MCO Test 4 Flashcards
1
Q
Abilities cells and organisms need to abide by
A
Structural integrity and
Receive and respond to stimuli
These are programmed during early development (ocellus are level)
2
Q
Proliferation
A
Increase in number
3
Q
Four processes for development
A
1 cell proliferation
2 cell specialisation
3 interaction of cells with other cells and environment
4. Cell movement and migration (tissues and organs)
4
Q
Common development stages
A
Egg
Cleavage first division
Gastrulation
Germ layers( groups of cells that will become differentiated into different cell types)
5
Q
Proteins important for cell development
A
Cell adhesion and signalling transmembrane proteins
Gene regulatory proteins
6
Q
Cell to cell adhere junctions
A
Actin filaments via cadherin proteins
7
Q
Cell to cell desmosome junctions
A
Intermediate filaments via Cadherin proteins
8
Q
Cell matrix anchoring junctions connect with intracellular cytoskeleton via integrin proteins
A
Actin linked is actin filaments and
hemidesmosomes intermediate
9
Q
Spermann and mangold 1924
A
Direct evidence of key cells and their products
By grafting small groups of cells into host embryo lead to conjoined tissue
10
Q
Embryo is divided into small number of broad regions
A
These will become future germ layers
-ectoderm
-mesoderm
-endoderm
11
Q
Fate
A
What a cell will normally develop into
12
Q
Two stages of commitment
A
Specification will differentiate into state but signalling could change its fate
Determination will become cell fate no matter what
13
Q
Undifferentiated tissue can be regionally determined as leg but not which part of leg therefore
A
Not fully committed
Gene regulatory proteins in leg and wing act differently so gene expression is altered
14
Q
Induction
A
Where a signal from one group of cells influences the developmental fate of another
15
Q
Inductive interaction
A
Determines pattern formation what drives cells with the same potential to follow a different path of development
16
Q
Morphogens
A
signaling molecules that emanate from a restricted region of a tissue and spread away from their source to form a concentration gradient. To trigger other cells
17
Q
Asymmetric division
A
Sister cells born differently
18
Q
Symmetric division
A
Sister cells become different as result of influences acting on them after birth
Based on how mRNA is spread
19
Q
anterioposteria patterning
A
-sequential zones along the body axis (orders of HOX genes on chromosomes same order that they are expressed during development)
20
Q
HOX proteins
A
transcription factors can activate or repress genes- determine type of structures formed in particular segment
21
Q
gene has highly conserved DNA region =
A
homeobox the protein expresses a homeodomain- binds to target DNA
22
Q
asymmetrical cell division
A
significant cell materials distributed differently
23
Q
HOX genes first discovered in
A
drosophilia
24
Q
what determines differences in one cells gene expression compared to another?
A
polarity shows the difference between the ectoderm and endoderm layers
25
before fertilisation there is already
polarity- maternally derived
animal and vegetal hemispheres contain different selections of mRNAs
26
after fertilisation
the outer cortex shifts 30 degrees
moving the important mRNA molecules
27
after cordial rotation competes
cleavage follows resulting in small cells called blastomeres without significant change in mass
28
ectoderm
predominantly animal blastomeres
29
esoderm
middle cells
30
encoderm
vegetal blastomeres
31
after cleavage the embryo becomes a hollow ball of cells
blastula
32
blastomeres predetermined to become 3
germ layers
33
gastrulation
lays down the tissue germ layers and body axis via a choreography of cells
34
morphogenic gradient coodinated by
dorsal lip
35
gastrulation
slight diffusion of proteins and mRNA creating slight axis
36
cell migration
during gastrulation cells are spatially rearranged some undergoing involution
cell shape also changes via convergence or elongation
37
neurulation
cadherins and other cell to cell adhesion molecules play a major role and in somite formation
as the central body axis is formed following gastrulation sections of MESODERM become apparent on either side of the body
38
somites
vertebrates, ribs and muscles
a notochord with ectoderm above(neural tube) and endoderm below
39
neural tube
Brain and spinal cord
40
congenital abnormality
can be inherited genetic (chromosomes or single genes)
environmental triggers (teratogens, chemical compounds)
pre-natal environment nutrition, alcohol
multifactorial (neural tube defects)
41
plant development model organism
Arabidopsis thaliana
small genome and short lifecycle
42
embryonic development in plants
-fertilsation
-division of zygote (asymmetry and polarity of embryo)
43
embryo proper
dense cytoplasm
44
suspensor
transports nutrients to embryo
45
diploid embryo has 2 groups
-one at suspensor end(root)
-one at opposite end(shoot)
46
seed development
-now there is rudimentary shoot and leaves - cotyledons
1)monocots
2)dicots
the structure is encased = seed
lays dormant until favourable conditions
47
meristems from the wuschel gene
groups of self renewing stem cells
capacity to divide
cells left behind then differentiate
48
plant morphogenesis
cell differentiation specialisation
cell growth (elongation)
cell division (meristem)
49
selective gene expression is?
essential
50
histology
the study of tissues
51
embryonic origin of mammalian tissues
oocyte and sperm once fertilised = zygote
morula = zona pellucida
blastocyst (embryonic stem cells= pluripotent) trophoblast
foetus
52
hypoblast =
yolk sac
53
epiblast
embryo proper-gastrulation and neurulation
54
progenitor/precursor cells
-connective tissue
-epithelia
-muscle
-nerve
55
ectoderm can become
skin, neutron of brain and pigment
56
germ
sperm and egg
57
mesoderm can become
cardiac, skeletal and tubule,RBC and smooth muscle in gut
58
endoderm
lung thyroid and pancreatic
59
epithelia tissue
-abundant and widely distributed throughout body
-epithelial cells are arranged in tightly packed continuous sheets in single or multiple layers
-line all internal surfaces and cover external surfaces
-cells are polarised and closely associated via cell junctions
60
epithelia tissue
-apical membrane face the body surface, body cavity the lumen or duct may be specialised with microvilli
-lateral surfaces may express tight junctions and gaps
-bottom layer will be anchored to a basement membrane and ECM composed of two layers
61
epithelia tissue
-protection, selective barriers, filtration, secretion, absorbtion and excretion
62
squamous
endothelial cells of blood vessels
63
cuboidal
cells of ovary and kidney tubules
64
columnar
lining of GIT
65
cell layers single
one layer (all of above)
66
cell layers pseudostratified
one layer but appears like several (ciliated lining of upper respiratory tract conciliated lining o epididymus)
67
cell layers stratified
2 layers
68
histology
staining tissue and preserving to study
69
connective tissue
-one of the most abundant and widely distributed tissue types of the body
-functions - bind, support, strengthen protect insulate and compartmentalise
-not present on body surfaces
-most types are innervated and vascular
-composed of EXTRACELULAR MATRIX AND CELLS
70
connective tissue types
-loose and dense CT = fibroblasts
cartilage CT = chondroblasts
bone = osteoblasts
71
muscle tissue
comprised of elongated muscle fibres myocytes
72
muscle tissue purpose
-movement and locomotion
-maintenance of posture
-controlled movement of substances
-thermogenesis
73
stem cells
-immature
-undifferetiated cells ability to divide
74
unbalanced regeneration of stem cells
can lead to disease states
75
the stem cell niche
microenvironment in vivo or in vitro regulates STC fate
76
embryonic SC
altering gene expression
77
adult SC
maintaining SC properties
78
stem cell two essential properties
-self renewal = proliferate indefinitely without limit
-potency = one or many differentiated cell types
79
totipotent
ALL
(it is the zygote)
80
pluripotent
limited range
81
oligopotent> unipotent
few or just once cell type
82
epidermis is continually renewed by
stem cells
83
asymmetrical division could lead to stem cells
as the necessary materials could end up in one cell
84
amplify terminally differentiated cell types to
get stem cells
85
identifying location of stem cells
-not all basal keratinocytes have potential
-those that DO have beta1 intern portein cell adhesion to BL
-those that DO NOT are not bound to matrix via integral and lose stem cell properties
86
stem cells incorporation of nucleotide analogues
BrdU(bromodeoxyuridine) thymine into newly synthesised DNA = pulse
87
label retaining cells still present weeks after the pulse (chase) =
stem cells found at tips of basal papillae
88
now live in post
genomic area
89
bacterial genetics
study of the mechanisms of heritable information in bacteria
-their chromosomes, plasmids, transpose and phages
90
bacterial genetics techniques
-defined media
-replica plating
-mutagenesis
-transformation
-conjugation
-transduction
91
bacterial DNA make up __% of DNA on earth
30
92
haploid
carry one copy of chromosome
93
bacterial genome
single circular double stranded DNA chromosome
-little inter-gene space
(not Borelia burgdorfei linear single chromosome)
94
related genes grouped in
operons
95
e coli that can grow without certain nurteints
prototroph
96
biosynthetic auxotrophs require
additional nutrients to grow
97
catabolic auxotrophs
have lost ability to catabolise some carbon source
98
housekeeping genes
genes that would be lethal if mutated
-DNA replication
-Transcription/lation
-Cell division
-Glycolysis
99
some mutations are conditional
for example in different temperatures
100
nomenclature- gene annotations
three lower case letter indicating biochemical pathway
then a capital letter denoting gene
sometimes filled by number to show the allele
letters and numbers in ITALICS
101
dnaA gene encodes the
DnaA protein
102
strain phenotype described using the same three letter mnemonic as genotype
first letter capitalised three letter not italicised
mutant shown with - power sign
103
leu to the power minus
requiring leu
104
leu to the power addition sign
not requiring leu
105
lamarkian evolution
(giraffe example with long neck)
-life is not fixed
-if an organism uses something more
-it will increase
-if not used it will shrink
106
darwinian evolution
change is spontaneous
natural selection ensures survival of the fittest
107
Exceptions to darwin in science community
-lysenkoism
-prokaryotes thought an exemption to evolution
108
lysenkoism
political interference in science (ideology over facts)
109
Luria-Delbruck experiment
-add a toxic agent to bacterial culture and the entire culture becomes resistant
-interpreted as the agent makes the cells resistant
-conclusion = bacteria unlike higher organisms follow lamarckian evolution
110
Luria delbruck hypothesis
-darwinian = random mutations predicts that mutants appear in culture prior to adding selective agent
-lamarkian = directed change predicts that mutants appear in the culture only after adding selective agent
111
Luria delbruck experiment
-e coli cultures grown
-aliquots plated on plates containing T1 phage
-T1 phage kills e coli
-small variation in number of resistant colonies
FOUND- big variation in number of resistant colonies SUPPORTING DARWINIAN
112
Newcombe experiment 1949
1)Ton^S strain sensitive to phage
2)split between two plates and incubate
3)one as control and other re-spread and incubate
4)spray with phage
5)grow
6)count colonies
RESULT-more colonies on A
113
lederberg and lederberg 1952
-replica plating
-amp on one and no on other
114
to understand which bacteria is each use 6 plates
2 plates with
-minimal media plus glucose
-minimal medium plus glucose and histidine
-minimal medium with lactose instead of glucose
GROW half at 30 and the other at 37
115
operon
-a group of genes under control of the same promotor
-operons are common in prokaryotes
-means genes can be regulated together
116
polycistronic mRNA
encodes more than one protein
117
constitutively expressed
housekeeping genes
118
making RNA and protein is
energy expensive
119
diauxic growth
two growth phases
120
lac genes encode
galactosides such as
lacy: beta gal permeate for protein transport
lacz codes beta gal for cleavage
lacA encodes galactoside acetyl transferase transfers an acetyl group t gals
121
lac operon Is normally turned
off by an operator (Op) this turns the operon off it is a rare sugar therefore to prevent wasting energy it is only turned on when required
122
inducer
molecule that turns genes on it disables the repressor
123
how can allolactose formation be catalysed by beta galactosidase if operon is reprssed
even in repressed state a small amount is made
124
allolactose is the
inducer
125
why is there a lag with the lac operon
lactose is not used when glucose is present
-lag phase = genes are transcribed and translated to proteins which are folded
BECAUSE GLUCOSE SUPPRESSES ACTIVATION
126
catabolite activator protein(CAP)
enhances transcription
by binding to promotor
-if CAP has cAMP can bind to promotor
127
adenylate cyclase
makes cAMP and
is inhibited by glucose
128
catabolite repression
-glucose inhibits adenylate cyclase no cAMP and lac operon is transcribed slowly
129
lac operon and biotehcnology
-what if we use this promotor to control other genes
FOR EXAMPLE INSULIN
130
lac promotor benefits and drawbacks
+want a strong promoter to make lots of mRNA
+dont want gene constitutively expressed due to energy cost
-growing on lactose is inconvenient. glucose is more convenient
131
induction of expression
-IPTG is a lactose analogue therefore tricking cells
132
variants of lac promotor used today
insensitive to glucose so can GROW on glucose
133
catabolite repression means that bacteria
prefer to grow on glucose and will not express genes to grow on other carbon sources if glucose is present
134
presence of lactose leads to
de-repression in lac operon
135
Griffith 1928
rough and smooth colony experiment SHOWING the TRANSFORMING PRINCIPLE
136
competence
the ability of a bacterial cell to take up extracellular (naked DNA) from the environment
137
can you induce competence
yes for example with E.coli
138
Recombination results in more fit organisms
for evolutionary advances like resistance
139
horizontal gene transfer three mthods
-transformation where cell takes up naked DNA
-bacterial transduction through phage
-bacterial conjugation cell makes contact and transfers plasmids
140
transformation requires competent cells
usually competence happens before stationary phase after exponential
141
why does transformation occur on entry to stationary phase
-relies on quorum sensing :the ability to regulate genes based on population density
-also depends on low nutrient levels
-stationary phase means cells are at risk of dying
142
mechanism for competence
-cells secrete ComX small pheromone
-cell density increases means ComX concentration increases
-binding of comX to ComP SETS OF A REACTIION of events leading to changes in gene expression
-changes means cells become competent
143
CFS pheremone
-promotes competence and sporulation
144
B. subtilise will take up DNA of
any origin
145
how can the bacteria distinguish between DNA from other bacteria of the same species from a different species
it recognises specific DNA sequences
146
how to recognise DNA from same organism
-contains recognition sequences these sequences are found more often than would happen by chance
147
uptake of DNA
-DNA bind to surface protein on cell
-depeninding on species single or double stranded DNA enters the cell
-bind to competence-specific protein
-RecA mediated integration
148
new train can be aquired
-gene fragment usually big enough to contain several genes
-if no recombination DNA will be degraded
-Recombination may convey new traits
149
phage ecology
phages are possibly the most abundant organism on earth
-co exist with hosts in all of environments
150
what do phages do
-influence characteristics of host
-population dynamics
-long term evolution
151
phage therapy
-species or even strain specific
-planktonic or biofilm
-seems safe but few good studies
152
antibiotics
-broad spectrum problem with resistance
-mosty poor on biofilms
-can cause anaphylaxis or organ toxicity
153
lytic phage
causes lysis of cell
154
lambda phage
-smaller genome about 48 kbp
tail important for interacting with e coli
-can be LYTIC or LYSOGENIC
155
lytic cycle
-attachment
-DNA inserted into cell
-circularises
-replication, transcription and translation
-new visions assembled
-lysis and release of new virions
156
phage restriction
-yields of virus are reduced by less than 1000 fold in E.coli the restrictive host
157
restriction modification system
-enzymatic cleavage of the phage DNA and the enzyme involved was therefore termed a restriction enzyme
-host DNA protected because it Is modified by methylation (many enzymes insensitive to methylation)
158
lysogeic cycle
-attachment
-DNA inserted into cell and integrated into genome
-stays there transmitted to daughter cells
UNTIL LYTIC CYCLE is triggered which is rare
159
lysogen
strain of bacteria carrying a lysogenic phage (e coli lambda
-lytic cycle is suppressed by expression of phage that stops lysis
-protein also suppresses lysis by other phage of same type infecting cell
160
prophage
phage in lysogenic state
161
lysogen sometimes carry other genes
-if repressor protein is activated by iron
-if iron concentration is low toxin production will be induced
162
generalised transduction
transfer of any DNA to the recipient cell by lytic or lysogenic phage
163
specialised transduction
-transfer of specific genes via lysogenic phages
164
generalised transduction
-infection
-replication of DNA host DNA degraded
-virions are packaged occasional bits of host DNA packaged by mistake
-virion inserts host DNA into recipient and DNA can be incorporated into recipients enome
165
specialised transduction
normal infective virions made
RARE EVENT -phage DNA incorrectly excised carries adjacent gene
166
conjugation
process of moving genetic material often but not always plasmids via direct cell to cell contact
167
lederberg and tatum 1946
-two strains looking at
-wash cells and plate on minimal media no growth because each strain needed certain nutrients
-WHEN MIXED the mixed colony could grow as they became phototrophic colonies (gained both ^+ )
168
Davies 1950
-used dual glass tube with semipermeable filter (allows small entities through no cells)
-one end cover with cotton wool
-other end apply suction or pressure to mix media via filter
-plate bacteria from both sides after no growth
-if FILTER REMOVED GROWTH occured
169
Davies ruled out
transduction, cross feeding and transformation
because the filter would of allowed metabolites, DNA and even phages to pass through but no reversion occurred therefore it must require cell to cell contact
170
plasmids
almost always double stranded DNA
-most are circular but they can be linear
-size 1kb to >1Mbp
-replicate independently of chromosomal DNA
-do not have extracellular form like phages
171
episomes
special plasmids that can integrate into the host genome
172
curing
plasmid is lost from host randomly or due to certain chemicals
173
conjugative plasmids
some plasmids are conjugative and others are not
-mostly they encode genes that will allow transfer to other cells
-some transfer only to same species
-other more promiscuous
174
f pilus
a matig pair connected by an f plus
sometimes called a sex pilus
unidirectional transfer of DNA from donor to recipient
-F stands for fertility factor
-F is integrative plasmid it can integrate in a. number of locations or exist as free plasmid
175
pilus process
1)donor looking for mate
2)contact is made
3) cells pull closer
4)transfer of plasmid via mating bridge
176
plasmid is copied by rolling circle replication (RCR)
LEADING STRAND
1)One strand is nicked at double stranded origin of replication 3 to 5
2)3' is primer for replication
3)once a full round has been completed old strand is released as ssDNA
4)new strand ligated to heal nick in conjugation this ssDNA has been transferred to acceptor cell
177
plasmid is copied by rolling circle replication (RCR)
LAGGING STRAND
1)single stranded DNA is circularised and ligated
2)replication initiated as single stranded organ of replication
RNA primer starts DNA polymerase off
3)once a full round has been completed new strand ligated to heal nick
178
both cells when they have f plasmid
-f plasmid can spread through f culture ensuring cells are converted to F+ whatever other genes are encoded by the plasmid will now be spread
-process takes about 2 minutes at 37 degrees celcius
sensitive to agitation
179
Her strains
High Frequency Recombination strain
derived from F+ strain
-F plasmid has integrated into genome through recombination
this is a rare event
F plasmid is an episome
still produces F pili but no plasmid to transfer
180
Her strains can transfer their genome
-just like plasmid copying
181
gene transfer stops when
mating pair breaks apart before C+ can be transferred
182
when mating pair breaks apart the strand is temporarily
diploid : merodiploid
merodiploid means the haploid stain that is diploid only in some genes
183
time of entry mapping
the further the gene is away from the origin the longer and less likely transfer is
184
Her strains can become
f' strains
-f plasmid can excise from genome to become f plasmid again
-occasionally excision is imprecise and some chromosomal genes end up in the plasmid this is an F' plasmid
185
F' strain can mate with F^- strain
-donor has some chromosomal genes on F plasmid
-recipient has its own copy of gene in genome
AND new copy on plasmid merodiploid
186
why might you want to clone/ express a gene
-determine its nucleotide sequence
-identify and analyse its control sequences (promoters, translational sequences)
-identify mutations in gene (that have led to a defect)
-investigate the structure and function of the encoded protein
-make tagged versions of the product for ease of purification
-investigate the intracellular targeting of the gene product
187
if you know a gene to be cloned sequence for prokaryotic
-perform PCR with primers with engineered restriction sites
188
but if you want to express a eukaryotic gene in E.coli
E coli cannot express this gene because it cannot remove the introns from the RNA
-need to convert mRNA from subject into cDNA (complementary DNA)
189
synthesis of cDNA first strand
1)hybridise a poly (dT) primer to the polyA tail in the mRNA
2)use an RNA-dependent DNA polymerase called reverse transcriptase to make a single strand DNA copy of the mRNA
3)remove mRNA strand by alkaline digestion
190
synthesis of cDNA second strand
4)add align(dG) to 3' end of cDNA using terminal transferase
5)hybrids oligo (dC)
6)use DNA pol to make second DNA strand
191
how do we make the construct
PCR Product with restriction sites at the end
-cut gene and put it in
192
restriction enzymes
-restriction modification system
-restriction is caused by an enzymatic cleavage of the phage DNA and the enzyme involved was therefore termed a restriction enzyme
-the host DNA is protected from the restriction enzyme because it is modified by methylation
193
four major classes of restriction enzymes TWO is most common
1)recognise specific sequences and cleave the DNA at sites remote from this require ATP and S-adenosyl-L-methionine
multifunctional proteins with both restriction and methyl's activities
2)typically cleave within their recognition site
most require magnesium
single function (restriction) enzymes independent of methyl's activities
3)cleave at sites a short distance from recognition site
require ATP (but do not hydrolyse it)
exist as part of a complex modification methylase
4)target modified DNA
194
type II restriction enzymes
typically recognise a 4, 6 or 8 bp sequence known as a restriction site and hydrolyse a phosphodiester and in each strand of DNA
-most Type II restriction sites are palindromic with cleavage sites symmetrically rearranged
195
nomenclature of type II restriction enzymes
italicised 3 letter abbreviation of species name
non-italicised strain designation (if needed)
non-italicised roman numeral
196
ligases
-remember replication ligases needed to join DNA on lagging strand
197
from plasmids to vectors FEATURES
-Origin of replication (ori) allows plasmid to be maintained in the host cell different vectors have different copy numbers
-selectable marker (antibiotic resistant gene)
-disruptable marker = allows selection of recombinant plasmids with DNA cloned into disruputable marker
-no conjugation ability = the plasmid vector cannot easily spread from cell to cell only transmitted via cell division to make CLONE
198
pUC plasmids
-small size, multi-copy
-ori
-bla (beta lactamase) conferring resistance to ampicillin this is SELECTABLE MARKER
-multiple cloning sites (MCS) an array of restriction site =s so that reading frame is not disrupted
-the lacZ gene : an engineered gene that expresses the N terminal domain of the lacZ protein this is the disreputable marker
-a host strain is engineered to express only the C terminal domain of lacZ
199
how does pUC cloning work? restriction and ligation
-recombinant plasmid with disrupted lacZ
200
rapidly growing E.coli cells treated with CaCL2 will take up plasmids
-collect bacteria with centrifuge
-add 50mM of CaCL2 then add DNA at 0 degrees for 30 minutes
then 42 degrees for 2 minutes
DNA TAKEN UP
201
surface of E coli is and DNA
negative
202
heat pulse
creat thermal imbalance on either side of the cell membrane which forces the DNA to enter the cells through either cell pores of the damaged cell wall
203
electroporation
DNA inserted to cell with electrical pulse (10-20 kV/cm)
creates pores in membrane for plasmid DNA to enter
204
the central dogma DNA makes RNA makes protein
-re-writing from one nucleic acid format to another with a substitution of U for T
-translating from one language nucleic acid to another protein
205
expression vector
ligated cDNA into special plasmid
206
example of clones
insulin
207
hex-histidine tag
his his his his followed by stop codon *
208
a hex-his tagged protein can bind nickel:a purification strategy
1)express H6 tagged protein in E.coli and extract proteins
2)extract proteins
3)load complex mix of proteins on to nickel-agarose column
4)wash
5)elute H6 tagged protein with imidazole
6) dialyse
209
GFP fusion proteins
for monitoring gene expression protein intracellular localisation mobility trafficking and interactions between proteins
210
recombination
-breaks and joins DNA into a new combination
-allows rapid evolution compared to accumulating mutations
-permits auxotroph to phototroph switch by complementation of deleted function
-necessary for all the methods of horizontal gene transfer we have discussed
211
homologous and non-homologous recombination
-switching DNA that is similar
non = repair of double stranded DNA break by simply joining with another piece of DNA
212
homologous recmobination
-requires extensive homology
-can result in replacement of faulty gene
-involved holiday junctions model proposed in 1964 by robin holliday
213
homologous recombination mechanism 1
-alignment = 2 homologous DNA helices align
-breakage = one strand is nicked often happen at specific sequences
-invasion = free 3' end invades the homologous helix DNA stabilised by SSb protein catalysed by RecA
214
RecA
has functional homologous in ALL known organisms including eukaryotes and archaea
-essential for DNA repair
-multiple functions = binds single stranded DNA and has two binding sites and catalyses branch migration
215
RecBCD
-has nuclease activity and catalyses initial nick in DNA needed for recombination - note requires specific DNA sequence so this is not a random event
-has helices activity so RecA can bind
216
homologous recombination mechanism 2
-cross strand exchange = second nick in the other piece of DNA
-branch migration = requires RuvAB helices extensive heteroduplexes can be 1000 bps long
217
Holliday junction
-rotate 90 degrees
-rotate to resolve crossover
-lower half 180
ISOMERISATION = crossing and uncrossing of strands
218
Holliday junction can result in two outcomes
LOOK ON PHONE
219
homologous recombination can rescue double stranded break
1)double stranded break
2)5' to 3' exonuclease activity
3)homologous DNA strand
4)strand invasion
5)DNA synthesis
6)ligation
7)branch migration
8)finally resolution of the Holliday junction
220
Hfr x F ^- mating
plasmid some
genome some
221
non-homologous recombination
-no sequence homology
-for example insertion and excursion of lambda phage
222
non-homologous recombination after DS breaks
-ends are rejoined
-if ends have been degraded sequence might be lost
-ends are joined to form wrong ends
223
insertion sequences (IS)
are small pieces (1000 bp) of DNA that can hop from one position to another
-hop is called transposition catalysed by transposes
-transposase is encoded by the insertion sequence
-carries no other genes
-also has tandem repeat at the ends needed for insertion
224
insertion sequences and phenotype
-carries no novel features
-can disrupt genes due to insertion
-high degree of reversion as IS simply moves somewhere else
-if housekeeping gene is disrupted this will be lethal
225
transposons
has same features as insertion sequences
but carries additional genes
-sometimes carry resistance genes
-some transposons carry tea genes and hence conjugate
-like insertion sequences they can knock out genes with insertion
226
mechanism of transposons
-transposase binds to ends
-transposon is cut out
-chromosome is repaired but may be changes to original sequence
-new target sequence is found elsewhere
-transposon insert
227
transposition can be conservative or replicative
replicative = the original copy is retained and a new copy is made which inserts elsewhere (more risk it will interfere)
