An Ordered NotI Fragment Map of Human Chromosome Band 11p15

Higgins, Michael J.; Smilinich, Nancy J.; Sait, Sheila N.J.; Koenig, Amanda; Pongratz, J.; Gessler, Manfred; Richard, Charles W.; James, M. R.; Sanford, Janet P.; Kim, B.-W.; Cattelane, J.; Nowak, Norma J.; Winterpacht, Andreas; Zabel, Bernard; Munroe, David J.; Bric, Eva; Housman, David E.; Jones, C. Hal; Nakamura, Yusuke; Gerhard, Daniela S.; Shows, Thomas B.

doi:10.1006/geno.1994.1479

Cited by 20 publications

(17 citation statements)

References 45 publications

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“…Current maps in the databases are still controversial on the order of 11p15.3 genes. The mapping data presented here settles the debate whether LMO1 is more centromeric compared to WEE1 as proposed by Redeker et al (1995), or more telomeric as proposed by Higgins et al (1994) and confirmed by our results.…”

Section: Resultssupporting

confidence: 77%

Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1)

Cichutek

Brueckmann²,

Seipel³

et al. 2001

Cytogenet Genome Res

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Human chromosome 11p15.3 is associated with chromosome aberrations in the Beckwith Wiedemann Syndrome and implicated in the pathogenesis of different tumor types including lung cancer and leukemias. To date, only single tumor-relevant genes with linkage to this region (e.g. LMO1) have been found suggesting that this region may harbor additional potential disease associated genes. Although this genomic area has been studied for years, the exact order of genes/chromosome markers between D11S572 and the WEE1 gene locus remained unclear. Using the FISH technique and PAC clones of the flanking markers we determined the order of the genomic markers. Based on these clones we established a PAC contig of the respective region. To analyse the chromosome area in detail the synteny of the orthologous region on distal mouse chromosome 7 was determined and a corresponding mouse clone contig established, proving the conserved order of the genes and markers in both species: “cen–WEE1–D11S2043–ZNF143–RANBP7–CEGF1– ST5–D11S932–LMO1–D11S572–TUB–tel”, with inverted order of the murine genes with respect to the telomere/centromere orientation. The region covered by these contigs comprises roughly 1.6 MB in human as well as in mouse. The genomic sequence of the two subregions (around WEE1 and LMO1) in both species was determined using a shotgun sequencing strategy. Comparative sequence analysis techniques demonstrate that the content of repetitive elements seems to decline from centromere to telomere (52.6% to 34.5%) in human and in the corresponding murine region from telomere to centromere (41.87% to 27.82%). Genomic organisation of the regions around WEE1 and LMO1 was conserved, although the length of gene regions varied between the species in an unpredictable ratio. CpG islands were found conserved in putative promoter regions of the known genes but also in regions which so far have not been described as harboring expressed sequences.

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Section: Resultssupporting

confidence: 77%

Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1)

Cichutek

Brueckmann²,

Seipel³

et al. 2001

Cytogenet Genome Res

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“…3). Furthermore, consistent with the previously determined map position of the 30a exon, we have been able to localize the 30a cDNA to a specific Not I fragment on human chromosome lip15.5 (26). We have found that the arrayed cDNA library pools are also amenable to screening by hybridization.…”

Section: Resultssupporting

confidence: 67%

“…5). In other experiments we have further mapped this cDNA to a specific Not I restriction fragment within lip15.5 (26).…”

Section: Resultsmentioning

confidence: 99%

Systematic screening of an arrayed cDNA library by PCR.

Munroe

Loebbert

Bric

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a PCR-based method for rapid and effective screening of arrayed cDNA libraries. This strategy directly addresses the limitations of conventional hybridization-based schemes and provides a more rapid, cost-effective, and sensitive method compatible with largescale and routine cDNA clone recovery. To prepare arrayed libraries, 1-2 x 106 cDNA clones were propagated as individual plaques on solid medium in 24-well culture dishes at -250 plaque-forming units per well. Phage suspensions were prepared from each well and transferred to a 96-well format. To screen the library, pools were generated that correspond to each individual 96-well plate and to each row and column within "blocks" of six plates each. Library screening for specific cDNA clones was conducted in a systematic and hierarchical fashion beginning with the plate pools. Next, the row/column pools corresponding to each positive plate pool were screened. Finally, isolated clones from within each positive well were identified by hybridization. We have applied this approach to the screening of an arrayed human brain cDNA library resulting in the recovery of cDNAs corresponding to >25 genes and expressed sequence tags.The efficient identification and recovery of expressed sequences from large segments of complex genomes are central to the development and integration of long-range physical and transcription maps. Construction of such maps facilitates the ongoing efforts of the genome initiative and heightens understanding of genome structure and organization as well as the molecular basis of development and disease.Three basic strategies have emerged that are amenable to large-scale identification of expressed sequences. (i) Exon amplification (1-4) employs in vivo splicing systems to identify and select for functional splice sites in genomic DNAs, resulting in the generation of cloned exon libraries. (ii) Hybrid selection (5, 6) relies on hybridization of cDNA fragments to immobilized genomic DNAs to enrich for cDNAs encoded by specific genomic clones. (iii) Automated partial DNA sequencing of random cDNA clones produces catalogs of expressed sequence tags (ESTs) (7-10). All three strategies result in the identification of short (100-400 bp) cDNA fragments that, when localized relative to markers of known map position, pinpoint the location of an expressed gene (8,9,(11)(12)(13)(14)(15)(16)(17) Here, we report an effective and high through-put system for arraying and screening cDNA libraries. This system directly addresses the limitations of phage/colony hybridization schemes and provides a more efficient and sensitive screening method compatible with large-scale and routine cDNA clone recovery.l MATERIALS AND METHODS Arraying and Pooling the Library. Approximately 250 phage were plated in 0.1 ml of NZYM low-melt top agar overlays in each well of 192 24-well culture dishes containing 1.0 ml of NZYM bottom agar. Following incubation at 4°C for 30 min and 37°C for 18-24 hr (until near-confluent lysis) phage suspensions were prepared from...

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“…Hybridization to cosmid probes from the human 11p15 region was detected with fluorescently labeled tyramide reagents (NEN Life Sciences Products) as described in reference 64. In most lines, we saw hybridization against the Ha-ras probe (29), located approximately 5 Mb from the ␤-globin locus toward the telomere, and against the J5-3 probe (40), located approximately 5 Mb from the ␤-globin locus toward the centromere. The few lines that gave a signal with only one of these probes hybridized with both the N159-H3 probe (29), located approximately 1,300 kb from the ␤-globin locus toward the telomere, and the N169-F10 probe (29), located approximately 1,600 kb from the ␤-globin locus toward the centromere.…”

Section: Methodsmentioning

confidence: 99%

The Locus Control Region Is Necessary for Gene Expression in the Human β-Globin Locus but Not the Maintenance of an Open Chromatin Structure in Erythroid Cells

Reik

Telling

Zitnik

et al. 1998

Molecular and Cellular Biology

153

117

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Studies in many systems have led to the model that the human ␤-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the ␤-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous ␤-globin locus in an erythroid background, transcription of all ␤-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the ␤-globin locus are not affected by the deletion. In contrast, deletion of 5HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on ␤-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an "open" conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the ␤-like globin genes.The human ␤-globin locus provides a model system to study the interplay between chromatin structure and transcriptional regulation. The locus is located on chromosome 11 (11p15.5) and contains five developmentally regulated erythroid cell-specific genes arranged in the order in which they are expressed during development (5Ј-ε-G␥-A␥-␦-␤-3Ј) and an upstream regulatory region characterized by five DNase I-hypersensitive sites (HSs; see Fig. 1). By convention, these 5ЈHSs are denoted 5ЈHS1, 5ЈHS2, etc., with 5ЈHS1 being the most 3Ј and located closest to the ε-globin gene. The importance of the upstream regulatory region was established by an analysis of the effects of a naturally occurring deletion which removes 5ЈHS2 to 5ЈHS5 and 25 kb of additional sequences 5Ј of the HSs (Hispanic thalassemia). The chromosome carrying the deletion was transferred from lymphocytes of the thalassemic patient into murine erythroleukemia (MEL) cells to create the thalassemia line T-MEL. It was found that the mutation abolishes transcription of the adult human ␤-globin gene, prevents formation of 5ЈHS1 and other HSs throughout the locus, and renders the chromatin of the locus resistant to DNase I, indicative of a "closed" chromatin structure (19). In addition, the replication timing of the locus is changed from early to late in S phase (19) and a different origin of replication is used, even though the normal origin lies more than 50 kb from the site of the deletion (1, 36). The region containing the five HSs was termed the locus control region (LCR), because of its global effects on the locus. In transgenic mice, the LCR permits the expression of linked genes in all lines independent of the integration site (26), and regions with LCR...

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An Ordered NotI Fragment Map of Human Chromosome Band 11p15

Cited by 20 publications

References 45 publications

Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1)

Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1)

Systematic screening of an arrayed cDNA library by PCR.

The Locus Control Region Is Necessary for Gene Expression in the Human β-Globin Locus but Not the Maintenance of an Open Chromatin Structure in Erythroid Cells

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