Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

392 Chapter 12 ● Structure of Nucleic Acids

3.X-ray diffraction studies indicate the existence of a novel dou- ble-stranded DNA helical conformation in which Z (the rise per base pair) 0.32 nm and P (the pitch) 3.36 nm. What are the other parameters of this novel helix: (a) the number of base pairs per turn, (b) (the mean rotation per base pair), and (c) c (the true repeat)?

4.A 41.5-nm-long duplex DNA molecule in the B-conformation adopts the A-conformation upon dehydration. How long is it now? What is its approximate number of base pairs?

5.If 80% of the base pairs in a duplex DNA molecule (12.5 kbp) are in the B-conformation and 20% are in the Z-conformation, what is the length of the molecule?

6.A “relaxed,” circular, double-stranded DNA molecule (1600 bp) is in a solution where conditions favor 10 bp per turn. What is the value of L0 for this DNA molecule? Suppose DNA gyrase introduces 12 negative supercoils into this molecule. What are the values of L, W, and T now? What is the superhelical density, ?

7.Suppose one double-helical turn of a superhelical DNA molecule changes conformation from B-form to Z-form. What are the changes in L, W, and T ? Why do you suppose the transition of DNA from B-form to Z-form is favored by negative supercoiling?

8.There is one nucleosome for every 200 bp of eukaryotic DNA. How many nucleosomes are in a diploid human cell? Nucleosomes

FURTHER READING

Adams, R. L. P., Knowler, J. T., and Leader, D. P., 1992. The Biochemistry of the Nucleic Acids, 11th ed. London: Chapman and Hall.

Arents, G., et al., 1991. The nucleosome core histone octamer at 3.1 Å resolution: A tripartite protein assembly and a left-hand superhelix. Proceedings of the National Academy of Sciences U.S.A. 88:10148–10152.

Axelrod, N., 1996. Of telomeres and tumors. Nature Medicine 2:158– 159.

Callandine, C. R., and Drew, H. R., 1992. Understanding DNA: The Molecule and How It Works. London: Academic Press.

Ferretti, L., Karnik, S. S., Khorana, H. G., Nassal, M., and Oprian, D. D., 1986. Total synthesis of a gene for bovine rhodopsin. Proceedings of the National Academy of Sciences U.S.A. 83:599–603.

Kornberg, A., and Baker, T. A., 1991. DNA Replication, 2nd ed. New York: W.H. Freeman and Co.

Luger, C., et al., 1997. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389:251–260.

Noller, H. F., 1984. Structure of ribosomal RNA. Annual Review of Biochemistry 53:119–162.

can be approximated as disks 11 nm in diameter and 6 nm long. If all the DNA molecules in a diploid human cell are in the B-con- formation, what is the sum of their lengths? If this DNA is now arrayed on nucleosomes in the “beads-on-a-string” motif, what is its approximate total length?

9.The characteristic secondary structures of tRNA and rRNA molecules are achieved through intrastrand hydrogen bonding. Even for the small tRNAs, remote regions of the nucleotide sequence interact via H-bonding when the molecule adopts the cloverleaf pattern. Using Figure 12.34 as a guide, draw the primary structure of a tRNA and label the positions of its various selfcomplementary regions.

10.Using the data in Table 11.3, arrange the DNAs from the following sources in order of increasing Tm: human, salmon, wheat, yeast, E. coli.

11.The DNAs from mice and rats have (G C) contents of 44% and 40%, respectively. Calculate the Tms for these DNAs in 0.2 M NaCl. If samples of these DNAs were inadvertently mixed, how might they be separated from one another? Describe the procedure and the results (hint: see the Appendix to this chapter).

12.Calculate the density ( ) of avian tubercle bacillus DNA from the data presented in Table 11.3 and the equation 1.660 0.098(GC), where (GC) is the mole fraction of (G C) in DNA.

Pienta, K. J., and Coffey, D. S., 1984. A structural analysis of the role of the nuclear matrix and DNA loops in the organization of the nucleus and chromosomes. In Cook, P. R., and Laskey, R. A., eds., Higher Order Structure in the Nucleus. Journal of Cell Science Supplement 1:123–135.

Rhodes, D., 1997. The nucleosome core all wrapped up. Nature 389:231– 233.

Rich, A., Nordheim, A., and Wang, A. H.-J., 1984. The chemistry and biology of left-handed Z-DNA. Annual Review of Biochemistry 53:791–846.

Wand, B.-C., et al., 1994. The octameric histone core of the nucleosome.

Journal of Molecular Biology 236:179–188.

Watson, J. D., Hopkins, N. H., Roberts, J. W., Steitz, J. A., and Weiner, A. M., 1987. The Molecular Biology of the Gene, Vol. I, General Principles, 4th ed. Menlo Park, CA: Benjamin/Cummings.

Watson, J. D., ed., 1983. Structures of DNA. Cold Spring Harbor Symposia on Quantitative Biology, Volume XLVII. New York: Cold Spring Harbor Laboratory.

Wu, R., 1993. Development of enzyme-based methods for DNA sequence analysis and their application in genome projects. Methods in Enzymology 67:431–468.

Appendix to Chapter 12

Isopycnic Centrifugation and

Buoyant Density of DNA

Density gradient ultracentrifugation is a variant of the basic technique of ultracentrifugation (discussed in the Appendix to Chapter 5). Density gradient centrifugation can be used to isolate DNA. The densities of DNAs are about the same as concentrated solutions of cesium chloride, CsCl (1.6 to 1.8 g/mL). Centrifugation of CsCl solutions at very high rotational speeds, where the centrifugal force becomes 105 times stronger than the force of gravity, causes the formation of a density gradient within the solution. This gradient is the result of a balance that is established between the sedimentation of the salt ions toward the bottom of the tube and their diffusion upward toward regions of lower concentration. If DNA is present in the centrifuged CsCl solution, it moves to a position of equilibrium in the gradient equivalent to its buoyant density (Figure A12.1). For this reason, this technique is also called isopycnic centrifugation.

Cesium chloride centrifugation is an excellent means of removing RNA and proteins in the purification of DNA. The density of DNA is typically slightly greater than 1.7 g/cm3, while the density of RNA is more than 1.8 g/cm3. Proteins have densities less than 1.3 g/cm3. In CsCl solutions of appropriate density, the DNA bands near the center of the tube, RNA pellets to the bottom, and the proteins float near the top. Single-stranded DNA is denser than double-helical DNA. The irregular structure of randomly coiled ssDNA allows the atoms to pack together through van der Waals interactions. These interactions compact the molecule into a smaller volume than that occupied by a hydrogen-bonded double helix.

The net movement of solute particles in an ultracentrifuge is the result of two processes: diffusion (from regions of higher concentration to regions of lower concentration) and sedimentation due to centrifugal force (in the direction away from the axis of rotation). In general, diffusion rates for molecules are inversely proportional to their molecular weight—larger molecules diffuse more slowly than smaller ones. On the other hand, sedimentation rates increase with increasing molecular weight. A macromolecular species that has reached its position of equilibrium in isopycnic centrifugation has formed a concentrated band of material.

Essentially three effects are influencing the movement of the molecules in creating this concentration zone: (1) diffusion away to regions of lower concentration; (2) sedimentation of molecules situated at positions of slightly lower solution density in the density gradient; and (3) flotation (buoyancy or “reverse sedimentation”) of molecules that have reached positions of slightly greater solution density in the gradient. The consequence of the physics of these effects is that, at equilibrium, the width of the concentration band established by the macro-

isopycnic ● same density

393

Chapter 13

Recombinant DNA:

Cloning and Creation of

Chimeric Genes

The Chimera of Arezzo, of Etruscan origin and probably from the 5th century B.C., was found near Arezzo, Italy, in 1553. Chimeric animals existed only in the imagination of the ancients. But the ability to create chimeric DNA molecules is a very real technology that has opened up a whole new field of scientific investigation.

(Scala/Art Resource, Chimera, Museo Archeologico, Florence, Italy)

In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged. In turn, these technologies led to the construction of DNA molecules composed of nucleotide sequences taken from different sources. The products of these innovations, recombinant DNA molecules,1 opened exciting new avenues of investigation in molecular biology and genetics, and a new field

1The advent of molecular biology, like that of most scientific disciplines, has generated a jargon all its own. Learning new fields often requires gaining familiarity with a new vocabulary. We will soon see that many words — vector, amplification, and insert are but a few examples — have been bent into new meanings to describe the marvels of this new biology.

. . . how many vain chimeras have you created? . . . Go and take your place with the seekers after gold.

LEONARDO DA VINCI, The Notebooks (1508 – 1518), Volume

II, Chapter 25

OUTLINE

13.1 ● Cloning

13.2 ● DNA Libraries

13.3 ● Polymerase Chain Reaction (PCR)

13.4 ● Recombinant DNA Technology: An

Exciting Scientific Frontier

395

amplification ● the production of multiple copies

was born—recombinant DNA technology. Genetic engineering is the application of this technology to the manipulation of genes. These advances were made possible by methods for amplification of any particular DNA segment, regardless of source, within bacterial host cells. Or, in the language of recombinant DNA technology, the cloning of virtually any DNA sequence became feasible.

4

PpaI

pBR322 (4363 bases)

3

2

● One of the first widely used cloning vectors, the plasmid pBR322. This 4363bp plasmid contains an origin of replication (ori) and genes encoding resistance to the drugs ampicillin (amp ) and tetracycline (tet ). The locations of restriction endonuclease cleavage sites are indicated.

Plasmid

vector

Cleavage at single specific site

site is a sequence of nucleotides representing one or more restriction endonuclease cleavage sites. Cloning sites are located where the insertion of foreign DNA neither disrupts the plasmid’s ability to replicate nor inactivates essential markers.

Virtually Any DNA Sequence Can Be Cloned

Nuclease cleavage at a restriction site opens, or linearizes, the circular plasmid so that a foreign DNA fragment can be inserted. The ends of this linearized plasmid are joined to the ends of the fragment so that the circle is closed again, creating a recombinant plasmid (Figure 13.2). Recombinant plasmids are hybrid DNA molecules consisting of plasmid DNA sequences plus inserted DNA elements (called inserts). Such hybrid molecules are also called chimeric constructs or chimeric plasmids. (The term chimera is borrowed from mythology and refers to a beast composed of the body and head of a lion, the heads of a goat and a snake, and the wings of a bat.) The presence of foreign DNA sequences does not adversely affect replication of the plasmid, so chimeric plasmids can be propagated in bacteria just like the original plasmid. Bacteria often harbor several hundred copies of common cloning vectors per cell. Hence, large amounts of a cloned DNA sequence can be recovered from bacterial cultures. The enormous power of recombinant DNA technology stems in part from the fact that virtually any DNA sequence can be selectively cloned and amplified in this manner. DNA sequences that are difficult to clone include inverted repeats, origins of replication, centromeres, and telomeres. The only practical limitation is the size of the foreign DNA segment: most plasmids with inserts larger than about 10 kbp are not replicated efficiently.

Bacterial cells may harbor one or many copies of a particular plasmid, depending on the nature of the plasmid replicator. That is, plasmids are classified as high copy number or low copy number. The copy number of most genetically engineered plasmids is high (200 or so), but some are lower.

Join free ends to ends of foreign DNA

Foreign DNA

● Foreign DNA sequences can be inserted into plasmid vectors by opening the circular plasmid with a restriction endonuclease. The ends of the linearized plasmid DNA are then joined with the ends of a foreign sequence, reclosing the circle to create a chimeric plasmid.

G

G

DNA ligase

Asp Pro Gly Asn Ser

GAT CCG GGG AAT TCA