Lecture 11: Genetics Flashcards
Microbial genetics
- Microbial genetics is the study of chromosome structure, gene structure, heredity, and horizontal transfer in bacteria
Many aspects of microbial genetics are substantially different from eukaryotic
Bacterial Genome
The genome is all the
DNA in the cell
Includes chromosomes, large, circular pieces of DNA only are transferred to daughter cells
Borrelia has linear
chromosome
Also includes plasmids
- smaller circular DNA- segments
Chromosome structure
The bacterial chromosome is one large loop of DNA
The chromosome is supercoiled, that is, tightly wound up, in order to pack it into the volume of the cell
E. coliE. coli chromosome length is about 4,600,000 base pairs (4.6 Mbp), and the typical genome contains about 4300 genes
- If the E. coli chromosome were stretched out it would be
1000 times longer than a single cell!
DNA replication
Semi-conservative
replication
Each daughter cell contains one new strand and one
parental strand
New DNA is made
by the DNA polymerase
holoenzyme
During binary fission, bidirectional replication occurs
Bacteriophage, plasmids in
conjugative transfer, use rolling circle replication
Accuracy of DNA Replication
- Mistakes are make by DNA polymerase about 1 in
every 10^10 bases
Since the genome of E. coli is only about 5 x 10^6
bases, this means that about every 5000 times a cell
divides, DNA polymerase makes one mistake
This is because of the proofreading capabilities of the
enzyme
These rare mistakes are important, because they cause mutations
mutations
Mutations affect the genotype of the microorganism directly
Mutations may affect the phenotype of the microbe as well
Mutations are changes in the genetic code
Mutations can be spontaneous
Mutations can be directed by experimenters
Mutations can be made by horizontal gene transfer
Cause of mutations
Exposure to radiation (UV, for example)
T-T dimers
Mutagenic chemicals
Ethidium Bromide, Acridine Orange
DNA Intercalators
Nucleoside analogues such as AZT
Error by DNA polymerase (very uncommon)
Mobile genetic elements
Bacteriophage
Transposon
Retrovirus
We’ve cultured a soil organism that degrades PCBs
We want to identify the genes that are responsible for this function
How do we do it?
- direct positive selection
Examine population for mutants that have an
overt phenotype that is selected for experimentally
Example - place population in medium containing penicillin, look for growth of penicillin resistant mutant strains
Advantage - efficient, positive result easy to identify
Disadvantage - limited to relatively straightforward, harsh selective processes
Can only use to find “gain of function” mutants
Indirect selection
- Use to identify mutants
containing “loss of function”
mutants
Method of choice is called
replica plating
Create a pool of mutants,
test mutants for loss of a
certain function (ex.certain function (ex. Auxotrophy)
The Ames Test
An application of the principle of indirect selection to detect mutagenic
chemicals
Bacteria incapable of living without histidine are used
The number of revertants is a measure of mutagenicity
We’ve cultured a soil organism that degrades PCBs
We want to identify the genes that are responsible for this function
How do we do it?
- place in rich, permissive media
- selective with PCB as a sole carbon source
- use sequencing
Direct (positive) selection
Examine population for mutants that have an
overt phenotype that is selected for experimentally
Example - place population in medium containing penicillin, look for growth of penicillin resistant mutant strains
Advantages and disadvantages of direct selection
Advantage - efficient, positive result easy to identify
Disadvantage - limited to relatively straightforward, harsh selective processes
Can only use to find “gain of function” mutants
Indirect selection: uses and method
Use to identify mutants
containing “loss of function”
mutants
Method of choice is called replica plating
Create a pool of mutants,
test mutants for loss of a
certain function (ex. Auxotrophy)
