Folic acid and chromosome breakage. III. Types and frequencies of spontaneous chromosome aberrations in proliferating lymphocytes

The types and frequencies of spontaneous chromosome aberrations were studied in human lymphocytes cultured for 96 h in minimal essential medium (MEM) or MEM without folic acid (MEM-FA). In both media, the most frequent aberrations were chromatid gaps, isochromatid gaps and chromatid breaks. Chromosome (isochromatid) breaks and dicentrics were seen less frequently. Neither of these less frequent aberrations was seen in 4000 cells from MEM, but both were seen in 4000 cells from MEM-FA.     

Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro

We used a predominantly diploid Chinese hamster cell line to test a number of naturally occurring and synthetic estrogens for their ability to arrest cells at metaphase, their potential for allowing anaphase recovery, and their capability of inducing aneuploid progeny. The chemicals employed included diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol. We also tested progesterone, estrone and testosterone in this regard. Only estrogens and their synthetic analogs caused mitotic arrest and aneuploidy, while progesterone, estrone and testosterone did not cause mitotic disturbances. Among the estrogens, DES was the most effective arrestant on a comparative molar basis, whereas dienestrol was most potent over a wide range of concentrations. Estriol was the least potent as an arrestant but was an effective inducer of aneuploidy. The addition of a metabolic activator (S9) did not alter the ability of DES to arrest mitosis. Following the removal of the drugs, cells were able to quickly reorganize a spindle apparatus and enter anaphase. Diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol caused significant increase in aneuploidy within a narrow range of high concentrations in recovering cell populations. Aneuploidy was induced in a non-random manner. Immunofluorescence studies with anti-tubulin antibody indicate that estrogens may have a mechanism of mitotic arrest similar to that of colchicine and colcemid, viz inhibiting the polymerization of tubulin to form microtubules. These data suggest that the interaction between estrogens and microtubules may mediate the induction of aneuploidy in somatic cells. Aneuploidy induction by DES and similar compounds may be related to their carcinogenic potential.     

A selenium-induced peroxidation of glutathione in algae

Two unicellular marine algae cultured in media containing sodium selenite were examined for glutathione peroxidase activity. The 400 g supernatant from disrupted cells of both the green alga Dunaliella primolecta and the red alga Porphyridium cruentum were able to enhance both the H₂O₂ and the tert-butyl hydroperoxide dependent oxidation of glutathione. The glutathione peroxidation activity of D. primolecta was reduced only slightly by heating the 400 g supernatant, a 30% decrease in the rate with H₂O₂ and 10% decrease in the rate with t-BuOOH being observed. Heating caused the H₂O₂ dependent activity in P. cruentum to be reduced by only 30%, but the activity with t-BuOOH was reduced by 90%. Freezing decreased the t-BuOOH dependent activity of P. cruentum by 90%, but did not lower the t-BuOOH dependent activity of D. primolecta or the H₂O₂ dependent activity of either alga. It was concluded that the heat and cold stable, glutathione peroxidation was non-enzymatic in nature. A variety of small molecules (ascorbate, Cu(NO₃)₂, selenocystine, dimethyldiselenide and selenomethionine) were shown to be able to enhance the hydroperoxide dependent oxidation of glutathione in the assay system employed in this study. Such compounds could be responsible for the activity observed in algae. The heat and cold labile t-BuOOH reductase activity of P. cruentumwas possibly enzymatic, but was not attributable to the presence of glutathione-S-transferase. Both algae, when cultured in the presence of added selenite, displayed an approximate doubling of the non-enzymatic H₂O₂ and t-BuOOH dependent glutathione oxidase activities. The heat and cold labile t-BuOOH reductase activity of P. cruentum was unaltered when the alga was grown in the presence of added selenite. These observations are consistent with the hypothesis that selenium compounds present in the algae are responsible for the selenium induced glutathione peroxidation.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Genome size and complexity in Azotobacter chroococcum

All of eight strains of Azotobacter chroococcum examined contained between two and six plasmids ranging from 7 to more than 200 MDal in size. Strain MCC-1, a derivative of NCIMB 8003, was cured of various of the four largest of its five plasmids and the phenotypes of the strains compared. all fixed nitrogen and exhibited uptake hydrogenase activity. No differences were observed in carbon source utilization or antibiotic, heavy metal or UV resistance. The genome sizes of two strains of A. chroococcum were determined by two-dimensional electrophoresis. Strain CW8, an isolate from local soil containing two small plasmids of 6 and 6.5 MDAl contained unique DNA sequences equivalent to 1.78 x 10(6) (+/- 20%) bp (1.2 x 10(9) Dal). In strain MDC-1, a derivative of MCC-1, containing a 190 MDal and 7 MDal plasmid, the genome size was 1.94 x 10(6) (+/- 20%) bp. In exponential batch cultures, both contained 20 to 25 genome equivalents per cell. MCD-1 exhibited complex UV kill kinetics with a marked plateau of resistance; CW8 showed a simple response inconsistent with the possibility of organization of its DNA into identical chromosome copies capable of independent segregation.     

CL-proteins and the regulation of lipoprotein lipase activity in locust flight muscle

Lipoprotein lipases in the flight muscles of Locusta migratoria show a marked substrate specificity: diacylglycerols associated with the adipokinetic hormone (AKH)-induced lipoprotein, A+, are hydrolysed at 4 to 5 times the rate of those associated with the lipoprotein in resting (non-hormone-stimulated) locusts, Ayellow. To determine the basis for this discrimination, the effect on the activity of flight muscle lipoprotein lipase of CL-proteins, a major constituent of lipoprotein A+, but not of Ayellow, has been investigated; they inhibit the flight muscle enzyme in a competitive manner whether activity is measured with a natural lipoprotein substrate, a lipid emulsion or a water soluble substrate. Experiments in vivo suggest that the flight muscle enzyme is normally inhibited in resting (non-AKH-stimulated) locusts but, interestingly, injection of synthetic AKH-I relieves the inhibition and increases the activity by 30 to 40%. This is not a direct effect of the hormone on the enzyme, but appears to be related to the hormone-induced formation of lipoprotein A+, so that the majority of CL-proteins in the haemolymph become bound to this lipoprotein and the concentration of free CL-proteins is markedly reduced. We suggest that CL-proteins play a major role in the regulation of lipoprotein lipase in locust flight muscle.     

DNA repair kinetics in irradiated undifferentiated and terminally differentiated cells

The brains of male Fisher 344 rats bearing 80-150 mg intracerebral 9L/Ro tumors were irradiated with doses of 1,250-5,000 rads of x- or gamma-rays. At various times after irradiation, the cerebellum and tumor were excised, dissociated into single cells and the DNA from these cells sedimented through alkaline sucrose gradients in zonal rotors with slow gradient reorienting capability. Quantitation of the DNA repair kinetics demonstrated that the process in both tumor cells and neurons has a fast and slow phase. Although all other alternatives cannot be completely eliminated, we suggest that these two phases are most reasonably interpreted as representing repair of lesions in very accessible and less accessible regions of the genome rather than 1) repair of different types of lesions such as single- or double-strand breaks or 2) removal of immediate breaks and breaks induced during excision repair of latent base damage. The slow repair phase is saturable, but not inducible in both tumor cells and neurons. The data suggest that tumor cells restore their chromosomal DNA structure to the unirradiated state faster than neurons because 1) they contain more of the repair system per unit of DNA and 2) a larger proportion of their genetic material is comprised of very accessible regions. The data also suggest that the entire tumor cell genome may be accessible to the repair enzyme(s), while it is possible that a portion of the neuronal genome may be completely inaccessible.