Gene expression

1
Gene expression

• The transcription involves synthesis of an RNA
  from the DNA template and an enzyme called RNA
  polymerase.
• In prokaryotes there is a single RNA polymerase,
  but in eukaryotes there are three types of RNA
  polymerase (I,II and III).
• Stages of transcription include (I) DNA/RNA
  polymerase binding (II) chain initation, (III)
  chain elongation and (IV) chain termination and
  relase of RNA.

2
The relase of RNA molecule may be immediately
available for translation (as in prokaryotes) or
processed and exported to the cytoplasm (as in
eukaryotes).Translation requires a mRNA
molecule a supply of charges t RNA (associated
with amino acid residues) and ribosomes (r RNA
and ribosomal proteins)
3
The ribosome acts as Jigwhich holds the mRNA
so that the codons may be matched up with the
appropriate anti-codon on the t RNA, thus
ensuring that the correct amino acid (a.a) is
inserted into the growing polypeptide chain. Fig 8
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5
Isolation of DNA and RNA

• The first step in any isolation protocol is the
  disruption of the cell, which may be viral,
  bacteria, plant or animal.
• The method used to open cells should be as
  gentle as possible to avoid the danger of
  mechanically shearing large DNA molecules.

6
Following cell disruption, a deproteinization
step is required.This is may be achieved by
one or more extractions using phenol or
phenol/chloroform mixtures.On the formation of
an emulsion and subsequent centrifugation to
separate the phases, protein molecules partition
into the phenol phase at the interface.
7
The nucleic acids remain mostly in the upper
aqueous phase, and may be precipitated using
isopropanol or ethanol.If a DNA preparation is
required, the enzymatic ribonuclease (RNase) can
be used to digest RNA.If mRNA is needed, a
further purification can be performed by
oligo(dt)-cellulose to bind the poly (A) tail of
eukaryotic mRNA (Fig 9)
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9
For preparing plasmid DNA (PDNA), a technique
of gradient ultracentrifugation is often used A
cesium chloride (Cscl) solution containing DNA is
spun at high speed over a long period, a density
gradient is formed and a band of DNA is then
taken off.
10
Handling and quantification of nucleic acids

• The concentration of a solution of nucleic acid
  can be determined by measuring the absorbance at
  260 nanometers (nm), using spectrophotometer.
• An A260 of 1.0 is equivalent to a concentration
  of 50 mg/ml for double- stranded DNA, or 40 mg/ml
  for single- stranded DNA or RNA.

11
The ration A260/A280 indicates if there are
contaminants present. The ration should be 1.8
for pure DNA and 2.0 for pure RNA. In the
presence of UV- absorbing contaminants, which
makes spectrophotometric measurements impossible,
DNA concentration is determined by the
fluorescence of ethidium bromide dye.
12
The dye intercalates between the DNA bases and
fluoresces orange when illuminated with UV
light.Precipitation of nucleic acids is an
essential technique, which may achieved by adding
ethanol or isopropanol to a DNA solution in a
ration by volume of 21 in the presence of 0.2M
salts.After precipitation the DNA is recovered
by centrifugation, the pellet can be dried and
the nucleic acid re-suspended in the buffer.
13
Radiolabelling of nucleic acids

• Labelling the nucleic acid with a radioactive
  molecule (usually deoxynucleoside triphosphate
  (dNTP) , labelled with 3H or 32p), enable keeping
  track of the small amounts of nucleic acid in any
  cloning procedure.

14
A second application of radiolabelling is the
production of high radioactive nucleic acid
molecules (Probes) for use in hybridization
experiments.
15
Some common methods of labelling are described
are described below(1) End labelling (2) Nick
translation (3) Primer extension.