BMS75 Advanced Tools in Molecular Biology | Master Biomedical Sciences Radboud University Nijmegen | Summary of all the lecture slides and additional information on explaining molecular principles and slides.
- To visualize (spatio-temporal) protein expression
- Tagging proteins
- Tagging peptides with a role in compartmentalization
- Expressing fluorescent cell cycle proteins
- To examine protein function by
- Changing protein amounts (overexpression, downregulation siRNA, shRNA, Crispr/Cas)
- Perturbing protein function (loss of function-or activating mutations)
- Applying function sensors
Actin branches are formed and they are contracting between the intra- and extracellular matrix. The
CRISPR/CAS approach is used in pathology and molecular therapy. CRISPR/CAS can cut of a loop where a
disease related gene is on.
The central dogma of protein synthesis is divided into several mechanisms:
Replication: DNA replication from 5’ 3’ from the non-coding strand. The coding strand is also
replicated, but the fragments are called Okazaki fragments (on the 3’ to 5’ strand).
Transcription: DNARNA, first there is the process of capping, elongation and splicing. Only the
exons will be used for the mRNA. mRNA consists of: cap-mRNA sequence-poly A tail (AAAA). The
complete mRNA is exported out of the nucleus into the cytosol (see figure).
Translation: mRNAProtein, proteins are made along with the reading frame, translation is done
from 5’ 3’. Translation starts at the startcodon (AUG) and stops at UAA, UGA or UAG. The N
terminus is translated first; the N terminus is located on the 5’ side.
,In the ER you have proteins which are used for exocytosis and the second class that is translated at the ER
are the transmembrane proteins. Those two classes are mainly translated at the RER. The other proteins
are translated in the cytosol.
During replication the polymerase opens the strand. DNA polymerase cannot connect the strands
anymore, but ligases close the DNA strand. The base pairs are C-G and A-T.
During transcription there is a promotor and RNA polymerase. Transcription starts at a transcription
initiation site (DATA/TATA box). Only translation starts at AUG. At the 5’ mRNA gets capped, which protects
it from degradation. At the 3’ end there is a poly-AAAAA tail. The splicing happens co-transcriptionally.The
poly-AAA tail is added because it is coded, the template strand has a lot of TTTTT. The cap is not coded,
this is a modification of an enzyme.
Translation is from RNA to protein. There is a ORF (open reading frame) which is the region between AUG
(ATG in DNA) and the stopcodon. The ORF is translated into a protein. The stopcodon does not code for
an amino acid in the protein.
In the nucleus splicing is taking place already, where introns are spliced out of the DNA and mRNA is
formed. Then the mRNA goes to the ribosome where it is translated. But it still has noncoding sequences
on the 5’ and 3’ end. This is very important when it is about viruses etc. The reading frame is from 5’ 3’.
The ribosome finds the first AUG.
Protein routing:
It is transported from nuclear pores from the nucleus, then it is transported across membranes and then
exocytosed by vesicles. There are differences in transmembrane domains. GPI anchors are on the outside
of the cell.
There are many types of RNAs:
mRNA – code for proteins
rRNA – form the core of the ribosome and catalyse protein synthesis
miRNA – regulate gene expression
tRNA – serve as adaptors between mRNA and amino acids during protein synthesis
There are also many types of RNA polymerases:
RNA polymerase I – most rRNA genes
RNA polymerase II – protein-coding genes, miRNA genes, plus genes for some small RNAs
RNA polymerase III – tRNA genes, 5S rRNA gene, and genes for many other small RNAs
The ribosome consists of proteins and RNA which are critical for some of the steps in translation. rRNA
catalyses reactions, they are also called ribosymes.
Micro RNA (miRNA) interacts with RNA to form dubblestranded RNA.
Cloning cutting a piece of DNA from one organism and inserting it into a vector where it can be
replicated by a host organism (subcloning).
OR Using nuclear DNA from one organism to create a second organism with the same nuclear DNA.
,Agents used for subcloning:
Plasmids – store DNA information
To make a DNA library:
Bacteriophages
Cosmid – most artificial, a derivative of phages and plasmids
Artificial chromosome vector
Plasmids:
Circular pieces of double stranded DNA found naturally in
bacteria
They can carry antibiotic resistance genes (genes from
receptors, toxins or other proteins)
They replicate separately from the genome of the organism
They can be engineered to be useful cloning vectors
Vector/construct = plasmid used for genetic engineering
Naturally occurring plasmids are never called a vector. Some genes
can undergo their own evolution. An example of a naturally occurring
vector is F plasmid.
Figure 7-3: General procedure for cloning a DNA fragment in a
plasmid vector
Although not indicated by colour, the plasmid contains a replication
origin and ampicillin-resistance gene. Uptake of plasmids by E.
coli cells is stimulated by high concentrations of CaCl 2. Even in the
presence of CaCl 2 , transformation occurs with a quite low frequency,
and only a few cells are transformed by incorporation of a single
plasmid molecule. Cells that are not transformed die on ampicillin-
containing medium. Once incorporated into a host cell, a plasmid can
replicate independently of the host-cell chromosome. As a
transformed cell multiplies into a colony, at least one plasmid
segregates to each daughter cell.
Plasmid characteristics:
Self-replicating, multiple copies
Replication origin site
(multiple) cloning site
Selectable marker gene
Small size: 1-10kb
Low molecular weight
Easily isolated and purified, also easily isolated into host cell
Control elements – promotor, operator, ribosome binding site
, Bacteriophages:
Phage lambda is a bacteriophage or phage (bacterial virus), that uses E. Coli as host
Head, tail and tail fibres
The bacteriophages used for cloning are the phage λ and M13 phage. It infects bacteria.
They follow either a lytic (the harmful cycle – similar to virus, creating lots of phages) or lysogenic
cycle (the lambda phage only invades the bacterium and does only insert the DNA, so the DNA is
stored, this is not harmful until you induce a lytic cycle that will change the conditions of the
bacteria which eventually makes them burst)
There are 2 kinds of lambda phage vectors: insertion vector and replacement vector
Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5-11 kb may be
inserted. The DNA is in the head. The physical size of the head limits the amount of DNA.
Bacteriophage λ is probably the most extensively studied
bacterial virus, and a great deal is known about its molecular
biology and genetics. A λ phage virion has a head, which
contains the viral DNA genome, and a tail, which functions in
infecting E. coli host cells (Figure 7-10a). When λ DNA enters
the host-cell cytoplasm following infection, it undergoes either
lytic or lysogenic growth (see Figure 6-19). In lytic growth, the
viral DNA is replicated and assembled into more than 100
progeny virions in each infected cell, killing the cell in the
process and releasing the replicated virions. In lysogenic
growth, the viral DNA inserts into the bacterial chromosome,
where it is passively replicated along with the host-cell
chromosome as the cell grows and divides.
Figure 7-11Assembly of bacteriophage λ virions
Empty heads and tails are assembled from multiple copies of
several different λ proteins. During the late stage of λ infection,
long DNA molecules called concatomers are formed;
these multimeric molecules consist of copies of the
λ genome linked end to end and separated by COS sites (red),
a protein-binding nucleotide sequence that occurs once in each
copy of the λ genome. Binding of the λ proteins Nu1 and A to
COS sites promotes insertion of the DNA between two adjacent
COS sites into an empty head. After the heads are filled with
DNA, preassembled λ tails are attached, producing complete λ
virions capable of infecting E. coli cells
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