The marked binding of antibodies specific for 5-methylcytidine to the short arm of chromosome 15 distinguishes this chromosome from the other human acrocentrics. This method has been used to study over 60 individuals including 12 who did not have Down's syndrome, but who did have an extra G-group sized acrocentric chromosome. In six cases the extra chromosome did not show intensive binding of anti-5-methylcytidine. In the other six cases, the extra chromosome contained a 5-methylcytidine rich band at each end indicating that both ends were derived from chromosome 15 and contained centromeric heterochromatin normally present on the short arm of chromosome 15. The duplication of short arm material in the abnormal chromosomes was confirmed in all cases by quinacrine staining, nucleolar organizer (Ag-AS) staining or C-banding. In three cases, the abnormal chromosome appeared to arise from two different chromosomes 15. Several possible mechanisms for the production of the abnormal chromosome are discussed. The individuals with this abnormal chromosome all showed some degree of mental retardation, but few common physical findings.
DNA amplification, manifested by homogeneously staining regions in chromosomes and by extrachromosomal, double minute bodies, is characteristic of many neuroblastoma cell lines. Sequences recruited from a specific domain on the short arm of chromosome 2 (2p) are amplified in advanced-stage primary neuroblastomas, whereas sequences from distinctly different regions of 2p are amplified in the neuroblastoma cell line IMR-32. Five different DNA segments, which include the oncogene N-myc, three other fragments derived from the homogeneously staining region of the neuroblastoma cell line IMR-32, and a fifth fragment, derived from the neuroblastoma cell line NB-9, showed differential and variable amplification in 24 advanced-stage neuroblastoma tumors out of 112 tested specimens. AU five fragments were mapped within the chromosomal region 2p23-2p25 by three different approaches. However, eight other fragments cloned from the homogeneously staining region of IMR-32 cells, which were not amplified in the tumor tissues examined, were mapped to two more proximal domains of 2p, thousands of kilobases apart from each other and from the chromosomal domain that is amplified in the tumors. These results establish the amplification, to different degrees, of a variable-sized segment of one domain near the terminus of 2p in advanced neuroblastomas. These tumors might ultimately be distinguished according to the pattern of amplification of DNA segments within this domain. The data presented also indicate the existence of a new and complex amplification mechanism in at least one neuroblastoma cell line (IMR-32), which involves not only relocation of DNA from specific genomic domains but also the formation of novel units by splicing together very distant DNA segments.
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...
Trisomy 22 is commonly found among spontaneous abortions, second in frequency of occurrence only to trisomy 16. Most earlier reports of surviving trisomy 22 cases in the literature are thought to represent the product of unbalanced 11;22 translocations or the result of undetected mosaicism, since this condition is thought to manifest early embryonic or fetal lethality. We present two strikingly similar cases of non-mosaic trisomy 22 surviving to late gestation. In this paper we emphasize the unique phenotype of this trisomy which included intrauterine growth retardation, microcephaly, broad flat nasal bridge with epicanthal folds and ocular hypertelorism, microtia, variable cleft palate, webbed neck, congenital heart defects involving anomalous great vessels, anorectal and renal anomalies, and hypoplastic distal digits with thumb anomalies. We also explore why some cases survive to late gestation. Confined placental mosaicism, a frequent finding in other lethal trisomies, has been ruled out in one of the cases. Molecular studies done to assess the parental origin of the extra chromosome in the other case showed that the non-disjunction originated during maternal meiosis II. Parental origin of the extra chromosome does not seem to play a role in late survival for trisomy 22.
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