Double Minute Chromosomes and the Homogeneously Staining Regions in Chromosomes of a Human Neuroblastoma Cell Line

103

authors request that the following correction be noted. All hybrids described in Fig. 5 contained at least part of the human X chromosome, and the box for each hybrid in the column corresponding to the human X should have been darkened. This clerical error does not alter any conclusions in the paper.Proc. Natt Acad. Sci. USA Vol. 80, pp. A number of human tumors (1-3), in particular neuroblastomas (4-6), exhibit homogeneously staining chromosomal regions (HSRs) and extrachromosomal double minute objects (DMs) which are probably cytological manifestations of amplified genes (see ref. 7 for a review). These H-SRs and DMs are often observed in tumor tissue removed prior to institution of therapy, and it seems reasonable to suggest that they may contain genetic information responsible for some of the phenotypic properties of the tumor cells. This hypothesis can be tested by isolation and characterization of DNA within the amplified chromosomal regions. In one study (8,9), DMs of a rat adrenal tumor cell line were isolated by differential centrifugation and cloned in a A bacteriophage. We have utilized a different approach to isolate sequences specific for the HSRs of a human neuroblastoma cell, exploiting the increased size imparted to metaphase chromosomes by these HSRs.The human neuroblastoma cell line IMR-32, which was established from tissue of a patient who had not been exposed to any chemotherapy (10), constitutes an excellent system for analysis of HSR-specific sequences. IMR-32 cells, which exhibit some karyotypic heterogeneity, possess, in addition to a normal chromosome 1, two abnormal chromosomes 1 in which an HSR is inserted in the short arm (in the middle of band lp3). The HSR-containing chromosome 1 is 50% larger than the normal chromosome 1 (11) and hence is easily separable from it and all other, smaller, human chromosomes by fluorescence-activated sorting of isolated IMR-32 metaphase chromosomes after they have been stained with a DNA-specific fluorochrome such as 33258 Hoechst (12).From the DNA of a few million metaphase chromosomes enriched for the HSR-containing chromosome I by flow sorting, we have prepared recombinant libraries in the Charon phage 21A (13) from which cloned sequences specific for the HSR have been identified both by DNA blotting (14) and by in situ hybridization (15, 16). These cloned sequences provide a unique opportunity to study the composition, formation, and functional significance of the amplified sequences in this and other neuroblastoma cell lines.MATERIAL AND METHODS IMR-32 neuroblastoma cells, originally obtained from the American Type Tissue Culture Collection and maintained in culture for several months, were used in these experiments. Cells were cultured in Ham F10 medium supplemented with 10% fetal calf serum. Cytological metaphase chromosome preparations were stained with quinacrine or chromomycin A3 plus methyl green as described (17).Nonconfluent cells were trapped at metaphase by treatment with Colcemid (45 ng/ml) for 20 hr. Cells were detached by shaking, d...

mentioning

confidence: 99%

Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: facilitation by fluorescence-activated flow sorting of metaphase chromosomes.

Kanda¹,

Schreck²,

Alt³

et al. 1983

103

“…The ovalbumin gene copies could be integrated into host cell sequences, as previously suggested (25), or they could be associated with the carrier DNA used in the transformation to form some sort of "double minute-like" chromosomes (26). At least some of the ovalbumin gene copies are transcribed.…”

Section: Discussionmentioning

confidence: 99%

Corrected splicing of a chicken ovalbumin gene transcript in mouse L cells.

Breathnach¹,

Mantei²,

Chambon³

1980

Mouse thymidine-kinase-negative L cells were transformed with a cloned chicken ovalbumin gene linked to the cloned thymidine kinase gene from herpes simplex virus type 1. Most thymidine-kinase-positive clones contained one or more copies of the ovalbumin gene, which were stably maintained for at least 6 months during continuous culture under selective conditions. Transcription of the ovalbumin gene was detected at a low level, producing RNA molecules that were correctly spliced and polyadenylylated by comparison with genuine ovalbumin mRNA. However, the 5' end of these RNA molecules does not correspond to that of ovalbumin mRNA. Molecular cloning has made possible the determination of the structure of the chicken ovalbumin gene (1), which is expressed under hormonal control in the oviduct tubular gland cells (2). By comparison with the structure of other genes, we have identified several conserved sequences that may play a role in the regulation of the expression of the ovalbumin gene and in the splicing of its primary transcript. However, for the true significance of these sequences to be evaluated, they, or mutants derived from them, must be tested in appropriate in vitro or in vivo systems (3, 4). We describe here our first approach to the development of such an in vivo biological system, which consists of introducing the ovalbumin gene into a heterologous cultured eukaryotic cell. Previous work (5-7) has indeed shown that it is possible to introduce foreign DNA into thymidinekinase-negative (TK-) mouse L cells by cotransformation with a thymidine kinase gene from herpes simplex virus type 1. Using this approach, we have introduced the chicken ovalbumin gene into L cells. MATERIALS AND METHODSPlasmids and Enzymes. pBovl and pBov2 were constructed as described in the legends to Figs. la and 3b and in the text. Other plasmids and enzymes have been described elsewhere (8-12). Ovalbumin mRNA (ov-mRNA) was a gift from M. LeMeur and A. Krust. Cell Culture and Transformation. LMTK-cells were grown in monolayer cultures as described (5). LMTK+ cells were maintained in the presence of hypoxanthine/aminopterin/ thymidine medium. Six micrograms of pBovl and 1 ,g of TK-M4 plasmid DNA (7,8,13) were cleaved at their single Sal I sites, extracted with phenol, and precipitated with ethanol.They were then ligated with Biolabs T4 DNA ligase (1 Al) in a volume of 50 Ml for 3 hr before phenol extraction and ethanol precipitation. The heterologous concatenates were used to transform LMTK-cells on five 100-mm petri dishes as described (5). The TK+ phenotype was selected as described (5), and 10 isolated colonies of cells were picked by using the sterile cylinder technique.Analysis of DNA. DNA was isolated from confluent cultures. After digestion with proteinase K (100 ,g/ml), phenol/chloroform extraction, and ethanol precipitation, the DNA was digested with a restriction enzyme by using adenovirus DNA as digestion marker (10). Electrophoresis, blotting, and hybridization to nick-translation probes were as described (10 (...

“…Conversely, minute-containing methotrexate-resistant cells characteristically rapidly lose both the resistance phenotype and minutes during growth in the absence of the inhibitor (6,7). Although one or the other of these karyotypic manifestations tends to predominate in a given cell line, both can coexist in a single cell (8,9).…”

mentioning

confidence: 99%

Ultrastructural features of minute chromosomes in a methotrexate-resistant mouse 3T3 cell line

Hamkalo

Farnham

Johnston

et al. 1985

The Miller spreading procedure was applied to mouse metaphase spreads of methotrexate-resistant 3T3 cells that contain large numbers of minute chromosomes and dihydrofolate reductase genes. There is substantial variation in both size and numbers of minutes in individual cells, the smallest of which (estimated as 5 X 103 kilobase pairs) would be undetected by standard light microscopic analyses. Minute chromosomes are composed of nucleosomal chromatin, which is organized into typical higher order fibers that are folded to form rosette-like structures characteristic of normal chromosome organization. There is no evidence that the DNA in minutes is linear. Minutes exist singly and in pairs, and members of a pair are connected by higher order chromatin fibers, sug-