Prof. Ketley Flashcards
What is a prokaryote?
An organism with no nucleus and no membrane bound organelles (Bacteria, blue-green algae + Archaea).
Why are prokaryote genetics important?
They have an affect on everything (Agriculture, environment and industry).Help in the development of genetics - Avery used phages to test what was the transmitting factor.Recombinant DNA technology - cloning and sequencing genomes.
How is genetic information passed on in prokaryotes?
Reproduction not sexual so transferred by horizontal gene transfer.
Transformation - extracellular DNA introduced into the cell.
Transduction - infection via phage or virus.
Conjugation - Joining of two cells via a pili and pass on using plasmids.
Transposition - movement of genes to a chromosome from a plasmid.
Where does prokaryote chromosome replication occur and where does it terminate?
oriC (origin of replication) and terC (termination of replication). Chromosome may undergo a number of division at the same time or one replication per cell cycle.
Describe a prokaryote gene.
Open reading frame with no introns. Transcription product is many genes in one strand of RNA (operon). This produces a polyprotein which is broken down by enzymes within the cell.
What is the role of a plasmid? Give examples.
Adaptation, pathogenesis (Ti-Plasmid found in Agrobacterium cause the development of tumours), evolution and antibiotic resistance (R-plasmid).
How is a plasmid transferred between cells?
Form a mating pair and pili forms between then connecting cytoplasm. DNA is then transferred from a unique origin (oriT) and a ssDNA transfer occurs in 1-2 minutes. (Conjugative plasmid must be present).
What is a bacteriophage?
‘Eaters of bacteria’ - protein/membrane coat containing phage genome with fibrils which bind to receptors on the membrane before injecting their genome into the host. Very small in size.
How does a bacteriophage cause infection and lysis?
Genome absorbed into cells and the DNA is replicated. The genes for phage assembly are then expressed and the new phages develop. The cell then dies releasing the new phages. (Is prone to errors with host DNA being taken up instead of phage DNA).
What is an insertion sequence/transposon?
A piece of DNA with inverted repeats on either end allowing for the insertion into target DNA. Can include 1-2 genes (may include antibiotic resistance).
Describe the composition of minimal medium
Source of Carbon and Nitrogen; salts; pH of 7 and kept at temperature of 37 degrees.
What is an auxotroph?
An individual who is unable to produce metabolites (eg His- or Lac- when grown on C source as unable to ferment sugar).
Why is resistance useful?
Bacteria will grow in presence of an inhibitor so allows for screening.
What is a lethal mutation and how can it be tested?
A mutation affecting essential function. Can be tested using sterilised velvet allowing for the transfer of bacterial cells from one plate to another (can look at growth in different temperatures). Replica plates mean many colonies can be tested simultaneously.
How are genes transferred into bacterial genome?
Transfer of linear DNA fragment from a donor source. This produces a merodiploid (partial diploid) with the homologous region on the recipient DNA and two reciprocal crosses occur. The linear crossing over product degrades and this recipient is now referred to as a recominant.
What is bacterial transformation?
Gene exchange by the uptake of naked DNA from outside the cell and is rare. Can be natural or artificial.
Describe Griffiths study on bacterial transformation.
Conducted study of mice looking at the virulence of Streptococcus pneumoniae. Wildtype had a smooth capsid (virulent) and mutant has no smooth capsid. Griffiths showed that when mixed together, dead smooth bacteria were able to transform the mutant form into the virulent wildtype.
How does transformation naturally occur? Why?
Occurs when bacteria cells die and split open releasing fragments of DNA into the environment. This DNA is then taken in by other bacteria and recombination occurs. Some bacteria will pick up any DNA, Bacillus subtilis, whereas others will only pick up specific DNA. Specific species recognise non-foreign DNA using a 12 bp recognition sequence.
This mechanism allows for difference in combination of genes leading to evolutionary adaptations (avoiding a hosts immune system). May also be used to repair DNA by switching in a working copy for a damaged copy.
How does artificial transformation occur?
Cells are made permeable to allow absorption of dsDNA plasmid using calcium chloride or electroporation. The DNA does not need to be homologous and can be any DNA sequence.
What is co-transduction and co-inheritance? How is it used in mapping?
This is the inheritance of more than one gene due to the closeness of the genes on the piece of DNA. From this a map can be produced giving the relative distance between genes rather than the actual distance.
Describe gene transfer via conjugation?
Cell to cell contact promoted by the presence of a fertility plasmid (polarity). The donor produces a pili connecting the cells and form a pore through which genetic information is exchanged. The plasmid is replicated via rolling circle replication which takes 1-2 minutes (both cells now fertile). At low frequencies the F plasmid can be inserted into a chromosome forming an episome.
How is time entry mapping carried out?
Transfer from ForiT at a constant rate of transfer. To map mating has to be interupted at intervals (cells have to vortex) and these cells are screened for appearance of marker genes (usually metabolites). Number of transconjugants with all characteristics will be low as strand is more readily degraded. In E.coli, F can be inserted at a number of different orientations so there are different directions of transfer. (PA, me, Luke and Late)
