Horizontal Gene Transfer in Prokaryotes and Recombinant DNA Technology Flashcards
What is horizontal gene transfer?
a non-reproductive transfer of genetic material from one organism to another. Horizontal gene transfer does not require cell division, and can occur between organisms of the same or different species
What is transformation?
the uptake of DNA from the environment, which is then incorporated into the bacterial genome
What are natural and artificial transformation?
In some bacteria, transformation is an active process (these bacteria are referred to as competent). Other bacteria are not naturally competent, but can be treated to allow for artificial transformation. Artificial transformation is widely used in genetic engineering (also works with eukaryotic cells).
What is conjugation?
the direct transfer of either plasmid or genomic DNA from one bacterium to another - two bacterial cells make contact and fuse. One cell (donor cell) transfers genomic DNA (usually part of the genome is transferred) or plasmids into the other cell.
Describe how conjugation is mediated by the F factor
F plasmid directs synthesis of pili that contact the recipient cell and draw it closer. The F DNA in the donor cell replicates (rolling circle replication), generating a single-stranded version that moves into the recipient cell. In the recipient cell, the other strand is synthesized - one copy remains in the donor, another one appears in the recipient
What is transduction?
the transfer of genetic material between bacterial cells by bacteriophages
How does generalised transduction take place?
During the lytic life cycle of a bacteriophage (many types of phages (virulent phages) can only undergo a lytic life cycle), the chromosomal DNA of the infected bacterium is broken up into small pieces. Occasionally, pieces of chromosomal DNA are incorporated into phage heads and can be transmitted to another cell. The piece of DNA might then recombine with the DNA of the recipient bacterium.
How does specialised transduction take place?
Some phages (e.g. lambda) act as specialized transducers and carry only certain host genes to recipient cells. Specialized transducers are phages that are temperate phages that integrate into the host cell genome and can remain within the host cell for a period without killing it (the phage exists in the host as a prophage, and the host is referred to as a lysogenic bacterium). Specialized transducers insert into the bacterial chromosome at only one position. When they exit it and the lytic phase starts, neighbouring genes can be incorporated into the outlooping (rare event). These genes can then be incorporated into phageheads that infect other bacteria.
Why is horizontal gene transfer evolutionarily beneficial for bacteria?
Horizontal transmission can spread DNA rapidly through a bacterial population by contact in much the same way that a disease spreads. For bacteria, horizontal transmission provides a powerful method by which they can adapt rapidly to changing environmental conditions
. Often, the genes discovered to be involved in horizontal transfer are those that also confer survival advantages to the recipient species. For example, one species may transfer antibiotic resistance genes to another species. Or genes conferring enhanced pathogenicity maybe be transferred.
What is recombinant DNA technology?
the creation of combinations of DNA sequences from different sources. It often involves the insertion of a DNA fragment into a DNA cloning vector (usually a modified bacterial plasmid) to generate a recombinant DNA molecule that is then amplified in the bacterium E.coli
How can you cut and join DNA fragments?
the DNA is cut at a recognition sequence using a restriction enzyme forming fragments with cohesive ends
DNA pieces with complementary cohesive ends can be hybridised
Annealing allows recombinant DNA molecules to form by complementary base pairing
DNA ligase seals the nicks in the DNA backbone, covalently bonding the two strands
What are cloning vectors?
DNA molecules that are used to hold a piece of DNA of interest, so that it can be maintained and replicated in an organism
The most common cloning vectors are modified bacterial plasmids. The origin of replication allows the replication of the vector within the bacterial cells (usually E.coli). Many cloning vectors can produce several hundred copies of the vector in a single bacterial cell. The multiple cloning site consists of many restriction sites that are not present elsewhere in the vector. This allows the vector to be cut for insertion of the DNA sequence of interest. A selectable marker (usually an antibiotic resistance gene) allows for selection of only those bacteria that carry the vector.
How do you carry out artificial transformation?
treating the cells with calcium ions and a brief heat shock, or by applying a brief high-voltage electrical pulse (electroporation). Both treatments lead to the opening of membrane pores through which the DNA molecule can enter the cell.
Why does a selection process need to be carried out?
During the transformation process, only some of the bacterial cells will take up the plasmid. Cloning vectors contain a selectable marker (usually an antibiotics resistance gene), so to kill cells without the vector, the cells can be plated on agar plates containing the respective antibiotic.
Some cells might contain the cloning vector without insert (e.g. if not all vector molecules were successfully cut when incubated with a restriction enzyme, or because of the vector religating with itself during the ligation step). To find out which cells contain the vector without the insert, blue/white selection can be carried out.
What is blue/white selection?
In some cloning vectors, such as pUC18, the multiple cloning site is located in the lacZ gene (without disrupting its function). The lacZ gene codes for beta-galactosidase, and enzyme that catalyses the hydrolysis of beta-galactosides. It can calalyse the reaction of the colourless substrate X-gal into a product that gets converted into a blue substance by oxidation.
Because the integration of an insert into the multiple cloning site will disrupt the function of the lacZ gene, colonies of cells that carry the vector with the insert will appear white on an agar plate treated with X-gal, whereas colonies of cells carrying the vector without plasmid will appear blue (because the lacZ gene is intact, beta-galactosidase is produced, which leads to the production of the blue substance).
