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Flashcards in lectures 12-17 Deck (63):

what are the key features which distinguish prokaryotes from eukaryotes?

-condensed free DNA
-circular chromosome
-unique DNA
-contiguous genes
-no introns
-co-transcribed groups of genes
-coupled transcription and translation
-chromosome segregation as an active process (not regulated)
-directional gene transfer
-diploids unstable


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)
-grow rapidly
-clonal growth


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?

-replicate autonomously
-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
-Resistance plasmids
-tumour induction plasmids
-symbiosis 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?

black spots


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
developmental pathways
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
stabilise mRNA


how do negatively-acting small RNAs result in post-transcriptional gene expression control?

inhibit translation by occluding ribosome binding site or stimulating degradation of mRNA