Samuel A. Latt

Transposition and amplification of oncogene-related sequences in human neuroblastomas

We have cloned a 2.0-kb EcoRI fragment of human genomic DNA (NB-19-21) which has homology to the v-myc oncogene but is distinct from the classical c-myc gene. This sequence is amplified from 25- to 700-fold in eight of nine tested human neuroblastoma cell lines which contain either homogeneously staining regions or double minutes (HSRs or DMs), the caryological manifestations of amplified genes. In the remaining line, the c-myc proto-oncogene is amplified approximately 30-fold. NB-19-21 hybridizes to a 3.2-kb cytoplasmic, poly(A)+ RNA species that is abundant only in lines in which the sequence is amplified. We propose that the gene encoding the NB-19-21-related RNA species may represent a new oncogene, which we call N-myc. NB-19-21 derives from chromosome 2; but in the five HSR-containing lines that have amplified this sequence, none has HSRs on chromosome 2. NB-19-21 is associated with DMs in a DM-containing line. A second, randomly cloned, amplified DNA segment from the HSR of one of the neuroblastoma lines is amplified in a subset of the lines in which NB-19-21 is amplified. In addition, this probe identifies a novel joint in the amplification unit of one line relative to that of the others. We suggest that, in the eight lines which have amplified NB-19-21, the amplification units are overlapping, but not identical, and that transposition of the common sequences may occur prior to amplification.

Optical studies of the interaction of 33258 Hoechst with DNA, chromatin, and metaphase chromosomes

The interaction of the bisbenzimidazole dye 33258 Hoechst with DNA and chromatin is characterized by changes in absorption, fluorescence, and circular dichroism measurements. At low dye/phosphate ratios, dye binding is accompanied by intense fluorescence and circular dichroism and exhibits little sensitivity to ionic strength. At higher dye/phosphate ratios, additional dye binding can be detected by further changes in absorptivity. This secondary binding is suppressed by increasing the ionic strength. A-T rich DNA sequences enhance both dye binding and fluorescence quantum yield, while chromosomal proteins apparently exclude the dye from approximately half of the sites available with DNA. Fluorescence of the free dye is sensitive to pH and, below pH 8, to quenching by iodide ion. Substitution of 5-bromodeoxyuridine (BrdU) for thymidine in synthetic polynucleotides, DNA, or unfixed chromatin quenches the fluorescence of bound dye. This suppression of dye fluorescence permits optical detection of BrdU incorporation associated with DNA synthesis in cytological chromosome preparations. Quenching of 33258 Hoechst fluorescence by BrdU can be abolished by appropriate alterations in solvent conditions, thereby revealing changes in dye fluorescence of microscopic specimens specifically due to BrdU incorporation.

SPECTRAL STUDIES ON 33258 HOECHST AND RELATED BISBENZIMIDAZOLE DYES USEFUL FOR FLUORESCENT DETECTION OF DEOXYRIBONUCLEIC ACID SYNTHESIS

Absorption, fluroescence and circular dichroism measrements on 33258 Hoechst-deoxyribonucleic acid (DNA) complexes are consistent with the existence of two types of dye-binding interactions. One type, which persists at elevated solution ionic strength, is highly specific for adenine-thymine-rich DNA. Dye bound under this condition exhibits efficient fluorescence and strong optical activity. A less specific, largely electrostatic interaction is associated with less intense fluorescence and weaker optical activity. The fluorescence of 33258 Hoechst and several other bisbenzimidazole dyes is less when bound to poly(deoxyadenylate-5-bromodeoxyuridylate) than when bound to poly(deoxyadenlyate-deoxythymidylate). Quenching of 33258 Hoechst fluorescence can also be used to detect biosynthetic incorporation of 5-bromodeoxyuridine into the DNA of living cells. This property of 33258 Hoechst should allow fluorescence-activated cell and chromosome sorting according to the extent of DNA synthesis, providing a bridge between biochemical and cytologic analyses of processes related to DNA replication.

Stimulated Human Phagocytes Produce Cytogenetic Changes in Cultured Mammalian Cells

CHRONIC inflammation can be associated with cancer.1 Phagocytes in inflammatory lesions enzymatically reduce oxygen to reactive metabolites, which include Superoxide anions, hydrogen peroxide, and ...

Sister chromatid exchanges.

Sister chromatid exchanges (SCEs) represent the interchange of DNA replication products at apparently homologous loci. These exchanges presumably involve DNA breakage and reunion, although little is known about the molecular basis of sister chromatid exchange formation, and information about the biological significance of exchanges is largely circumstantial. In spite of these uncertainties, analysis of sister chromatid exchange formation in cytological systems has already provided information about chromosome structure and has been used to detect the effects of clastogens and to differentiate between chromosome fragility diseases.

Localization of Sister Chromatid Exchanges in Human Chromosomes

The bromodeoxyuridine; sensitivity of 33258 Hoechst fluorescence allows microfluorometric analysis of sister chromatid exchanges in human metaphase chromosomes. The frequency of sister chromatid exchanges among chromosomes correlates with chromosome length. Exchanges appear to occur predominantly in interband regions, as defined by quinacrine fluorescence, or very near band-interband junctions. A few regions are involved unusually frequently.

Thermolysin: A zinc metalloenzyme

Metal analyses and inhibitor studies have shown that thermolysin, a neutral protease from B. thermoproteolyticus, is a zinc metalloenzyme. The relevance of this finding to the active site characteristics of other bacterial neutral proteases and to those of alkaline proteases is considered.

A simplified technique for in vivo analysis of sister-chromatid exchanges using 5-bromodeoxyuridine tablets

A small tablet of 5-bromodeoxyuridine (BrdU), implanted subcutaneously in a mouse, provides sustained release of base analog sufficient to effect substitution of DNA throughout an entire replication period. As illustrated by studies of mouse bone-marrow and spleen cells in the presence or absence of cyclophosphamide, this depot method of BrdU administration greatly simplifies in vivo analysis of sister-chromatid-exchange formation.

Mapping of the X-linked agammaglobulinemia locus by use of restriction fragment-length polymorphism.

A molecular linkage analysis in 11 families with X-linked agammaglobulinemia (XLA) localized the XLA gene to the proximal part of the long arm of the human X chromosome. Significant linkage was detected between XLA and loci defined by two polymorphic DNA probes called 19-2 for the DXS3 locus and S21 for the DXS17 locus. Both localize to the region Xq21.3-Xq22. Most likely recombination distances (theta) and associated logarithm of the odds (lod) scores for the XLA-DXS3 and XLA-DXS17 pairs were theta = 0.04 morgans (lod, 3.65) and theta = 0 (lod, 2.17), respectively. Tight linkage between XLA and the locus DXS43 defined by the X short arm probe D2 (localized to Xp22-Xp21) was strongly excluded and we obtained no evidence for significant linkage between XLA and any other X short arm probe. The probe pair 19-2 and S21 should be informative for molecular linkage-based analysis of XLA segregation in the majority of families afflicted with this disorder.

Bromodeoxyuridine tablet methodology for in vivo studies of DNA synthesis.

Abstract5-Bromodeoxyuridine (BrdU) tablets with different physical characteristics are useful in a wide variety of studies requiring detection of DNA replication in vivo. These tablets can effect a high substitution of BrdU in DNA, thereby permitting sister chromatid differentiation in chromosomes stained with 33258 Hoechst alone or in conjunction with Giemsa. Baseline and cyclophosphamide-induced in vivo sister chromatid exchange frequencies in mouse spleen, marrow, and thymus were measured and found to be significantly greater than those in spermatogonia. Sister chromatid exchange analysis was also extended to mouse liver and to Chinese hamster and Armenian hamster marrow cells. Sister chromatid differentiation was observed in Armenian hamster meiotic tissue, and evidence for interhomolog chromatid exchange obtained.