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.     

The isolation of a yeast artificial chromosome (YAC) contig extending for 2 megabases in the vicinity of the Von Hippel Lindau disease gene

Von Hippel LIndau disease (VHL) is a rare autosomal dominantdisease associated with tumors and cysts in multiple organ systems.The VHL disease gene is tightly linked to the polymorphic DNAmarker 233E2 (D3S720) and flanked by 479H4 (D3S719) on its telomericand RAF1 on its centromeric side. Two additional markers, D3S1038and D3S601, have also been identified, and these markers, likeD3S720, are very tightly linked to VHL. Previously 93 cosmidclones were mapped to the larger region, 3p24.2 - pter, surroundingthe VHL disease gene (1). Using a Southern-based screening strategyon pools of YAC clones we have Isolated a contig of overlappingYAC clones that extends about 0.7 megabase centromeric, andabout 1.3 megabases telomeric of D3S720 and contains all threetightly linked VHL markers. Individual YACs In this contig werehybridized to grids containing cosmids localized between 3p24.2-pterand to several cosmids localized by fluorescent in situ hybridization(FISH) to 3p25. A total of 28 cosmids were positioned on thiscontig of overlapping YAC clones. We have also identified homologousYAC clones to many additional cosmid clones localized between3p24.2–p25, although these have not yet been preciselylocalized relative to the contig of YAC clones. This contigof YAC clones probably contains the VHL disease gene and shouldfacilitate the isolation and characterization of this gene.     

Integrated YAC Contig Map of the Prader–Willi/Angelman Region on Chromosome 15q11–q13 with Average STS Spacing of 35 kb

Prader–Willi syndrome and Angelman syndrome are associated with parent-of-origin-specific abnormalities of chromosome 15q11–q13, most frequently a deletion of an ~4-Mb region. Because of genomic imprinting, paternal deficiency of this region leads to PWS and maternal deficiency to AS. Additionally, this region is frequently involved in other chromosomal rearrangements including duplications, triplications, or supernumerary marker formation. A detailed physical map of this region is important for elucidating the genes and mechanisms involved in genomic imprinting, as well as for understanding the mechanism of recurrent chromosomal rearrangments. An initial YAC contig extended from D15S18 to D15S12 and was comprised of 23 YACs and 21 STSs providing an average resolution of about one STS per 200 kb. To close two gaps in this contig, YAC screening was performed using two STSs that flank the gap between D15S18 and 254B5R and three STSs located distal to the GABRA5–149A9L gap. Additionally, we developed 11 new STSs, including seven polymorphic markers. Although several groups have developed whole-genome genetic and radiation hybrid maps, the depth of coverage for 15q11–q13 has been somewhat limited and discrepancies in marker order exist between the maps. To resolve the inconsistencies and to provide a more detailed map order of STSs in this region, we have constructed an integrated YAC STS-based physical map of chromosome 15q11–q13 containing 118 YACs and 118 STSs, including 38 STRs and 49 genes/ESTs. Using an estimate of 4 Mb for the size of this region, the map provides an average STS spacing of 35 kb. This map provides a valuable resource for identification of disease genes localized to this region as well as a framework for complete DNA sequencing.     

Physical map of a YAC contig containing the region of the human gene (HYRC) complementing hyper-radiosensitivity of the scid mouse mutation

Summary We previously mapped the putative humanHYRC (the hyper-radiosensitivity of the scid mutation, complementing gene) to human chromosome 8q11.1 by fluorescencein situ hybridization (FISH) using Alu-based PCR products from a mouse-human scid radiation cell hybrid (RD15/5) as probes. From a cosmid library constructed from RD15/5, 57 cosmid clones containing human DNA inserts were isolated, 18 of which were mapped to 8q11. Based on the sequences of plasmid subclones of the 18 cosmids, five novel sequence-tagged-sites (STSs) were made. By a screening of the CEPH-YAC library with these STSs, five yeast artificial chromosome, (YAC) clones were isolated. All these YAC clones were confirmed not to be chimeric by FISH, but two of them showed deleted human insert DNAs. Using the other 3 non-deleted YACs, we constructed a physical map covering theHYRC region. We confirmed that the recently isolated gene (the DNA-PK_cs gene) which is a strong candidate forHYRC is located within the present contig and spans less than 200 kb. This map will be useful for the analysis of the genomic structure of the DNA-PK_cs gene and for isolation of other complementing genes in theHYRC region.     

Physical mapping of a 670-kb region of chromosomes XVI and XVII from the human protozoan parasite Trypanosoma cruzi encompassing the genes for two immunodominant antigens

As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 and jl8 loci, we used YAC, cosmid, and lambda phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.     

Angelman syndrome associated with a maternal 15q11-13 deletion of less than 200 kb

Angelman syndrome (AS) is a neurogenetic disorder arising froma lack of genetic contribution from the maternal chromosome15q11–13. To date, the AS critical region has been definedby an inherited deletion of approximately 1.5Mb, spanning the3–21 (D15S10), LS6–1 (D15S113) and GABRB3 loci.We have Identified an individual with the typical features ofAS who has a deletion of the maternal chromosome which encompassesLS6–1, but does not extend to either flanking marker.This deletion, initially detected by (CA)n repeat analysis,was further characterised by fluorescence In situ hybridisation(FISH) using cosmids derived from a 260 kb LS6–1 yeastartificial chromosome (YAC). Neither end cosmid from this YACclone falls within the deletion, suggesting that the minimalAS region Is less than 200 kb. We also studied three loci within15q11–13 which detect parent-of-origin specific DNA methylationimprints, and found that both normal maternal and paternal patternswere present in this patient.     

Characterization of a 1.0 Mb YAC contig spanning two chromosome breakpoints related to Menkes disease.

Menkes disease, an X-linked recessive disorder of copper metabolism, has recently been mapped to Xq13.3 by two Menkes patients carrying chromosome rearrangements within this region. The breakpoints have been investigated by nonisotopic in situ suppression hybridization using YACs isolated from this region with the flanking markers DXS56 and PGK1. Three YACs were extending over the breakpoints at Xq13.3 and were shown to be overlapping by partial digest restriction maps, IRS-PCR fingerprinting and by the presence of common cosmid clones. These cosmids were subcloned and one of the single copy probes detected both breakpoints using rare-cutting restriction enzyme digests of the patients. All the results together localize the breakpoints to about 100 kb within the overlapping region of the YACs. Mapping of both breakpoints in a 1 Mb YAC contig implies that these YACs contain at least partially, the gene responsible for Menkes disease.     

The genes for X-linked ocular albinism (OA1) and microphthalmia with linear skin defects (MLS): cloning and characterization of the critical regions

We have used cell lines from patients with deletions and transiocationsinvolving the Xp22 region to map the genes for two X-linkeddisorders, ocular albinism type 1 (OA1) and microphthalmia withlinear skin defects (MLS). Using existing and newly isolatedDNA markers, the map position within Xp22 of key patient breakpoints,defining the boundaries of the genomic regions involved in thesedisorders (the critical regions), has been precisely determined.A 2.6 Mb yeast artificial chromosome (YAC) contig, spanningthe critical regions for these two disorders, was assembled.Detailed long-range restriction analysis of the contig establishedthe sizes of the critical regions to be 200 kb for OA1 and 800–925kb for MLS. Ten potential CpG-islands, representing candidatesites for genes, have been mapped within the 2.6 Mb region.Our data should greatly facilitate efforts aimed at cloningthe genes for these developmental defects.     

Characterization of an 800 kb region at 3p22-p21.3 that was homozygously deleted in a lung cancer cell line

We have characterized a homozygous deletion at 3p22 –p21.3 found in a lung cancer cell line ACC-LC-5. Three yeastartificial chromosomes (YACs) were Isolated from a YAC libraryby hybridization with two cosmid probes (cCI3–1994 andcCI3–1999) representing loci that were homozygously deletedIn five lung cancer cell lines. Cloning both ends of the regiondeleted In the cell line ACC-LC-5 revealed the deletion wasan interstitial deletion within the chromosomal arm. A cosmidcontig map covering the entire region corresponding to the homozygousdeletion was constructed by means of Southern hybridizations.From these results and analyses by pulsed-field gel electrophoresis,this interstitial deletion on chromosome 3p22–21.3 inthis cell line was estimated to be nearly 800 kb long and issmallest among five cell lines containing the homozygous deletion.The cosmid clones representing this region will contribute importantnew resources for isolating the putative tumor suppressor gene(s)on chromosome 3p22 – 21.3.     

Identification of a 100-kb region of common allelic loss on chromosome bands 10q25–q26 in human endometrial cancer

In human endometrial cancer, we have previously identified a 790-kb region of common allelic loss in chromosome bands 10q25–q26, flanked by D10S587 and D10S1723. We constructed a contig covering the entire deleted region using YACs, PACs, and BACs. Five overlapping cosmid clones derived from YAC clones completely covered the entire deleted region: its size was estimated to be no larger than 200 kb. We further performed two-color fluorescence in situ hybridization (FISH) analysis to confirm the deletion and narrowed down the deleted region to 100 kb or less; it was covered by three overlapping cosmid clones that were included in one BAC clone. Restriction endonuclease mapping identified a region in which NotI, SalI, SmaI, and XhoI were clustered, suggesting the possible existence of a CpG island. Genes Chromosomes Cancer 23:74–77, 1998. © 1998 Wiley-Liss, Inc.     

A YAC contig in Xp21 containing the adrenal hypoplasia congenita and glycerol kinase deficiency genes.

The gene loci for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GK) map in Xp21 distal to Duchenne muscular dystrophy (DMD), and proximal to DXS28 (C7), by analysis of patient deletions. We have constructed a yeast artificial chromosome (YAC) contig encompassing a 1.2 Mb region extending distally from DMD, and containing DXS708 (JC-1), the distal junction clone of a patient with GK and DMD. A pulsed-field gel electrophoresis map of the YAC contig identified 3 potential CpG islands. Whole YAC hybridization identified cosmids both for construction of cosmid contigs, and isolation of single copy probes. Thirteen new single copy probes and DXS28 and DXS708 were hybridized on a panel of patients; the deletion mapping indicates that the YAC contig contains both GK and at least part of AHC, and together with the physical map defines a GK critical region of 50-250 kb. In one AHC patient with a cytogenetically detectable deletion we used the new probes to characterize a complex double deletion. Non-overlapping deletions observed in other unrelated AHC patients indicate that the AHC gene is large, extending over at least 200-500 kb. This mapping provides the basis for the identification of the AHC and GK genes.     

A proposed new contiguous gene syndrome on 8q consists of Branchio-Oto-Renal (BOR) syndrome, Duane syndrome, a dominant form of hydrocephalus and trapeze aplasia; implications for the mapping of the BOR gene

The analysis of a de novo 8q12.2–q21.2 deletion led tothe identification of a proposed previously undescribed contiguousgene syndrome consisting of Branchio-Oto-Renal (BOR) syndrome,Duane syndrome, hydrocephalus and trapeze aplasia. This is thefirst reported localization of the genes responsible for Duanesyndrome and this dominant form of hydrocephalus. In contrast,we report a new localization for the gene responsible for BORsyndrome which is more telomeric to an initial placement. Linkageanalysis of affected families consistently mapped the gene responsiblefor BOR and Branchio-Oto (BO) syndromes to within the deletion.Using new algorithms, a YAC contig was constructed and usedto localize the breakpoint of another chromosomal rearrangementassociated with BO syndrome to a 500 kb interval within thedeletion. The 8q12.2–q21.2 deletion suggests that reduceddosage of the relevant genes is sufficient to cause Duane syndrome,BOR syndrome and this dominant form of hydrocephalus.     

PAC and Cosmid Contig Spanning the HOXA Cluster on Human Chromosome 7p15

To construct the PAC and cosmid contig map spanning the HOXA cluster on human chromosome 7, we used 9 DNA markers (D7S2243, D7S3010, HOXA1, EVX1, 750, pBH8, p60, p8.0, and HOXA11), among which the final 4 were generated in this study by shotgun cloning strategy. From the libraries, 5 PAC and 35 cosmid clones were screened and as a result, an overlapping continuous array of cosmid and PAC clones covering the genomic region (about 200 kb) spanning the entire cluster were constructed. The isolated cosmids contained several consecutive HOX genes of regional group, probably sharing the regulatory processes such as alternative splicing or polyadenylation, and thus could be used as useful materials for elucidating the molecular mechanism of HOX gene expression in the future.     

Construction of a YAC contig and a STS map spanning at least seven megabasepairs in chromosome 5q34-35

We have constructed a YAC contig containing 54 clones and aminimum of 7 Mbp of human DNA, that maps to bands q34–35on chromosome 5. The contig was nucleated using FISH mappedcosmid clones shown to flank the t(2; 5)(p23;q35) translocationbreakpoint in a CD30–positive large cell lymphoma cellline. Thirty of the 54 YAC clones are non-chimeric and six spanthe translocation breakpoint, as determined by FISH analysis.A total of 28 YAC clone end fragments, 14 non-polymorphic YACend STS probes and 13 polymorphic microsatellite STS markershave been used to order clones within the contig. The most distalgenetic markers (D5S498 and D5S619) are separated by 15 cM basedon multipoint linkage analysis. This map of overlapping clonesand the set of densely spaced physical markers will promoteour understanding of the 5q34–35 reglon and its associatedgenes.     

Genetic and physical characterization of the early-onset Alzheimer's disease AD3 locus on chromosome 14q24.3

Genetic linkage studies have provided significant evidence thata major gene defect, AD3, for familial early-onset Alzheimer'sdisease (EOAD) is located at chromosome 14q24.3, between theshort tandem repeat (STR) markers D14S52 and D14S53 defininga genetic size of 22.7 cM for the AD3 candidate region. We constructeda physical map of the AD3 region using yeast artificial chromosomes(YACs) selected from both the CEPH and megaCEPH YAC librariesusing the AD3 linked STR markers as well as new sequence-taggedsites (STSs) designed based on YAC terminal sequences. The YACmap is contiguous in the region between D14S258 and D14S53,a region of 8.2 cM, and has an estimated physical size of 4–8Mb. The YAC contig map was used as a framework to localize threeknown genes, a pseudogene and two brain expressed sequence tags(ESTs). Linkage analysis studies in two Belgian chromosome 14EOAD families AD/A and AD/B, identified obligate recombinantsin family AD/A with D14S289 and D14S61 reducing the geneticsize of the candidate AD3 region substantially. The minimalAD3 candidate region measured 6.4 cM on the genetic map andis contained within six overlapping megaCEPH YACs that covereda physical distance estimated between 2 and 6 Mb. These YACsas well as other YACs in the YAC contig map are valuable resourcesin gene cloning efforts or genomic sequencing experiments aimingat isolating the AD3 gene.     

Bacterial Contig Map of the 21q11 Region Associated with Alzheimer’s Disease and Abnormal Myelopoiesis in Down Syndrome

We present a high-resolution bacterial contig map of 3.4 Mb of genomic DNA in human chromosome 21q11–q21, encompassing the region of elevated disomic homozygosity in Down Syndrome-associated abnormal myelopoiesis and leukemia, as well as the markers, which has shown a strong association with Alzheimer’s Disease that has never been explained. The map contains 89 overlapping PACs, BACs, or cosmids in three contigs (850, 850, and 1500 kb) with two gaps (one of 140–210 kb and the second <5 kb). To date, eight transcribed sequences derived by cDNA selection, exon trapping, and/or global EST sequencing have been positioned onto the map, and the only two genes so far mapped to this cytogenetic region, STCH and RIP140 have been precisely localized. This work converts a further 10% of chromosome 21q into a high-resolution bacterial contig map, which will be the physical basis for the long-range sequencing of this region. The map will also enable positional derivation of new transcribed sequences, as well as new polymorphic probes, that will help in elucidation of the role the genes in this region may play in abnormal myelopoiesis and leukemia associated with trisomy 21 and Alzheimer’s Disease.