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D Sheer

Publications and source records attributed to D Sheer.

At least 73 records · Page 4Linked to original sources
PubMed1994

Cloning of the region between HLA-DMB and LMP2 in the human major histocompatibility complex.

The human MHC is one of the most extensively mapped regions of the human genome. Almost all of the class II region of the MHC has already been cloned in cosmids but a gap remained between the DMB and LMP2 genes. Previously, screening of several complete cosmid libraries had failed to bridge this gap, which may contain novel antigen processing or presentation genes. We constructed cosmid libraries from two different sources in order to clone the region: (a) a library with fourfold coverage made from flow-sorted human chromosome 6 DNA and (b) a library derived from a yeast artificial chromosome clone spanning the region. Using this saturation approach, cosmid clones were eventually isolated over the region of interest. A single bacteriophage P1 clone was also obtained spanning the region. The YAC, cosmid, and P1 physical maps were consistent and the distance between the DMB and LMP2 genes was measured as 70 kb. It is not clear why DMB to LMP2 is infrequently represented in cosmid libraries, but the clones that we have obtained will now enable us to search for new coding sequences.

Bacteriophage P1↗
PubMed1994

Rapid detection of prognostic genetic factors in neuroblastoma using fluorescence in situ hybridisation on tumour imprints and bone marrow smears. United Kingdom Children's Cancer Study Group.

A number of biological factors have been identified which correlate with prognosis in neuroblastoma. Among these are genetic aberrations, including ploidy, deletions of chromosome 1p and N-myc amplification. Conventional methods of detecting these changes, such as tissue culture for karyotyping and Southern blotting, are time-consuming and yield interpretable results in only a small proportion of cases. We have developed interphase fluorescence in situ hybridisation for use on tumour imprints and bone marrow smears, allowing rapid visualisation of the relevant genetic changes. Valuable prognostic information is therefore available in a few days: the results in our cases were later confirmed by conventional methods. In the foreseeable future it will be possible to define distinct prognostic categories on the basis both of this genetic information and other parameters, and separate therapeutic strategies may then be employed for the different patient groups.

Bone Marrow↗
PubMed1994

Olfactory receptor gene cluster on human chromosome 17: possible duplication of an ancestral receptor repertoire.

A gene superfamily of olfactory receptors (ORs) has recently been identified in a number of species. These receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated transduction of odorant signals. Previously, OR genes cloned in different species were from random locations in the respective genomes. We report here the cloning of 16 human OR genes, all from chromosome 17 (17p13.3). The intronless coding regions are physically mapped (on 35 cosmids) in one 0.35Mb long contiguous cluster, with an average intergenic separation of 15kb. The human OR genes in the cluster belong to four different gene subfamilies, displaying as much sequence variability as any randomly selected group of ORs. This suggests that the cluster identified may be one of several copies of an ancestral OR gene repertoire whose existence may predate the divergence of mammals. The latter may have duplicated in some species to form the present mammalian OR gene repertoire, with several hundred genes. The human chromosome 17 OR gene cluster may thus be a good model for understanding human olfaction, as well as the ontogeny and phylogeny of the OR gene superfamily.

Amino Acid Sequence↗
PubMed1994

Released chromatin: linearized DNA for high resolution fluorescence in situ hybridization.

Free DNA was prepared from routinely harvested and fixed cells for high resolution FISH mapping using either a sodium hydroxide/ethanol mixture or 70% formamide. Hybridization signals from cosmid probes appeared as extended lines. The average length of signals on DNA prepared with sodium hydroxide was significantly greater than with formamide. A set of overlapping cosmids from the HLA class II region was used to determine how precisely the actual overlap or gap between probes can be calculated from the measured overlap or gap between their signals. Lengths of the probe signals and their known kilobase lengths were used as an internal ruler. The mean values calculated from the measured length from 30 or more signals for each probe pair showed remarkable conformity with the known kilobase lengths of their overlaps and gaps. Immediately adjacent probes could also be ordered on the released DNA. These simple procedures dramatically increase the speed with which relationships between probes can be determined during contig construction.

Chromatin↗
PubMed1993

Combinatorial generation of variable fusion proteins in the Ewing family of tumours.

Balanced translocations involving band q12 of human chromosome 22 are the most frequent recurrent translocations observed in human solid tumours. It has been shown recently that this region encodes EWS, a protein with an RNA binding homologous domain. In Ewing's sarcoma and malignant melanoma of soft parts, translocations of band 22q12 to chromosome 11 and 12 result in the fusion of EWS with the transcription factors FLI-1 and ATF1, respectively. The present analysis of 89 Ewing's sarcomas and related tumours show that in addition to the expected EWS-FLI-1 fusion, the EWS gene can be fused to ERG, a transcription factor closely related to FLI-1 but located on chromosome 21. The position of the chromosome translocation breakpoints are shown to be restricted to introns 7-10 of the EWS gene and widely dispersed within introns 3-9 of the Ets-related genes. This heterogeneity generates a variety of chimeric proteins that can be detected by immuno-precipitation. On rare occasions, they may be associated with a truncated EWS protein arising from alternate splicing. All 13 different fusion proteins that were evidenced contained the N-terminal domain of EWS and the Ets domain of FLI-1 or ERG suggesting that oncogenic conversion is achieved by the linking of the two domains with no marked constraint on the connecting peptide.

Amino Acid Sequence↗
PubMed1993

A long-range restriction map of human chromosome 5q21-q23.

A long-range restriction map encompassing the APC (adenomatous polyposis coli) gene has been constructed. The map includes 35 DNA markers and consists of two segments of 10 and 2.5 Mb. Published genetic markers have been connected using additional, nonpolymorphic DNA probes. The map clarifies marker order and allows comparison of physical and genetic data.

Adenomatous Polyposis Coli↗
PubMed1993

New vector for transfer of yeast artificial chromosomes to mammalian cells.

A modification vector has been constructed to facilitate the transfer of yeast artificial chromosomes (YACs) to mammalian cells in culture by targeting a dominant selectable marker (G418 resistance) to the right arm of pYAC4 clones. The ADE2 gene is used for yeast selection with consequent disruption of the URA3 gene, allowing direct modification of YACs within the common host strain AB1380, and providing a simple test for correct targeting. This vector has been tested by modification of a 550-kb YAC containing part of the human MHC class II region and transfer to CHO cells by protoplast fusion. Analysis of 15 independent G418-resistant CHO lines obtained following fusion suggests the majority contain a complete YAC with moderate amplification in some lines.

Animals↗
PubMed1993

"Junction trapping". A simple PCR-based method for the isolation of YAC-insert termini.

A new and potentially reliable method for the isolation of yeast artificial chromosome (YAC)-insert termini, termed junction trapping, is described. This method is based on simple partial digestions of the YAC-containing yeast strain, ligation to a plasmid vector, and selection of the termini by two rounds of polymerase chain reaction (PCR). To date, the method has generated both terminal sequences from each of nine YACs (100%) that have been examined.

Chromosome Mapping↗
PubMed1993

Diagnosis of Ewing's sarcoma and peripheral neuroectodermal tumour based on the detection of t(11;22) using fluorescence in situ hybridisation.

Fluorescence in situ hybridisation (FISH) has been used increasingly for gene mapping and ordering probes on interphase and metaphase preparations. The association of consistent chromosomal aberrations with certain malignancies allows the possibility of using interphase cytogenetics as a diagnostic tool. In small round cell tumours of children accurate diagnosis may be difficult using existing methods. We have therefore evaluated the diagnostic potential of this technique when applied to the characteristic t(11;22) found in Ewing's sarcoma and peripheral neuroectodermal tumour (ES and PNET). Interphase nuclei were prepared from normal human foreskin fibroblasts (HFF), two Ewing's sarcoma cell lines and several fresh tumour biopsies. DNA probes each side of the breakpoint at 22q12 were labelled with biotin and digoxygenin, hybridised to chromosomes in interphase and detected in different colours. Measurements between pairs of signals arising from each copy of chromosome 22 were taken and statistical analysis performed. There was a highly significant difference (P < 0.0001) between the two populations of measurements obtained (from nuclei with and without the t(11;22)). Studying four tumours and one further ES line (blind) it was found that median values from 30 nuclei could correctly identify which samples contained the t(11;22). This application of interphase cytogenetics contributes a reliable, accurate and conceptually simple diagnostic test for ES and PNET. It may now be applied to other tumours with characteristic translocations, amplifications or deletions when suitable probes are available. This approach is likely to become a routine in clinical diagnosis.

Chromosomes, Human, Pair 11↗
PubMed1993

The organization and conservation of the human Surfeit gene cluster and its localization telomeric to the c-abl and can proto-oncogenes at chromosome band 9q34.1.

The mouse Surfeit locus contains an unusually tight cluster of six housekeeping genes (Surf-1 to -6) which are unrelated by sequence homology. Using a mouse Surfeit locus probe, a 16 kb clone has been isolated which contains the human Surf-1 and Surf-3 genes and regions of the human Surf-2 and Surf-5 genes. The organization and juxtaposition of these human Surfeit locus genes are the same as found in the mouse. Using the human clone as a biotinylated probe for fluorescence in situ hybridization (FISH) we have confirmed the location of the human Surfeit locus to chromosome band 9q34. Metaphase spreads of human chronic myeloid leukemic cells containing the t(9;22)(q34;q11) translocation involving The c-abl gene at 9q34.1 an acute nonlymphocytic leukemic cells containing the t(6;9)(q34;p23) translocation involving the can gene at 9q34.1 were analyzed by FISH using the human Surfeit clone as a probe. These analyses locate the human Surfeit locus telomeric to the c-abl and can genes at chromosome band 9q34.1.

Animals↗
PubMed1993

Localization of the genes encoding the catalytic subunits of protein phosphatase 2A to human chromosome bands 5q23-->q31 and 8p12-->p11.2, respectively.

The gene for the alpha isoform of the catalytic subunit of human protein phosphatase 2A (PPP2CA) was localized to chromosome 5 using somatic cell hybrids, and then more finely mapped to chromosome region 5q23-->q31 by in situ hybridization using a tritiated cDNA probe. The gene for the beta isoform of the catalytic subunit of this enzyme (PPP2CB) was mapped by the polymerase chain reaction to human chromosome 8 using somatic cell hybrids. Fluorescence in situ hybridization was then used to localize the PPP2CB gene to 8p12-->p11.2, using a mixture of three genomic probes that ranged from 3.5 to 8 kb in size. Finally, Southern blot analysis of somatic cell hybrid DNA suggested that a PPP2CB catalytic subunit pseudogene (PPP2CBP) is on chromosome 16.

Animals↗
PubMed1993

Fine mapping of the human MHC class II region within chromosome band 6p21 and evaluation of probe ordering using interphase fluorescence in situ hybridization.

Eight previously well-characterized and mapped probes derived from the human major histocompatibility complex (MHC) class II region were used to investigate the advantages and limitations of fluorescence in situ hybridization (FISH) techniques for fine mapping. The class II region of the MHC was localized within subband 6p21.31 by in situ hybridization on metaphase chromosomes that were banded by immunofluorescence staining with an antibody against 5-bromodeoxyuridine (BrdU). Ordering of probes that were separated by up to 900 kb was achieved by simultaneous hybridization of two or three probes on interphase nuclei. Three-color FISH proved to be an excellent method for ordering of probes within distances of 200-1,000 kb. Under certain conditions, closer probes could be ordered by comparing measured distances between their hybridization signals in interphase nuclei. A linear correlation between measured interphase distances and kilobase distances was observed up to 500 kb. With increasing distances, the measurements become more inaccurate due to chromatin folding.

Chromosome Mapping↗
PubMed1993

Ordering of probes surrounding the Ewing's sarcoma breakpoint on chromosome 22 using fluorescent in situ hybridization to interphase nuclei.

Eight probes were localized by fluorescent in situ hybridization to the region surrounding the Ewing's sarcoma breakpoint on chromosome 22. Three of these were initially ordered by pair-wise hybridization to metaphase chromosomes with differential detection of the probes. These and the remaining probes were then ordered by hybridizing two or three probes simultaneously to interphase nuclei. In the two probe experiments and some of the three probe experiments, the order was derived by comparing mean interphase distances between signals from the probes. In the three probe experiments, either two probes were detected with one fluorochrome and the third with another or all three probes were individually distinguished by detecting one probe with fluorescein isothiocyanate (FITC), one with Texas Red, and one with both fluorochromes to give a mixed color. The order of the signals was then noted. Greater than 60 percent of configurations with a discrete order were shown or deduced to be correct. These approaches are assessed and we demonstrate a more obvious predominating order when all three probes are differentially detected. The order of the probes was deduced to be centromere: D22S271: D22S260: lambda S1: D22S262: cosK1831: Ewing's sarcoma breakpoint: cosLIF: D22S261: lambda S15: telomere.

Cell Nucleus↗
PubMed1992

Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24.

Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.

Amino Acid Sequence↗
PubMed1992

Complex translocation involving Ph chromosome in a patient with typical chronic myelogenous leukemia.

We report a cytogenetic study of a patient with chronic myelogenous leukemia (CML) who, while displaying a Philadelphia (Ph) chromosome, resulting from a standard t(9;22) at diagnosis, during the chronic phase (CP) showed disappearance of the Ph and occurrence of new chromosome changes, including a marker probably arising from a translocation involving chromosome 17 and the Ph. In situ hybridization confirmed the cytogenetic appearance and demonstrated that the breakpoint on the Ph marker occurred below the BCR-ABL fusion gene.

Chromosome Deletion↗