Flashcards in lectures 12-17 Deck (63):
what are the key features which distinguish prokaryotes from eukaryotes?
-condensed free DNA
-co-transcribed groups of genes
-coupled transcription and translation
-chromosome segregation as an active process (not regulated)
-directional gene transfer
what are merozygotes/merodiploids?
a prokaryotic cell which is temporarily a diploid, when DNA is transferred in and there is temporarily 2 copies of a gene
what is: transformation, transduction and conjugation?
transformation: movement of extracellular DNA into the cell
transduction: bacteriophage dependent genetic exchange
conjugation: plasmid dependent exchange involving transposons
what are the technical advantages of using bacteria?
-easy to grow
-powerful selection systems (easy to measure change)
what is meant by clonal growth?
all cells in the colony are identical
give the features of the E.coli chromosome
-single dsDNA molecule in a 430mm circle
-4.639 x10^6 bp
-DNA is supercoiled
describe the features of nucleoids
-non membrane bound
-found in growing cells in centre
-replication causes them to 'explode' in loops
-blockage of replication causes them to compact
where does chromosome replication begin and terminate?
oriC and terC
what is the difference between slow growth chromosome replication and fast growth?
slow growth: single replication fork
fast growth: multiple replication forks
what are the properties of plasmids?
-genes encode protein and RNA
-used for recombinant DNA technology
what are the types of plasmids?
-F plasmids: involved in transmission of plasmids from one cell to another
-tumour induction plasmids
-virulence plasmids: responsible for enterotoxin and haemolysin
what is a replicon?
a self-regulating, freely-replicating element
what are the key features of plasmid replication?
characteristic fixed copy number per cell
copy control genes located next to oriV
related plasmids cannot coexist in the cell
outline phage infection and lysis
adsorption to cell -> injection of DNA -> DNA replication + gene expression -> assembly of new phage particles -> cell lysis and release
what is the basis for transduction?
mistakes in phage packaging; DNA from host packaged into phage, upon injection into new host, this DNA becomes part of host genome
describe the lysogenic lifecycle
adsorption -> injection -> DNA circularises by ligation of cohesive ss ends -> site-specific recombination between phage genome and cell chromosome -> phage genome incorporated into chromosome with phage genes switched off
what does stimulation of prophage cause?
-accurate excision of phage genome
-aberrant excision causing phage genome to be excised with flanking chromosomal DNA, so that phage particle carries bacterial genes
what are insertion sequences? what enzyme do they require?
discrete DNA segments with inverted repeats
what is meant by the term 'operon'?
when multiple genes are expressed at the same time via a single mRNA
how does coupled transcription and translation occur in prokaryotes?
1) RNA polymerase binds, initiating the promoter. transcription occurs
2) ribosomes immediately attach as soon as the shine-delgarno sequence is recognised, translation occurs immediately
explain how bacterial transformation occurs
1) transfer of DNA from donor to recipient produces a partial diploid
2) homologous DNA on transferred DNA pairs with homologous regions of recipient DNA
3) 2 reciprocal crossovers occur causing a region of recipient DNA to be replaced with donor DNA
4) linear reciprocal recombinant product is degraded
explain how plasmid conjugation occurs
pili/fimbriae on the surface allow mating pairs to form. genes are transferred from oriT in ssDNA transfer in 1-2 minutes
explain how artificial transformation works
-cells permeabilised for DNA uptake using CaCl2, spheroplasts or electroporation
-homology is not required, any gene can be inserted
what are the steps for gene cloning?
cut DNA with enzyme -> insert into a vector -> transport into host
what are palindromes?
recognition sequences for restriction endonucleases
what is the role of modification enzymes?
protects own DNA from cleavage by methylation of bases in the restriction endonuclease sites
describe the features of restriction endonucleases
-recognise and cut up specific DNA sequences
-have 2-fold rotational symmetry
-produce staggered cuts, leaving cohesive ssDNA ends
how is the DNA rejoined after it is cut with a restriction endonuclease?
-5' sticky ends are left
-bases anneal and ligation occurs via ligase
-leaving recleavable sites
what is the role of ligase (restriction endonuclease)?
facilitates the joining of 2 DNA strands by catalysing the formation of phosphodiester bonds
what are the essential features of cloning vectors?
-must be able to replicate in the host cell, maintaining appropriate copy number
-must have an appropriate restriction enzyme site
-must have an appropriate method of introduction into the host cell
-must have a genetic selection marker
-must identify recombinants
what is blue-white screening?
allows us to differentiate between recombinants and vectors alone using LacZ gene for B-Galactosidase
How does blue-white screening identify recombinants?
-LacZ encodes B-galactosidase, which converts colourless X-Gal into a blue compound
-recombinant contains fragments which insertionally inactivate LacZ = colourless plate
-vectors still produce blue compound
how is a genomic library made?
take total genomic DNA -> cut into random fragments -> cut vector with same enzyme to give sticky ends -> anneal + ligate
what are the advantages and disadvantages of large inserts when building a genomic library?
-fewer clones required for the library to be representative
-plasmids have small vector capacity, other vectors must be used
what are the advantages and disadvantages of small inserts when building a genomic library?
-plasmids can be used
-more clones required for it to be representative
what type of organism is yeast artificial chromosome useful as a vector for building a genomic library? why?
eukaryotes; yeast is eukaryotic, plus vector capacity is high, 200-2000kb compared to 10kb for a regular plasmid. useful for creating a representative library in organisms with a large genome
how is the number of clones for a representative genomic library calculated?
N= ln(1-P) / ln(1-f/n)
why are more clones required than the minimum amount to cover all the bases in a chromosome?
plasmid insertion is not 100% effective
what do PHYSICAL methods of identifying clone of interest rely on?
base sequence complimentarity; you must know the identity of the gene you are investigating
what do BIOLOGICAL methods of identifying clone of interest rely on?
screen for phenotype by complementation
explain hybridisation as a method of identifying clone of interest and state what factors influence it
complementary strands find each other e.g. a probe and the clone.
influenced by temperature, salt concentration and sequence
what result of a colony blot is indicative of positive hybridisation?
how is a 'specialist library' made?
mRNA -> spliced mRNA -> reverse transcriptase used to make a DNA copy of mRNAs -> remove mRNAs -> dsDNA made from DNA using a polymerase
why are 'enriched/specialist libraries' not used for prokaryotes?
prokaryotes do not have introns; removing non-coding DNA offers no advantage
how is immunoblotting used for specific bacterial genome libraries?
1) transfer colonies (clones) to nylon filter
2) lyse bacteria
3) bind proteins from lysed bacteria to nylon
4) add labelled antibody-sticks to desired protein
5) detect label
outline sanger sequencing
1) shear bacterial chromosome so it forms many fragments of different sizes which overlap
2) fragments inserted into a vector- forming a library
3)add individual clones to individual wells
4) sequence each individual clone
what is de novo assembly?
when overlapping fragments are are assembled like a jigsaw until the whole genome is complete
aside from de novo, which other types of assembly are used?
assembly to a reference genome: when fragments are aligned against a known reference
sequence assembly: when a computer is used to assemble fragments
outline WGS by Next Generation Sequencing
1) make a fragment library, no vector required. attach an adapter to fragments
2) adapter sticks to solid matrix
3) amplify individual genome fragments on the matrix
4) all fragments sequenced at the same time
what is the goal of annotating the genome (WGS)?
determining the position of coding genes and other sequence motifs
how does computational analysis result in annotation of the genome?
computer recognises start, stop and sense codons, alongside shine-delgarno sequences. compares them to other sequences on database to establish homology
why do bacteria need tightly controlled gene expression?
economy- efficient RNA/protein synthesis
preventing clashes in physiological processes
responding to environmental conditions
at which points can gene expression be controlled in bacteria?
transcriptional: via transcription factors
post-transcriptional: via sRNA
post-translational: via enzymes
which elements are responsible for control of gene expression?
regulatory genes: produce proteins which control transcription
transcription factors: act on promoters of regulated genes
effectors: inducers and co-repressors which control a regulon
what is a regulon?
group of genes controlled by specific transcription factors
why does diauxic growth occur when E.coli is grown in a substrate containing glucose and lactose?
lac genes are repressed by glucose, when glucose is used up, lactose catabolic enzymes are produced and lactose is metabolised
which enzymes are required for lactose utilisation in E.coli?
permease: transports lac into cell, encoded by lacY
B-galactosidase: breaks down lac into glucose and galactose, encoded by lacZ
transacetylase: unknown function, encoded by la
what is the structure of the lac operon?
what is the role of lac| and LacO in controlling activation of the lac operon?
LacI: DNA binding protein (repressor)
LacO: DNA consensus sequence where a repressor binds, blocking RNA polymerase and thus, transcription `
explain positive control of the lac operon
catabolite activating protein senses glucose levels and binds to the promoter to activate transcription in the presence of cAMP (low glucose)
briefly outline how two component signal transduction systems can alter gene expression
1) signal sensed by histidine protein kinase
2) conformational change causes them to dimerise and phosphorylate a his
3) His passed to a conserved aspartate on a DNA binding protein
4) DNA binding proteins dont bind unless phos.
how do positively-acting small RNAs result in post-transcriptional gene expression control?
stimulate translation by unmasking ribosome binding sites