Detailed Physical Map Research Articles

Construction of a High-Resolution Physical and Transcription Map of Chromosome 16q24.3: A Region of Frequent Loss of Heterozygosity in Sporadic Breast Cancer

A breast cancer tumor suppressor gene has been localized to chromosome 16q24.3 by loss of heterozygosity (LOH) studies of breast tumor DNA. To identify candidate genes for this suppressor function, we have constructed a detailed physical map extending approximately 940 kb from the telomere of the long arm of chromosome 16 that encompasses the minimum LOH interval. This contig consists of a minimum overlapping set of 35 cosmids and a single PAC clone that were aligned by restriction enzyme site mapping. Cosmids were initially identified by screening filters with markers localized to the region by physical mapping using mouse/human somatic cell hybrids, and subsequently cosmid ends were used to complete the contig. A total of seven known genes, includingPRSM1, PISSLRE,and the recently cloned Fanconi anemia A (FAA) gene, and potential transcripts from exon-trapping experiments have been located to this contig. A minimum of 14 new transcripts have been identified based on homology of trapped exons with database sequences. This contig and expressed sequence map will form the basis for the identification of the breast cancer tumor suppressor gene in this region.

Refined Genetic Mapping of the Darier Locus to a

Darier disease (DD) (MIM 124200) is an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells and by abnormal keratinization. We present linkage analysis showing, in four families, key recombination events that refine the location of the DD locus on chromosome 12q23-24.1 to a region of <1 cM. We have constructed a YAC/P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC)-based physical map that encompasses this refined DD region. The map consists of 35 YAC, 69 PAC, 16 BAC, and 2 cosmid clones that were ordered by mapping 54 anonymous sequence-tagged sites. The critical region is estimated to be 2.4 Mb in size, with an average marker resolution of 37.5 kb. The refinement of the critical interval excludes the ALDH2, RPL6, PTPN11, and OAS genes, as well as seven expressed sequence tags (ESTs) previously mapped in the DD region. The three known genes (ATP2A2, PPP1CC, and SCA2) and the 10 ESTs mapped within the critical region are not obvious candidates for the DD gene. Therefore, this detailed integrated physical, genetic, and partial transcript map provides an important resource for the isolation of the DD gene and, possibly, other disease genes.

Rapid mapping of markers applying vectorette technology to YAC fragmentation allows easy assembly of a high-density STS bacterial clone contig spanning the markers D6S1260-D6S1918.

We have generated a detailed physical map of the 6p21.3/p22.1 boundary, using a combination of yeast artificial chromosome (YAC) fragmentation and high-resolution sequence tagged site (STS) content mapping. YACs from the CEPH, St. Louis, and ICRF libraries have been used to construct a 4.5-Mb contig spanning the markers D6S306 to D6S1571. YAC insert sizes were determined by pulsed field gel electrophoresis (PFGE). Chimerism of YACs was determined by fluorescent in situ hybridization (FISH), and their integrity was determined by fingerprinting with Alu-PCR. We have identified 10 new CA repeat loci in this region as well as over 50 novel STSs, several tRNA genes, a new histone H2B gene and the phospholipase D gene. Using these new markers, we have rapidly generated a bacterial clone contig of over 250 kb, spanning the markers D6S1260 to D6S1918 (WI-3111) with STSs spaced on average every 6 kb.

Cytogenetics for the model system Arabidopsis thaliana.

A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 microns, which is 20-25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm-ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.

An improved physical and genetic map of Campylobacter jejuni NCTC 11168 (UA580).

Campylobacter jejuni is recognized as the major cause of food-borne gastrointestinal disease in the developed world. To facilitate the molecular genetic analysis of this pathogen, an approximately 18-fold redundant Tropist3 cosmid library was constructed from C. jejuni NCTC 11168 genomic DNA. The cosmid library was partially ordered by hybridization to 15 pulsed-field electrophoresis (PFGE) restriction fragments. This analysis confirmed the estimated size of the genome to be 1730 kb, but suggested discrepancies in some regions of the published physical map. The precise locations of two of the three rRNA gene clusters were mapped using a combination of restriction fingerprinting, sample sequencing and riboprobing. Additionally, 15 further genes were located on the revised map. A more detailed physical and genetic map of C. jejuni NCTC 11168 is presented.

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 approximately 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.

A 370-kb Cosmid Contig of the Serpin Gene Cluster on Human Chromosome 14q32.1: Molecular Linkage of the Genes Encoding α1-Antichymotrypsin, Protein C Inhibitor, Kallistatin, α1-Antitrypsin, and Corticosteroid-Binding Globulin

The human genes encoding α1-antitrypsin (α1AT, gene symbol PI), corticosteroid-binding globulin (CBG), α1-antichymotrypsin (AACT), and protein C inhibitor (PCI) are related by descent, and they all map to human chromosome 14q32.1. This serine protease inhibitor (serpin) gene cluster also contains an antitrypsin-related sequence (ATR, gene symbol PIL), but the precise molecular organization of this region has not been defined. In this report we describe the generation and characterization of an ∼370-kb cosmid contig that includes all five serpin genes. Moreover, a newly described serpin, kallistatin (KAL, gene symbol PI4), was also mapped within the region. Gene order within this interval is cen–CBG–ATR–α1AT–KAL–PCI–AACT–tel. The genes occupy ∼320 kb of genomic DNA, and they are organized into two discrete subclusters of three genes each that are separated by ∼170 kb. The distal subcluster includes KAL, PCI, and AACT; it occupies ∼63 kb of DNA, and all three genes are transcribed in a proximal-to-distal orientation. Within the subcluster, there is ∼12 kb of intergenic DNA between KAL and PCI and ∼19 kb between PCI and AACT. The proximal subcluster includes α1AT, ATR, and CBG; it occupies ∼90 kb of genomic DNA, with ∼12 kb of DNA between α1AT and ATR and ∼40 kb between ATR and CBG. These genes are all transcribed in a distal-to-proximal orientation. This represents the first detailed physical map of the serpin gene cluster on 14q32.1.

Gene structure and allelic expression assay of the human GLI3 gene.

The GLI3 gene encodes a putative zinc finger transcription factor that is important in early vertebrate development. Haploinsufficiency of this gene has been associated with the Greig cephalopolysyndactyly syndrome and truncation mutations cause Pallister-Hall syndrome. In the course of studies to determine the etiology of Pallister-Hall syndrome, we required knowledge of the fine structure of GLI3 to perform detailed genetic and physical mapping and mutation screening of this gene. The coding region of GLI3 is composed of 14 exons, including a large exon of more than 2500 bp. In addition, the gene contains two intragenic dinucleotide repeats, and four single-base pair polymorphisms in the coding region. We have used these coding region polymorphisms to design an allele-specific expression study that will be useful for studying patients with Greig cephalopolysyndactyly syndrome. In addition, GLI3 should be considered a candidate gene for related developmental anomalies of humans. Such hypotheses will be more readily addressed with the availability of the fine structure of the gene and the allele-expression assay.

Detailed comparative mapping of cereal chromosome regions corresponding to the Ph1 locus in wheat.

Detailed physical mapping of markers from rice chromosome 9, and from syntenous (at the genetic level) regions of other cereal genomes, has resulted in rice yeast artificial chromosome (YAC) contigs spanning parts of rice 9. This physical mapping, together with comparative genetic mapping, has demonstrated that synteny has been largely maintained between the genomes of several cereals at the level of contiged YACs. Markers located in one region of rice chromosome 9 encompassed by the YAC contigs have exhibited restriction fragment length polymorphism (RFLP) using deletion lines for the Ph1 locus. This has allowed demarcation of the region of rice chromosome 9 syntenous with the ph1b and ph1c deletions in wheat chromosome 5B. A group of probes located in wheat homoeologous group 5 and barley chromosome 5H, however, have synteny with rice chromosomes other than 9. This suggests that the usefulness of comparative trait analysis and of the rice genome as a tool to facilitate gene isolation will differ from one region to the next, and implies that the rice genome is more ancestral in structure than those of the Triticeae.

Analysis of 1;17 translocation breakpoints in neuroblastoma: implications for mapping of neuroblastoma genes.

Deletions and translocations resulting in loss of distal 1p-material are known to occur frequently in advanced neuroblastomas. Fluorescence in situ hybridisation (FISH) showed that 17q was most frequently involved in chromosome 1p translocations. A review of the literature shows that 10 of 27 cell lines carry 1;17 translocations. Similar translocations were also observed in primary tumours. Together with the occurrence of a constitutional 1;17 translocation in a neuroblastoma patient, these observations suggest a particular role for these chromosome re-arrangements in the development of neuroblastoma. Apart from the loss of distal 1p-material, these translocations invariably lead to extra copies of 17q. This also suggested a possible role for genes on 17q in neuroblastoma tumorigenesis. Further support for this hypothesis comes from the observation that in those cell lines without 1;17 translocations, other chromosome 17q translocations were present. These too lead to extra chromosome 17q material. Molecular analysis of 1;17 translocation breakpoints revealed breakpoint heterogeneity both on 1p and 17q, which suggests the involvement of more than 2 single genes on 1p and 17q. The localisation of the different 1p-breakpoints occurring in 1;17 translocations in neuroblastoma are discussed with respect to the recently identified candidate tumor suppressor regions and genes on 1p. In this study, we focused on the molecular analysis of the 17q breakpoints in 1;17 translocations. Detailed physical mapping of the constitutional 17q breakpoint allowed for the construction of a YAC contig covering the breakpoint. Furthermore, a refined position was determined for a number of 17q breakpoints of 1;17 translocations found in neuroblastoma cell lines. The most distal 17q breakpoint was identified in cell line UHG-NP and mapped telomeric to cosmid cCI17-1049 (17q21). This suggests that genes involved in a dosage-dependent manner in the development of neuroblastoma map in the distal segment 17q22-qter. Future studies aim at the molecular cloning of 1;17 translocation breakpoints and at deciphering the mechanisms leading to 1;17 translocations and possibly to the identification of neuroblastoma genes at or in the vicinity of these breakpoints.

A 1-Mb Physical Map and PAC Contig of the Imprinted Domain in 11p15.5 That Contains TAPA1 and the BWSCR1/WT2 Region

We have constructed a 1-Mb contig in human chromosomal band 11p15.5, a region implicated in the etiology of several embryonal tumors, including Wilms tumor, and in Beckwith–Wiedemann syndrome. Cosmid, P1, PAC, and BAC clones were characterized byNotI/SalI digestion and hybridized to a variety of probes to generate a detailed physical map that extends from D11S517 to L23MRP. Included in the map are the CARS, NAP2, p57/KIP2, KVLQT1, ASCL2, TH, INS, IGF2, H19, and L23MRP genes as well as end probes isolated from PACs. The TAPA1 gene, whose protein product can transmit an antiproliferative signal, was also localized in the contig. However, Northern blot analysis demonstrated that its expression did not correlate with tumorigenicity in G401 Wilms tumor hybrids, suggesting that TAPA1 is not responsible for the tumor suppression associated with 11p15.5. Genomic clones were used as probes in FISH analysis to map the breakpoints from three Beckwith–Wiedemann syndrome patients and a rhabdoid tumor. Interestingly, each of the breakpoints disrupts the KVLQT1 gene, which is spread over a 400-kb region of the contig. Since 11p15.5 contains several genes with imprinted expression and one or more tumor suppressor genes, our contig and map provide a framework for characterizing this intriguing genetic environment.

Structure and Organization of GABRB3 and GABRA5

The genes encoding the γ-aminobutyric acid (GABA) type-A receptor subunits β3 (GABRB3), α5 (GABRA5), and γ3 (GABRG3) map to chromosome 15q11–q13. The three genes are contained within roughly 800 kb of the distal part of the imprinted Prader–Willi and Angelman syndrome region. A 570-kb contig encompassing GABRB3 and GABRA5 has been constructed in P1, λ phage, and PAC clones. GABRB3 spans 250 kb of DNA and is organized into 9 exons that range from 68 to 504 bp, while GABRA5 is encoded by 11 exons (65 to 924 bp in length) within 86 kb. The exon/intron borders for both genes have been characterized, and primers have been designed to amplify each of the individual exons. Two reference STR markers have been positioned in the contig. The reference STR for GABRB3 is in fact located at least 60 kb beyond the 3′ terminus of GABRB3, while D15S97 is contained within intron 4 of GABRB3. The detailed physical map of this GABAAreceptor subunit gene cluster should not only be useful in genetic studies of the 15q11–q13 region, but will also be important for investigating the evolution and expression of the GABAAreceptor gene superfamily.

Comprehensive physical map of the Cydia pomonella granulovirus genome and sequence analysis of the granulin gene region.

A cloned strain of Cydia pomonella granulovirus, CpGV-M1, was obtained using successive rounds of an in vivo limiting dilution method. A detailed physical map of the genome was constructed using 11 restriction enzymes. The region containing the granulin gene and an open reading frame immediately upstream of the granulin gene was sequenced. This region showed a high degree of homology to the equivalent region from Cryptophlebia leucotreta granulovirus with 98% amino acid identity for the granulins and 68% identity for the putative polypeptides encoded by the upstream ORFs. These latter polypeptides contained two zinc finger-like motifs and showed a low degree of homology to ME53 from Autographa californica nucleopolyhedrovirus (AcMNPV). Evidence is presented for a similar upstream ORF in Artogeia rapae GV also. Hybridization studies showed that the CpGV genome had a similar overall organization to the Artogeia rapae GV genome. Hybridization between CpGV and AcMNPV was limited to fragments spanning about 15% of each genome suggesting that very few genes are highly conserved between GVs and NPVs.

Genomic organization and chromosomal localization of the human casein gene family.

Five yeast artificial chromosome (YAC) clones containing the human casein gene family were isolated and characterized to study the control mechanisms for the expression of these genes. Partial restriction analysis in conjunction with the chromosomal fragmentation method and fluorescence in situ hybridization (FISH) analysis were performed to construct a detailed physical map of the casein gene family and to determine the chromosomal localization of these genes. The isolated YAC clones 748F3, 750D11, 882G11, 886B3 and 960D2 were 1.2 Mb, 860 kb, 800 kb, 1.5 Mb and 1.5 Mb in size, respectively. The clones 748F3, 882G11, 886B3 and 960D2 contained the entire casein gene family, while the kappa-casein gene was absent in 750D11. The human alphaS1-, beta- and kappa-casein genes were found to be closely linked and arranged in the order alphaS1-beta-kappa. The distance between alphaS1 and beta, and between alphaS1 and kappa was approximately 10 and 300 kb, respectively. The beta-casein gene was oriented in the opposite direction to the alphaS1- and kappa-casein genes. The casein gene family was localized to chromosome 4q21.1 by FISH analysis.

Toward Cloning of a Novel Ataxia Gene: Refined Assignment and Physical Map of the IOSCA Locus (SCA8) on 10q24

Infantile onset spinocerebellar ataxia (IOSCA) is a progressive neurological disorder of unknown etiology. It is inherited as an autosomal recessive trait and has so far been reported in just 19 Finnish patients in 13 separate families. We have previously assigned the IOSCA locus (HGMW-approved symbol SCA8) to chromosome 10q, where no previously identified ataxia loci are located. Haplotype analysis combined with genealogical data provided evidence that all the IOSCA cases in Finland originate from a single 30- to 40-generation-old founder mutation. By analyzing extended disease haplotypes observed today, the IOSCA locus can now be restricted to a region between two adjacent microsatellites, D10S192 and D10S1265, with no genetic intermarker distance. We have constructed a detailed physical map of this 270-kb IOSCA region and cytogenetically localized it to 10q24. We have also assigned two previously known genes, PAX2 and CYP17, more precisely into this region, but the sequence analysis of coding regions of these two genes has not revealed mutations in an IOSCA patient. The obtained long-range clones will form the basis for the isolation of a novel ataxia gene.

Human Mucin Genes Assigned to 11p15.5: Identification and Organization of a Cluster of Genes

Four distinct genes that encode mucins have previously been mapped to chromosome 11p15.5. Three of these genes (MUC2, MUC5AC,andMUC6) show a high level of genetically determined polymorphism and were analyzed in the CEPH families. Linkage analysis placed all three genes on the genetic map in a cluster betweenHRASandINS,and more detailed analysis of recombinant breakpoints revealed thatMUC6is telomeric toMUC2.Using these recombinantsD11S150was mapped close toMUC2.Ten of the 11 recombinant chromosomes studied in detail were paternal, and the recombinant events were distributed throughout the 11p15 region, suggesting that the high level of recombination observed in 11p15.5 is not due to a particular recombinational hot spot. Pulsed-field gel electrophoresis was used to make a detailed physical map of theMUCcluster and to integrate the physical and genetical maps. The gene order was determined to beHRAS–MUC6–MUC2–MUC5AC–MUC5B–IGF2.TheMUCgenes span a region of some 400 kb and the map extends 770 kb and contains 15 putative CpG islands. The order of theMUCgenes on the map corresponds to the relative order of their expression along the anterior–posterior axis of the body, suggesting a possible functional significance to the gene order.

Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo.

The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) has high potential for development as a bio-insecticide for control of the beet armyworm (S. exigua). It is highly infectious for S. exigua larvae and its host range is very narrow. A prerequisite for such application is the possibility of growing this virus in large quantities, e.g. in insect cell lines. It was observed, however, that polyhedra of SeMNPV plaque-purified in Se-UCR1 cells did not cause larval mortality or morbidity when fed to S. exigua larvae. As this suggested a genetic alteration in in vitro produced SeMNPV, comparative restriction analysis of in vitro and in vivo produced SeMNPV DNA was performed. The restriction patterns of viral DNA from several different plaques always differed from that of the wild-type in the same way, suggesting that a large, single deletion had occurred in the in vitro produced viral genome. In order to localize this deletion more precisely a detailed physical map of the wild-type SeMNPV genome was constructed, using the restriction endonucleases XbaI, BamHI, Bg/II, PstI, SstI, HindIII and SpeI. In addition, the entire SeMNPV genome was cloned into a library containing five overlapping cosmids and a plasmid library. About 80 restriction sites were located and the orientation of the map was set according to the location of the polyhedrin and p10 genes. The approximate size of the viral genome was 134 kbp. Based on this map it could be established that mutant SeMNPV, obtained by passage in cell culture, contained a single deletion of approximately 25 kbp between map units 12.9 and 32.3.

Identification of Genes from a 500-kb Region at 7q11.23 That Is Commonly Deleted in Williams Syndrome Patients

Williams syndrome (WS) is a multisystem developmental disorder caused by the deletion of contiguous genes at 7q11.23. Hemizygosity of the elastin (ELN) gene can account for the vascular and connective tissue abnormalities observed in WS patients, but the genes that contribute to features such as infantile hypercalcemia, dysmorphic facies, and mental retardation remain to be identified. In addition, the size of the genomic interval commonly deleted in WS patients has not been established. In this study we report the characterization of a 500-kb region that was determined to be deleted in our collection of WS patients. A detailed physical map consisting of cosmid, P1 artificial chromosomes, and yeast artificial chromosomes was constructed and used for gene isolation experiments. Using the techniques of direct cDNA selection and genomic DNA sequencing, three known genes (ELN, LIMK1, and RFC2), a novel gene (WSCR1) with homology to RNA-binding proteins, a gene with homology to restin, and four other putative transcription units were identified. LIMK1 is a protein kinase with two repeats of the LIM/double zinc finger motif, and it is highly expressed in brain. RFC2 is the 40-kDa ATP-binding subunit of replication factor C, which is known to play a role in the elongation of DNA catalyzed by DNA polymerase δ and ϵ. LIMK1 and WSCR1 may be particularly relevant when explaining cognitive defects observed in WS patients.

Locations of Human and Mouse Genes Encoding theRFX1andRFX2Transcription Factor Proteins

RFX transcription factors constitute a highly conserved family of site-specific DNA binding proteins involved in the expression of a variety of cellular and viral genes, including major histocompatibility complex class II genes and genes in human hepatitis B virus. Five members of the RFX gene family have been isolated from human and mouse, and all share a highly characteristic DNA binding domain that is distinct from other known DNA binding motifs. The humanRFX1andRFX2genes have been assigned byin situhybridization to chromosome 19p13.1 and 19p13.3, respectively. In this paper, we present data that localizeRFX1andRFX2precisely within the detailed physical map of human chromosome 19 and genetic data that assignRfx1andRfx2to homologous regions of mouse chromosomes 8 and 17, respectively. These data define the established relationships between these homologous mouse and human regions in further detail and provide new tools for linking cloned genes to phenotypes in both species.

Framework for a Physical Map of the Human 22q13 Region Using Bacterial Artificial Chromosomes (BACs)

Detailed physical maps of entire chromosomes based on combined genetic, cytogenetic, and structural information are essential components for positional cloning and genomic sequencing. Despite the wealth of genetic information of the known diseases in the chromosome 22q13, the construction of a detailed physical map of the terminal region is difficult due to the sparsity of the genetic markers. We present here a map of bacterial artificial chromosome (BAC) contigs that cover a number of genetic loci in the 22q13 region. One hundred thirty-six BACs with an average insert size of 140 kb are assembled into 35 contigs defined by 64 markers in 22q13–qter. Twenty-three anonymous markers are now linked to the previously mapped genetic anchor points.