C r u c i f o r m D N A 493 A. tumefaciens strains that are sensitive to the antagonist. Other prophylactic strategies include maintaining clean propagation nurseries free of crown gall diseased plants, and sanitary cultural practices. The recent rise of genetically engineered crop technology has opened the way for developing crown gall resistant lines of fruit and nut trees, including grapevines and canes.
T-DNA Transfer Mechanism Depending on the Ti plasmid type, the T-DNA is located as one or more adjacent DNA segments on the Ti plasmid; for example, the T-DNA is one contiguous segment in nopaline-type Ti plasmids while the T-DNA can occur in three adjacent segments in octopine-type Ti plasmids. Regardless of the Ti plasmid type, the T-DNA is recognized by its nucleotide sequences at its borders. These border sequences are composed of 25-bp repeats that are recognized by processing enzymes that cleave at the left and right borders, releasing a single-stranded T-DNA molecule on to which a pilot protein called VirD2 is covalently attached at the 50 end. T-DNA processing is initiated by A. tumefaciens recognizing specific phenolic compounds and simple sugars that promote the expression of virulence (vir) genes located near the T-DNA on the Ti plasmid. The processed T-DNA bearing VirD2 protein is transferred by means of a transmembrane nucleoprotein transport system composed of VirB proteins. There are 11 proteins encoded by the virB operon, 10 of which comprise the nucleoprotein secretion system. The remaining VirB protein, VirB2, is cleaved by a signal peptidase and the remaining peptide is cyclized into a circular peptide that is the subunit used in the biogenesis of an extracellular appendage called the T-pilus. The T-pilus is a long flexuous filament of 10 nm diameter. The T-pilus forms when A. tumefaciens cells interact with plant cells and is essential for T-DNA transfer.
Braun AC (1982) A history of the crown gall problem. In: Kahl G and Schell JS (eds) Molecular Biology of Plant Tumors, pp. 155±210. New York: Academic Press. Das A (1998) DNA transfer from Agrobacterium to plant cells in crown gall tumor disease. Subcellular Biochemistry 29: 343±363. Kado CI (1998) Agrobacterium-mediated horizontal gene transfer. Genetic Engineering 20: 1±24. Schell J, van Montague M, De Beuckeleer et al. (1979) Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host. Proceedings of the Royal Society of London Series B 204: 251±266.
Cavara F (1897) Tubercolosi della Vite. Le Stazioni Sperimentale Agrarie Italiane 30: 483±487. Fabre E and Dunal F (1853) Observations sur les maladies reÂgnantes de la vigne. Bulletin de la SocieÂteÂ Centrale d'Agriculture du DeÂpartement de l'HeÂrault 40: 46. Hedgcock GG (1905) Some of the results of three years' experiments with crown gall. Science 22: 120±122. Smith EF and Townsend CO (1907) A plant-tumor of bacterial origin. Science 25: 671±673.
See also: Agrobacterium; Horizontal Transfer; Ti Plasmids; Transfer of Genetic Information from Agrobacterium tumefaciens to Plants
Cruciform DNA D M J Lilley Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0296
A cruciform structure contains a helical branchpoint of four double-stranded helical segments joined by the covalent continuity of the four strands (formally defined as a 4H junction). The strands pass between adjacent helices in a cyclical manner around the junction. This junction is equivalent to the Holliday junction formed by homologous genetic recombination and by the integrase family of site-specific recombination events. A cruciform structure is often taken to mean a twin-hairpin structure formed by intrastrand pairing of the strands at an inverted-repeat sequence, and indeed this was the original meaning of the term (Figure 1). Such a structure is invariably less stable than the perfect duplex from which it forms, but can be stabilized in a negatively supercoiled DNA molecule.
Structure of Four-Way DNA Junction Like many nucleic acid species, the structure of the four-way DNA junction is highly dependent on the presence or absence of metal ions (Figure 2). In the absence of added metal ions, the junction adopts an open structure in which the axes of the four helices are directed toward the corners of a square. This conformation is probably approximately planar, though it is unlikely to be exactly so, since the two sides have a different character, with major and minor groove characteristics. On addition of divalent metal ions, the junction undergoes a folding transition based upon the pairwise coaxial stacking of helices. The structure adopted is termed `the stacked X-structure.' Folding reduces the fourfold pseudosymmetry of the junction,
C ru c i f o r m D NA 2
Figure 1 Formation of a cruciform structure from an inverted repeat. The structure is extruded by intrastrand base-pairing, forming two stem-loop structures. Inverted repeats are often referred to as `palindromes' ± this term is incorrect and should be avoided. dividing the strands into two distinct types. Two continuous strands have single axes that run the length of the stacked helices, while two exchanging strands pass between axes at the junction. The point where the strands exchange is variously called `the crossover' or `the point of strand exchange.' The resulting structure is antiparallel, and thus the two continuous strands run in opposite directions. However, the axes are not exactly antiparallel and lie at a right-handed angle of 408±608. Like the extended structure, the stacked X-structure has dissimilar sides, with major and minor groove characteristics. The structure of the fourway junction was deduced in the late 1980s by the application of biophysical methods, but the stacked X-structure has recently been confirmed by X-ray crystallography. Two alternative conformers are possible for the stacked X-structure, which depend on the choice of
4 Extended, square structure
Inverted repeat sequence
Folded structure, alternative stacking conformers
Figure 3 Formation of alternative stacking conformers by the four-way junction. stacking partner (Figure 3). If the arms were numbered 1±4 sequentially around the junction, then one conformer would be formed by stacking helix 1 on 4, and 2 on 3. Alternatively, a distinct conformer could be formed by stacking helix 2 on 1, and 3 on 4. The nature of the strands becomes exchanged if the stacking partners are changed ± exchanging strands become continuous strands and vice versa in a transition between the two conformers. The relative stabilities of the two forms depends on local sequence, and most junctions consist of populations of both forms with dynamic interconversion.
Branch Migration When a junction is formed by strand exchange between two homologous duplexes, it can undergo a sequential exchange of base-pairing in which the branchpoint becomes effectively displaced along the DNA sequence. This is termed `branch migration.' When the junction is folded into the stacked X-structure in the presence of divalent metal ions, this process is
Extended, low-salt structure
Ion-dependent folding of the four-way DNA junction into the stacked X-structure.
C r y p t i c Sp l i c e S i t e s a n d C r y p ti c S p l i c i n g 495 relatively slow, with a rate of a few steps per second. Thus the process requires protein-mediated acceleration inside the cell.
Interaction with Proteins Four-way DNA junctions are subject to structurespecific recognition by a number of proteins. These include the junction-resolving enzymes (junctionselective nucleases that resolve the junction into component duplexes) and branch migration proteins. The former have been obtained from a wide variety of sources that include bacteriophage, eubacteria, yeast, and mammalian viruses.
Cruciform Structures in Supercoiled DNA Cruciform structures (twin hairpin-loop structures) can enjoy a stable existence in negatively supercoiled DNA molecules, but there is little or no evidence that they do so inside the living cell. Indeed, the instability of long inverted repeats in bacteria suggest that their formation may be strongly deleterious. In addition to their low stability relative to the duplex form (cruciform structures are characterized by a large and positive free energy of formation from duplex DNA 14 kcal mol 1), there is a large kinetic barrier to the extrusion of most cruciform structures (with alternating adenine±thymine sequences as a prominent exception). Extrusion occurs by one of two contrasting mechanisms. Most sequences extrude by the S-type mechanism, in which the center of the cruciform forms intrastrand base pairs, followed by branch migration. C-type cruciform formation occurs in AT-rich sequences at low ionic strength and involves the opening of a large region of DNA and the formation of the cruciform in a single step.
Murchie AIH and Lilley DMJ (1992) Supercoiled DNA and cruciform structures. Methods in Enzymology 211: 158±180. Lilley DMJ (2000) Structures of helical junctions in nucleic acids. Quarterly Reviews of Biophysics 33: 109±159. White MF Giraud-Panis M-JE PoÈhler JRG and Lilley DMJ (1997) Recognition and manipulation of branched DNA structure by junction-resolving enzymes. Journal of Molecular Biology 269: 647±664.
See also: DNA Supercoiling; Holliday Junction; Site-Specific Recombination
Cryptic Satellite Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1814
A cryptic satellite is a satellite DNA sequence not identified as a separate peak on a density gradient but remaining present in main-band DNA. See also: DNA Structure
Cryptic Splice Sites and Cryptic Splicing T Scholl Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0297
A cryptic splice site is a consensus recognition sequence for the cellular RNA splicing machinery that is used, or used more prevalently, due to genetic variation. A cryptic splice site shares homology with the splice donor, the splice acceptor, or the branch point which are all consensus sequences utilized in the course of RNA splicing. Most commonly, cryptic splice sites are utilized when a point mutation occurs within one of the above consensus sequences that are used to create the normal splice junction. These mutations reduce the fitness of the normal site for recognition by the splicing system and result in the activation or increased use of the cryptic site. While mutations that reduce the fitness of normal splice sites cause the majority of splicing at cryptic sites, mutations that increase the fitness of cryptic sites also induce cryptic splicing. In these cases, a point mutation creates a site with strong homology to the consensus sequence. This results in the preferential recognition of the new site by the cellular splicing system and the formation of abnormally spliced transcripts. Genetic variants outside of splice consensus sites can also result in the activation of cryptic splicing. This can occur with mutations nearby, but outside of the recognition sequences themselves. This effect presumably occurs through changes in RNA secondary structure that interfere with accessibility to the normal sites by the splicing machinery. The use of alternative cryptic sites is thereby favored. All of the preceding examples of cryptic splicing involve mutations that occur within the RNA molecule in question. Genetic variants that occur elsewhere and operate to induce cryptic splice