Cloning Strategies

Figure 40-2. Results of restriction endonuclease digestion. Digestion with a restriction endonuclease can result in the formation of DNA fragments with sticky, or cohesive, ends (A) or blunt ends (B).This is an important consideration in devising cloning strategies.

A very similar cloning strategy has been used for the 48-kDa subunit of the glycine-receptor channel by Betz and collaborators [4]. Strychnine was used as an affinity ligand, and synthetic oligopeptides were constructed from the purified material. They have been used to screen cDNA libraries. 

Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T. Signal sequence trap a cloning strategy for secreted proteins and type I membrane proteins. Science 1993 261 600-603. 

Systematic cloning strategies based on the conserved motor domain sequences have identified a remarkable number of KRPs expressed in brain. Members of several KRP families expressed in brain have been implicated in forms of MBO transport. Kinesin-2 family members have been implicated in assembly and maintenance of cilia and... 

Procedures for construction of the expression clone follow classical molecular cloning methods. The overall cloning strategy is shown in Fig. 2. 

Deletion mutation of the initiating codon (especially A ) has sometimes been observed for this cloning strategy. 

SCNT is a cloning strategy, originally reported by Campbell, et al. [33], in which nuclei are isolated from a donor s somatic cells, such as fibroblasts, and are transferred into enucleated oocytes from female donors [33]. By mechanisms yet to be uncovered, the cytoplasm of the oocytes reprograms the chromosomes of the somatic cell nucleus and the cloned cells develop into blastocysts, from which ESCs can be derived [34]. One of the landmarks of SCNT is the potential to generate isogeneic ESCs, carrying a set of chromosomes identical to that of an individual, and therefore unlikely to be rejected after transplantation into that individual [35]. 

Miller, A.N., Ofori, K., Lewis, F. and Knight, M. (1996) Schistosoma mansoni use of a subtractive cloning strategy to search for RFLPs in parasite-resistant Biomphalaria glabrata. Experimental Parasitology 84,... 

Besides the abundant nodulins like Lb, GS, uricase and sucrose synthetase, many other enzymes involved in nodule metabolism are induced during the course of nodule development (see Verma Nadler, 1984). However, owing to the relatively low abundance of these enzymes and their mRNAs in nodules, none of the cognate genes have been cloned and new cloning strategies need to be employed to isolate these relatively less abundantly expressed nodulin sequences. 

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