Cytoplasmic DNA

Since hematin inhibits Taq polymerase, it is absolutely essential to eliminate red blood cell contamination. Selective lysis of red blood cells can be accomplished with a buffer mixture consisting of 155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM EDTA adjusted to pH 7.4. Alternatively, the cytoplasmic membrane of all cells can be dissolved with a buffer mixture containing the non-ionic detergent Triton-X 100, leaving behind nuclei of white blood cells from which DNA can be extracted. However, this technique will result in the loss of cytoplasmic DNA to the supernatant, and hence will not be able to extract mitochondrial DNA (B11). 

A useful modification of the basic clip-and-paste process involves inserting the DNA fragments into a DNA molecule, which has the power of self-replication. Many bacteria contain small circular cytoplasmic DNA molecules called plasmids, which are capable of self-replicalion inside I he bacterial cell. The characteristics of rapid bacterial growth and multiplication allow quantity replication of the recombinant plasmids in short periods of lime. This technique Ihus offers an obvious advantage over the slow and laborious chemical methods. 

Autoradiography and staining methods are based on detection of cytoplasmic DNA (in the former case replicating cytoplasmic DNA). For this reason there will always be a low background caused by mitochondrial DNA and samples to be tested should be compared with positive and negative controls. Such prefixed controls are available from Moredun Ltd. (Appendix 3) ready for staining. 

An alternative to proceeding with a crude DNA preparation19 at this point is to employ a cytoplasmic DNA (CNA) preparation.18 We present protocols for both. 

Zhang J, Peny G, Smith MA, Robertson D, Olson SJ, Graham DG, Montine TJ (1999) Parkinson s disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons. Am J Pathol 154 1423—1429. 

D. hnRNA is produced in the nucleus and converted to mRNA, which migrates to the cytoplasm. DNA is found in the nucleus and in mitochondria. 

Nakai GS, Guganig ME, Kelley RO, et al. 1971. Cytoplasmic DNA in 90Sr-induced rat chloro leukemia. Rev Eur Etud Clin Biol XVL560-563. 

A cytoplasmic DNA that is capable of autonomous replication. Pneumococcus ... 

FIG. 15 Cellular entry and intracellular kinetics of the cationic lipid-DNA complexes. Cationic lipid-DOPE liposomes form electrostatic complexes with DNA, and, in this case, also transferrin (Tf) is incorporated. Cellular uptake by endocytosis and endosomal acidification can be blocked with cytochalasin B and bafilomycin A, respectively. DNA is proposed to be released at the level of endosomal wall after fusion of the carrier lipids with endosomal bilayer. This process is facilitated by the formation of inverted hexagonal DOPE phase as illustrated in the lower corner on the right. After its release to the cytoplasm DNA may enter the nucleus. (From Ref. 253. By permission of Nature Publishing Group.)... 

One of the main tasks of the DNA is to initiate the synthesis of proteins as and when they are needed. Proteins are synthesised in the ribosomes of the cell cytoplasm. DNA, however, is found in the cell nucleus. So how is the information contained in the DNA passed out of the cell nucleus and into the cytoplasm First, the DNA helix unfolds, and, in a process called transcription, a complementary strand of RNA is synthesised along a crucial part of one of the single DNA strands. This is the messenger RNA (mRNA) which leaves the cell nucleus and is transported into the manufacturing centres for proteins, the ribosomes. In the ribosome, transfer RNA (tRNA) delivers the amino acids required for polypeptide synthesis. The sequence of each group of three bases on the mRNA determines which amino acid is next in the peptide sequence. For example, the sequence AGC in the mRNA specifies the incorporation of the amino acid serine. This process is referred to as translation (Fig. 1.27). The genetic code, i.e. which sequence of bases in the DNA strand refers to which amino acid is given in Table 1.5. 

Other novel cytosolic PRR detectors ofviral nucleic acid are have also been discovered recendy. Takaoka et al characterised the Z-DNA binding protein (ZBP-1) as a cytoplasmic DNA receptor capable of IRF3 activation and IFNp induction and as such renamed it as DNA-dependent activator of IRFs (DAI). DAI associates with IRF3 and TBKl upon stimulation with DNA, but further study is needed to determine all the other signalling components of this pathway and how viruses might evade it. Of interest is the fact that the poxvirus virulence factor E3 which binds dsRNA (see Section 2) also has a DNA-binding motif shown to contribute to virulence. Thus E3 might bind to poxvirus DNA and prevent its detection by DAI or a related cytosolic DNA PRR. 

While the synthesis of mitochondrial DNA observed in cultured animal cells in the G2 phase may result from the method used to synchronize the cells (Koch and Stokstad, 1967 Watson and Kidson, 1970), it remains that the initiation of DNA synthesis in various cell organelles can take place at different times during the cell division cycle. Differences in the patterns of nuclear and cytoplasmic DNA synthesis found in various eukaryotes may mean that no rule exists to describe the time in the cell cycle that ach occurs. Important here is the conclusion that organelle-specific mechanisms must exist to regulate the initiation of DNA synthesis at these different times in the cell cycle. 

A. Bassel, and H. Stern. 1965. Nuclear DNA and cytoplasmic DNA from tissues... 

Koch, J. 1969. The cytoplasmic DNAs of cultured human cells. Europ. J. Biochem., 11 296-304. 

These studies have led to the conclusion that the factor responsible for inducing DNA synthesis, unlike the cleavage factor, is not present within the GV prior to its dissolution, but is perhaps synthesized at this time in response to hormone stimulation (Gurdon, 1967 Gurdon and Woodland, 1968). The additional studies on injected, purified DNA have suggested that the inability of oocyte cytoplasm (prior to GV dissolution) to support DNA synthesis is either because of the absence of cytoplasmic DNA polymerase or the presence of an inhibitor of DNA polymerase (Gurdon and Speight, 1969). 

The term cytoplasmic DNA applies to DNA species found outside a nucleus either by morphological methods or biochemically, after homogenization and differential centrifugation. In the latter case the cytoplasmic DNA may be contaminated with degraded nuclear DNA. Except for comparatively short polydeoxyribonucleotide components attached to cytochrome b, and the DNA components (I-DNA) described by Bell and co-workers which occur in the cytoplasm of embryonic cells (cf. Section, II,C) extranuclear DNA is mainly localized within the mitochondria and chloroplasts of eukaryotic cells. Since mitochondrial DNA (mt DNA) is the only form of cytoplasmic DNA which is well characterized at the moment we shall concentrate on this. 

In all animal cells studied so far mt DNA has been found. Although its presence had been suggested before (Chevremont et al., 1959), the first conclusive demonstration of cytoplasmic DNA was the identification of intramitochondrial fibrous structures (see Fig. 1) as DNA by electron microscopy (Nass and Nass, 1962, 1963a,b) and their sensitivity to DNase. In a short footnote these authors also reported on a chemical identification of this mitochondrial DNA. Using biochemical methods, the DNA content of mitochondria from different sources was accurately measured by Schatz et al. (1964a,b). Extranuclear DNA was also found in chloroplasts by Gibor and Izawa (1963). 

Besides the demonstration of DNA in mitochondria and chloroplasts there are reports on two additional types of cytoplasmic DNA ... 

Since in base composition I-DNA clearly resembles n DNA, it certainly does not represent a cytoplasmic DNA in the usual sense. DNA synthesis in the cytoplasm also occurs after infection of mammalian cells with RNA tumor viruses (Hatanaka et al., 1971), but since this product of the viral genome apparently becomes associated with the nuclear DNA in some stable form, it also cannot be considered mammalian cytoplasmic DNA. 

It was noted several decades ago that the early embryonic stages of several animals contain much more DNA than would correspond to a haploid, or later diploid, chromosome complement. It was suggested that most of this DNA is located within the cytoplasm. The pioneering work of Brachet and his group is especially noteworthy. The earlier results in this area have been reviewed (Brachet, 1967). At that time, the cytoplasmic DNA was believed to be associated with the yolk platelets and was assumed to be a storage form. More recent studies have established beyond doubt that most of this cytoplasmic DNA represents mt DNA. However, unequivocal identification and reliable quantitative data on mt DNA are available only for a few species such as sea urchins starfish, echiuroid worms, and frogs. 

The enzyme associated with the microsomal-ribosomal fraction of the testis showed a considerable level of activity in the absence of added DNA. However, if calf-thymus DNA was added to the assay mixture, three to four times more stimulation of the activity was observed. Furthermore, the experiments carried out with DNAase 1 strongly suggest that the activity observed in this cytoplasmic fraction was due to the presence of DNA. Therefore, a pertinent question remains as to the nature of this cytoplasmic DNA responsible for the residual activity of the enzyme. To answer this, further work will be necessary. 

In eukaryotic cells, the replication of cytoplasmic DNA is more susceptible to the action of intercalating dyes than the replication of chromosomal DNA. Well-known examples of this selective efficiency are (a) action of ethidium bromide on the kinetoplast of the trypanosomes, and (b) action of acriflavine on the mitochondrial DNA in yeasts (leading to petite ctAonie mutants). Such dyes are now used to study the importance of cytoplasmic DNA during morphogenesis. 

Shmerling (1967) showed that cytoplasmic DNA in enucleated frog oocytes is used as templates for cytoplasmic RNA synthesis. She suggests that some of this DNA may consist of additional replicas of genes required for the early stages of embryogenesis. 

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