ABSTRACTnaked DNA, and that it is the cooperative binding of histone Hi that is responsible for the folding of the thin chromosome fiber. The results of four independent experimental approaches provide evidence that supports this view: (i) competition between long and short nucleosome chains for histone Hi analyzed by filter binding; (ii) the distribution of histone Hi in mixtures of long and short chromosome fibers separated by sucrose gradient velocity sedimentation; (iii) the sedimentation behavior of long chromosome fiber fragments as a function of NaCl concentration in the range of the transition; (iv) electron microscopy of chromosome fibers above and below the transition. MATERIALS AND METHODSCell nuclei were isolated from bovine lymphocytes (13) and stored in 2 mM MgCl2, 5 mM Tris.HCl (pH 7.5), and 66% (vol/vol) glycerol at -600 until needed, but not longer than 10 days. Radioactively labeled nuclei were obtained from bovine lymphocytes that had been stimulated by phytohemagglutinin P (Difco) in medium containing [3H]thymidine after 60 hr of incubation.Fragmentation of chromatin in nuclei by micrococcal nuclease (Boehringer or Worthington) was carried out at 00 in 0.2 M sucrose, 1 mM CaCl2, 5 mM Tris-HCI (pH 7.5), and either 80 or 60 mM NaCl at a concentration of 2.4-108 nuclei per ml. The digestion reaction was terminated and nuclei were lysed by gently adding the same volume of a solution containing 5 mM EDTA, 5 mM Tris-HCl (pH 7.5), and 80 or 60 mM NaCl. The nuclear debris was pelleted at 5000 X g for 8 min. The supernatant contained 50-80% of the nuclear DNA in the form of fragmented chromosome fibers. Histone-HI-depleted fiber fragments were made by the aid of tRNA (14). Histone HI was prepared by the trichloroacetic acid extraction procedure (15).The filter binding assay was carried out as described earlier (12). Stock solutions of histone H1 were diluted into 0.5 ml samples of 5 mM Tris-HCl (pH 7.5) and 40 mM NaCl containing equal weights of labeled and unlabeled histone-Hldepleted nucleosomes. After incubation for 30 min at 00, the reaction mixture was filtered through nitrocellulose membrane filters at a flow rate of 0. 1 ml-sec-'. The filters were washed three times with 0.7 ml of buffer, dried, and monitored for radioactivity. The values given are the means of three experiments; the standard deviations were less than 10% of the mean values. Filters retained 20 (+5)% of the histone-Hi-depleted nucleosome trimer (background).Sucrose gradient analyses were made by layering fragmented chromosome fiber samples (0.5 ml) on preformed linear gradients from 10 to 30% sucrose containing 1 mM sodium phosphate (pH 6.8), 0.2 mM EDTA, and NaCl at the same concentration as in the samples. Nitrocellulose tubes with 0.5 ml cushions of 86% (vol/vol) glycerol were spun in a Beckman SW 40 rotor at 3°. The gradients were analyzed with the use of a turbulence-free flow cell (ISCO).DNA sizes were analyzed electrophoretically on 1.4% agarose gels as previously described (16

NALM-6-M1, one of eight leukemia cell lines cultured from the blood of a 19-year-old boy with non-T, non-B acute lymphoblastic leukemia (ALL) i n relapse, was characterized. This cell line was found t o be more than 90% cytoplasmic lmmunoglobulin positive (clg+) for both mu heavy and lambda light chains, but surface immunoglobulin (slg) and complement receptor (CR) negative. NALM-6-M1 was clg-for alpha, delta, and gamma heavy chains and kappa light chain. About

CD4(+) CD56(+) lineage-negative malignancies are difficult to diagnose and classify. Recent studies have suggested that these malignancies may derive from plasmacytoid dendritic cells (pDC). In this report, we examine 10 cases of CD4+, CD56+ lineage-negative malignancies that presented in various tissue sites. The goal was to identify the morphologic, immunophenotypic, and genotypic findings to devise a diagnostic approach to tissue biopsies of these lesions and to confirm the proposed cell of origin. The mean age was 66 years (range, 45-80 years) with a male predominance (8 males/2 females). Frequent sites of disease included skin (60%) and peripheral blood/bone marrow (70%). Tumor cells were positive for CD45, CD43, CD4, and CD56 (9 of 10). The pDC markers, CD123 (9 of 10) and CD45RA (10 of 10), were detected by immunoperoxidase staining. Also noted was CD2 positivity (1 case), weak CD7 positivity (4 of 8 cases), weak CD33 (4 of 9 cases), TdT (2 cases), and CD68 (2 cases). All cases were otherwise negative for EBV (EBER), B-cell, T-cell, myeloid, and NK cell markers. T-cell receptor-gamma gene rearrangement was negative in all cases. Complex structural chromosomal abnormalities were seen in 3 of 5 cases, a subset of which may be recurrent in pDC malignancy. Overall prognosis was poor despite multiagent chemotherapy and/or radiation. Our study confirms that CD4+/CD56+ lineage-negative tumors are derived from pDC and have characteristic clinical, histopathologic, and immunophenotypic features. Furthermore, these rare neoplasms can be readily diagnosed using recently developed immunoperoxidase techniques.

Etoposide, a topoisomerase II inhibitor widely used in cancer therapy, is suspected of inducing secondary tumors and affecting the genetic constitution of germ cells. A better understanding of the potential heritable risk of etoposide is needed to provide sound genetic counseling to cancer patients treated with this drug in their reproductive years. We used a mouse model to investigate the effects of clinical doses of etoposide on the induction of chromosomal abnormalities in spermatocytes and their transmission to zygotes by using a combination of chromosome painting and 4 ,6-diamidino-2-phenylindole staining. High frequencies of chromosomal aberrations were detected in spermatocytes within 64 h after treatment when over 30% of the metaphases analyzed had structural aberrations (P < 0.01). Significant increases in the percentages of zygotic metaphases with structural aberrations were found only for matings that sampled treated pachytene (28-fold, P < 0.0001) and preleptotene spermatocytes (13-fold, P < 0.001). Etoposide induced mostly acentric fragments and deletions, types of aberrations expected to result in embryonic lethality, because they represent loss of genetic material. Chromosomal exchanges were rare. Etoposide treatment of pachytene cells induced aneuploidy in both spermatocytes (18-fold, P < 0.01) and zygotes (8-fold, P < 0.05). We know of no other report of an agent for which paternal exposure leads to an increased incidence of aneuploidy in the offspring. Thus, we found that therapeutic doses of etoposide affect primarily meiotic germ cells, producing unstable structural aberrations and aneuploidy, effects that are transmitted to the progeny. This finding suggests that individuals who undergo chemotherapy with etoposide may be at a higher risk for abnormal reproductive outcomes especially within the 2 months after chemotherapy.

Chromosome fibers isolated from lymphocyte nuclei and prepared for electron microscopy by techniques designed to preserve their native structure have a distinctly knobby appearance, suggesting that DNA and protein are not distributed evenly along the fiber axis. Individual knobs (superbeads) are arranged in tandem and have an average diameter of about 200 A. Mild nuclease digestion of isolated nuclei releases apparent monomer superbeads that are composed of nucleohistone particles with the properties of nucleosomes. The kinetics of digestion indicate that the superbead is a discrete structural unit containing, on the average, about eight nucleosomes.

The mouse lymphoma L5178Y TK+/-3.7.2C cell line allows quantitation of induced TK+/--

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Analysis of immunologic cross-reacting material in Chinese hamster-human somatic cell hybrids allowed assignment of the structural gene for glucocerebrosidase (glucosylceramidase; 8-D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) to chromosome 1 bands q21-q32. In situ hybridization of a radiolabeled human glucocerebrosidase cDNA to high resolution human chromosomes demonstrated that a single locus encoding glucocerebrosidase is on 1q21, adjacent to a region of chromosome 1 (lqh) abundant in structural heteromorphisms. We also have identified a hydrophobic leader polypeptide encoded by this locus, permitting a more complete description of the biosynthesis of the enzyme. These results suggest that the type-specific protein polymorphisms in Gaucher disease result from mutations at this single locus, whose segregation might be followed by linkage to visible chromosomal heteromorphisms.