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Results for “positional cloning”

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At least 127 records · Page 7Linked to original sources
PubMed2000

Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.

Animals↗
PubMed1988

Karyotype evolution and multilineage involvement of Philadelphia chromosome-positive clones in blastic transformation of two patients with chronic myelocytic leukemia.

Simultaneous analysis of the cell morphology and karyotypes on single colonies was carried out in two patients with Philadelphia chromosome (Ph1)-positive chronic myelocytic leukemia in blastic transformation in order to clarify the origin of leukemic cells involved. Patient no. 1 was in a typical myeloblastic transformation and patient no. 2 in "basophilic transformation." Both patients exhibited karyotype evolution in blastic phase (BP), so that we could differentiate BP clones with additional chromosomal abnormalities from chronic phase (CP) clones with only Ph1 among single colonies. The number of single colonies yielding two or more analyzable metaphases was 18 in patient no. 1, and 19 in patient no. 2. Among these colonies, only three in patient no. 1 and none in patient no. 2 were from CP clones and 15 in patient no. 1 and 19 in patient no. 2 were from BP clones. Morphological examination revealed that not only blasts but also mature neutrophils, eosinophils, basophils, macrophages, and erythroblasts were derived from BP clones. These results suggested that (1) BP clones developed at the pluripotent stem cell level, (2) additional chromosomal abnormalities were not restricted to occur in a specific cell line representative in BP; and (3) BP clones, if not all, may retain capacity for maturation and differentiation.

Blast Crisis↗
PubMed1996

Positional cloning of the Drosophila melanogaster mei-9 gene, the putative homolog of the Saccharomyces cerevisiae RAD1 gene.

Mutation of the Drosophila melanogaster mei-9 gene causes enhanced mutagen sensitivity, DNA excision repair incision deficiency, germ line hypermutability, mitotic chromosome breakage and instability, and, in females, increased levels of meiotic non-disjunction and decreased levels of meiotic recombination. This paper reports on the utilization of a positional mapping strategy combined with a differential expression assay to clone the mei-9 gene. We report that mei-9 is homologous to the yeast repair gene, RAD1, and demonstrate that the mei-9 gene transcript is absent in the mei-9AT3 mutant.

Adenosine Triphosphatases↗
PubMed1986

I-J-positive cloned macrophages as accessory cells for the induction of suppressor T cells in vitro.

I-A+/I-J+ cloned macrophages, SL-1, played the role of APC in the in vitro induction of Ts against DTH to BCG. By treating SL-1 cells with various antibodies, it was shown that I-J antigens on SL-1 cells are essential for Ts induction, but not for the effector cell induction for DTH. Ia-negative cloned macrophages, SL-4, did not show any APC activity either in suppressor or effector cell induction. The precursors of the Ts were also I-J-positive, and I-J restriction resided between T cells and macrophages in the Ts induction. Thus, it is suggested that the pre-Ts recognizes the antigenic determinants of BCG presented on the APC in association with the I-J antigen and differentiates into the Ts. This pathway seems analogous to that of helper or effector T induction, where the antigenic determinant is recognized by a T cell in association with the I-A antigen on APC.

Animals↗
PubMed1990

Selection of HTLV-I positive clones is prevented by prostaglandin A in infected cord blood cultures.

Type A prostaglandins (PGA1 and 16,16-dimethyl-PGA2-methyl ester) were found to block the proliferation of HTLV-I infected cord blood lymphocytes (CBL) in vitro, thus preventing the clonal immortalisation that is considered as a predisposing condition to HTLV-I positive leukaemia. PGA1 and di-M-PGA2 did not affect the long-term survival of normal non-infected CBL, whereas they suppressed the proliferation of an established cord-blood derived HTLV-I positive cell line, MT-2. As shown by the number of HTLV-I infected p19+ cells, the block of the selection of immortalised, infected clones by PGAs did not appear to be due to an inhibition of early stages of HTLV-I infection. The possibility that the effect of PGAs could be mediated by an action on the immune response was also examined. PGAs regulated the cell-mediated cytotoxic function of CBL to a different extent when normal non-infected or HTLV-I exposed CBL were compared. In fact, PGAs down-regulated the natural killing and macrophage/lymphocyte cytotoxic response of normal CBL, whereas they did not modify the already depressed immune response of CBL challenged with HTLV-I. These results suggest that the protective effect of PGAs against HTLV-I infection in vitro is mostly related to the direct suppression of the clonal expansion of virus-infected cells, rather than to the anti-viral activity or modulation of the cell-mediated immunity.

Cells, Cultured↗
PubMed1989

Production and characterisation of immunoreactive calcitonin gene-related peptide (CGRP) from a CGRP receptor-positive cloned osteosarcoma cell line (UMR 106.01).

A subclone of an osteoblast-like osteosarcoma cell line (UMR 106.01) has recently been shown to possess specific binding sites for calcitonin gene-related peptide (CGRP) linked to adenylate cyclase. The present study provides the first demonstration for the production of immunoreactive CGRP from CGRP-receptor positive osteosarcoma cells. Mean immunoreactive CGRP levels were 15 pmol/g and 1 pmol/l for acid extracts of cells and cell-exposed media respectively. On gel filtration and high performance liquid chromatography, a major proportion of immunoreactive CGRP was found to co-elute with synthetic rat CGRP(1-37). Only negligible quantities of calcitonin were detected in cell extracts or cell-exposed supernatant. The production of authentic CGRP from a CGRP-receptor positive tumour suggests that the peptide may have autocrine effects on its producer cell.

Animals↗
PubMed2005

Exploitation of a marker dense linkage map of potato for positional cloning of a wart disease resistance gene.

A marker-saturated linkage map of potato was used to genetically map a locus involved in the resistance against wart disease Synchytrium endobioticum race 1. The locus mapped on the long arm of chromosome 4 and is named Sen1-4 in contrast to a Sen1 locus on chromosome 11. The AFLP markers from the Sen1-4 interval enabled the isolation of BAC clones from an 11 genome equivalent BAC library. This was achieved via fingerprinting of BAC pools with the AFLP primer pairs that resemble the genetic marker loci. With non-selective AFLP primers, fingerprints of individual BAC clones were generated to analyse the overlap between BAC clones using FPC. This resulted in a complete contig and a minimal tiling path of 14 BAC clones enclosing the Sen1-4 locus. The BAC contig has a genetic length of approximately 6 cM and a physical length of approximately 1 Mb. Our results demonstrate that map-based cloning of Sen1-4 can be pursued on the basis of a strategy of marker saturation alone. Genetic resolution achieved by screening large numbers of offspring for recombination events may not be required. Together with the construction of the BAC contig, a physical map with the position of the markers is accomplished in one step. This provides proof of concept for the utility of the marker saturation that is offered by the ultra dense AFLP map of potato for gene cloning.

Chromosome Mapping↗
PubMed1993

Positional cloning of the hereditary renal carcinoma 3;8 chromosome translocation breakpoint.

The chromosome (p14.2;q24.1) translocation t(3;8) has been associated with hereditary renal cancer in one family. Based on cytogenetic analyses and loss-of-heterozygosity experiments, the 3p14 region has been independently implicated as harboring a tumor suppressor gene critical to kidney and lung cancer development. The 3p14.2 region also contains FRA3B, the most sensitive fragile site induced by aphidicolin. A chromosome 3 probe, R7K145, derived from a radiation-reduced hybrid was positioned between the t(3;8) breakpoint and an aphidicolin-induced 3p14 breakpoint. A yeast artificial chromosome (YAC) contig containing R7K145 was developed that crossed the aphidicolin-induced breakpoint on its telomeric side. A subsequent chromosome walk identified a YAC that crossed the 3;8 translocation breakpoint. A lambda sublibrary allowed isolation of clones spanning the rearrangement. Unique and evolutionarily conserved DNA sequences were used to screen a kidney cDNA library. We have identified a gene, referred to as HRCA1 (hereditary renal cancer associated 1), that maps immediately adjacent to the breakpoint. On the basis of its chromosomal position, HRCA1 may be a candidate tumor suppressor gene.

Animals↗
PubMed1986

Effect of cholera toxin on the induction of gamma-glutamyl transpeptidase-positive clones by benzo(a)pyrene in embryonic mouse tongue cells in culture.

The utility of gamma-glutamyl transpeptidase (GGT) as a histochemical marker to study the carcinogen-induced changes and its possible modulation by cholera toxin was explored in primary cell cultures. It was observed that cholera toxin not only promotes growth of fibroblasts in primary cell culture, but it also enhances the GGT-positive foci induced by benzo(a)pyrene. It is interesting to speculate that cholera toxin may be acting as a tumor promoter in primary cell cultures of embryonic mouse tongue.

Animals↗