(80)
11. Forklare hvad der menes med ekspression af den klonede DNA-sekvens
(både i bakterier og i eukaryote celler)
Devlin, s. 309-311, fig. 7.22
Stryer, s.157; s.160-fig.6.28
Eukaryotic genes, in a simplified form, can be introduced into bacteria and the bacteria can be used as factories to produce the desired protein product. It is also possible to introduce DNA into higher organisms.
Expression of eukaryotic genes in prokaryotes:
Many plasmid and bacteriophage vectors have been constructed to permit the expression of eukaryotic genes in bacterial cells. Rapidly replicating bacteria can serve as biological factory to produce large amounts of specific proteins that have research and clinical value.
Several important aspects have to be considered:
The inserted
foreign gene must be in the form of cDNA from its corresponding mRNA since the
bacterial systems can not remove the introns on the pre-mRNA transcript. This
is achieved by using reverse transcriptase. Complementary DNA molecules can be
inserted into vectors that favour their efficient expression in the
host cells, called expression vectors.
The cDNA is
inserted into the vector in the correct reading frame, that is insertion must
occur after a triplet codon of the bacterial protein and at the beginning of
the triplet codon of the eukaryotic gene protein in order to ensure proper
translation
The cDNA has to be
inserted near a bacterial promoter site in order to maximise transcription.
Stronger promoter – more mRNAs.
Eukaryotic
proteins synthesized within bacteria are often unstable and are quickly
degraded by intracellular proteases. Fusion protein products,
however, are usually stable.
They are not degraded and are send out of the cell.
The fusion protein amino-acids encoded by the prokaryotic genome may be
cleaved from the purified protein of interest by enzymatical or chemical
procedures. An alternative can be creating a foreign protein that can be
secreted, by cloning a foreign gene in a vector, such that the fusion protein
synthesized contains a signal peptide that can be recognized by the bacterial
signal peptidase that properly process the protein for secretion.
Expression of eukaryotic genes in eukaryotic cells
Mammalian genetic diseases result from missing or defective intercellular proteins. To utilize recombinant techniques to treat these diseases, vectors have to be constructed that can be incorporated into mammalian cells. In addition, these vectors have to be selective for tissue or cells containing the aberrant protein. They also have to be inserted at specific places in the genome so that they can be transcribed and translated. (must have promotores and enchancers)
Some expression vectors become integrated into the host cell genome while others remain as extra chromosomal small entities called episomes.
The vectors can be introduced into the mammalian cell in several ways:
The foreign DNA
molecules are precipitated with calcium phosphate and taken up by the cell.
This procedure is also called transfection. 10-20% of cells in a culture can
be transfected.
DNA can be
microinjected into the cells nucleus. 2% of all cells contain the foreign DNA.
Viruses are used to bring the new genes into cells. The most effective vectors are retroviruses, which use reverse transcriptase. The double helical DNA produced by the retroviruses can be incorporated randomly in the host chromosome. This DNA version of the viral genome is called proviral DNA and can be efficiently expressed by the host cell and replicated along with normal cellular DNA.
The creation of transgenic animals has demonstrated that a foreign eukaryotic gene containing introns can be correctly transcribed and later spliced, with correct removal of introns, to create a functional mRNA into another eukaryotic organism. In order to get a lot of copies of the protein, the gene needs to be inserted in front of a powerful promoter.
tilbage til molekylær biologi