Lecture 2 Flashcards
Simplistic Definition of Virulence by Koch’s Postulates
- Microbe found in diseased tissue but not in normal tissue
– Some pathogens can be colonizers and only sometimes cause disease (Staph. aureus, Helicobacter pylori) - Microbe can be isolated from diseased tissue as pure culture
-Some organisms are non-culturable (T. pallidum – causative agent of syphilis) - Disease may require multiple organisms (Hepatitis D disease)
- Microbe can cause disease when inoculated in animal or man
– Gonococcus has no animal model
Expansion on Koch’s original postulates: Therapeutic or preventative measures can eliminate disease
What is metagenomics?
bulk sequencing from the environment (16s rRNA or random) to define new species, new genes, and/or new pathways
What is the importance of inbred animals in the study of disease?
You can’t really study the disease really well unless you have inbred animals in order to ensure that genetics isn’t affecting the results
What is the Surrogate end point?
Instead of looking for a specific disease features, you can look for things that are indicator of the occurrence of the disease
Why is arabidopsis thaliana useful?
Small flowering plant related to cabbage and mustard.
The first plant to have its entire genome sequenced.
Checked to see which is the insertion into the genes cause damage from the mutant. With transposons inserted, they stopped this damage, and were able to identify a number of site within pseudomonas genome could cause damage to the plant
Why is Caenorhabditis Elegans useful?
Easy to maintain a population in the laboratory
Worms can be frozen and subsequently thawed
The complete cell lineage of the species has been determined
C. elegans is a simplest multicellular eukaryote that contains only 1031 cells. During development, 131 cells are eliminated by programmed cell death (apoptosis).
In addition, C. elegans is one of the simplest organisms with a nervous system, comprising 302 neurons whose pattern of connectivity has been completely mapped out
Relatively straightforward to disrupt the function of specific genes in nematode by RNA interference (RNAi). The nematode can be simply soaked in (or injected with) a solution of double stranded RNA.
Susceptible to several pathogens
Worms can be fed on genetically-modified bacteria which express the genes of interest.
Why is Dictyostelium amoebae useful?
Cellular slime moulds that Normally feed on bacteria.
Has been used as a model organism in molecular biology and genetics, and in studies of cell communication, differentiation, and programmed cell death.
Grow as separate, independent cells but interact to form multicellular structures when challenged by adverse conditions such as starvation.
Entire genome of Dictyostelium has been sequenced
Why is Dictyostelium melanogaster useful?
Small and easy to grow in the laboratory; Cheap to use for studies
ability to produce offspring in great numbers (females can lay >800 eggs in one day
It has only 4 pairs of chromosomes including 1 sex chromosome pair; its entire genome has been sequenced.
Because many of the basic pathway mechanisms used by the fly have similarities to mammalian organisms, the fly is being used to study mechanisms underlying different aspects of genetics, neurological disorders, immunity, diabetes, and cancer
Why is Zebrafishr useful?
Common and useful model organism for studies of vertebrate development and gene function
Not amenable to sophisticated genetic manipulation
Zebrafish embryos develop rapidly within 3 days
The embryos are large, robust, and transparent and develop externally
Drugs may be administered by adding directly to the tank
Useful in the study of microbial toxins
What does horizontal transfer allow for?
Horizontal transfer occurs to create Genomic islands which carry genes that carry specific functions in the bacteria
What is the difference between fitness, ecological and saprophytic Islands?
Fitness islands allow the bacteria to survive better
Ecological islands are produced when the bacteria changes to fit better for the environment around them
Saprophytic island refers especially to bacteria that can decompose organic matter
How do you find virulence factors?
Isolate proteins, fractionate and study in the appropriate detection system
Sequence the protein, go back to DNA to find a gene
To find a gene, make a library of genome fragments
Use cloning vector to insert the fragments into and transfer the vector into the appropriate host
Select clones expressing the virulence factor in the appropriate test system.
What is the process of cloning a DNA library in a plasmid vector?
Plasmid bacterial have cloning sites that can be cut by an enzyme and insert pieces of genes inside
selectable markers are antibiotic resistance on the plasmid, so the bacteria keeps the plasmid around to survive the antibiotic.
Each plasmid has some piece of genomic DNA inside of it, and you can see which one of the pieces containing plasmid is causing some sort of virulence factor phenotype that you are scoring for.
What is the process of complementation screen?
Make library from wild-type organism
Transform library into new non-pathogenic host or a mutant host
Select for “+” transformants
Recover transforming fragment
What is the process of Nefarious Misuse; Cloning of Diphtheria Toxin into Yersinia by Former Soviet Scientists to Create a Highly Virulent Biological Weapon?
- Isolate bacteria from patient
- Extract DNA from that bacteria
- Digest DNA W/RESTRICTION ENZYME
- Make E.COLI library of plasmids
- Test individual E.COLI clones created from extracting plasmid DNA from each of these clones
- Transform another Yersinia specifies
- Put individual clones into pseudotuberculosis and infected the guinea pigs
- Look to see which clones/plasmids were causing lethal expression
- Do a reduction approach, and diluted it out
- Fraction it out, until you get a single clone identified that was causing the sickness
- Take that plasmid and sequenced it to see what the gene is
