The origin of a morphologically unidentifiable human supernumerary minichromosome traced through sorting, molecular cloning, and in situ hybridisation.

Raimondi, Elena; Ferretti, Luca; Young, Brian; Sgaramella, Vittorio; Carli, L. De

doi:10.1136/jmg.28.2.92

Cited by 21 publications

(16 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…human supernumerary chromosome monochromosomic hybrid (MCH cells) were previously described (Raimondi et al, 1991). We verified by fluorescence in situ hybridization (FISH) the presence of the correct human chromosomes in monochromosomal hamsterϾhuman cell hybrids (our unpublished results).…”

Section: Cell Linesmentioning

confidence: 83%

“…After heat shock, stress bodies were detectable in both cell hybrids, thus mapping the region involved in this process to the p13 to q13 of HSA9 shared by the two chromosomal fragments (our unpublished results). To confirm this conclusion, we took advantage of the fact that a human supernumerary mini-chromosome spanning the 9p13-q13 region had been previously characterized in our laboratory (Raimondi et al, 1991). MCH cells containing a single mini-chromosome as the only human chromosome (Raimondi et al, 1996) were therefore challenged for the ability to form stress bodies after heat shock.…”

Section: Stress-induced Snbs Colocalize With the Pericentromeric Hetementioning

confidence: 99%

See 1 more Smart Citation

Human Chromosomes 9, 12, and 15 Contain the Nucleation Sites of Stress-Induced Nuclear Bodies

Denegri

Moralli

Rocchi

et al. 2002

MBoC

View full text Add to dashboard Cite

We previously reported the identification of a novel nuclear compartment detectable in heatshocked HeLa cells that we termed stress-induced Src-activated during mitosis nuclear body (SNB). This structure is the recruitment center for heat shock factor 1 and for a number of RNA processing factors, among a subset of Serine-Arginine splicing factors. In this article, we show that stress-induced SNBs are detectable in human but not in hamster cells. By means of hamsterϾhuman cell hybrids, we have identified three human chromosomes (9, 12, and 15) that are individually able to direct the formation of stress bodies in hamster cells. Similarly to stress-induced SNB, these bodies are sites of accumulation of hnRNP A1-interacting protein and heat shock factor 1, are usually associated to nucleoli, and consist of clusters of perichromatin granules. We show that the p13-q13 region of human chromosome 9 is sufficient to direct the formation of stress bodies in hamsterϾhuman cell hybrids. Fluorescence in situ hybridization experiments demonstrate that the pericentromeric heterochromatic q12 band of chromosome 9 and the centromeric regions of chromosomes 12 and 15 colocalize with stress-induced SNBs in human cells. Our data indicate that human chromosomes 9, 12, and 15 contain the nucleation sites of stress bodies in heat-shocked HeLa cells.

show abstract

Section: Cell Linesmentioning

confidence: 83%

Section: Stress-induced Snbs Colocalize With the Pericentromeric Hetementioning

confidence: 99%

Human Chromosomes 9, 12, and 15 Contain the Nucleation Sites of Stress-Induced Nuclear Bodies

Denegri

Moralli

Rocchi

et al. 2002

MBoC

View full text Add to dashboard Cite

show abstract

“…The sorting of microchromosomes has been reported previously only for microchromosomes larger than 20 Mb in size (5)(6)(7)(8)20). The potential of large scale purification of MACs for gene-therapy applications (8,13,14,(20)(21)(22)(23) has generated the need for a better method for the preparation and isolation of chromosomes of smaller microchromosomes with improved purity.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, flow cytometry has been applied successfully to the isolation of microchromosomes greater than 20 Mb in size (5)(6)(7)(8). To date, we are not aware of any reports on the use of flow cytometry to isolate small (<3 Mb in size), experimentally generated, microchromosomes, or mammalian artificial chromosomes, MACs (9)(10)(11)(12), which are a useful research tool for the functional characterization of genes as well as potential gene carriers for somatic gene therapy (12)(13)(14).…”

mentioning

confidence: 99%

Flow analysis and sorting of microchromosomes (<3 Mb)

Yang

Carter

2007

Cytometry Pt A

View full text Add to dashboard Cite

Background: The analysis and isolation of high numbers of chromosomes smaller than 3 Mb in size (microchromosomes) with good purity is dependent primarily on the detection sensitivity of the flow cytometer and the precision of the sort unit. The aim of this study was to investigate the capability of using a conventional flow cytometer for the detection and sorting at high purity microchromosomes with an estimated size of 2.7 Mb. Methods: Chromosomes were isolated from a human cell line containing a pair of X‐derived microchromosomes, using a modified polyamine isolation buffer. The chromosome preparation was labeled with Hoechst and Chromomycin and analyzed and purified using a MoFlo sorter (DAKO) configured for high‐speed sorting. The purity of the flow‐sorted microchromosomes was assessed by reverse chromosome painting. Results: Improved resolution of the peak of microchromosomes in a bivariate plot of Hoechst versus Chromomycin fluorescence was obtainable after discriminating clumps and debris based on gating data within a FSC versus pulse width plot. Conclusions: Chromosomes of smaller size, less than 3 Mb, can be detected with high resolution and flow‐sorted with high purity using a conventional flow sorter. © 2007 International Society for Analytical Cytology

show abstract

“…In the present paper we report data on the cytological and molecular characterisation of a chromosome 9-derived X-ray reduced accessory chromosome Raimondi et al, 1991) and on the integration of a loxP sequence into its pericentromeric region, with the purpose of generating a human chromosome vector (HCV). The genetically modified minichromosome was characterised for its mitotic stability, and ability to bind the centromeric antigens CENP-B, CENP-C, CENP-E. Site-specific insertion efficiency of a controlled number of copies of exogenous DNA into this vector was tested using the histone H2B-GFP reporter gene.…”

confidence: 99%

Insertion of a <i>lox</i>P site in a size-reduced human accessory chromosome

Moralli

Vagnarelli

Bensi

et al. 2001

Cytogenet Genome Res

View full text Add to dashboard Cite

The generation in vitro of mammalian artificial chromosomes, in view of the possibility of developing new technologies for gene therapy, is still an ambitious goal. Mammalian artificial chromosomes, to be used as cloning and expression vectors, have been constructed either by de novo synthesis or by reduction of pre-existing chromosomes. In the work here reported, we introduced a loxP sequence into the pericentromeric region of a chromosome 9-derived X-ray-reduced minichromosome, with the purpose of generating a human chromosome vector (HCV). The modified accessory chromosome is linear and mitotically stable, has lost at least 1400 kb of alpha satellite DNA and normally binds CENP-B, CENP-C and CENP-E. The efficiency of gene targeting via loxP mediated homologous recombination was tested using the histone H2B-Green Fluorescent Protein chimaeric gene as a reporter. The frequency of site-specific insertion of the exogenous sequence was found to be about 50% and to occur in a controlled way with regard to the number of copies. The expression level of the fusion protein was stable over prolonged time in culture.

show abstract

The origin of a morphologically unidentifiable human supernumerary minichromosome traced through sorting, molecular cloning, and in situ hybridisation.

Cited by 21 publications

References 18 publications

Human Chromosomes 9, 12, and 15 Contain the Nucleation Sites of Stress-Induced Nuclear Bodies

Human Chromosomes 9, 12, and 15 Contain the Nucleation Sites of Stress-Induced Nuclear Bodies

Flow analysis and sorting of microchromosomes (<3 Mb)

Insertion of a <i>lox</i>P site in a size-reduced human accessory chromosome

Contact Info

Product

Resources

About