbe stably cloned and maintained in YAC vectors in yeast.     

Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.

The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.     

A cre-lox recombination system for the targeted integration of circular yeast artificial chromosomes into embryonic stem cells

The ability to produce embryonic stem (ES) cell lines containing different yeast artificial chromosomes (YACs) integrated into the same location in the genome provides a system for comparing the bio-logical effects of YAC transgenes without the confounding influences of integration site and copy number. A targeting system was developed for the directed integration of circular YACs into mouse ES cells. The system combines Cre-lox recombination technology, specifically a positive-selection integration system, with circular YAC lipofection technology to achieve single copy targeted integration of a transgene. Three independent germline competent ES cell lines [lox-containing ES lines (designated LES)] were created that contain a '-neo-lox' cassette integrated at different sites within the ES genome. A plasmid containing YAC vector sequences and a complementary '-neo-lox' cassette was used to circularize two linear YACs containing genomic DNA from human chromosome 21. The circularized YACs were then targeted to the lox sites of the LES cell lines. Polymerase chain reaction and Southern analysis demonstrated that 21% (5 of 24) of lox-recombinants contain a full-length intact YAC. This system will make the study of YAC transgenic mice more reliable and reproducible, allowing the potential for direct comparison of different transgenes expressed from the same site within the genome.     

Expression of the human CFTR gene from episomal oriP-EBNA1-YACs in mouse cells

Plasmids carrying the origin of plasmid replication ( oriP ) and expressing the EBNA-1 protein from the Epstein-Barr virus replicate and segregate in human cells and are thus potentially useful vectors for gene therapy. As very large circular molecules, up to 660 kb in size, can be maintained episomally using this system, it is possible to include intact human genes with all their long-range controlling elements which might give high levels of tissue-specific and controlled gene expression. We have shown previously that a 320 kb yeast artificial chromosome (YAC) carrying the intact human CFTR gene can complement the Cambridge null cystic fibrosis mice as a transgene. We have now modified this YAC to a circular molecule carrying both oriP and the EBNA-1 gene. We show that this oriP-EBNA1-YAC can be stably maintained as unrearranged episomes in mouse LA-9 cells, which do not express endogenous cftr, and in mouse CMT-93 cells, which do express endogenous cftr. The human CFTR gene is expressed in some of the cell lines, but the level of expression is very variable between cell lines and is not related to the copy number of the elements.     

Five skeletal myosin heavy chain genes are organized as a multigene complex in the human genome

Myosin heavy chain (MyHC) isoforms are encoded by a multigenefamily in vertebrates. We used genomic DNA mapping by pulsefield gel electrophoresis to demortstrate that, in humans, theembryonic, fetal, fast IIB and IIX MyHC genes and a gene codingfor a non-identified striated muscle MyHC fast-type isoform(NI), are contained within a 320 kb SalI genomic fragment. Thelocus is flanked by two CpG islands, separated by 580 kb. Inorder to further characterize the MyHC genes, a human genomiclibrary constructed in yeast artificial chromosomes (YAC) wasscreened and five independent clones were Isolated. Characterizationof these YACs revealed that one of them contains at least fiveMyHC genes, based on partial sequencing of their conserved thirdcoding exons. Three of these genes correspond to those encodingthe embryonic, fetal and fast IIB MyHC isoforms. Moreover, inthis YAC done the embryonic and fetal genes, on the one hand,and the adult fast (IIB, IIX and NI) genes, on the other hand,are contained within two different ClaI fragments. This resultsuggests that the genes encoding the two developmental formsare adjacent In the human genome and that temporal regulationof the MyHC genes might be related to their organization withinthe locus. These data represent the first direct evidence forthe existence in the human genome of a MyHC multigene locusthat contains at least five genes.     

Characterization of a complex chromosome rearrangement involving 6q in a melanoma cell line by chromosome microdissection

Deletion of 6q is one of the most frequent chromosomal alterations in human malignant melanoma. Recently, we used chromosome painting probes of 6p and 6q to study 21 melanoma cell lines. A reciprocal translocation between chromosomes 6q and 17p was detected in one cell line (UACC-930). Upon further characterization of the translocation marker using the micro fluorescence in situ hybridization (FISH) technique, a complex rearrangement including an inversion of 6q and a translocation between the inverted 6q and 17p, [der(6)inv(6)(q16q27)t(6;17)(q26;p13)], was detected. A yeast artificial chromosome (YAC) clone spanning the breakpoint at 6q16 was isolated by the FISH screen. Loss of one or more copies of the YAC clone was also detected in 10 of 12 melanoma cell lines. This result implies that the YAC clone may contain a putative tumor suppressor gene related to the pathogenesis of malignant melanoma. Further characterizations of the breakpoint at 6q16 and molecular cloning breakpoints at 6q27 and 17p13 are in progress.     

Introduction of YACs containing a putative mammalian replication origin into mammalian cells can generate structures that replicate autonomously

Yeast artificial chromosomes (YACs) containing or lacking a biochemically defined DNA replication origin were transferred from yeast to mammalian cells in order to determine whether origin-dependent autonomous replication would occur. A specialized YAC vector was designed to enable selection for YACs in mammalian cells and for monitoring YAC abundance in individual mammalian cells. All of eight clones made with linear and circularized YACs lacking the origin and seven of nine clones made with linear and circularized YACs containing the origin region contained single copies of the transfected YAC, along with various amounts of yeast DNA, integrated into single but different chromosomal sites. By contrast, two transformants derived from circularized YACs containing the putative replication origin showed very heterogeneous YAC copy number and numerous integration sites when analyzed after many generations of in vitro propagation. Analysis of both clones at an early time after fusion revealed variously sized extrachromosomal YAC/yeast structures reminiscent of the extrachromosomal elements found in some cells harboring amplified genes. The data are consistent with the interpretation that YACs containing a biochemically defined origin of replication can initially replicate autonomously, followed by integration into multiple chromosomal locations, as has been reported to occur in many examples of gene amplification in mammalian cells.     

Three genes that escape X chromosome inactivation are clustered within a 6 Mb YAC contig and STS map in Xp11.21-p11.22

In order to study the distribution of genes that escape X chromosomeinactivation, a high density yeast artificial chromosome (YAC)contig and STS map spanning approximately 6 Mb has been constructedin Xp11.21-p11.22. The contig contains 113 YACs mapped with53 markers, including 10 genes. Four genes have been assayedfor their expression status on both the active and inactivehuman X chromosomes, and these data have been combined withprevious results on two other genes in the contig. Three ofthese genes escape X inactivation and have been localized toa single YAC clone of {small tilde}1075 kb. The other threegenes are subject to inactivation, with two of them lying amongthe genes that escape inactivation. These results suggest thatthere are both regional control signals as well as gene-specificelements that determine the X inactivation status of genes onthe proximal short arm of the human X chromosome.     

Localization of the synovial sarcoma t(X;18)(p11.2;q11.2) breakpoint by fluorescence in situ hybridization.

A high proportion of synovial sarcomas contain a chromosome translocation t(X;18)(p11.2;q11.2). We have previously used somatic cell hybrids derived from an established cell line, SS255, to map the X chromosome breakpoint to the interval flanked by the markers DXS14 and DXS146. In this study we have examined these hybrids with thirteen additional markers located at Xp11.3-Xcen, by Southern hybridization. Based on these results we have delimited the breakpoint as follows Xpter-DXS228-(UBE1-OATL1-TIMP-DXS226 )-(DXS255-TFE3-ELK1-DXS146)-OATL2- X;18-(DXS14-DXS422-DXS423-DXS674-DXS679)-+ ++Xcen. Confirmation of the breakpoint location has been obtained by analysis of two synovial sarcoma cell lines, SS255 and HA2243, using fluorescence in situ hybridization. A 350kb YAC probe spanning the DXS423 locus hybridized only to the derivative X chromosome, showing that it maps proximal to the breakpoint. Two YAC probes of 300kb and 450kb, containing the OATL2 locus, hybridized to both derivative chromosomes, indicating that these YACs span the translocation breakpoint. Similar results were obtained with both cell lines. The identification of YACs that span the t(X;18) breakpoint now facilitates a strategy for cloning candidate genes from this precisely defined region.     

Homozygous deletion on the chromosomal region 5q12.3 in human lines of small-cell lung cancers

 Loss of heterozygosity at the polymorphic loci on the long arm of chromosome 5 is observed in about 80% of human small-cell lung cancer (SCLC). Absence of inactivating mutations in the APC gene on 5q14 suggests the involvement of another tumor suppressor gene. We found a homozygous deletion of sequence tagged site sequence G73332 on 5q12.3 in 2 of 12 human SCLC cell lines, Lu130 and Lu134. One copy of chromosome 5q was lost in these cell lines, and the remaining allele had a deletion in a more restricted region. A polymerase chain reaction-based analysis of yeast artificial chromosome, bacterial artificial chromosome (BAC), and lambda-phage clones narrowed the region of homozygous deletion to a fragment cloned within one BAC. Sequencing analysis revealed that a DNA fragment of approximately 25 kb was deleted interstitially, probably because of recombination through Alu repetitive sequences in Lu130 and Lu134 cells. This deletion was not detected in normal lymphocyte DNA from 98 unrelated individuals. No candidate genes, however, were detected within this region or in the adjacent 150-kb fragment. The absence of microsatellite instability and the presence of an interstitial deletion as well as gross chromosomal aberration suggest that the genomic integrity of Lu130 and Lu134 cells might possibly be affected by Alu-mediated recombination in addition to chromosomal instability. The identical breakpoints in Lu134 and Lu135 cells as well as the same genotypes at all 33 polymorphic loci examined on various chromosomes strongly suggest that these cell lines share the same genetic materials, at least in part, during the establishment or propagation of cell lines. Received: January 30, 2002 / Accepted: March 25, 2002     

Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes

The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. CFTR expression is controlled temporally and cell specifically by mechanisms that are poorly understood. Insight into CFTR regulation could be facilitated by the successful introduction of the entire 230 kb human CFTR and adjacent sequences into mammalian cells. To this end, we have introduced two different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells. Clonal cell lines containing human CFTR were identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described. Integration of the human CFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescence in situ hybridization (FISH). Southern blot analysis suggested that on the order of one copy of human CFTR was integrated per CHO cell genome. Fiber-FISH and restriction analysis suggested that CFTR remained grossly intact. Northern analysis showed full-length, human CFTR mRNA. Immunoprecipitation followed by phosphorylation with protein kinase demonstrated mature, glycosylated CFTR. Finally, chloride secretion in response to cAMP indicated the functional nature of the human CFTR. This study provides several novel results including: (i) functional human CFTR can be expressed from these YACs; (ii) CHO cells are a permissive environment for expression of human CFTR; (iii) the level of human CFTR expression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH of CFTR integrity correlates with functional gene expression. These YACs and the cell lines derived from them should be useful tools for the study of CFTR expression.      

Rescue of Targeted Regions of Mammalian Chromosomes by in Vivo Recombination in Yeast

In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in ~1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system.     

A YAC-based binning strategy facilitating the rapid assembly of cosmid contigs: 1.6 Mb of overlapping cosmids in Xp22

We have applied a yeast artificial chromosome (YAC)-based cosmidisolation and binning strategy to convert a YAC contig in Xp22into 1.6 Mb of overlapping cosmids. This strategy is based onthe screening of a high-density arrayed X chromosome-specificcosmid library with large YAC-derived restriction fragmentsand entire YAC probes. Cosmids selected in this way were griddedon dot blots and further mapped into bins defined by the overlapintervals of the YACs and YAC fragments. This rapid binningof cosmids simplified the subsequent assembly of cosmid contigsby restriction fingerprint hybridization. In total, we identified139 cosmids spanning the entire 1.6 Mb region with a minimaloverlap set of 53 clones. These cosmids were assigned to 17bins and 9 contigs. One of the contigs is 665 kb in length andis one of the largest uninterrupted cosmid contigs in humansreported to date. The gaps between the contigs are minor and,together, they represent less than 7% of the region covered.Two previously identified genes are contained in these cosmids,the gene for amelogenin (AMG) and the recently isolated putativechloride channel gene CICN4. In addition, two disease loci havebeen mapped to this region: X-linked ocular albinism type 1(OA1) and the microphthalmia with linear skin defects (MLS)syndrome. The assembly of the cosmid maps allowed us to determinethe size of the deletion intervals for these two loci, whichwere estimated to be 110 kb for OA1 and 570 kb for MLS. Thesecosmid contigs will greatly facilitate the positional cloningof the OA1 and MLS disease genes. Together with the Huntingtondisease gene region on chromosome 4, this region in Xp22 representsone of the best characterized large regions in the human genome.     

Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2) to different regions of the X chromosome and assignment of two PRPS1-related genes to autosomes

Complementary DNA clones for phosphoribosylpyrophosphate synthetase subunits I and II (PRS I and PRS II) were used to determine the chromosomal localization of the corresponding human genes. Southern blot analysis of genomic DNAs isolated from human placenta and a panel of humanmouse somatic cell hybrids revealed that the rat PRS I cDNA probe detected at least five human specific DNA segments (23, 20, 14.5, 6.7, and 4.3 kb) in BamHI digests. The 23-, 14.5-, and 6.7-kb DNA segments were detected only if the hybrids contained human chromosome X or translocation chromosome 7p ⁺ (7qter>7p22::Xq21>Xqter), indicating the location of these segments to Xq21-qter (PRPS1). The 20- and 4.3-kb DNA segments did not cosegregate with the other three segments, and spot blot hybridization analysis using flow-sorted human chromosomes indicated that these are the PRPS1-related genes (PRPS1L1 and PRPS1L2) and could be assigned to chromosomes 7 and 9, respectively. The human-specific PRS II cDNA probe revealed a BamHI DNA segment (17 kb), which segregated condordantly with the X chromosome but not with the PRPS1 gene. We surmise that the gene for PRS II (PRPS2) is located at a different region of the X chromosome, namely Xpter-q21.Preliminary report of this research was presented at Ninth International Workshop on Human Gene Mapping, Abstract supplement p. 5 (1987).