Nuclear DNA

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

Patterns of nuclear DNA degeneration over time--a case study in historic teeth samples.

Abstract: The amount of nuclear DNA extracted from teeth of 279 individual red fox Vulpes vulpes collected over a period spanning the last three decades was determined by quantitative polymerase chain reaction (PCR). Although teeth were autoclaved during initial collection, 73.8% of extracts contained sufficient DNA concentration (> 5 pg/ micro L) suitable for reliable microsatellite genotyping but the quantity of nuclear DNA decayed significantly over time in a nonlinear pattern. The success of PCR amplification across four examined canine microsatellites over time was dependent on fragment size. By including data from two different tests for human contamination and from frequencies of allelic dropout and false alleles, the methodological constraints of population genetic studies using microsatellite loci amplified from historic DNA are discussed.

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster.

Abstract: In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with 3H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.

Characterization of target nuclear DNA from faeces reduces technical issues associated with the assumptions of low-quality and quantity template

Abstract: Faecal material has increasingly become an important non-invasive source of DNA for wildlife population genetics. However, DNA from faecal sources can have issues associated with quantity (low-template and/or low target-to-total DNA ratio) and quality (degradation and/or low DNA-to-inhibitor ratio). A number of studies utilizing faecal material assume and compensate for the above properties with minimal characterization of quantity or quality of target DNA, which can unnecessarily increase the risk of downstream technical problems. Here, we present a protocol which quantifies faecal DNA using a two step approach: (1) estimating total DNA concentration using a PicogreenTM fluorescence assay and (2) estimating target nuclear DNA concentration by comparing amplification products of field samples at suspected concentrations to those of control DNA at known concentrations. We applied this protocol to faecal material collected in the field from two species: woodland caribou (Rangifer tarandus) and swift fox (Vulpes velox). Total DNA estimates ranged from 6.5 ng/μl to 28.6 ng/μl (X = 16.2 ng/μl) for the caribou extracts and 1.0–26.1 ng/μl (X = 7.5 ng/μl) for the swift fox extracts. Our results showed high concordance between total and target DNA estimates from woodland caribou faecal extracts, with only 10% of the samples showing relatively lower target-to-total DNA ratios. In contrast, DNA extracts from swift fox scat exhibited low target DNA yields, with only 38% (19 of 50) of the samples showing comparative target DNA amplification of at least 0.1 ng. With this information, we were able to estimate the amount of target DNA entered into PCR amplifications, and identify samples having target DNA below a lower threshold of 0.2 ng and requiring modification to genotyping protocols such as multiple tube amplification. Our results here also show that this approach can easily be adapted to other species where faeces are the primary source of DNA template.

7-Methylguanine adducts in DNA are normally present at high levels and increase on aging: analysis by HPLC with electrochemical detection.

Abstract: The 7-methylguanine adduct in the DNA of rat liver is determined as an indicator of exposure to exogenous and endogenous methylating agents. A method for the analysis of 7-methylguanine adducts has been developed by combining the selectivity of separation of reversed-phase HPLC with the specificity and high sensitivity of electrochemical detection. The sensitivity of the method is about 10,000-fold that of optical methods and is sufficient to determine the endogenous background of DNA methylation. DNA from the liver of normal young rats (6 months old) contains 7-methylguanine at a level of 1 residue per 31,000 bases in mitochondrial DNA and 1 residue per 105,000 bases in nuclear DNA. These levels increase about 2.5-fold in old rats (24 months old). We attribute this strikingly high level of adducts to endogenous methylation, which could contribute to aging and cancer.

Integration of mitochondrial gene sequences within the nuclear genome during senescence in a fungus

Abstract: Cellular senescence in the ascomycete fungus Podospora anserina is associated with the appearance of an altered mitochondrial genome. Discrete mitochondrial DNA sequences are excised and amplified and isolated as multimerically arranged, head-to-tail repetitions. We have referred to the most frequently observed excision/amplification product as alpha-event senDNA. It is a 2.6-kilobase pair (kbp) monomeric unit (see refs 1, 3, 7) and is often found in senescent mitochondria in conjunction with other excision products. At the final stage of senescence these plasmids constitute virtually all of the DNA present in senescent mitochondria; they have replicated to high copy number at the expense of the young native genome. Because P. anserina is characterized by race-specific timing of senescence (that is, a programme of senescence), we have begun to contrast rapidly and slowly senescing races in terms of senDNA. Here we present evidence that young mitochondria of the rapidly senescing race, A+, possess an extremely high copy number of alpha-event senDNA plasmid in contrast to the more slowly senescing races s+ or s-. Moreover, we observe that during senescence the alpha-event senDNA and the beta-event senDNA (a 9.8-kbp monomer) are transposed to the nucleus and integrated into nuclear DNA. These plasmids contain the coding information for subunits I and III (respectively) of the mitochondrial cytochrome c oxidase. This constitutes the first clear evidence for the active mobilization of genetic elements from the mitochondrion to the nucleus.