Biology Reference
In-Depth Information
Molecular biology became a further success story when it joined forces with
biochemistry and microbiology and became modern biotechnology. First, it was
discovered that many organisms make enzymes that cut DNA with specific
nucleotide sequences. By not having those base sequences themselves, those
organisms could protect themselves against invading viruses. These 'restriction
enzymes' were used by scientists to put genes of interest into organisms that did
contain those sequences. By growing these organisms and then again applying
the restriction enzyme to isolated DNA, pieces of DNA corresponding to
genes could be 'cloned', i.e. purified and their amounts greatly amplified. The
resulting material could then be introduced into other living cells which would
then express that DNA into protein. If those cells altered their functioning this
helped in establishing the function of the gene. The amplified amount of DNA
also enabled that DNA to be sequenced, first by tedious methodology, but in a
demand-driven mode this led to the development of new and rapid sequencing
methodology. (The methodology to amplify DNA also became much more
effective when the polymerase chain reaction (PCR) was developed, allowing for
the amplification to occur in vitro .) The result was that the nucleotide sequence
of each gene of interest could be determined. Because of the 64-to-20 mapping
of DNA sequence to protein sequence, this implied that the amino acid sequence
of the corresponding protein was also determined. Through the above cloning
procedure, larger amounts of proteins could be obtained enabling structure deter-
mination through X-ray crystallography and NMR. At present the structure of
almost any soluble protein can be determined, albeit at relatively low throughput.
It also became possible to determine whether any given gene was expressed
in an organism. Here the base-pairing phenomenon that underpins DNA and
mRNA function served molecular biology. Tagged DNA or RNA molecules that
were complementary in terms of nucleotide sequence were synthesized and made
to react ('hybridize') with mRNA isolated from living organisms. If a certain
mRNA was expressed then the hybridization would betray this. Because so many
genes are expressed in any organism and because of background reactivity, the
mRNAs first had to be separated from each other, which was accomplished
by gel electrophoresis. A corresponding methodology was developed for the
measurement of expression at the level of protein, by using specific antibodies
for the proteins. The separation power of these methods is however limited, and
therefore they were not suitable for genome wide measuring of gene expression.
Another powerful tool came from genetics applied to rapidly growing
microorganisms. Mutations were made in the DNA of these organisms and the
consequences for their functioning was determined. Through the above method-
ologies, mutations could be related to proteins. Deleting genes and observing the
consequences, pathways could be constructed that should be responsible for cer-
tain aspects of cellular behaviour. When this was done for different organisms
and combined with nucleotide sequencing, an astonishing phenomenon turned up.
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