Ligation

There exist a variety of vectors for cloning into eukaryotic systems, ranging from yeast (Saccharomyces as well as Pichia) through insect cells (Baculovims) and plants (Ti plasmid from Agrobacterium tumefaciens) to mammalian cells (transfected by viral or mammalian vectors). As expression in eukaryotic hosts is less efficient than bacterial expression in terms of yield and time and more complicated in terms of vector structure and culture conditions, such eukaryotic expression systems are only used for genes whose proteins require posttranslational modification which is not possible in bacteria. Yeast is the preferred option as a relatively easily culturable single-cell system but posttranslational modification capabilities is limited. The additional complexity can be circumvented in part by exploiting the ability of eukaryotic vectors to act as shuttle vectors, which can be shuttled between two evolutionarily different hosts. Thus, eukaryotic vectors can be replicated and analyzed in bacteria and transfected into eukaryotic cells for expression of the recombinant product. 

These vectors are dealt with in connection with Section 4.3. 

Before blunt-end ligation of an insert, the vector needs to be dephosphorylated because resealing of the two ends of the linear vector to form a circular plasmid representing an intramolecular reaction is entropically favored, and thus faster (Chapter 2, Section 2.2.3), than the intermolecular insertion of the blunt-end frag- 

Other catalysts, sensitive to environmental conditions [110-112], were designed by placing ionic residues at the e and g positions of the helical heptad repeats of the coiled coil. With glutamic acids at these positions, the duplex is stable under acidic conditions. Under physiological conditions, the side chain acids are negatively charged and the coiled coil is destabilized. With lysine residues at these positions, either basic conditions. 

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Plenary 7 7. P M Champion et al, e-mail address champ neu.edu (TRRRS). Femtosecond impulsive preparation and timing of ground and excited state Raman coherences in heme proteins. Discovery of coherence transfer along a de-ligation coordinate. See above for fiirther connnent. 

Crystal stmctures of Grignard reagents do not necessarily correspond to their stmcture in solution. In general, the crystal stmctures (61—64) indicate the reagents are ligated with THF or diethyl ether and are frequentiy observed to be dimers. The Mg atoms in the dimers do not have a Mg—Mg bond instead the dimers are typically held together by a haUde bridge. 

Fig. 10. The receptor—G-protein sequence. An activated receptor interacts with the trimeric GDP-ligated receptor to cause an interchange of GDP by GTP and dissociation into the activated Ga—GTP (left) and G y (right) subunits. These then interact with a variety of effectors. The purpose of the activated...

Fig. 4. Steps in making a cDNA library. Cellular mRNA is used as a template to make a complementary DNA. This cDNA is then ligated to a plasmid,...

The automated method differs from the ICSH method chiefly in that oxidation and ligation of heme iron occur after the hemes have been released from globin. Therefore, ferricyanide and cyanide need not diffuse into the hemoglobin and methemoglobin, respectively. Because diffusion is rate-limiting in this reaction sequence, the overall reaction time is reduced from approximately three minutes for the manual method to 3 —15 seconds for the automated method. Reaction sequences in the Coulter S + II and the Technicon H 1 and H 2 are similar. Moreover, similar reactions are used in the other Coulter systems and in the TOA and Unipath instmments. 

Affinity Chromatography. This technique involves the use of a bioselective stationary phase placed in contact with the material to be purified, the ligate. Because of its rather selective interaction, sometimes called a lock-and-key mechanism, this method is more selective than other lc systems based on differential solubiHty. Affinity chromatography is sometimes called bioselective adsorption. 

Gouaux, J.E., Lipscomb, W.N. Crystal structures of phosphonoacetamide ligated T and phosphono-acetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8 A resolution and neutral pH. Biochemistry 29 389-402, 1990. 

FIGURE 13.4 Blunt-end ligation using phage T4 DNA ligase, which catalyzes the ATP-dependent ligation of DNA molecules. AMP and PP are by-products. 

It is clear that some boranes are amphoteric Lewis acid/bases — that is they can act either as electron-pair donors as above or as electron-pair acceptors (e.g. in L.BH3 and L.B1H7). It follows that a borane donor could conceivably ligate... 

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