Containing RNA

Virus particles vary widely in size and shape. As we have stated, some viruses contain RNA, others DNA. We have discussed nucleic acids in previous chapters and have noted that the DNA of the cell genome is in the double-stranded form. Some viruses have double-stranded DNA whereas others have single-stranded DNA (Figure 6.3). 

Not all the cellular DNA is in the nucleus some is found in the mitochondria. In addition, mitochondria contain RNA as well as several enzymes used for protein synthesis. Interestingly, mitochond-rial RNA and DNA bear a closer resemblance to the nucleic acid of bacterial cells than they do to animal cells. For example, the rather small DNA molecule of the mitochondrion is circular and does not form nucleosomes. Its information is contained in approximately 16,500 nucleotides that func-tion in the synthesis of two ribosomal and 22 transfer RNAs (tRNAs). In addition, mitochondrial DNA codes for the synthesis of 13 proteins, all components of the respiratory chain and the oxidative phosphorylation system. Still, mitochondrial DNA does not contain sufficient information for the synthesis of all mitochondrial proteins most are coded by nuclear genes. Most mitochondrial proteins are synthesized in the cytosol from nuclear-derived messenger RNAs (mRNAs) and then transported into the mito-chondria, where they contribute to both the structural and the functional elements of this organelle. Because mitochondria are inherited cytoplasmically, an individual does not necessarily receive mitochondrial nucleic acid equally from each parent. In fact, mito-chondria are inherited maternally. 

Hofmann, I., Casella, M., Schnolzer, M., Schlechter, T., Spring, H., and Franke, W. W. (2006). Identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing RNA-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules. Mol. Biol. Cell 17, 1388—1398. 

AIDS was initially described in the U.S. in 1981, although sporadic cases probably occurred for at least two decades prior to this. By 1983, the causative agent, now termed HIV, was identified. HIV is a member of the lentivirus subfamily of retroviruses. It is a spherical, enveloped particle, 100-150 nm in diameter, and contains RNA as its genetic material (Figure 13.13). 

Blanche et al. [45] showed that the P-CAC technology is very promising for the purification of Plasmid DNA at preparative scale especially when resins with low binding capacities for the product of interest are used. The aim of the study was to purify the Plasmid DNA out of a clear lysate of E. coli. The lysate containing RNA, nicked DNA, as well as the Plasmid DNA was loaded onto the annular column filled with Poros 20 R2 beads as the stationary phase. The chromatographic process for the purification is shown in Fig. 7. 

Sequier JM, Richards JG, Malherbe P, Price GW, Mathews S, et al. 1988. Mapping of brain areas containing RNA homologous to cDNAs encoding the a and P subunits of the rat GABAa gamma-aminobutyrate receptor. Proc Natl Acad Sci USA 85 7815-7819. 

Immunonutril is an enteral feed produced by Sandoz diat contains RNA. The nucleosides that enter die blood after digestion of the RNA can be taken up by die immune cells to provide nucleotides required for their proliferation to improve the immune response. Hence the name of the feed. 

Davis KA, Lin Y, Abrams B et al (1998) Staining of a cell surface human CD4 with 2 F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic Acids Res 26 3915-3924... 

The term oncogene was coined in association with the search for the tumor-causing principle in retroviruses. Retroviruses contain RNA as the genetic material and can transcribe RNA into DNA with the help of the virus s own enzyme reverse transcriptase. The DNA form of retroviruses can integrate into the DNA of the host cell and, during cell division, is passed on to the daughter cells as a provirus. From the provirus, viral RNA and complete virus particles may be formed. 

RNA is only a small fraction of total RNA, but it is expressed in high concentrations in tissues that express the target protein. Therefore messenger RNA isolated from enriched sources is likely to contain RNA sequences that encode for the target protein. 

We now turn our attention briefly from DNA structure to the expression of the genetic information that it contains. RNA, the second major form of nucleic acid in cells, has many functions. In gene expression, RNA acts as an intermediary by using the information encoded in DNA to specify the amino acid sequence of a functional protein. 

RNA is also nonenzymatically degraded by hydroxyl ions and, at high temperatures, by divalent cations. Hence, solutions that contain RNA should not exceed a pH value of 9 and should not be heated for too long periods of time if divalent cations are present. 

Fig. 7.9. Backscattered ICP Raman (/R + /L) and ROA (/R — /L) spectra measured for (a) the empty cowpea mosaic virus (CPMV) protein capsid (top pair), (b) the intact capsid containing RNA-2 (middle pair), and (c) the difference spectra obtained by subtracting the top from the middle spectra to reveal the spectra of the viral RNA-2 (bottom pair)...

Ueda, T., Tohda, H., Chikazumi, N., Eckstein, F. and Watanabe, K. (1991) Phosphorothioate-containing RNAs show mRNA activity in the prokaryotic translation systems in vitro. Nucleic Acids Res., 19, 547-552. 

Besides the CTE binding motif, TAP also contains an essential C-terminal domain that functions as a nucleocytoplasmic shuttle domain (Figure 13.3). The C-terminal domain of TAP was shown to directly and specifically interact with several nucleoporins (Bachi et al., 2000 Kang et al., 2000 Katahira et al., 1999). These latter activities have proven to be mutationally inseparable. Therefore, the ability of TAP to exit the nucleus, and hence to export CTE containing RNAs, seems to be mediated by a direct interaction of TAP with constituents of the NPC (Kang et al., 2000). 

Ernst, R.K., Bray, M., Rekosh, D. and Hammarskjold, M.-L. (1997b) A structural retroviral element that mediates nucleocytoplasmic export of intron-containing RNA. Mol. Cell. Biol, 17,135-144. 

Guzyk, B.W., Levesque, L., Prasad, S., Bor, Y.-C., Black, B.E., Pashal, B.M. etal. (2001) NXT1 (pi 5) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells. Mol. Cell. Biol, 21,2545-2554. 

Figure 7.4. Isolation of poly-A+ RNA by biotin-streptavidin affinity matrix. Poly-A+ RNA is captured as a hybrid between poly-A+ RNA and biotinylated oligo(dT) by streptavidin matrix. Most mRNAs carry poly-A+ stretch at their 3 end, and hence poly A-containing RNA can be enriched substantially by this affinity capture method. Poly-A+ RNA can be eluted from the beads by low salt or water. The eluted RNA can be ethanol precipitated.

Why can we isolate DNA from the precipitate, which also contains RNA and proteins, by the simple spooling procedure ... 

We conclude that the replication paradigm has not been able, so far, to account for chemical evolution, but could be valid for postchemical evolution, and this, it will be remembered, is also Dyson s hypothesis (metabolism first, replication second). Let us examine therefore the evolutionary potential of primitive vesicles containing RNAs that could behave both as genes and enzymes. 

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