Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

146
The problem of gene expression
Immunological screening depends on the cloned gene being expressed so that the
protein translation product is present in the recombinant cells. However, as will be
discussed in greater detail in Chapter 13, a gene from one organism is often not
expressed in a different organism. In particular, it is very unlikely that a cloned animal
or plant gene (with the exception of chloroplast genes) will be expressed in E. coli cells.
This problem can be circumvented by using a special type of vector, called an expression
vector (p. 227), designed specifically to promote expression of the cloned gene in
a bacterial host. Immunological screening of recombinant E. coli colonies carrying animal
genes cloned into expression vectors has been very useful in identifying genes for
several important hormones.
Further reading
FURTHER READING
Part I The Basic Principles of Gene Cloning and DNA Analysis
Benton, W.D. & Davis, R.W. (1977) Screening lgt recombinant clones by hybridization to
single plaques in situ. Science, 196, 180–182.
Feinberg, A.P. & Vogelstein, B. (1983) A technique for labelling DNA restriction fragments
to high specific activity. Analytical Biochemistry, 132, 6–13. [Random priming labeling.]
Grunstein, M. & Hogness, D.S. (1975) Colony hybridization: a method for the isolation of
cloned cDNAs that contain a specific gene. Proceedings of the National Academy of
Sciences of the USA, 72, 3961–3965.
Gubler, U. & Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA
libraries. Gene, 25, 263–269.
Southern, E.M. (2000) Blotting at 25. Trends in Biochemical Science, 25, 585–588. [The
origins of Southern hybridization.]
Thorpe, G.H.G., Kricka, L.J., Moseley, S.B. & Whitehead, T.P. (1985) Phenols as enhancers
of the chemiluminescent horseradish peroxidase–luminol–hydrogen peroxide reaction:
application in luminescence-monitored enzyme immunoassays. Clinical Chemistry, 31,
1335–1341. [Describes the basis to a non-radioactive labeling method.]
Young, R.A. & Davis, R.W. (1983) Efficient isolation of genes by using antibody probes.
Proceedings of the National Academy of Sciences of the USA, 80, 1194–1198.