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PubMed1998

High-resolution YAC-cosmid-STS map of human chromosome 13.

We have assembled a high-resolution physical map of human chromosome 13 DNA (approximately 114 Mb) from hybridization, PCR, and FISH mapping data using a specifically designed set of computer programs. Although the mapping of 13p is limited, 13q (approximately 98 Mb) is covered by an almost continuous contig of 736 YACs aligned to 597 contigs of cosmids. Of a total of 10,789 cosmids initially selected from a chromosome 13-specific cosmid library (16,896 colonies) using inter-Alu PCR probes from the YACs and probes for markers mapped to chromosome 13, 511 were assembled in contigs that were established from cross-hybridization relationships between the cosmids. The 13q YAC-cosmid map was annotated with 655 sequence tagged sites (STSs) with an average spacing of 1 STS per 150 kb. This set of STSs, each identified by a D number and cytogenetic location, includes database markers (198), expressed sequence tags (93), and STSs generated by sequencing of the ends of cosmid inserts (364). Additional annotation has been provided by positioning 197 cosmids mapped by FISH on 13q. The final (comprehensive) map, a list of STS primers, and raw data used in map assembly are available at our Web site (genome1.ccc.columbia.edu/ approximately genome/) and can serve as a resource to facilitate accurate localization of additional markers, provide substrates for sequencing, and assist in the discovery of chromosome 13 genes associated with hereditary diseases.

Animals↗
PubMed1996

Generation of sequence-tagged sites from Xp22.3 by isolating common Alu-PCR products of radiation hybrids retaining overlapping human X chromosome fragments.

Several human diseases have been mapped to Xp22.3 on the distal short arm of the human X chromosome, and many genes in this area have been found to be expressed from the inactive X chromosome. To facilitate physical mapping and characterization of this interesting region, we have constructed a battery of radiation hybrids containing human X chromosomal fragments, and isolated two hybrid clones A with overlapping fragments of Xp22.3. Alu-PCR on these hybrids and identification of sequences common to both hybrids allowed the isolation of six sequences-tagged sites (STSs) from Xp22.3. Five of the STSs were mapped+ to individual YACs comprising a recently constructed contig of this region. These novel STSs are useful markers for further physical characterization of this part of the genome.

Animals↗
PubMed1995

Techniques in mammalian genome mapping.

Increasing emphasis is being given to genomic cloning using Escherichia coli vectors of intermediate insert capacity, such as bacteriophage P1, P1-derived artificial chromosomes and the F factor based bacterial artificial chromosomes. These vectors are being used in addition to yeast artifical chromosomes (YACs) in recognition of the difficulties encountered with YAC stability and with handling of YAC DNAs (problems that will not easily be overcome). Nonetheless, YACs remain the most practical cloning system for global contig building. Efforts are currently under way to produce YAC contigs that represent the human and mouse genomes, and these will increasingly exploit extensive anchoring to detailed genetic maps. Intermediate capacity clone collections based on YAC contigs will follow, enabling the compilation of mapped gene catalogues. In this way, the era of big gene hunts will draw to a close.

Animals↗
PubMed1996

Degeneracy in human multicopy RBM (YRRM), a candidate spermatogenesis gene.

In order to search for mutations in the multicopy RBM genes that might be associated with male infertility, we have used sequence data from the reported cDNA clone to determine the intron exon boundaries of the YRRM 1 gene. This gene has 12 exons, three of which encode the putative RNA binding domain of the protein. Different copies of the gene contain sequence variations and, additionally, give rise to transcripts with different numbers of copies of the repeated SRGY motif. Since mutations in the RNA binding domain would seem likely to have an effect on the activity of the protein, we have scanned these exons for mutations by SSCP on DNA from normal and infertile men. Sequence differences in the exon encoding the N-terminal part of the RNA binding domain account for at least four different classes of the gene and give rise to different SSCP conformers. Sequence analysis shows that one of these classes is a pseudogene and that the members of another class are nonfunctional. RT-PCR shows that all classes are transcribed and that the A class is most abundant. We have found a point mutation that alters the highly conserved RNP2 motif in one infertile patient. This mutation is also found in his father. We have used PCR followed by SSCP analysis to map RBM on a Y Chromosome (Chr) YAC contig and have demonstrated a distribution that spans a major part of this chromosome's euchromatin.

Amino Acid Sequence↗
PubMed1993

Physical linkage of the murine Hox-b cluster and nerve growth factor receptor on yeast artificial chromosomes.

Evidence derived from sequence comparisons and the genomic organization of the murine Antennapedia-class homeobox gene clusters suggest that they arose from a primordial cluster through a process of gene duplication and divergence followed by cluster duplication. A large chromosomal domain surrounding the ancestral homeobox cluster also appears to have been duplicated and has remained relatively stable since the divergence of humans and rodents. To test the extent of the duplicated chromosomal domain, we have initiated physical mapping studies of the regions surrounding the four murine homeobox clusters using pulsed-field gel electrophoresis and yeast artificial chromosome cloning. In this study, we present a long-range restriction map of mouse chromosome 11 spanning 1500 kb in the region surrounding the Hox-b cluster. We have determined that the gene for the nerve growth factor receptor is tightly linked to the Hox-b complex and is located within 50 kb of the Hox-b 1 gene at the 3' end of the cluster. Four yeast artificial chromosomes have been isolated and characterized by the polymerase chain reaction, long-range restriction mapping, and Southern blotting. Two clones of 150 and 300 kb contain the entire Hox-b cluster and the nerve growth factor receptor gene. A 440-kb clone contains the 3' end of the Hox-b cluster, the nerve growth factor receptor gene, and extends downstream. A 210-kb clone contains the 5' end of the Hox-b cluster and extends upstream. These clones confirm the pulsed-field restriction map of uncloned mouse DNA and represent a contig of approximately 600 kb of cloned material from mouse chromosomes 11.

Animals↗
PubMed2004

Comparative physical mapping reveals features of microsynteny between Glycine max, Medicago truncatula, and Arabidopsis thaliana.

To gain insight into genomic relationships between soybean (Glycine max) and Medicago truncatula, eight groups of bacterial artificial chromosome (BAC) contigs, together spanning 2.60 million base pairs (Mb) in G. max and 1.56 Mb in M. truncatula, were compared through high-resolution physical mapping combined with sequence and hybridization analysis of low-copy BAC ends. Cross-hybridization among G. max and M. truncatula contigs uncovered microsynteny in six of the contig groups and extensive microsynteny in three. Between G. max homoeologous (within genome duplicate) contigs, 85% of coding and 75% of noncoding sequences were conserved at the level of cross-hybridization. By contrast, only 29% of sequences were conserved between G. max and M. truncatula, and some kilobase-scale rearrangements were also observed. Detailed restriction maps were constructed for 11 contigs from the three highly microsyntenic groups, and these maps suggested that sequence order was highly conserved between G. max duplicates and generally conserved between G. max and M. truncatula. One instance of homoeologous BAC contigs in M. truncatula was also observed and examined in detail. A sequence similarity search against the Arabidopsis thaliana genome sequence identified up to three microsyntenic regions in A. thaliana for each of two of the legume BAC contig groups. Together, these results confirm previous predictions of one recent genome-wide duplication in G. max and suggest that M. truncatula also experienced ancient large-scale genome duplications.

Arabidopsis↗
PubMed2002

The human gamma-aminobutyric acid A receptor delta (GABRD) gene: molecular characterisation and tissue-specific expression.

Terminal deletions of 1p36 result in a specific and common syndrome characterised by the following: growth delay, distinctive facial anomalies, hearing and visual deficits, heart defects, body asymmetry, moderate to severe psychomotor retardation, epilepsy, and self-abusive behaviour. The human gamma-aminobutyric acid A receptor delta-subunit gene (GABRD) encodes for one of at least 15 ligand-gated chloride channels for gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Recently we have mapped this gene by radiation hybrid mapping to the critical region of gene loss of the 1p36 deletion syndrome within 1p36.33. The complete complementary DNA (cDNA) sequence of GABRD was generated using assembled sequence of cDNA fragments already available, and 5'-rapid amplification of cDNA ends products. Fine physical mapping of the GABRD gene within this genomic interval was performed by screening bacterial artificial chromosome contigs spanning the critical region of the 1p36 deletion syndrome. The GABRD gene maps immediately proximal to the PRKCZ gene that is located between marker D1S243 and cosmid D1Z2--a region thought to be critical for cognition and speech development. The GABRD gene is expressed most abundantly in brain and has three alternative exons (1A-C) with alternative start codons at the 5'-end. Genomic localisation, function, and expression would suggest that the GABRD gene represents a good candidate for the neurodevelopmental and neuropsychiatric anomalies seen in the 1p36 deletion syndrome.

Amino Acid Sequence↗
PubMed2002

Comparative genomic analysis of sequences sampled from a small region on soybean (Glycine max) molecular linkage group G.

Eight DNA markers spanning an interval of approximately 10 centimorgans (cM) on soybean (Glycine max) molecular linkage group G (MLG-G) were used to identify bacterial artificial chromosome (BAC) clones. Twenty-eight BAC clones in eight distinct contiguous groups (contigs) were isolated from this genome region, along with 59 BAC clones on 17 contigs homoeologous to those on MLG-G. BAC clones in four of the MLG-G contigs were also digested to produce subclones and detailed physical maps. All of the BAC-ends were sequenced, as were the subclones, to estimate proportions in different sequence categories, compare similarities among homoeologs, and explore microsynteny with Arabidopsis. Homoeologous BAC contigs were enriched in repetitive sequences compared with those on MLG-G or the soybean genome as a whole. Fingerprint and cross-hybridization comparisons between MLG-G and homoeologous contigs revealed cases of highly similar physical organization between soybean duplicates, as did DNA sequence comparisons. Twenty-seven out of 78 total sequences on soybean MLG-G showed significant similarity to Arabidopsis. The homologs mapped to six compact genome segments in Arabidopsis, with the longest containing seven homologs spanning two million base pairs. These results extend previous observations of large-scale duplication and selective gene loss in Arabidopsis, suggesting that networks of conserved synteny between Arabidopsis and other angiosperm families can stretch over long physical distances.

Arabidopsis↗
PubMed1992

Physical mapping of a 950-kb region surrounding a locus (D10S102) tightly linked to the MEN2A gene.

We have constructed a long-range contig of cosmid and YAC clones around D10S102, a locus that is tightly linked to the gene responsible for multiple endocrine neoplasia type 2A (MEN2A). With D10S102 as a starting point, a 360-kb cosmid contig was constructed by bidirectional genomic walking, and at least six fragments from these cosmids showed high sequence homology to other species. Five YAC clones were also isolated at the D10S102 locus, and they formed a contig covering 950 kb of genomic DNA. Furthermore, we obtained six RFLP systems from the contig, which will serve as new resources for fine-scale genetic linkage mapping of the MEN2A locus.

Base Sequence↗
PubMed1996

Physical mapping of the human neurotensin gene (NTS) between markers D12S1444 and D12S81 on chromosome 12q21.

Neurotensin (NTS) is an endogenous tridecapeptide of the central nervous system and the gastrointestinal tract of different mammalian species including human. The human gene encoding neurotensin has previously been assigned to chromosome 12 but no regional localization was available. We now confirm this assignment and place the NTS gene on the physical and cytogenetic maps. The NTS gene is located on a yeast artificial chromosome contig that contains several polymorphic markers and is close to a polymorphic marker located at 95.8 cM on the Généthon linkage map. NTS is immediately proximal to four polymorphic markers, including D12S81 (AFM102xg9) and D12S88 (AFM158yb4). Using fluorescence in situ hybridization, we map the gene cytogenetically to band 12q21.

Animals↗
PubMed1995

A high-resolution map of genes, microsatellite markers, and new dinucleotide repeats from UBE1 to the GATA locus in the region Xp11.23.

Several new genes and markers have recently been identified on the proximal short arm of the human X chromosome in the area of Xp11.23. We had previously generated a YAC contig in this region extending from UBE1 to the OATL1 locus. In this report two polymorphic dinucleotide repeats, DXS6949 and DXS6950, were isolated and characterized from the OATL1 locus. A panel of YAC deletion derivatives from the distal portion of the contig was used in conjunction with the rest of the YAC map to position the new microsatellites and order other markers localizing to this interval. The marker order was determined to be DXS1367-ZNF81-DXS6849-ZNF21-DXS6616-DXS 6950-DXS6949. In the proximal region below OATL1, we have isolated a pair of YACs from the GATA locus, B1026 and C01160. Mapping within these YACs indicates the orientation of DXS1126 and DXS1240, while a cosmid near the OATL1 region reveals the overlap between the YAC contigs from the two loci. This cosmid contains the gene responsible for Wiskott-Aldrich syndrome (WAS) and localizes the disease gene between OATL1 and GATA. These data enable the expansion of the present physical map of the X chromosome from UBE1 to the GATA locus, covering a large portion of the Xp11.23 region. Genetic cross-overs in Xp11.23 support the marker orientation and the position of WAS, contrary to previous reports. With the integration of both physical and genetic maps we have predicted the following marker order: Xpter-UBE1-SYN1/ARAF1/ TIMP1-DXS1367-ZNF81-DXS.6849-ZNF21-DXSy6616++ +-(OATL1, DXS6950-DXS6949)- WAS-(GATA, DXS1126)-DXS1240-Xcen.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗
PubMed1995

Alignment of a 1.2-Mb chromosomal region from three strains of Rhodobacter capsulatus reveals a significantly mosaic structure.

High-resolution physical maps of the genomes of three Rhodobacter capsulatus strains, derived from ordered cosmid libraries, were aligned. The 1.2-Mb segment of the SB1003 genome studied here is adjacent to a 1-Mb region analyzed previously [Fonstein, M., Nikolskaya, T. & Haselkorn, H. (1995) J. Bacteriol. 177, 2368-2372]. Probes derived from the ordered cosmid set of R. capsulatus SB1003 were used to link cosmids from the St. Louis and 2.3.1 strain libraries. Cosmids selected this way did not merge into a single contig but formed several unlinked groups. EcoRV restriction maps of the ordered cosmids were then constructed using lambda terminase and fused to derive fragments of the chromosomal map. In order to link these fragments, their ends were transcribed to produce secondary probes for hybridization to gridded cosmid libraries of the same strains. This linking reduced the number of subcontigs to three for the St. Louis strain and one for the 2.3.1 strain. Hybridization of the same probes back to the ordered cosmid set of SB1003 positioned the subcontigs on the high-resolution physical map of SB1003. The final alignment of the restriction maps shows numerous large and small translocations in this 1.2-Mb chromosomal region of the three Rhodobacter strains. In addition, the chromosomes of the three strains, whose fine-structure maps can now be compared over 2.2 Mb, are seen to contain regions of 15-80 kb in which restriction sites are highly polymorphic, interspersed among regions in which the positions of restriction sites are highly conserved.

Chromosomes, Bacterial↗
PubMed1997

Molecular studies of an ependymoma-associated constitutional t(1;22)(p22;q11.2).

We previously described a patient with a de novo constitutional translocation, t(1;22)(p22;q11.2), who developed a malignant ependymoma at age 5, and we proposed that the translocation predisposed the child to the development of the tumor. As a step toward isolation of a putative cancer gene, we have characterized the breakpoints of the (1;22) translocation at the molecular level. The chromosome 22 breakpoint has been narrowed to a region between ARVCF and D22S264. The chromosome 1 breakpoint has been mapped onto a doubly-linked Whitehead YAC contig by PCR analysis of the STS contents of the patient's derivative chromosomes isolated in somatic cell hybrids. Loss-of-heterozygosity (LOH) studies of the patient's ependymoma and of sporadic ependymomas showed no evidence of consistent loss in the breakpoint regions, suggesting that activation of an oncogene, rather than inactivation of a tumor suppressor gene, is the more likely molecular mechanism involved in this case. The gene for Edg-1, a neurally expressed, seven-segment transmembrane receptor, maps to the region of the chromosome 1 breakpoint but does not appear to be interrupted by the translocation. Molecular characterization of the breakpoint regions reported here represents an important step in the identification of the gene(s) affected by this translocation.

Animals↗
PubMed1998

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5.

B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc-finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B-CLL rearrangements.

Amino Acid Sequence↗
PubMed1997

Localization of 16 exons to a 450-kb region involved in the autoimmune polyglandular disease type I (APECED) on human chromosome 21q22.3.

As a step toward identifying the pathogenic genes for autoimmune polyglandular disease type I (APECED) and other disorders mapped to the PFKL locus on chromosome 21q22.3, we have constructed a cosmid/BAC (bacterial artificial chromosome) contig of 450 kb covering markers D21S1460-D21S25-PFKL-D21S154 and performed exon trapping. We isolated 22 distinct exons including 6 exons derived from two known genes (PFKL and EHOC-1). Among 16 novel exons, 2 exons matched with human expressed sequence tags (EST) and 7 exons showed homology at predicted amino acid sequence level with proteins from other species. These 16 exons were mapped back to the cosmid contigs, 12 of which were confirmed for their expression by polymerase chain reaction (PCR) screening of human cDNA libraries of various tissues. These exon sequences and a transcript map will aid for isolation of corresponding genes which will be identified as candidate genes involved in the pathogenesis of disorders mapped to the 21q22.3 region.

Amino Acid Sequence↗
PubMed2002

Fine genetic mapping and physical delimitation of the lesion mimic gene Spl11 to a 160-kb DNA segment of the rice genome.

The rice lesion mimic mutant spl11 was previously found to confer broad-spectrum disease resistance to both Magnaporthe grisea and Xanthomonas oryzae pv. oryzae. To better understand the molecular basis underlying cell death and disease resistance in rice, a map-based cloning strategy has been employed to isolate Spl11. Five Spl11-linked RAPD markers were developed and four of them were mapped to rice chromosome 12. A high-resolution genetic map was developed using a segregating population consisting of 1138 lesion mimic individuals. Recombination suppression was observed in the vicinity of Spl11. Three molecular markers tightly linked to Spl11 were identified and used to screen a BAC library. A contig spanning the Spl11 locus was constructed and physical mapping delimited Spl11 to a 160-kb DNA segment within a single BAC clone. These results provide the essential information for the final isolation of this important gene in the rice defense pathway.

Apoptosis↗
PubMed1997

A 1200-kilobase transcription map encompassing the D6S105 locus at 6p21.3.

The gene content of the MHC class I telomerically adjacent region, in linkage disequilibrium with hereditary hemochromatosis, has not been well characterized yet. In the present work, we established three bacterial clone contigs, including mainly P1-derived artificial chromosomes. These contigs cover 89% of the 1.2-Mb 6p-subtelomeric region encompassing locus D6S105. Terminal exon trapping was applied to selected clones from these contigs. Forty-six independent terminal exons were identified and mapped within the region, 2 of which matched perfectly to expressed sequence tags. These 3' exons are all expressed in human fetal brain but differentially expressed in four tissues and two cell lines. The high number of exons identified indicates that the high gene density observed in the MHC class I region extends to this telomerically adjacent region.

Animals↗
PubMed2000

Characterization of a 500-kb contig spanning the region between c-Ha-Ras and MUC2 on chromosome 11p15.5.

The 11p15.5 region is associated with a broad range of diseases, including childhood acute myeloid leukemia; non-small cell lung carcinoma; arthrogryposis multiplex congenita, distal type 2B; and bladder cancer. Since targets for these diseases are unknown, we have constructed a physical map consisting of BAC and PAC clones spanning the region from the HRAS1 gene to the cluster of mucin genes on 11p15.5. The contig spans approximately 500 kb and includes 13 genes (9 novel), 9 STSs (5 novel), and 1 SNP and builds upon a published physical map spanning the region from the telomere to the HRAS gene. In addition, we expand the mucin gene cluster located on 11p15.5 to include a novel mucin-like gene (MUCDHL) located less than 250 kb telomeric to MUC6. The identification of potential disease genes within an organizational and evolutionary context provides valuable clues to function and as such will benefit our understanding of this region of the genome.

Chromosomes, Artificial, Bacterial↗