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PubMed1994

Physical mapping of 38 highly informative genetic markers to 10 intervals of chromosome 11q: integration of the physical and genetic maps.

A large number of highly polymorphic microsatellite markers particularly suitable for genetic linkage analysis have recently been developed. In order to facilitate integration of the genetic maps of chromosome 11q generated using these types of markers with the physical maps of 11q currently being assembled, we have regionally assigned the Genethon markers and the 11q designated index plus other commonly used polymorphic markers to ten physical intervals of 11q. These intervals are defined by translocation breakpoints immortalized in somatic cell hybrid lines and can therefore serve as readily accessible and stable landmarks for detailed map integration and facilitate the derivation and placement of new markers and cloned contigs.

Chromosome Mapping↗
PubMed1994

High resolution ordering of YAC contigs using extended chromatin and chromosomes.

The general usefulness of fluorescence in situ hybridization (FISH) for the physical mapping of the human genome is greatly enhanced by improved DNA resolution. Several techniques have been described for decondensing or stretching interphase chromatin in a linear fashion, allowing long-range FISH mapping in finer detail. Highly extended linear chromatin can be hybridized over physical distances of at least several megabases, possibly over the whole length of a chromosome. By multi-color FISH, we have determined the order and overlaps of YACs from a 5q34-35 contig. Cosmids can be localized within larger YAC clones. Extended chromatin mapping can be applied as an adjunct ordering technique in genome studies.

Amsacrine↗
PubMed1994

A 1.2-Mb YAC contig spans the quaking region.

We describe here a 1.2-Mb yeast artificial chromosome (YAC) contig within the region of mouse chromosome 17 between Brachyury (T) and D17Rp17e, and spanning the quaking (qk) region. We describe six new probes distributed across 1.2 Mb: D17Leh502, D17Leh503, D17Leh504, D17Leh505, D17Leh506, and D17Leh507. Probes D17Leh502 and D17Leh507 are at the extreme ends of the YAC contig. With the exception of D17Leh507, all of these probes are within a deletion associated with the quaking(viable) (qkv) allele of quaking. We have positioned these probes on a detailed YAC physical map together with two previously published probes, D17Leh508 and D17Aus119. We show here that D17Leh508 is also within the qkv deletion. Genetic mapping of D17Leh504 and D17Leh507 on two high-resolution genetic crosses carrying qkv and quaking(lethal-1) (qkl-1) alleles shows that these probes do not recombine with quaking and are therefore within 0.04 cM of qkv and 0.05 cM of qkl-1 mutations. The deletion breakpoint contained within the YAC contig has been positioned to within 90 kb by restriction mapping of wildtype and mutant DNA. This contig will form the basis for identification and mapping of expressed sequences and for an investigation of genome organization.

Alleles↗
PubMed1998

Comparative mapping of two adjacent regions of MMU19 with their human counterpart on HSA11q13.

High resolution physical maps of two adjacent regions of MMU19 were constructed in order to establish a comparative map between the pericentromeric region of MMU19 and its human counterpart on HSA11q13. These two physical maps span 2.5 and 0.5 megabases on MMU19. Long range restriction analysis and YAC contigs have been built, five genes were located on MMU19 and eight new STSs were generated. The 0.5-Mb map which has been positioned close to the centromere of MMU19, based on dual-color FISH experiments and genetic data, includes eight genes (Type I markers), three microsatellites (Type II markers) and five new STSs. The 2.5-Mb map is located more telomeric and contains seven genes, four microsatellites and four new STSs. Gene order and physical distances appear to be similar in human and in mouse in this 2.5-Mb region. Strikingly, the 0.5-Mb region has a similar size in human but gene order is shuffled. The overall comparative map shows that these two regions are inverted on MMU19 when compared with HSA11q13.

Animals↗
PubMed1998

The INSL4 gene maps close to WI-5527 at 9p24.1-->p23.3 clustered with two relaxin genes and outside the critical region for the monosomy 9p syndrome.

The insulin like growth factor 4 (INSL4) gene belongs to the insulin gene superfamily and has been mapped by fluorescent in situ hybridization to 9p24. Expression of INSL4, of unknown function, has been recently detected in the perichondrium and ligaments of the human embryo. Here we have mapped INSL4 within the framework of a partial YAC contig covering the distal part of 9p to find out whether this gene lies within the critical region defined for the monosomy 9p syndrome. The gene was also located using a human-rodent radiation hybrid panel. INSL4 was found to be distal to the del(9p) critical region and excluded as a candidate for the syndrome. In addition, the positions of two relaxin genes (RLN1, RLN2) that belong to the same superfamily as INSL4 and which have also been mapped to chromosome 9, were refined. We have shown that the three genes are clustered in the same region.

Animals↗
PubMed2004

A first generation physical map of the medaka genome in BACs essential for positional cloning and clone-by-clone based genomic sequencing.

In order to realize the full potential of the medaka as a model system for developmental biology and genetics, characterized genomic resources need to be established, culminating in the sequence of the medaka genome. To facilitate the map-based cloning of genes underlying induced mutations and to provide templates for clone-based genomic sequencing, we have created a first-generation physical map of the medaka genome in bacterial artificial chromosome (BAC) clones. In particular, we exploited the synteny to the closely related genome of the pufferfish, Takifugu rubripes, by marker content mapping. As a first step, we clustered 103,144 public medaka EST sequences to obtain a set of 21,121 non-redundant sequence entities. Avoiding oversampling of gene-dense regions, 11,254 of EST clusters were successfully matched against the draft sequence of the fugu genome, and 2363 genes were selected for the BAC map project. We designed 35mer oligonucleotide probes from the selected genes and hybridized them against 64,500 BAC clones of strains Cab and Hd-rR, representing 14-fold coverage of the medaka genome. Our data set is further supplemented with 437 results generated from PCR-amplified inserts of medaka cDNA clones and BAC end-fragment markers. Our current, edited, first generation medaka BAC map consists of 902 map segments that cover about 74% of the medaka genome. The map contains 2721 markers. Of these, 2534 are from expressed sequences, equivalent to a non-redundant set of 2328 loci. The 934 markers (724 different) are anchored to the medaka genetic map. Thus, genetic map assignments provide immediate access to underlying clones and contigs, simplifying molecular access to candidate gene regions and their characterization.

Animals↗
PubMed2002

Organization, expression and evolution of a disease resistance gene cluster in soybean.

PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar "Williams 82" [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca(2+)-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.

Amino Acid Sequence↗
PubMed2001

Smallest region of overlapping deletion in 1p36 in human neuroblastoma: a 1 Mbp cosmid and PAC contig.

In human neuroblastomas, the distal portion of 1p is frequently deleted, as if one or more tumor suppressor genes from this region were involved in neuroblastoma tumorigenesis. Earlier studies had identified a smallest region of overlapping deletion (SRO) spanning approximately 23 cM between the most distally retained D1S80 and by the proximally retained D1S244. In pursuit of generating a refined delineation of the minimally deleted region, we have analyzed 49 neuroblastomas of different stages for loss of heterozygosity (LOH) from 1pter to 1p35 by employing 26 simple sequence length polymorphisms. Fifteen of the 49 tumors (31%) had LOH; homozygous deletion was not detected. Seven tumors had LOH at all informative loci analyzed, and eight tumors showed a terminal or an interstitial allelic loss of 1p. One small terminal and one interstitial deletion defined a new 1.7 cM SRO, approximately 1 Mbp in physical length, deleted in all tumors between the retained D1S2731 (distal) and D1S2666 (proximal). To determine the genomic complexity of the deleted region shared among tumors, we assembled a physical map of the I Mbp SRO consisting predominantly of bacteriophage P1-derived artificial chromosome (PAC) clones. A total of 55 sequence-tagged site (STS) markers (23 published STSs and short tandem repeats and 32 newly identified STSs from the insert ends of PACs and cosmids) were assembled in a contig, resulting in a sequence-ready physical map with approximately one STS per 20 Kbp. Twelve genes (41BB, CD30, DFFA, DJ1, DR3, FRAP, HKR3, MASP2, MTHFR, RIZ, TNR2, TP73) previously mapped to 1p36 are localized outside this SRO. On the basis of this study, they would be excluded as candidate genes for neuroblastoma tumorigenesis. Ten expressed sequence tags were integrated in the contig, of which five are located outside the SRO. The other five from within the SRO may provide an entrance point for the cloning of candidate genes for neuroblastoma.

Bacteriophage P1↗
PubMed1995

High-resolution alignment of a 1-megabase-long genome region of three strains of Rhodobacter capsulatus.

A detailed restriction map of the genome of Rhodobacter capsulatus SB1003 was constructed recently by using an ordered set of overlapping cosmids. Pulsed-field gel electrophoresis-generated restriction patterns of the chromosomes of 14 other R. capsulatus strains were compared. Two of them, St. Louis and 2.3.1, were chosen for high-resolution alignment of their genomes with that of SB1003. A 1-Mb segment of the R. capsulatus SB1003 cosmid set was used as a source of ordered probes to group cosmids from the other strains. Selected cosmids were linked into one 800-kb contig and two smaller contigs of 100 kb each. EcoRV and BamHI restriction maps of the newly ordered cosmids were constructed by using lambda terminase. Long-range gene order in the new strains was mainly conserved for the regions studied. However, one large genome rearrangement inverted a 470-kb DNA fragment of the St. Louis strain between the rrnA and rrnB operons. A 50-kb deletion covering three SB1003 probes was found in strain 2.3.1 near rrnB. Conservation of about 50% of the positions of restriction sites in all these strains and nearly 80% for the pair 2.3.1- St. Louis made it possible to produce high-resolution alignment of the contiguous 800-kb genome segment. Ten deletions of 2 to 27 kb, one 30-kb inversion, and three translocations were found in this region. Strong clustering of the positions of polymorphic restriction sites was observed. For a 50-kb size interval, two patterns of the distribution of restriction sites were found, one with about 90% and the other with 5 to 30% conservation of sites. This structure may be explained by independent acquisition of these divergent regions from other Rhodobacter strains.

Chromosome Inversion↗
PubMed1995

Cosmid assembly and anchoring to human chromosome 21.

A human chromosome 21-specific cosmid library from the Lawrence Livermore National Laboratory has been analyzed by two complementary methods, fingerprinting and hybridization; 40% coverage of the entire chromosome 21 has been achieved. To prepare a contig pool, approximately 9300 cosmid clones randomly selected from the library were fingerprinted and automatically assembled into 467 overlapping sets by the fluorescence-tagged restriction fragment method. The average size of the overlapping sets was 9.5 cosmids with minimal tiling paths consisting of 5.4 cosmids with a 10-kb extension each. However, as many as 10% of overlaps within members were estimated to be false. For regional localization, we hybridized gridded arrays of cosmids with inter-Alu-PCR probes obtained from YAC clones and somatic cell hybrids and assigned 592 cosmids to 26 subregions of 21q. Of these, 371 clones were incorporated into 139 contigs, anchoring the total 1864 cosmids to the subregion. The remaining 221 clones were mapped as orphans. To correlate the cytogenetic, YAC, and cosmid maps on 21q, the translocation breakpoints of the chromosomes contained in the somatic cell hybrids were mapped with respect to the STS content of the YACs. From the gene cluster regions, 176 ribosomal and 25 alphoid clones were isolated by hybridization. Together, these sets of anchored contigs and cosmids will provide a valuable resource for construction of a high-resolution map and for isolation of genes of interest from chromosome 21.

Animals↗
PubMed1997

12p rearrangement and DNA amplification mapped by comparative genomic hybridization in a patient with secondary myeloid leukemia.

Rearrangements of the short arm of chromosome 12 (12p) are a common finding in hematologic malignancies. There has recently been considerable interest in chromosome 12 abnormalities in view of the mapping of the TEL gene to 12p13 and frequent 12p interstitial deletions. Overrepresentation of 12p sequences is, on the other hand, a consistent finding in testicular germ cell tumor (TGCT), and the 12p11.2-p12.1 subregion has been found to be specifically involved. We have studied a secondary leukemic patient whose cells contained 12p rearrangements with a view to clarifying the underlying molecular events. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) have revealed the presence of 12p11 breakpoints on both 12 homologs as well as amplification of 12p11-p12-derived sequences. Six YACs and a cosmid probe have been used in an attempt to map the amplification unit on 12p. The two YACs contigs WC-1468 and WC-985 were not amplified, and our results suggested a small amplicon localized in the 12p11.2-p12 subregion. We speculate that this region harbors gene(s) which are critical in tumor formation and could be involved in both TGCT and our patient. Whether the same gene(s) are involved in both amplification and translocation is unknown.

Adult↗
PubMed1996

Physical map of the HLA-A/HLA-F subregion and identification of two new coding sequences.

As part of an effort to characterize the hemochromatosis gene, we selected three non-chimeric yeast artificial chromosomes (YACs) overlapping with the YAC B30 previously described and forming an 800 kilobase contig covering the HLA-A/HLA-F region. The precise physical map of these YACs and of the corresponding genomic region were established. Nine concentrated sites of CpG cutter elements, potentially HTF islands, were mapped. In addition, several probes have been generated as tools for mapping and examining transcripts produced in the region. This allowed for the characterization and localization of two new coding sequences, provisionally named HCG (for hemochromatosis candidate gene) and numbered VIII and IX.

Blotting, Northern↗
PubMed1991

Optimized strategies for sequence-tagged-site selection in genome mapping.

The physical mapping of complex genomes is based on the construction of a genomic library and the determination of the overlaps between the inserts of the mapping clones in order to generate an ordered, cloned representation of nearly all the sequences present in the target genome. Evaluation of the relative efficiency of experimental procedures used to accomplish this goal must minimally include a comparison of the fraction of the genome covered by the ordered arrays (or "contigs"), the average size of the contigs, and the cost, in terms of time and resources, required to generate the map. Sequence-tagged-site (STS) content mapping is one strategy that has been proposed and is being utilized for this type of experiment. This paper describes three STS selection schemes and presents computer simulations of contig-building experiments based on these procedures. The results of these simulations suggest that a nonrandom STS strategy that uses paired probes requires one-third to one-fourth as many STS assays as are required in random and nonpaired approaches, and also results in a map that has both greater genome coverage and a larger average contig size. This strategy promises to reduce the time and cost required to build a high-quality physical map.

Base Sequence↗
PubMed1999

Genomic mapping of chromosomal region 2p15-p21 (D2S378-D2S391): integration of Genemap'98 within a framework of yeast and bacterial artificial chromosomes.

The region of chromosome 2 encompassed by the polymorphic markers D2S378 (centromeric) and D2S391 (telomeric) spans an approximately 10-cM distance in cytogenetic bands 2p15-p21. This area is frequently involved in cytogenetic alterations in human cancers. It also harbors the genes for several genetic disorders, including Type I hereditary nonpolyposis colorectal cancer (HNPCC), familial male precocious puberty (FMPP), Carney complex (CNC), Doyne's honeycomb retinal dystrophy (DHRD), and one form of familial dyslexia (DYX-3). Only a handful of known genes have been mapped to 2p16. These include MSH2, which is responsible for HNPCC, FSHR, the gene responsible for FMPP, EFEMP-1, the gene mutated in DHRD, GTBP, a DNA repair gene, and SPTBN1, nonerythryocytic beta-spectrin. The genes for CNC and DYX-3 remain unknown, due to lack of a contig of this region and its underrepresentation in the existing maps. This report presents a yeast- and bacterial-artificial chromosome (YAC and BAC, respectively) resource for the construction of a sequence-ready map of 2p15-p21 between the markers D2S378 and D2S391 at the centromeric and telomeric ends, respectively. The recently published Genemap'98 lists 146 expressed sequence tags (ESTs) in this region; we have used our YAC-BAC map to place each of these ESTs within a framework of 40 known and 3 newly cloned polymorphic markers and 37 new sequence-tagged sites. This map provides an integration of genetic, radiation hybrid, and physical mapping information for the region corresponding to cytogenetic bands 2p15-p21 and is expected to facilitate the identification of disease genes from the area.

Chromosome Mapping↗
PubMed1995

Yeast artificial chromosome cloning of the Xq13.3-q21.31 region and fine mapping of a deletion associated with choroideremia and nonspecific mental retardation.

Microscopically detectable deletions and X;autosome translocations have previously facilitated the construction of a high-resolution interval map of the Xq21 region. Here, we have generated three yeast artificial chromosome contigs spanning approximately 7 megabases of the Xq13.3-q21.31 region. In addition, a novel deletion associated with choroideremia and mental retardation was identified and mapped in detail. The proximal deletion endpoint was positioned between the loci DXS995 and DXS232, which enabled us to confirm the critical region for a locus involved in mental retardation. The distal deletion endpoint is situated in the Xq21.33 band, which allowed us to refine the order of several markers in this region.

Base Sequence↗
PubMed1999

High-resolution physical and genetic mapping of the critical region for Meckel syndrome and Mulibrey Nanism on chromosome 17q22-q23.

Previously, we assigned the genes for two autosomal recessive disorders, Meckel syndrome (MKS; MIM 249000) and Mulibrey Nanism [MUL (muscle-liver-brain-eye Nanism); MIM 253250] that are enriched in the Finnish population, to overlapping genomic regions on chromosome 17q. Now, we report the construction of a bacterial clone contig over the critical region for both disorders. Several novel CA-repeat markers were isolated from these clones, which allowed refined mapping of the MKS and MUL loci using haplotype and linkage disequilibrium analysis. The localization of the MKS locus was narrowed to <1 cM between markers D17S1290 and 132-CA, within an approximately 800-kb region. The MUL locus was refined into an approximately 1400-kb interval between markers D17S1290 and 52-CA. The whole MKS region falls within the MUL region. In the common critical region, the conserved haplotypes were different in MKS and MUL patients. A trancript map was constructed by assigning expressed sequence tags (ESTs) and genes, derived from the human gene map, to the bacterial clone contig. Altogether, four genes and a total of 20 ESTs were precisely localized. These data provide the molecular tools for the final identification of the MKS and the MUL genes.

Chromosome Mapping↗
PubMed2002

Physical and transcript map of the region between D6S264 and D6S149 on chromosome 6q27, the minimal region of allele loss in sporadic epithelial ovarian cancer.

We have previously shown a high frequency of allele loss at D6S193 (62%) on chromosomal arm 6q27 in ovarian tumours and mapped the minimal region of allele loss between D6S297 and D6S264 (3 cM). We isolated and mapped a single non-chimaeric YAC (17IA12, 260-280 kb) containing D6S193 and D6S297. A further extended bacterial contig (between D6S264 and D6S149) has been established using PACs and BACs and a transcript map has been established. We have mapped six new markers to the YAC; three of them are ESTs (WI-15078, WI-8751, and TCP10). We have isolated three cDNA clones of EST WI-15078 and one clone contains a complete open reading frame. The sequence shows homology to a new member of the ribonuclease family. The other two clones are splice variants of this new gene. The gene is expressed ubiquitously in normal tissues. It is expressed in 4/8 ovarian cancer cell lines by Northern analysis. The gene encodes for a 40 kDa protein. Direct sequencing of the gene in all the eight ovarian cancer cell lines did not identify any mutations. Clonogenic assays were performed by transfecting the full-length gene in to ovarian cancer cell lines and no suppression of growth was observed.

Amino Acid Sequence↗
PubMed1996

Treacher Collins syndrome.

Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. In the absence of a candidate gene, a positional cloning approach has been used to isolate the mutated gene which maps to chromosome 5q31.3-32. Flanking markers were identified and a yeast artificial chromosome and cosmid contig of the region defined by these markers was created as a prelude to the creation of a transcript map of the region. Analysis of genes isolated using this approach resulted in the identification of the mutated gene. While the function of the gene remains unknown, the identification of 20 mutations spread throughout the gene, all of which would result in the insertion of a premature termination codon into the reading frame, suggests that the mechanism underlying the disease is haploinsufficiency.

Animals↗