Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

ANALYZING AND MANIPULATING DNA
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gene complement of several thermophilic bacteria, for example, does not reveal
why these bacteria thrive at temperatures exceeding 70°C. And examination of the
genome of the incredibly radioresistant bacterium Deinococcus radiodurans does
not explain how this organism can survive a blast of radiation that can shatter
glass. Further biochemical and genetic studies, like those described in the other
sections of this chapter, are required to determine how genes, and the proteins
they produce, function in the context of living organisms.
DNA Cloning Allows Any Protein to be Produced in Large
Amounts
In the last section, we saw how protein-coding genes can be identified in genome
sequences. Using the genetic code (and provided the intron and exon boundaries
are known), the amino acid sequence of any protein coded in a genome can be
deduced. As was discussed earlier, this sequence can often provide an important
clue to the protein’s function if found to be similar to the amino acid sequence of
a protein that has already been studied (see Figure 8–23). Although this strategy
is often successful, it typically provides only the likely biochemical function of the
protein; for example, whether the protein resembles a kinase or a protease. It usually
remains for the experimenter to verify (or refute) this assignment and, most
importantly, to discover the protein’s biological function in the whole organism;
that is, to what attributes of the organism does the kinase or the protease contribute
and in what molecular pathways does it function? Nowadays, most new proteins
are “discovered” through genome sequencing, and it often remains a great
challenge to ascertain their functions.
An important approach in determining gene function is to alter the gene (or in
some cases, its expression pattern), to put the altered copy back into the germ line
of the organism, and to deduce the function of the normal gene by the changes
caused by its alteration. Various techniques to implement this strategy are discussed
in the next section of this chapter. But it is equally important to study the
biochemical and structural properties of a gene product, as outlined in the first
part of this chapter. One of the most important contributions of DNA cloning to
cell and molecular biology is the ability to produce any protein, even the rare ones,
in nearly unlimited amounts—as long as the gene coding for it is known. Such
high-level production is usually carried out in living cells using expression vectors
(Figure 8–41). These are generally plasmids that have been designed to produce a
large amount of stable mRNA that can be efficiently translated into protein when
the plasmid is introduced into bacterial, yeast, insect, or mammalian cells. To prevent
the high level of the foreign protein from interfering with the cell’s growth,
the expression vector is often designed to delay the synthesis of the foreign mRNA
and protein until shortly before the cells are harvested and lysed (Figure 8–42).
Because the desired protein made from an expression vector is produced
inside a cell, it must be purified away from the host-cell proteins by chromatography
following cell lysis; but because it is such a plentiful species in the cell (often
1–10% of the total cell protein), the purification is usually easy to accomplish
in only a few steps. As we saw in the first part of this chapter, many expression
Figure 8–41 Production of large amounts of a protein from a proteincoding
DNA sequence cloned into an expression vector and introduced
into cells. A plasmid vector has been engineered to contain a highly active
promoter, which causes unusually large amounts of mRNA to be produced
from an adjacent protein-coding gene inserted into the plasmid vector.
Depending on the characteristics of the cloning vector, the plasmid is
introduced into bacterial, yeast, insect, or mammalian cells, where the inserted
gene is efficiently transcribed and translated into protein. If the gene to be
overexpressed has no introns (typical for genes from bacteria, archaea, and
simple eukaryotes), it can simply be cloned from genomic DNA by PCR. For
cloned animal and plant genes, it is often more convenient to obtain the gene
as cDNA, either from a cDNA library (see Figure 8–32) or cloned directly by
PCR from RNA isolated from the organism (see Figure 8–37). Alternatively, the
DNA coding for the protein can be made by chemical synthesis (see p. 472).
promoter
sequence
CUT DNA WITH
RESTRICTION NUCLEASE
INSERT PROTEIN-
CODING DNA SEQUENCE
INTRODUCE
RECOMBINANT DNA
INTO CELLS
overexpressed
mRNA
expression vector
overexpressed
protein