Eukaryotic genes cloning

Contreras, R., Cheroutre, H., Degrave, W., and Fiers, W. (1982). Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes. Nucl. Acids Res. 10, 6353—6362. 

If the end goal of cloning is to have a cloned gene expressed in a cell, the entire coding sequence must be doned intact. Furthermore, if a cloned eukaryotic gene is to be expressed in bacteria (to make recombinant proteins), the gene must not contain introns, which could not be processed in a prokaryotic cell. In these cases it is more convenient to done cDNA rather than DNA restriction fragments. 

Vectors that include replication systems derived from more than one host species are known as shuttle vectors. Such vectors commonly include a replication system able to function in E. coli and one that works in a second host, which may be bacterial or eukaryotic. Initial cloning and amplification of the DNA segment to be studied is often carried out in E. coli because it is easier to make large quantities when culturing in E. coli. The recombinant DNA molecule, consisting of the bifunctional vector plus the cloned segment of DNA, is then introduced into the second host, where the purpose is usually to measure the expression of the genes carried by the vector. Shuttle vectors that can replicate in both E. coli and yeast are the most common. 

Because many eukaryotic genes have been cloned during the last few years, it has become possible to compare the DNA sequences preceding genes that may act as promoterlike signals for RNA polymerase II. One feature that stands out is a common sequence, TATAAA, called the TATA box, found usually 25-30 bp before the transcription start site in many but not all PolII-transcribed genes. 

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