Recombination Up Close
BIO. "Biotechnology in Perspective." Washington, D.C.:
Biotechnology Industry Organization, 1990.
Manipulating the Gene
The attractions of genetically-engineered products are clear. A wide
range of materials for use in medicine, agriculture, and industry that
are currently in short supply could be produced in much greater
quantities if the genes regulating their production by higher plants
and animal cells could be incorporated into the microbial genome.
Growing bacteria in bioreactors under conditions that favor the
synthesis of the material is relatively straightforward. Industries
such as baking and brewing have a wealth of experience in just these
areas. Why not simply grow the cells of the higher organisms that
make the product in the first place? That is clearly a potential
alternative solution, but in practice many such cells cannot readily
be grown in vitro.
By contrast, very effective methods are available for growing
micro-organisms rapidly and at low cost. If we examine the case of
insulin, for example, we find that cells of the human pancreas - the
source material - cannot be grown easily in isolation, but genetic
engineering can result in cells that will readily manufacture a
precursor of insulin, just as our own bodies do, and then
enzymatically remove a polypeptide to give recombinant human insulin.
Although certain ground rules for genetic engineering have been
established, after over a decade of intensive effort it is still the
case that few industrial problems in this area can be solved by
applying a standard formula. The method adopted in a given case will
depend on the gene in question and the microbe which is to receive it.
Nevertheless, four main stages can be recognized. The first of these
involves obtaining the gene which codes for the product. This
isolated gene must then be inserted into the microorganism, often a
bacterium. In many ways these are the simplest stages to achieve.
Conditions must be established under which the microbes will
synthesize the required product in appropriate amounts. This may
require particular ingenuity, and the same is true at the final stage
in which the product is collected, isolated, and purified.
Isolation of Genes
Human cells contain thousands of genes, so the key initial step is to
isolate the gene. Rather than search through the mass of DNA in the
genome (only a fraction of which has been sequenced to date),
genetic engineers have turned to gene expression as a way to isolate
specific genes. Using this method to isolate the insulin gene, for
example, human pancreas cells are examined to find the messenger RNA
molecules that have been copied from that gene as part of the process
of protein synthesis. There will be other kinds of RNA as well as
that coding for insulin, but already the scale of the problem is
greatly reduced. Until recently, there was no way to connect the
genetic information in mRNA back to DNA. It was then discovered that
some viruses can do precisely this. This was something of a surprise,
since it involved a reversal of the "central dogma" of molecular
biology (DNA makes RNA makes protein).
The enzyme that achieves this is known as reverse transcriptase.
Viruses consist simply of a core of genetic material wrapped in a
protein coat, and are much smaller than bacterial cells. They can
reproduce themselves only when they invade cells. Some viruses employ
RNA as their genetic material, and in the case of retroviruses,
reverse transcriptase inside the virus is used to generate DNA. In
this way, retroviruses convert their RNA 'genes' into a DNA form, and
because of the universality of the genetic code, the host cell is
deceived into replicating new viruses. Biotechnologists can adopt a
similar strategy by mixing reverse transcriptase with human mRNAs in
vitro, thereby producing the complementary or copy (cDNA) sequence for
the required gene, e. g., insulin.
Plasmids Help to Transfer Genes between Organisms
The insertion of genes is facilitated by bacterial plasmids (small
circles of DNA), which are smaller than the bacterial chromosome.
Some plasmids can pass readily from one cell to another, even when the
cells are clearly from different species far apart on the evolutionary
scale. By inserting the human cDNA gene into the plasmid ring, it can
be readily introduced into its microbial host for a biotechnological
application. Plasmids used in this way are known as vectors and
certain viruses can perform the same role. The stitching in place of,
say, a human gene, is achieved by a family of remarkable
'cut-and-paste' enzymes, the restriction endonucleases and ligating
With the recombinant DNA
molecule successfully inserted into the bacterial host, another
property of plasmids can be exploited - their capacity to replicate
themselves. Once inside a bacterium, the plasmid containing the human
cDNA can multiply to yield several dozen replicas. With cells
dividing rapidly (every 20 minutes), a bacterium containing human cDNA
(encoding for, say, insulin) will in a relatively short time produce
many millions of similar cells (clones) containing the same human
Recombinant DNA Technology
Recombinant DNA technology
requires DNA extraction, purification, and fragmentation. This
molecular dissection is achieved using specific 'restriction' enzymes
and is followed by sorting and isolation of fragments containing a
particular gene. This portion of the DNA is then coupled to a carrier
molecule and the hybrid DNA is introduced into a chosen cell for
reproduction and synthesis. Modern laboratories even have access to a
DNA synthesizer (gene machine), which provides a desired DNA sequence.
Recombinant DNA allows the
genome to be manipulated much more readily than by classical breeding
methods and can therefore circumvent incompatibility between species.
Already human genes have been incorporated into bacteria, permitting
large-scale synthesis of rare biochemicals, such as the hormone
insulin and the natural anti-viral agent interferon.
Technology Produces Human Products
Fifteen or more years ago if you posed the question "which is the most
difficult cellular component to analyze?," any biochemist would have
undoubtedly answered "DNA." Its enormous size and its chemical
monotony (only four repeated nucleotides) meant that its sequence
could be approached only by indirect procedures of protein or RNA
sequencing; genetic analysis was an alternative approach, but was time
consuming and inefficient.
When the same question is posed today, a very different answer is
forthcoming. DNA is by now one of the simplest molecules in the cell
to examine, and specific regions can be obtained in unlimited
quantities. Sequencing of DNA can now be achieved at a rate of
several hundred nucleotides per day.
That such a transformation has been achieved is a direct result of recombinant DNA technology.
Such has been the progress in this field that this approach has
superseded conventional methods for determining the amino acid
sequence of proteins. It is this technology that is offering industry
the possibility of large-scale production of protein hormones and
vaccines at economically viable costs. Recombinant DNA technology is
not a single technique - it is a combination of methods, some quite
new, others borrowed from established microbial genetic procedures.
Recombinant DNA techniques
allow even the minor proteins in a cell to be studied. This is a
major advance over conventional methods, which require several hundred
grams of cells to purify even a major protein (i.e., one that makes up
1% or more of the total cell protein). From such source material,
conventional procedures (chromatographic and electrophoretic methods)
would yield perhaps negligible quantities of pure protein. Since many
of the chemicals that would be of great commercial value are present
in small quantities in cells, it is instructive to follow the sequence
of steps that enable purification using recombinant DNA technology.
- Cell fractionation by conventional chromatographic methods to
yield a microgram of protein, which can be obtained in a pure form by
- The protein is analyzed to yield the identity of the first 30
amino acids - its amino terminus.
- The genetic code is used to predict the nucleotide sequence
corresponding to this amino acid sequence, and rapid chemical
synthesis of this DNA fragment is achieved.
- These short DNA fragments are used to identify the complementary
mRNA from which cDNA is produced using reverse transcriptase.
- Cloning produces large amounts of cDNA.
- DNA produced from each clone is hybridized to total cellular
mRNA. Purified mRNA corresponding to each cloned cDNA sequence is
- Having checked by cell-free protein synthesis procedures which
mRNA codes for the desired protein, the appropriate cDNA is then
sequenced and used to determine the protein's amino acid sequence.
- Finally, the cloned cDNA is incorporated into a plasmid vector
and transferred to bacterial or yeast cells. This is the starting
point for scaled-up production of large amounts of the purified
Go to next story: Speaking the Language of Recombinant DNA
See Graphics Gallery:
Transfer and Cloning of the Insulin Gene
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