Cambridge International A Level Biology Revision Guide

Chapter 19: Genetic technology
QUESTION
19.3 Would all the bacteria that fluoresce definitely have
taken up the gene that it is hoped was inserted into
them? Explain your answer.
Promoters
Bacteria contain many different genes, which make many
different proteins. But not all these genes are switched
on at once. The bacteria make only the proteins that are
required in the conditions in which they are growing.
For example, as we saw in Chapter 16, E. coli bacteria
make the enzyme β-galactosidase only when they are
growing in a medium containing lactose and there is no
glucose available.
The expression of genes, such as those in the lac
operon, is controlled by a promoter – the region of DNA
to which RNA polymerase binds as it starts transcription.
If we want the gene that we are going to insert into a
bacterium to be expressed, then we also have to insert
an appropriate promoter. When bacteria were first
transformed to produce insulin, the insulin gene was
inserted next to the β-galactosidase gene so they shared
a promoter. The promoter switched on the gene when
the bacterium needed to metabolise lactose. So, if the
bacteria were grown in a medium containing lactose but
no glucose, they synthesised both β-galactosidase and
human insulin.
The promoter not only allows RNA polymerase to
bind to DNA but also ensures that it recognises which
of the two DNA strands is the template strand. Within
the sequence of nucleotides in the promoter region is the
transcription start point – the first nucleotide of the gene
to be transcribed. In this way, the promoter can be said
to control the expression of a gene and can ensure a high
level of gene expression. In eukaryotes, various proteins
known as transcription factors are also required to bind
to the promoter region or to RNA polymerase before
transcription can begin (Chapter 16, page 391).
Gel electrophoresis
Gel electrophoresis is a technique that is used to separate
different molecules. It is used extensively in the analysis
of proteins and DNA. This technique involves placing
a mixture of molecules into wells cut into agarose gel
and applying an electric field. The movement of charged
molecules within the gel in response to the electric field
depends on a number of factors. The most important are:
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net (overall) charge – negatively charged molecules
move towards the anode (+) and positively charged
molecules move towards the cathode (–); highly
charged molecules move faster than those with less
overall charge
size – smaller molecules move through the gel faster
than larger molecules
composition of the gel – common gels are
polyacrylamide for proteins and agarose for DNA; the
size of the ‘pores’ within the gel determines the speed
with which proteins and fragments of DNA move.
Electrophoresis of proteins
The charge on proteins is dependent on the ionisation
of the R groups on the amino acid residues. You will
remember from Chapter 2 that some amino acids have R
groups that can be positively charged (−NH 3+ ) and some
have R groups that can be negatively charged (−COO − ).
Whether these R groups are charged or not depends on
the pH. When proteins are separated by electrophoresis,
the procedure is carried out at a constant pH using a
buffer solution. Usually proteins have a net negative charge
(Figure 19.8).
Figure 19.8 Gel electrophoresis of proteins. The gel was
placed in the tank containing a suitable buffer solution.
Protein samples stained red have been added to wells along
the top of the gel. They are migrating downwards towards
the anode.
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