Genetics Flashcards
Where do regulators bind?
Main subgrooves in inverted repeats, as dimers
Negative gene regulation involves what?
protein repressor that blocks transcription
Anabolic genes
subject to end-product repression (need co-repressor)
Catabolic genes
induced by starting substrate (which inhibit repressor proteins)
Where are operators?
Downstream of promotor = block RNA polymerase
What do activators bind to?
Activator binding site upstream of promotor
Does regulon or operon have multiple dispersed gens regulated by same protein?
Regulon
Diauxic growth
i.e. 2 exponential growth rates
Example of an autokinase
sensor (bacterial)- can phosphorylate itself
When does DNA taken up by bacteria not need to be recombined in host?
if DNA is a self-replicating plasmid
Example of selection system for measuring DNA uptake
Auxotrophic marker, antibiotic resistance
How is transformed DNA taken up?
As single stranded molecule then recombined
What are the 2 types of transduction?
Generalised and specialised
4 Properties of transducing phage
1- can go between circular and linear
2- genes for site specific integration
3- replicates via rolling circle replication
4- phage stays in genome as PROPHAGE
Specialaised transduction means…
.. prophage excises incorrectly = acquires neighbouring host DNA and loses some of its own
What is oriV and OriT replication of F plasmid
vegetative and transfer replication
What do F+ cells always have? and what does it form?
Sex Pillus
Forms mating junction
What does Tral do in transfer of F+ plasmid?
nicks DNA and has helicase activity to unwind DNA fro transfer
What is an episome?
plasmid that also integrates into the chromosome
Is F plasmid an episome?
Yes
what is clockwise/counterclockwise orientation of F dependant on?
Original recombination
Do archaea do transforamtion?
some
what happens in genome following insertion of transposable element?
duplication of a few base pairs after insertion
What are the 2 types of transpostion?
Conservative- non replicated, just transpired
Replicative- stays in host DNA and new location
What are the 2 types of transpostion?
Conservative- non replicated, just transpired
Replicative- stays in host DNA and new location
how can you screen for mutants followign transposition?
using biofilm formation and staining assay following by sequencing
How does sanger sequencing work?
Add chain-terminating dideoxynucleotides usign DNA polymerase THEN electrophoresis
Sequence analysis of Sanger sequencing…
… use diff fluorescent indicators for each molecule and detect using laster in capillary tube
How do we determine position of promotor, introns, exons ect ?
compare cDNA and genomic clone sequences
What is a use of Southern blot analysis?
see how many fragments are similar to the gene on interest
what are the 2 methods of gene expression analysis?
Northern blot analysis and in situ hybridization
What is the benefit of using dual-antibody technique to detect proteins?
allows signal amplification so better sensitivity
what do His- tagged proteins bind to?
Nickel column
so can be purified using this method
What technique is immunohistochemistry very similar to?
Western blot analysis (used tissue section instead of gel blot)
RNA expression patterns can be investigated by what techniques?
northern blotting or in situ hybridisation
protein accumulation patterns can be investigated by what techniques?
western blotting or in sutu in tissue sections by immunohistochemistry
what strategies can be used to analyse genomic DNA?
souther blotting or FISH to determine copy number
What vector delivery system can not be used with transient transformation?
Targeted integration, knock in/out
What is the main difference between transient snd stable DNA transformation ?
Transient- not integrated into host genome
Stable- Integrated into host genome
Give an advantage and disadvantage of calcium phosphate precipitation method of transformation
+ cheap, quick, no vector required
- only for mammalian cell line
How does electroporation cell transformation work?
Electric field causes cell polarization = pores in membrane for DNA entry
Where is transient DNA injected to during microinjection?
Male pronucleus
What particles does microprojectile bombardment use for DNA transformation?
Gold (tungsten) particle coated in DNA
Name 2 mammalian viruses adapted as vectors
Adenovirus (transient)
Lentivirus (stable)
2 Risks of using viral vectors fro mammalian expression
1- integrating vectors may activate cellular oncogenes
2- non-integrating vectors offer less stable expression
What parasitic bacterium is used in the most common method for transforming plants?
A. tumefaciens
What is a common promotor in mammalian, drosophila and plant cells?
Actin
What is ultimate outcome of RNA interference (RNAi) mediated suppression of gene expression?
mRNA degradation
What does genome editing using designer nucleases facilitate?
Gene insertion, correction or knockout of a specific gene
Name 2 examples of agricultural application of cell transformation
Golden rice- express vitamin A
Glyphosphate resistant crops- Roundup ready soybeans
Blight resistant potato
What is a medical application of transformed cells?
Use as ‘factories’ to make insulin, HGH, Hep. B vaccine…
What is non-mendelian inheritance?
traits determined by genes outside chromosomes in organelles
What is the name of the structures mitochondrial DNA exists in?
nucleoids - mostly circular DNA
How many genes in the human mitochondrial genome?
37 and no introns
Does chloroplast DNA have introns?
YES
What bigger chloroplast or mitochondrial genome?
Chloroplast
How might genes have been lost in endosymbiotic organnelles?
genes transferred to nucleus then redundant sequences lost
What is the key concepts of the hYdrogen hypothesis?
Host- used H and CO2 to produce methane
Future mitochondrion - produced H and CO2 as by product of anaerobic respiration as facultatively anaerobic eubacterium
What is autophagy?
Autophagy is the body’s way of cleaning out damaged cells, in order to regenerate newer, healthier cells
Difference between heteroplasmic and homoplasmic cells?
Hetero= mixture of organelle genomes Homo= 1 type
Why might mutations in mitochondrial genomes be maternally inherited?
Maternal gametes usually bigger so more organelles donated.( + some cells degrade male organelles)
What is good about sticky ends when restriction enzymes cut DNA?
overhang makes easier to ligate and more directional
What are the 2 steps of joining cut DNA?
hydrogen bind formation then phosphodiester bond formation (ATP-dependant)
What are the 2 types genetic libraries?
genomic and cDNA
What is the simplest vector (for small insertion)?
plasmids
What retroviRal enzyme is used to coNvert mRNA to cDNA?
reverse transcriptase
What are the 3 steps of PCR?
denature, anneal, extend
What are the 4 steps of the hierarchical genome sequencing approach?
1) clone target genome in BAC vectors
2) identify minimal set of overlapping clones
3) shotgun
4) sequencing and assembly
What genome sequencing approach did the public human genome project use?
hierarchical genome sequencing
What is the approach used by celera genomics?
whole genome shotgun approach
What does 454 pyrosequencing use and measure?
emulsion PCR and measures light intensity
What is required once sequencing has been performed?
Bioinformatics e.g. BLAST
WHAT IS THE MOST EFFECTIVE TYPE OF 2ND GENERATION GENOME SEQUENCING?
illumina sequencing
uses glass flow cells and bridge amplification
