References Periannan Senapathy, M. B. (1987). RNA splice

 

 

References

Kenneth J.Livaka, T. D. (2001).
Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR
and the 2???CT Method. Methods , 402- 408 Vol 25, Is 4.
Mark
D. Curtis, D. G. (2016). Plant Biotechnology and Genetics: Principles,
Techniques, and Applications. In J. C. Neal Stewart, Recombinant DNA,
Vector Design, and Construction (pp. 181 – 235).
Nikolaus
Blin, D. W. (1976 ). A general method for isolation of high molecular weight
DNA from eukaryotes. Nucleic Acids Research, 2303 – 2308, Vol 3, Is 9.
Periannan
Senapathy, M. B. (1987). RNA splice junctions of different classes of
eukaryotes: sequence statistics and functional implications in gene
expression. Nucleic Acids Research, 7155 – 7174 , Vol 15, Is 17.
Southern,
E. (2006). Southern Blotting . Nature Blottings , 518 – 525 .
 

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

The gene can then be tested for its expression by Southern Blotting.
Southern Blotting is a technique used in laboratories to detect whether a
specific fragment of DNA has the gene that you are interested in expressed, it
was developed in 1975 by Edwin M. Southern. It is used to look at genomic DNA,
it often uses radioactive DNA hybridization probes, also uses radiography. The DNA
is then placed on a gel (after being stained with the ethidium bromide, so they
can be visible under UV illumination) – with a southern blot you are checking
for the whole sequence to see if the gene is present. Radio label the probes to
check whether the DNA binds giving an indication to whether your gene would be
detected. The gel with the DNA, the transfer buffer which moves the DNA through
the gel and up onto the membrane, there are layers of tissue that wick the
buffer through to draw the DNA through to the cellulose, and then stick the
radiolabelled probe to see whether the gene is present (Southern, 2006).

Agrobacterium tumefaciens can be used as the vector
itself, so the T-DNA is what will be copied into the plant genome: by removing
the native bacterial genes within the region, you can replace them with genes
you wish to express. The T-DNA is integrated by illegitimate recombination.  Firstly, the Ti-plasmid is removed from the Agrobacterium tumefaciens, the genes we
don’t want inserted a cut out using a restriction enzyme which leaves behind a
sticky overhang. The DNA taken from the eukaryote we wish to insert also have
the same restriction enzyme overhangs, producing complimentary overhangs for
the Ti-plasmid. By adding a ligase enzyme, the gene of interest will anneal to
the plasmid itself. The plant cells are then taken from the parent plant – so
in this case a cell plant, and placed on an agar medium with hormones present
which will allow the development of callus. The transformed bacteria are added
to the callus culture where the bacteria would infect the plant cells with the
modified DNA, after a couple of days the antibacterial agent is added to kill
the bacteria as it is no longer needed. Markers can be added to the agar as
well – they are used to track whether the plant cells have successfully taken
up the genes that we wanted to express, by inserting an herbicide resistance
gene alongside the desired gene, the plant cells that fail to take up the gene
are killed off.

You can use Agrobacterium
tumefaciens to insert a certain Ti Plasmid into a plant genome to test
for the gene. Agrobacterium tumefaciens are
a type of plant pathogen that is found in soil around plant roots. It can
mutate any plant, by transforming the plant cells by inserting a section of its
tumour inducing (Ti) plasmid into the plant cell genome. Agrobacterium tumefaciens has a 200kb mega plasmid containing a Vir region which is made up of 8
operons. VirG binds to the vir-box regions of each operon enabling
transcription of the Vir­-region. The
activity of the vir genes is
regulated by wound associated signals – acetosyringone is released from plant
wound sites and was found to activate the vir region.

This DNA produced can then be
amplified up using Polymerase Chain Reaction to produce a larger sample of the
DNA with the gene of interest. PCR is split into three different section:
Denaturation, Annealing and Extension. Denaturation involved breaking the
hydrogen bonds between the DNA helicase using heat or melting the template DNA,
typically 95oC, Annealing is where the DNA is cooled to about 55oC,
primers are added which will anneal before the DNA template can anneal to its
own strand. The reaction is then heated to 72oC (the temperature at
which polymerase works) and copying begins- where complementary base pairing
takes place with the free nucleotides, producing more strands. The growth of
the amplified DNA is exponential, so you start of with 2 strands, after 1 cycle
you have 4 and then after your 2nd cycle you have 16 strands. This
will amplify the DNA with the particular gene present which can then be
inserted into a plant cell (Kenneth J.Livaka, 2001).

The complex
is now bound to the start point in the mRNA and the first amino acid protein
chain is in place ready for attachment of the second. tRNA and mRNA move
through the ribosome in the same direction, with there being three sites: A
site (Acceptor site), P Site (Donor site) and E site (Exit site). The tRNAs
rapidly enter and exit the A site until the right match is found between the
anticodon and the codon on the mRNA. It is then joined to the existing amino
acid by peptide bonding forming a short peptide chain. After a large number of
amino acids are joined together by the peptide bonds, once a stop codon is
added on, the amino acid chain would leave the ribosome – producing a protein,
where it would under go additional changes forming into a certain type of protein
(Fibrous or Globular).

Next step if the addition of the
Poly-A tail which occurs when transcription is still on going, it requires a set
of enzymes bound to the RNA polymerase II which recognises the signal motif
within the 3′ end of the UTR. This signal is recognised by an enzyme complex
called cleavage/poly-adenylation specificity factor (CPSF) which is bound to
the recognition sequence, the cleavage specificity factors are bound to the GU
rich region further down the stream, once the CPSF is bound , it also recruits
another enzyme known as polyadenylate polymerase (PAP) which binds to it, CPSF
will clip off- mRNA 30 nucleotides
downstream, leaving the discarded run on transcript which will be degraded by
nucleases and process – mRNA. PAP will then transfer onto the – mRNA and begin
adding the poly-a tail and move along the 5′ to the 3′ region. The next stage
is splicing to remove the introns as they are non-coding. Introns contain
conserved recognition sequences at 5′ and 3′ ends of the sequence know as donor
and receptor sequences, there is also a branchpoint sequence just upstream of
the spliceosomes – s complex of 5 small nuclear riboproteins (snRNPs). The spliceosomes
cleave the 5′ site and folds the end over to join the branchpoint motif
creating a lasso type structure which is then cleaved away, the proteins hold
the 5′ and 3′ ends of the mRNA and the 2 exons in place and then ligates the
donor and receptor sequences together to join the 2 exons into 1 whole. For
translation to take place numerous eukaryotic accessory initiation factors
(eIFs) are required, eIF-2 binds to form a ternary complex with the initiator tRNA. This bind small subunits of ribosomes
(40s). eIF-3 maintains 40s subunit in free state until it can bid not the
ternary complex and the complex binds to the 5′ cap. (Periannan Senapathy,
1987)

Docking is then enabled for the final
enzyme – TFII Helicase which acts to unwind the DNA (Similar to DNA helicase)
it opens up the double helix and separates out the single strands forming an
open complex which allows the template strand to feed though the RNA polymerase
and acts as a substrate for the copying into RNA. Once the TFII helicase is
added and docks onto the RNA polymerase molecule, it transfers a phosphate
group onto the carboxy terminal domain of the polymerase and activates the RNA
polymerase enzyme for elongation – once elongation begins, release of the
general transcription factors takes place. The Carboxy terminal domain forms a
group for which the DNA fits as it enters the RNA polymerase complex, once this
is active, it allows the RNA polymerase to move along and the helicase to begin
unwinding the DNA as the complex moves along the gene. Straight after transcription has occurred, mRNA is still in the form of
pre- mRNA with a 5′ untranslated region (UTR) with the condign regions still
containing introns which are non -coding and the 3′ UTR – as there is no
specific termination factor within the gene itself, there is a long run on
transcript. The 5′ and 3′ untranslated regions are not processed by themselves,
they contain signals which allow the processing to occur, sites within the 5′
UTR bind to proteins that promote and inhibit translation whereas the 3′ UTR contains
signals for the addition of the Poly-A tail. The first stage if the addition of
a 5′ cap where a 5′ terminal guanine residue is enzymatically added by guanylyl
transferase in a reverse orientation – this cap is then used as a substrate for
several methylation reactions. The Guanylyl transferase if capable of binding
the two sets of tri-phosphate groups in a very different orientation, the
structure of mRNA the 5′ end has three phosphate residues (known as guanosine
tri-phosphate) which is the base that is formed into mRNA or DNA (Mark D. Curtis, 2016).

The process by which to isolate the
coding regions within the eukaryote gene starts off with the Transcription
process, where proteins in the basal transcription complex interacts with the
RNA polymerase II to initiate transcription. Starts off with the TATA box
binding protein, binding it the TATA box which then recruits another
transcription factor known as TFIA which enhances the binding onto the DNA
template, this then recruits another transcription factor known as TFIIB which
binds to the RNA polymerase II. The complex of transcription factor IIF and the
RNA polymerase complex then binds onto the growing initiation complex.

Enhancer elements bind transcription
factors together to the gene, they will bind to the enhancer motifs in the DNA,
these proteins then serve to unwind the nucleosomes allowing access to the RNA
polymerase and arioso other transcription factors to the minimal promoter in
order to initiate transcription. There are variable sub stream sites which vary
depending on the particular type of expression system on the gene (whether it
is tissue specific or inducible). The most common motifs found are SRE and GC
regions which lie far upstream in the mammalian genes and bins to transcription
factor complexes which interact with the RNA polymerase as well as unwind the
nucleosomes (Nikolaus Blin, 1976 ).

The Upstream sequences can trigger
expressions – many of the ‘upstream’ regulatory sequences control tissues specific
or inducible expressions. The gene itself has a minimal promoter which is used
for basal expression in any cell type – it has a similar structure to
prokaryotes. The further upstream you go there are elements known as enhancers
which bind to specialised transcription factors which are involved in tissue
specificity and induction of the genes under environmental conditions. They are
variable on the distance from the start site on the gene as they can lie a
random distance form the start site, as they are bi-directional in function, so
genes can exist on both sides of the DNA – they can be transcribed on either
side of the DNA strand so if they are inserted in a reverse direction they will
still work. (Periannan Senapathy, 1987)

Eukaryotic
cells have their DNA packed inside the nucleus which is surrounded by a nuclear
membrane which separates transcription from translation which occurs in the
cytoplasm. The mRNA which is produced is longer lived as it has features that
allow it to be transported out of the nucleus and to protect it from any
degradation that may take place by nucleosomes in the cytoplasm. The structure
of the Eukaryotic gene is similar to that of a prokaryotic gene however there
are some differences as-well. The main differences include, there being
regulatory sequences upstream (at the 5′ end) of the minimal promoter – so
where the CAAT and the TATA boxes are, the introns – which are the non-coding
parts of the gene which are transcribed into mRNA but spliced out prior to
translation taking place- are present and the last difference is there is a
polyadenylation signal which allows the addition of a poly-a tail to take place
{figure 1 shows this in more detail}

There are two types of cells that are
present within organisms Prokaryotes and Eukaryotes, prokaryotes are typically
found within bacterial cells how ever eukaryotic cells are found within living organisms
such as protozoa, fungi, plants and
animals. These organisms are grouped into the biological domain Eukaryota. Eukaryotic cells are larger and more
complex than prokaryotic cells which are found in Archaea and Bacteria, the
other two domains of life.