In contrast to DNA molecules, molecules of single-stranded (ss) RNA, as found in the genomes of many RNA viruses, are susceptible to chemical degradation at relatively mild pH ranges (<3 and >9). Further, ribonucleases that readily degrade RNA are notoriously difficult to inactivate and are often excreted by bacteria and fungi, which can contaminate laboratory reagents. Indeed, a very potent ribonuclease ("finger nuclease") is found in sweat and is carried on the
Fig. 11.9 Use of a method similar to that shown in Fig. 11.6 to spread HSV DNA for comparative contour length measurement. One full-length DNA molecule is extended and its length can be measured and compared to the length of the circular OX174 replicative form (RF) DNA molecules included as size standards. A second DNA molecule (or molecules) has formed a tangle around a contaminating protein fragment in the solution.
hands. Despite the difficulties in working with RNA, with appropriate care, RNA molecules as large as 15,000 bases can be isolated with relative convenience. This means that the size range of ssRNA virus genomes (2—15 kb) is well within the bounds of experimental manipulation.
Unfortunately, ssRNA is not very easy to work with in the electron microscope because its flexibility and ability to form hairpin loops makes it very difficult to spread for accurate measures of length. The size of viral genomes, however, is just the size that is convenient for rate zonal (velocity) sedimentation in sucrose gradients, and conversely, for size fractionation on low-density acrylamide gels using electrophoresis.
The same principles applied to protein fractionation generally can be applied to nucleic acid fractionation. Since all ssRNA and ssDNA molecules will have essentially the same shape in solution, their sedimentation rate in a centrifugal field will be only a function of their molecular size. Thus, each specific size of ssRNA or ssDNA macromolecule will sediment at a specific rate in a centrifugal field under standard conditions. The sedimentation rate under such standard conditions is termed the sedimentation constant (s value) for that macromolecule. This s value also determines the rate of migration under standard conditions in acrylamide gel electrophoresis. The s value is related to molecular size by a logarithmic function. For example, prokaryotic 16s ribosomal RNA (rRNA) and 23s rRNA are 1.5 kb and 2.3 kb, while eukaryotic 18s rRNA and 28s rRNA are 2 kb and 5.2 kb, respectively.
Interestingly, the size range of RNA (and ssDNA) molecules found as viral genomes is just that range that is readily separable by gel electrophoresis or rate zonal centrifugation. Further, bacterial and eukaryotic rRNAs provide readily available internal size standards of just the right general values for measuring the sizes of mRNA and viral genomes. For this reason, many scientific reports define species of RNA by s value, which is just a shorthand way of listing its molecular size.
The principles of rate zonal centrifugation for measure of molecular size also can be applied to dsDNA molecules, but the inflexibility of dsDNA in solution and its - generally - larger size require considerably different experimental techniques. Often determining the size of dsDNA molecules requires using analytical ultracentrifuges that generate very high centrifugal fields and very sophisticated optical methods for measuring sedimentation. It is very important to remember that the mathematical relationship between the s value of a dsDNA molecule and its size is quite different from one relating the size and sedimentation rates of ssRNA or ssDNA molecules.
While most double-stranded viral genomes are too large for easy gel electrophoresis, the ability to cut DNA molecules into specific pieces using restriction endonucleases (see Chapters 8 and 14) allows one to partially resolve this problem. If purified virion DNA is digested with a restriction endonuclease that does not cut it too often, fragments of a convenient size can be produced and fractionated on high-porosity agarose gels.
An example is shown in Fig. 11.10, which shows the electrophoretic separation of DNA fragments produced when the 48-kbp bacteriophage X genome was cut with the restriction endonuclease BstEII, which cleaves DNA at locations where the seven-base sequence GGTNACC occurs (N represents any base). Mobility of the fragments is roughly proportional to a logarithmic function, but this function is not constant throughout the whole size range of fragments produced. This can be seen by looking at the semilog plot of migration versus log of fragment size, which is also shown in Fig. 11.10. The only way that gel electrophoresis can be used to measure accurately the total size of all fragments produced by digestion of a mid-sized to large DNA genome is to include numerous different size markers and use several different agarose concentrations in the gel. Still, the method is very convenient, and is often used to compare the size of specific restriction fragments produced by digesting related viruses.
Fig. 11.10 Electrophoretic separation of bacteriophage X restriction fragments. Bacteriophage DNA was digested with the restriction enzyme BstEII, which is so named because it was derived from Bacillus stearothermophilus (a hot springs-loving organism or extremophile). The DNA fragments were fractionated by electrophoresis on 1% agarose gel, and visualized by viewing under ultraviolet light following the addition of ethidium bromide, which specifically binds dsDNA and produces orange fluorescence under ultraviolet light. The migration rate of individual fragments, whose sizes are shown, is plotted against a log of fragment size.
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