Nuclear DNA

About: Nuclear DNA is a research topic. Over the lifetime, 3933 publications have been published within this topic receiving 185830 citations.

Determining Ploidy Level and Nuclear DNA Content in Rubus by Flow Cytometry

Abstract: Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifica- tions. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r 2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).

Effects of chronic exposure to microcystin-LR on hepatocyte mitochondrial DNA replication in mice.

Abstract: Microcystins (MCs) are produced by cyanobacterial blooms, and microcystin-LR (MC-LR) is the most toxic among the 80 MC variants. Data have shown that the liver is one of the specific target organs for MC-LR, which can cause mitochondrial DNA (mtDNA) damage, resulting in mitochondrial dysfunction. However, the underlying mechanism is still unclear. In the present study, we evaluated the genetic toxicity of MC-LR in mice drinking water at different concentrations (1, 5, 10, 20, and 40 μg/L) for 12 months. Our results showed that long-term and persistent exposure to MC-LR increased the 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels of DNA in liver cells, damaged the integrity of mtDNA and nuclear DNA (nDNA), and altered the mtDNA content. Notably, MC-LR exposure can change the expression of mitochondrial genes and nuclear genes that are critical for regulating mtDNA replication and repairing oxidized DNA. They also further impaired the function of mitochondria and liver cells.

Adenovirus chromatin structure at different stages of infection.

Abstract: We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

The spatial distribution of exposed nuclear DNA in normal, cancer, and reverse-transformed cells.

Abstract: The malignant CHO-K1 cell is reverse-transformed by cAMP, regaining the phenotype of a normal fibroblast. During this reaction, much of its DNA re-acquires sensitivity to hydrolysis by DNase I in a way characteristic of the normal fibroblast. Exposed DNA forms a rim about the nucleus in both the normal and reverse-transformed cell but not in the malignant CHO-K1. Reacquisition of the nuclear rim requires an organized cytoskeleton. Sequestered DNA forms families of different degrees of sequestration. In accordance with previous theoretical developments it is proposed that (i) genes specific to a given differentiation state are stored in the nuclear rim, whereas genes specific to other states are sequestered within the nucleus; (ii) only exposed genes are active, and their activity is modulated by regulatory molecules in the fluid medium; (iii) exposure and sequestration are regulated by cytoskeletal and nuclear protein structures; (iv) in at least several types of cancer the regulatory defect lies in the genome exposure process so that the specific DNA sequences and their associated growth regulatory loci have been transferred from the exposed to the sequestered condition with consequent loss of the nuclear rim of exposed DNA. The methodology described should be generally applicable to examining the accessibility state of subsets of DNA during various physiological modulations of cell function.