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MCO > MCO Test 4 > Flashcards

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)

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2
Q

Proliferation

A

Increase in number

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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)

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4
Q

Common development stages

A

Egg

Cleavage first division

Gastrulation

Germ layers( groups of cells that will become differentiated into different cell types)

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5
Q

Proteins important for cell development

A

Cell adhesion and signalling transmembrane proteins

Gene regulatory proteins

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6
Q

Cell to cell adhere junctions

A

Actin filaments via cadherin proteins

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7
Q

Cell to cell desmosome junctions

A

Intermediate filaments via Cadherin proteins

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8
Q

Cell matrix anchoring junctions connect with intracellular cytoskeleton via integrin proteins

A

Actin linked is actin filaments and
hemidesmosomes intermediate

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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

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10
Q

Embryo is divided into small number of broad regions

A

These will become future germ layers
-ectoderm
-mesoderm
-endoderm

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11
Q

Fate

A

What a cell will normally develop into

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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

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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

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14
Q

Induction

A

Where a signal from one group of cells influences the developmental fate of another

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15
Q

Inductive interaction

A

Determines pattern formation what drives cells with the same potential to follow a different path of development

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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

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17
Q

Asymmetric division

A

Sister cells born differently

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18
Q

Symmetric division

A

Sister cells become different as result of influences acting on them after birth

Based on how mRNA is spread

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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)

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20
Q

HOX proteins

A

transcription factors can activate or repress genes- determine type of structures formed in particular segment

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21
Q

gene has highly conserved DNA region =

A

homeobox the protein expresses a homeodomain- binds to target DNA

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22
Q

asymmetrical cell division

A

significant cell materials distributed differently

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23
Q

HOX genes first discovered in

A

drosophilia

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24
Q

what determines differences in one cells gene expression compared to another?

A

polarity shows the difference between the ectoderm and endoderm layers

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25
Q

before fertilisation there is already

A

polarity- maternally derived

animal and vegetal hemispheres contain different selections of mRNAs

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26
Q

after fertilisation

A

the outer cortex shifts 30 degrees
moving the important mRNA molecules

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27
Q

after cordial rotation competes

A

cleavage follows resulting in small cells called blastomeres without significant change in mass

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28
Q

ectoderm

A

predominantly animal blastomeres

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29
Q

esoderm

A

middle cells

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30
Q

encoderm

A

vegetal blastomeres

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31
Q

after cleavage the embryo becomes a hollow ball of cells

A

blastula

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32
Q

blastomeres predetermined to become 3

A

germ layers

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33
Q

gastrulation

A

lays down the tissue germ layers and body axis via a choreography of cells

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34
Q

morphogenic gradient coodinated by

A

dorsal lip

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35
Q

gastrulation

A

slight diffusion of proteins and mRNA creating slight axis

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36
Q

cell migration

A

during gastrulation cells are spatially rearranged some undergoing involution

cell shape also changes via convergence or elongation

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37
Q

neurulation

A

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

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38
Q

somites

A

vertebrates, ribs and muscles

a notochord with ectoderm above(neural tube) and endoderm below

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39
Q

neural tube

A

Brain and spinal cord

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40
Q

congenital abnormality

A

can be inherited genetic (chromosomes or single genes)

environmental triggers (teratogens, chemical compounds)

pre-natal environment nutrition, alcohol

multifactorial (neural tube defects)

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41
Q

plant development model organism

A

Arabidopsis thaliana
small genome and short lifecycle

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42
Q

embryonic development in plants

A

-fertilsation
-division of zygote (asymmetry and polarity of embryo)

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43
Q

embryo proper

A

dense cytoplasm

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44
Q

suspensor

A

transports nutrients to embryo

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45
Q

diploid embryo has 2 groups

A

-one at suspensor end(root)
-one at opposite end(shoot)

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46
Q

seed development

A

-now there is rudimentary shoot and leaves - cotyledons
1)monocots
2)dicots
the structure is encased = seed
lays dormant until favourable conditions

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47
Q

meristems from the wuschel gene

A

groups of self renewing stem cells
capacity to divide
cells left behind then differentiate

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48
Q

plant morphogenesis

A

cell differentiation specialisation
cell growth (elongation)
cell division (meristem)

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49
Q

selective gene expression is?

A

essential

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50
Q

histology

A

the study of tissues

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51
Q

embryonic origin of mammalian tissues

A

oocyte and sperm once fertilised = zygote

morula = zona pellucida

blastocyst (embryonic stem cells= pluripotent) trophoblast

foetus

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52
Q

hypoblast =

A

yolk sac

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53
Q

epiblast

A

embryo proper-gastrulation and neurulation

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54
Q

progenitor/precursor cells

A

-connective tissue
-epithelia
-muscle
-nerve

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55
Q

ectoderm can become

A

skin, neutron of brain and pigment

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56
Q

germ

A

sperm and egg

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57
Q

mesoderm can become

A

cardiac, skeletal and tubule,RBC and smooth muscle in gut

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58
Q

endoderm

A

lung thyroid and pancreatic

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59
Q

epithelia tissue

A

-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

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60
Q

epithelia tissue

A

-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

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61
Q

epithelia tissue

A

-protection, selective barriers, filtration, secretion, absorbtion and excretion

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62
Q

squamous

A

endothelial cells of blood vessels

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63
Q

cuboidal

A

cells of ovary and kidney tubules

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64
Q

columnar

A

lining of GIT

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65
Q

cell layers single

A

one layer (all of above)

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66
Q

cell layers pseudostratified

A

one layer but appears like several (ciliated lining of upper respiratory tract conciliated lining o epididymus)

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67
Q

cell layers stratified

A

2 layers

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68
Q

histology

A

staining tissue and preserving to study

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69
Q

connective tissue

A

-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

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70
Q

connective tissue types

A

-loose and dense CT = fibroblasts
cartilage CT = chondroblasts
bone = osteoblasts

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71
Q

muscle tissue

A

comprised of elongated muscle fibres myocytes

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72
Q

muscle tissue purpose

A

-movement and locomotion
-maintenance of posture
-controlled movement of substances
-thermogenesis

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73
Q

stem cells

A

-immature
-undifferetiated cells ability to divide

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74
Q

unbalanced regeneration of stem cells

A

can lead to disease states

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75
Q

the stem cell niche

A

microenvironment in vivo or in vitro regulates STC fate

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76
Q

embryonic SC

A

altering gene expression

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77
Q

adult SC

A

maintaining SC properties

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78
Q

stem cell two essential properties

A

-self renewal = proliferate indefinitely without limit
-potency = one or many differentiated cell types

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79
Q

totipotent

A

ALL

(it is the zygote)

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80
Q

pluripotent

A

limited range

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81
Q

oligopotent> unipotent

A

few or just once cell type

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82
Q

epidermis is continually renewed by

A

stem cells

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83
Q

asymmetrical division could lead to stem cells

A

as the necessary materials could end up in one cell

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84
Q

amplify terminally differentiated cell types to

A

get stem cells

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85
Q

identifying location of stem cells

A

-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

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86
Q

stem cells incorporation of nucleotide analogues

A

BrdU(bromodeoxyuridine) thymine into newly synthesised DNA = pulse

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87
Q

label retaining cells still present weeks after the pulse (chase) =

A

stem cells found at tips of basal papillae

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88
Q

now live in post

A

genomic area

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89
Q

bacterial genetics

A

study of the mechanisms of heritable information in bacteria
-their chromosomes, plasmids, transpose and phages

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90
Q

bacterial genetics techniques

A

-defined media
-replica plating
-mutagenesis
-transformation
-conjugation
-transduction

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91
Q

bacterial DNA make up __% of DNA on earth

A

30

92
Q

haploid

A

carry one copy of chromosome

93
Q

bacterial genome

A

single circular double stranded DNA chromosome
-little inter-gene space
(not Borelia burgdorfei linear single chromosome)

94
Q

related genes grouped in

A

operons

95
Q

e coli that can grow without certain nurteints

A

prototroph

96
Q

biosynthetic auxotrophs require

A

additional nutrients to grow

97
Q

catabolic auxotrophs

A

have lost ability to catabolise some carbon source

98
Q

housekeeping genes

A

genes that would be lethal if mutated
-DNA replication
-Transcription/lation
-Cell division
-Glycolysis

99
Q

some mutations are conditional

A

for example in different temperatures

100
Q

nomenclature- gene annotations

A

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
Q

dnaA gene encodes the

A

DnaA protein

102
Q

strain phenotype described using the same three letter mnemonic as genotype

A

first letter capitalised three letter not italicised
mutant shown with - power sign

103
Q

leu to the power minus

A

requiring leu

104
Q

leu to the power addition sign

A

not requiring leu

105
Q

lamarkian evolution

A

(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
Q

darwinian evolution

A

change is spontaneous
natural selection ensures survival of the fittest

107
Q

Exceptions to darwin in science community

A

-lysenkoism
-prokaryotes thought an exemption to evolution

108
Q

lysenkoism

A

political interference in science (ideology over facts)

109
Q

Luria-Delbruck experiment

A

-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
Q

Luria delbruck hypothesis

A

-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
Q

Luria delbruck experiment

A

-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
Q

Newcombe experiment 1949

A

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
Q

lederberg and lederberg 1952

A

-replica plating
-amp on one and no on other

114
Q

to understand which bacteria is each use 6 plates

A

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
Q

operon

A

-a group of genes under control of the same promotor
-operons are common in prokaryotes
-means genes can be regulated together

116
Q

polycistronic mRNA

A

encodes more than one protein

117
Q

constitutively expressed

A

housekeeping genes

118
Q

making RNA and protein is

A

energy expensive

119
Q

diauxic growth

A

two growth phases

120
Q

lac genes encode

A

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
Q

lac operon Is normally turned

A

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
Q

inducer

A

molecule that turns genes on it disables the repressor

123
Q

how can allolactose formation be catalysed by beta galactosidase if operon is reprssed

A

even in repressed state a small amount is made

124
Q

allolactose is the

A

inducer

125
Q

why is there a lag with the lac operon

A

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
Q

catabolite activator protein(CAP)

A

enhances transcription
by binding to promotor
-if CAP has cAMP can bind to promotor

127
Q

adenylate cyclase

A

makes cAMP and

is inhibited by glucose

128
Q

catabolite repression

A

-glucose inhibits adenylate cyclase no cAMP and lac operon is transcribed slowly

129
Q

lac operon and biotehcnology

A

-what if we use this promotor to control other genes
FOR EXAMPLE INSULIN

130
Q

lac promotor benefits and drawbacks

A

+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
Q

induction of expression

A

-IPTG is a lactose analogue therefore tricking cells

132
Q

variants of lac promotor used today

A

insensitive to glucose so can GROW on glucose

133
Q

catabolite repression means that bacteria

A

prefer to grow on glucose and will not express genes to grow on other carbon sources if glucose is present

134
Q

presence of lactose leads to

A

de-repression in lac operon

135
Q

Griffith 1928

A

rough and smooth colony experiment SHOWING the TRANSFORMING PRINCIPLE

136
Q

competence

A

the ability of a bacterial cell to take up extracellular (naked DNA) from the environment

137
Q

can you induce competence

A

yes for example with E.coli

138
Q

Recombination results in more fit organisms

A

for evolutionary advances like resistance

139
Q

horizontal gene transfer three mthods

A

-transformation where cell takes up naked DNA
-bacterial transduction through phage
-bacterial conjugation cell makes contact and transfers plasmids

140
Q

transformation requires competent cells

A

usually competence happens before stationary phase after exponential

141
Q

why does transformation occur on entry to stationary phase

A

-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
Q

mechanism for competence

A

-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
Q

CFS pheremone

A

-promotes competence and sporulation

144
Q

B. subtilise will take up DNA of

A

any origin

145
Q

how can the bacteria distinguish between DNA from other bacteria of the same species from a different species

A

it recognises specific DNA sequences

146
Q

how to recognise DNA from same organism

A

-contains recognition sequences these sequences are found more often than would happen by chance

147
Q

uptake of DNA

A

-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
Q

new train can be aquired

A

-gene fragment usually big enough to contain several genes
-if no recombination DNA will be degraded
-Recombination may convey new traits

149
Q

phage ecology

A

phages are possibly the most abundant organism on earth
-co exist with hosts in all of environments

150
Q

what do phages do

A

-influence characteristics of host
-population dynamics
-long term evolution

151
Q

phage therapy

A

-species or even strain specific
-planktonic or biofilm
-seems safe but few good studies

152
Q

antibiotics

A

-broad spectrum problem with resistance
-mosty poor on biofilms
-can cause anaphylaxis or organ toxicity

153
Q

lytic phage

A

causes lysis of cell

154
Q

lambda phage

A

-smaller genome about 48 kbp
tail important for interacting with e coli

-can be LYTIC or LYSOGENIC

155
Q

lytic cycle

A

-attachment
-DNA inserted into cell
-circularises
-replication, transcription and translation
-new visions assembled
-lysis and release of new virions

156
Q

phage restriction

A

-yields of virus are reduced by less than 1000 fold in E.coli the restrictive host

157
Q

restriction modification system

A

-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
Q

lysogeic cycle

A

-attachment
-DNA inserted into cell and integrated into genome
-stays there transmitted to daughter cells

UNTIL LYTIC CYCLE is triggered which is rare

159
Q

lysogen

A

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
Q

prophage

A

phage in lysogenic state

161
Q

lysogen sometimes carry other genes

A

-if repressor protein is activated by iron
-if iron concentration is low toxin production will be induced

162
Q

generalised transduction

A

transfer of any DNA to the recipient cell by lytic or lysogenic phage

163
Q

specialised transduction

A

-transfer of specific genes via lysogenic phages

164
Q

generalised transduction

A

-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
Q

specialised transduction

A

normal infective virions made
RARE EVENT -phage DNA incorrectly excised carries adjacent gene

166
Q

conjugation

A

process of moving genetic material often but not always plasmids via direct cell to cell contact

167
Q

lederberg and tatum 1946

A

-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
Q

Davies 1950

A

-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
Q

Davies ruled out

A

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
Q

plasmids

A

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
Q

episomes

A

special plasmids that can integrate into the host genome

172
Q

curing

A

plasmid is lost from host randomly or due to certain chemicals

173
Q

conjugative plasmids

A

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
Q

f pilus

A

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
Q

pilus process

A

1)donor looking for mate
2)contact is made
3) cells pull closer
4)transfer of plasmid via mating bridge

176
Q

plasmid is copied by rolling circle replication (RCR)
LEADING STRAND

A

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
Q

plasmid is copied by rolling circle replication (RCR)
LAGGING STRAND

A

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
Q

both cells when they have f plasmid

A

-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
Q

Her strains

A

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
Q

Her strains can transfer their genome

A

-just like plasmid copying

181
Q

gene transfer stops when

A

mating pair breaks apart before C+ can be transferred

182
Q

when mating pair breaks apart the strand is temporarily

A

diploid : merodiploid

merodiploid means the haploid stain that is diploid only in some genes

183
Q

time of entry mapping

A

the further the gene is away from the origin the longer and less likely transfer is

184
Q

Her strains can become

A

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
Q

F’ strain can mate with F^- strain

A

-donor has some chromosomal genes on F plasmid
-recipient has its own copy of gene in genome
AND new copy on plasmid merodiploid

186
Q

why might you want to clone/ express a gene

A

-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
Q

if you know a gene to be cloned sequence for prokaryotic

A

-perform PCR with primers with engineered restriction sites

188
Q

but if you want to express a eukaryotic gene in E.coli

A

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
Q

synthesis of cDNA first strand

A

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
Q

synthesis of cDNA second strand

A

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
Q

how do we make the construct

A

PCR Product with restriction sites at the end
-cut gene and put it in

192
Q

restriction enzymes

A

-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
Q

four major classes of restriction enzymes TWO is most common

A

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
Q

type II restriction enzymes

A

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
Q

nomenclature of type II restriction enzymes

A

italicised 3 letter abbreviation of species name
non-italicised strain designation (if needed)
non-italicised roman numeral

196
Q

ligases

A

-remember replication ligases needed to join DNA on lagging strand

197
Q

from plasmids to vectors FEATURES

A

-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
Q

pUC plasmids

A

-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
Q

how does pUC cloning work? restriction and ligation

A

-recombinant plasmid with disrupted lacZ

200
Q

rapidly growing E.coli cells treated with CaCL2 will take up plasmids

A

-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
Q

surface of E coli is and DNA

A

negative

202
Q

heat pulse

A

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
Q

electroporation

A

DNA inserted to cell with electrical pulse (10-20 kV/cm)

creates pores in membrane for plasmid DNA to enter

204
Q

the central dogma DNA makes RNA makes protein

A

-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
Q

expression vector

A

ligated cDNA into special plasmid

206
Q

example of clones

A

insulin

207
Q

hex-histidine tag

A

his his his his followed by stop codon *

208
Q

a hex-his tagged protein can bind nickel:a purification strategy

A

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
Q

GFP fusion proteins

A

for monitoring gene expression protein intracellular localisation mobility trafficking and interactions between proteins

210
Q

recombination

A

-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
Q

homologous and non-homologous recombination

A

-switching DNA that is similar

non = repair of double stranded DNA break by simply joining with another piece of DNA

212
Q

homologous recmobination

A

-requires extensive homology
-can result in replacement of faulty gene
-involved holiday junctions model proposed in 1964 by robin holliday

213
Q

homologous recombination mechanism 1

A

-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
Q

RecA

A

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
Q

RecBCD

A

-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
Q

homologous recombination mechanism 2

A

-cross strand exchange = second nick in the other piece of DNA
-branch migration = requires RuvAB helices extensive heteroduplexes can be 1000 bps long

217
Q

Holliday junction

A

-rotate 90 degrees
-rotate to resolve crossover
-lower half 180
ISOMERISATION = crossing and uncrossing of strands

218
Q

Holliday junction can result in two outcomes

A

LOOK ON PHONE

219
Q

homologous recombination can rescue double stranded break

A

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
Q

Hfr x F ^- mating

A

plasmid some
genome some

221
Q

non-homologous recombination

A

-no sequence homology
-for example insertion and excursion of lambda phage

222
Q

non-homologous recombination after DS breaks

A

-ends are rejoined
-if ends have been degraded sequence might be lost
-ends are joined to form wrong ends

223
Q

insertion sequences (IS)

A

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
Q

insertion sequences and phenotype

A

-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
Q

transposons

A

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
Q

mechanism of transposons

A

-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
Q

transposition can be conservative or replicative

A

replicative = the original copy is retained and a new copy is made which inserts elsewhere (more risk it will interfere)

MCO (2 decks)

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