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M R James

Publications and source records attributed to M R James.

At least 19 recordsLinked to original sources
PubMed2001

Sequences adjacent to oriP improve the persistence of Epstein-Barr virus-based episomes in B cells.

Epstein-Barr virus (EBV) oriP and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spans oriP and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing oriP alone.

Attachment Sites, Microbiological↗
PubMed2001

Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo.

Many human breast tumors are driven by high intratumor concentrations of 17beta-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [(3)H]estrone sulfate to [(3)H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein.h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein.h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein.h). 17beta-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G(2)/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17beta-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17beta-estradiol sulfate appears more effective than 17beta-estradiol when both are administered at comparable concentrations. This effect, which is seen only in STS Clone 20 cells, may reflect differences in the cellular pharmacology of exogenous estrogens compared with those released by the activity of intracellular STS. These studies directly demonstrate that intratumor STS activity can support estrogen-dependent tumorigenicity in an experimental model and may contribute to the promotion of human breast tumors.

Animals↗
PubMed2000

Characterization of newly developed SSLP markers for the rat.

We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27-36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F2 progeny of (OLETFxBN)F2 crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species.

Animals↗
PubMed2000

Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene.

Persistent expression of a transgene at therapeutic levels is required for successful gene therapy, but many small vectors with heterologous promoters are prone to vector loss and transcriptional silencing. The delivery of genomic DNA would enable genes to be transferred as complete loci, including regulatory sequences, introns, and native promoter elements. These elements may be critical to ensure prolonged, regulated, and tissue-specific transgene expression. Many studies point to considerable advantages to be gained by using complete genomic loci in gene expression. Large-insert vectors incorporating elements of the bacterial artificial chromosome (BAC) cloning system, and the episomal maintenance mechanisms of Epstein-Barr virus (EBV), can shuttle between bacteria and mammalian cells, allowing large genomic loci to be manipulated conveniently. We now demonstrate the potential utility of such vectors by stably correcting a human genetic deficiency in vitro. When the complete hypoxanthine phosphoribosyltransferase (HPRT) locus of 115 kilobases (kb) was introduced into deficient human cells, the transgene was both maintained as an episome and expressed stably for six months in rapidly dividing cell cultures. The results demonstrate for the first time that gene expression from an episomal genomic transgene can correct a cell culture disease phenotype for a prolonged period.

Cell Line↗
PubMed2000

Chemical protective clothing; a study into the ability of staff to perform lifesaving procedures.

OBJECTIVE: To investigate the ability of medical and nursing staff to perform certain tasks while wearing a chemical protection suit with a respirator. Tasks chosen were those that would be required before decontamination. METHODS: Ten experienced accident and emergency doctors (middle grade and consultants) and 10 nurses were asked to perform certain tasks that were judged to be life saving, relevant to triage, or necessary to confirm death, on an advanced life support manikin, while wearing a TST-Sweden chemical protection suit. The operators were objectively assessed by one of the authors for achieving each task, then asked to make a subjective assessment of the difficulty experienced. RESULTS: Medical staff were asked to ventilate the manikin using a bag-valve-mask, intubate within 30 seconds, apply monitor electrodes and cables and check cardiac rhythm, apply gel pads and defibrillate safely, and finally, fold the cruciform triage card to show "RED", and attach it to the manikin. All the doctors completed these tasks, except for one, who could only intubate the manikin after several attempts. Nursing staff were asked to open and apply an oxygen mask, adjust oxygen flow, size and insert an oropharyngeal airway, ventilate the manikin using a bag-valve-mask, apply a pressure bandage to a limb, and fold the cruciform triage card to show "YELLOW", and attach it to the manikin. All the nurses completed these tasks. Operators reported varying degrees of difficulty, the most difficult tasks were those requiring fine movements or delicate control. Generally, operators found the butyl rubber gloves cumbersome. Communication difficulties were frequently reported. Although only intubation was formally timed, tasks were perceived to take longer. Some operators found the suits too warm and uncomfortable. CONCLUSION: Should the need arise, the TST-Sweden chemical protection suits would enable experienced doctors and nurses to perform lifesaving measures effectively, without significant impairment to their skills. Tasks would be easier to accomplish with better fitting gloves.

Accidents↗
PubMed2000

An anchored YAC-STS framework for the rat genome.

We report here the first YAC-STS framework for the rat genome. A total of 417 anchor microsatellite markers were used to screen a 10-fold redundant YAC library. One or more unambiguous YACs were identified for 372 markers. Assuming the genetic length of the rat genome to be 2,000 cM (Bihoreau et al. 1997b), the YAC-STS framework will provide, on average, one informative YAC clone every 5.4 cM. A total of 111 anchor markers used in this study were derived from known gene regions. We also demonstrated one of the important and immediate uses of this YAC-STS framework, which is to establish a correlation between the genetic and cytogenetic maps in the rat through FISH analysis.

Animals↗
PubMed2000

The ATM gene in the pathogenesis of mantle-cell lymphoma.

BACKGROUND: Mantle-cell lymphoma (MCL) is genetically characterized by the translocation t(11;14)(q13;q32) leading to an overexpression of cyclin-D1, but additional chromosomal abnormalities appear to be required for MCL pathogenesis. PATIENTS AND METHODS: Deletions involving chromosome 11q, which were recently found as recurrent aberrations in MCL, were analyzed at the molecular level in a series of 81 MCL by fluorescence in situ hybridization (FISH) with probes from a contiguous set of yeast artificial chromosomes (YACs) spanning bands 11q14-q24. RESULTS AND CONCLUSIONS: Loss of chromosome 11 material was observed in 37 of the 81 MCL cases (46%). The consensus deletion comprised YAC 801e11 containing the ATM gene. The minimal region of loss was further narrowed with P1-derived artificial chromosome (PAC) probes. This allowed the identification of a deletion confined to the genomic region of ATM, which, together with intragenic mutations found in the coding sequence, suggests a role of ATM as a tumor suppressor gene in MCL.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1999

Molecular characterization of 11q deletions points to a pathogenic role of the ATM gene in mantle cell lymphoma.

Deletions involving the long arm of chromosome 11 (11q) have been recently found as recurrent chromosome aberrations in mantle cell lymphoma (MCL). In the current study, the incidence and molecular extent of 11q deletions were analyzed in a series of 81 MCL by fluorescence in situ hybridization with probes from a contiguous set of yeast artificial chromosomes (YACs). Loss of chromosome 11 material was observed in 37 of 81 cases (46%). The minimally deleted segment comprised YAC 801e11 containing the ATM gene. To further narrow the minimal region of loss, P1-derived artificial chromosomes mapping to the critical region were isolated and used as probes in cases without aberrations detectable with YACs. This allowed the identification of an ATM deletion that was beyond the resolution of YAC probes. The identification of a minimally deleted segment affecting ATM suggests a pathogenic role of ATM as a tumor suppressor gene in MCL.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1999

Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells.

We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay. The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication. This element provides nuclear retention in cells expressing the EBV protein EBNA-1. We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line. Episomal mitotic stability was quantitatively analysed using flow cytometry. The episomes were also tracked by time course photography of expanding colonies. A 117 kb episome was retained at approximately 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth. Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days. Such a trackable E.coli /human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.

Cell Line, Transformed↗
PubMed1999

11q23.1 and 11q25-qter YACs suppress tumour growth in vivo.

Frequent allelic deletion at chromosome 11q22-q23.1 has been described in breast cancer and a number of other malignancies, suggesting putative tumour suppressor gene(s) within the approximately 8 Mb deleted region. In addition, we recently described another locus, at the 11q25-qter region, frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this approximately 2 Mb deleted region. An 11q YAC contig was accessed and three YACs, one containing the candidate gene ATM at 11q23.1, and two contiguous YACs (overlapping for approximately 400-600 kb) overlying most of the 11q25 deleted region, were retrofitted with a G418 resistance marker and transfected into murine A9 fibrosarcoma cells. Selected A9 transfectant clones (and control untransfected and 'irrelevant' alphoid YAC transfectant A9 clones) were assayed for in vivo tumorigenicity in athymic female Balb c-nu/nu mice. All the 11q YAC transfectant clones demonstrated significant tumour suppression compared to the control untransfected and 'irrelevant' YAC transfected A9 cells. These results define two discrete tumour suppressor loci on chromosome 11q by functional complementation, one to a approximately 1.2 Mb region on 11q23.1 (containing the ATM locus) and another to a approximately 400-600 kb subterminal region on 11q25-qter.

Animals↗
PubMed1999

A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Animals↗
PubMed1999

Localization of Jacobsen syndrome breakpoints on a 40-Mb physical map of distal chromosome 11q.

Jacobsen syndrome is a haploinsufficiency disorder caused, most frequently by terminal deletion of part of the long arm of chromosome 11, with breakpoints in 11q23.3-11q24.2. Inheritance of an expanded p(CCG)n trinucleotide repeat at the folate-sensitive fragile site FRA11B has been implicated in the generation of the chromosome breakpoint in several Jacobsen syndrome patients. The majority of such breakpoints, however, map distal to this fragile site and are not linked with its expression. To characterize these distal breakpoints and ultimately to further investigate the mechanisms of chromosome breakage, a 40-Mb YAC contig covering the distal long arm of chromosome 11 was assembled. The utility of the YAC contig was demonstrated in three ways: (1) by rapidly mapping the breakpoints from two new Jacobsen syndrome patients using FISH; (2) by demonstrating conversion to high resolution PAC contigs after direct screening of PAC library filters with a YAC clone containing a Jacobsen syndrome breakpoint; and (3) by placing 23 Jacobsen syndrome breakpoints on the physical map. This analysis has suggested the existence of at least two new Jacobsen syndrome breakpoint cluster regions in distal chromosome 11.

Abnormalities, Multiple↗
PubMed1998

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

Animals↗
PubMed1998

Bipolar affective disorder partially cosegregates with a balanced t(9;11)(p24;q23.1) chromosomal translocation in a small pedigree.

Analysis of an extended pedigree in which a balanced t(9;11)(p24;q23.1) translocation was found to cosegregate with bipolar affective disorder revealed that five of 11 translocation carriers had bipolar affective disorder and one carrier had unipolar depression. There were no affected individuals in the pedigree without the balanced translocation. We hypothesized that gene(s) or gene regulatory regions disrupted by the translocation might be contributing to the bipolar affective disorder in a dominant fashion. To test this hypothesis, we isolated the derivative chromosome 9 and derivative chromosome 11 in somatic cell hybrids and identified the nearest flanking markers on chromosome 9 (D9S230 and D9S2011E/HRFX3) and chromosome 11 (EST00652 and CRYA2). YAC contigs were constructed in the region of flanking markers for both chromosomes 9 and 11. Chromosome 11 breakpoint was localized within an 8-kb region in a small insert (100 kb) YAC. Chromosome 9 breakpoint was localized within approximately 2 Mb region. Several genes and ESTs including EST00652, CRYA2, DRD2, 5HTR3 on chromosome 11 and VLDLR and SLC1A1 on chromosome 9 were mapped within the vicinity of the breakpoint but were shown not to be disrupted by the translocation breakpoint. Although several possibilities exist regarding the role of the balanced translocation in developing bipolar affective disorder in this pedigree, including a chance cosegregation, identification of a disrupted gene or gene regulatory region with the help of physical mapping resources described in this study may help to identify the presence of a susceptibility gene for this disorder.

Bipolar Disorder↗
PubMed1997

A high-resolution STS, EST, and gene-based physical map of the hereditary paraganglioma region on chromosome 11q23.

The genes responsible for hereditary paragangliomas (glomus tumors, MIM No. 168000) have been mapped to two distinct loci on the long arm of chromosome 11. Most of the informative families appear to be linked to the distal locus on chromosome 11q23 (PGL1), which has been previously confined to a 2-cM interval by haplotype analysis in an extended Dutch pedigree. To facilitate the identification of the PGL1 disease gene, we constructed an approximately 4-Mb ordered clone contig map of Sequence tagged sites, expressed sequence tags (ESTs), and known genes that spans the PGL1 critical region on chromosome 11q23. Among 29 new positional candidate ESTs, only two (EST100999 and EST241777) mapped within the PGL1 critical region. We further characterized the genomic organization of the promyelocytic leukemia zinc finger (PLZF) gene that maps within the PGL1 critical region and physically excluded the serotonin receptor type 3 (5HT3R) gene. Finally, we identified a common, silent, single-base substitution polymorphism in the 5HT3R gene and characterized the allele sets of two new highly polymorphic microsatellite repeats within the PGL1 critical region.

Alleles↗
PubMed1997

11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis.

Deletions of the long arm of chromosome 11 (11q) are one of the most frequent structural chromosome aberrations in various types of lymphoproliferative disorders. However, in most conventional chromosome banding studies of B-cell chronic lymphocytic leukemia (B-CLL), 11q deletions were not identified as a frequent aberration. The objective of this study was to analyze the frequency and clinical impact of 11q deletions in B-CLL by interphase cytogenetics using fluorescence in situ hybridization (FISH). Mononuclear cells from 214 patients with B-CLL were studied by FISH using the yeast artificial chromosome (YAC) clone 755b11 from chromosome region 11q22.3-q23.1; we previously showed that this clone was contained within a 2- to 3-Mb sized segment of 11q commonly deleted in lymphoproliferative disorders. Forty-three of the 214 (20%) tumors exhibited 11q deletions; 11q deletions were the second most frequent chromosome aberration following 13q14 (RB1 and/or D13S25) deletions (45%); they were more frequent than trisomy 12 (15%) or deletion of 17p (TP53 gene) (10%). Patients with 11q deletions were younger (P = .01) and had more advanced clinical stages (P = .01). 11q deletions were associated with extensive peripheral, abdominal, and mediastinal lymphadenopathy (P < .001). Patients with 11q deletions had a more rapid disease progression as shown by a shorter treatment-free interval (9 months v 43 months; P < .001). The prognostic effect of 11q deletion on survival strongly depended on the age: in patients less than 55 years old, the median survival time was significantly shorter in the deletion group (64 months v 209 months; P < .001), whereas in patients > or = 55 years old there was no significant difference (94 months v 111 months; P = .82). 11q deletions identify a new clinical subset of B-CLL characterized by extensive lymph node involvement. In younger B-CLL patients, this aberration is an important predictor of survival.

Adult↗