Biomedical Engineering Reference
In-Depth Information
5'
Pi
lacI
P
O
lacZ
lacY
lacA
3'
repressor
region
control
region
structural genes
Figure 3.8
The
lac
operon houses three structural genes:
lacZ
,
lacY
and
lacA
. These code for three enzymes
required for lactose metabolism (
β
-galactosidase, galactose permease and a transacetylase). Immediately
upstream of these structural genes is a control region that houses a promoter (P) and operator (O) sequence.
The operator represents a binding site for a 'repressor' protein that is in turn coded for by a repressor gene (
Pi
)
found nearby but upstream of the
lac
operon. The repressor gene is in turn controlled by its own promoter. In
the absence of the sugar lactose (or, more accurately, an isomer of lactose called 1,6-allolactose, which acts
as an inducer) the repressor gene product is bound to the
lac
operator site, preventing transcription of the
lac
operon. In the presence of lactose (and hence the inducer), the inducer binds the repressor and the in-
ducer-repressor complex disassociates from the operator, allowing transcription to go ahead. A polycystronic
mRNA is produced, but the operon also houses translational start and stop sites that allow for independent
ribosomal production of the three gene products
regulatory sequences, DNA molecules also invariably house additional non-coding sequences in
the form of various kinds of repeat sequences. For example, genes account for only some 30 per
cent of the total human genome sequence.
The detail and arrangement of gene structure is also normally different in prokaryotes and eu-
karyotes. In prokaryotes, genes of related function are often clustered together in operons, which
are usually under the control of a single promoter/regulatory region. An example is the well-known
'lac operon' described in Figure 3.8. Transcribed operon mRNA thus usually contains coding se-
quence information for several polypeptides, and such mRNA is termed polycistronic. Although
common in prokaryotes, the presence of polycistronic operons is infrequent in lower eukaryotes
and essentially absent from higher eukaryotes, where virtually all protein-encoding genes are
transcribed separately. Eukaryotic genes, however, usually contain coding sequences (exons) that
are interrupted by non-coding intervening sequences (introns), and in many cases exons represent
a minor proportion of the entire gene length (Figure 3.9, and also see Figure 4.3). For example, of
the 30 per cent of the human genome believed to be taken up by genes, an estimated 28.5 per cent
is accounted for by introns with only some 1.5 per cent being accounted for by exons.
mRNA transcripts in eukaryotes undergo substantial editing. The introns are enzymatically
removed from (spliced out of) the primary transcript, and further characteristic modifi cations in-
clude the addition of a cap at the mRNA's 5
'
end and the addition of a polyadenine nucleotide tail
(poly A tail) at the molecule's 3
'
terminus.
3.2.2 Nucleic acid purifi cation
A prerequisite step to any rDNA work is the initial isolation of DNA or RNA from the source
material (which can be microbial, plant, animal or viral). Numerous methodologies have been de-
veloped to achieve nucleic acid purifi cation, and some of these methodologies have been adapted
for use in a variety of commercially available purifi cation kits. Although details vary, the general
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