lectures 32-43 Flashcards
(205 cards)
1
Q
what processes of development are determined by selective gene expression?
A
1 - cell proliferation
2 - cell specialisation
3 - interaction of cells with other cells and their environment
4 - cell movement and migration to form tissues and organs
2
Q
what sequence of events in the basic body plan are shared by all animals?
A
egg -> cleavage -> gastrulation -> germ layers
3
Q
what percentage of genes are generally conserved between species?
A
50%
4
Q
of the conserved genes, what are the two main protein types they predominantly code for, and what is their importance?
A
1 - cell adhesion and signalling transmembrane proteins
2 - gene regulatory proteins
- importance in the development of multicellular organisms
5
Q
what are the two main cell adhesion junctions and what are their general functions?
A
- cell to cell anchoring junctions connect with the intracellular cytoskeleton
- cell matrix adhering junctions also connect with the intracellular cytoskeleton
6
Q
what are the two main cell to cell anchoring junctions and what are they comprised of?
A
- adherens junctions - actin filaments via cadherin proteins
- desmosome junctions - intermediate filaments via cadherin proteins
7
Q
describe the structure of cadherin proteins
A
- 2 types
- classical cadherins possess a transmembrane domain, a cytosolic domain, and extracellular domains
- there are other cadherins which differ in structure but all play a similar role in adhesion
8
Q
describe the structure and function of integrins
A
- they contain an underlying talin structure which binds to the transmembrane domains, which then bind to the extracellular matrix
- a conformational change in the talin causes a force to be pulled on the extracellular matrix, providing a mechanical signalling process
9
Q
what are DNA regulatory proteins called?
A
transcription factors
10
Q
what is non-coding regulatory DNA called?
A
enhancers
11
Q
what is the impact of differences in transcription factors and enhancers
A
it leads to variations in body plan, shape, and structure
12
Q
does the same regulatory protein in different organisms produce the same protein transcription?
A
no, it results in different downstream protein transcription because of altered regulatory enhancers, leading to different environmental influences of the cell
13
Q
what occurred in the first embryology experiment conducted to test early development?
A
- an early egg cell was fertilised and then ligated almost into two using a hair loop
- it was found that the region that was allowed to communicate is likely to be the area of abnormality in the offspring
14
Q
what occurred in the 1924 experiment carried out by Mangold?
A
- a fertilised egg was allowed to undergo a couple of divisions and then a small group of egg cells were grafted into the host embryo
- it caused conjoined offspring
15
Q
how is an embryo segmented and what occurs in each region?
A
- the embryo is divided into a small number of broad regions which will become future germ layers - they are called the ectoderm, the mesoderm, and the endoderm
- the cells within these regions will become more and more committed to their fate
16
Q
what are the two stages of commitment?
A
1 - specification
2 - determination
17
Q
what occurs in specification?
A
- a cell is specified when it can be cultured in a neutral environment and differentiate according to its fate
- if it is cultured in different environments the fate can change
18
Q
what occurs in determination?
A
- the cell can differentiate according to its fate even in a different environment
19
Q
what does it mean if undifferentiated tissue is not fully committed?
A
- undifferentiated tissue can be regionally determined as a body part but not which specific section
- as gene regulatory proteins in different areas of the body act differently, gene expression is altered
- e.g. tissue that would form thigh tissue can be grafted into the end of a wing bud, and an upper wing but with toes will be produced
20
Q
define induction
A
where a signal from one group of cells influences the developmental fate of another
21
Q
what is the main influence on a cell’s behaviour in development?
A
the environmental signals which can determine gene expression
22
Q
what are morphogens?
A
inductive signals that work cell to cell, short range, or long range
23
Q
how does asymmetrical cell division determine cell fate?
A
- the sister cells will be born differently (e.g. there will be more mRNA present in one cell than another) and then these sister cells will go onto differentiate differently again
24
Q
what do HOX genes determine?
A
- involved in the animal body plan, especially anterioposteria patterning
25
in what organism were HOX genes first identified?
drosophila
26
what are HOX genes?
transcription factors
27
what does a homeobox protein express?
- a homeobox protein expresses a homeodomain which binds to the target DNA
28
what polarity does the xenophus leavis egg exhibit?
- the animal pole is 'on the top' of the egg and will become the ectoderm layers
- the vegetal pole will become the endoderm layers
- it exhibits maternally derived polarity as the animal and vegetal hemispheres contain differing selections of mRNA
29
what occurs in the cleavage of vertebrate cells?
- occurs around 1hr after corticol rotation
- it makes blastomeres, causing the first differences in cell fate
- the molecular components become differently distributed in the cell: the ectoderm is made up of predominantly animal blastomeres, the endoderm is made up of vegetal blastomeres, and the mesoderm is made up of the middle cells
30
what occurs in gastrulation of vertebrate cells?
- gastrulation lays down the tissue layers by 3 axes: the anteroposterior axis, the dorsoventral axis, and the mediolaterial axis
- it causes differing gradients in mRNA and proteins, and forms an organiser
- the organiser causes the cells to move inwards into the hollow space - this generates organs involved in the digestive pathway
- convergence and elongation of cell shape also occurs
31
what happens in neuralation of vertebrate cells?
- the creation of the spinal column
- a tube is formed via elongation, forming the central body axis - through this, distinct sections of mesoderm become apparent on either side of the body: a notochord with the ectoderm above and the endoderm below
32
what happens in neuralation of vertebrate cells?
- the creation of the spinal column
- a tube is formed via elongation, forming the central body axis - through this, distinct sections of mesoderm become apparent on either side of the body: a notochord with the ectoderm above and the endoderm below
33
what are formed either side of the neural tube?
somites are formed, which make up vertebrates, ribs, and muscles
34
how is vertebrate limb development controlled?
- cells on limb buds are regionally determined to turn into digits through morphogenic gradients caused by Sonic Hedgehog protein
- the type of digit produced is determined by local concentrations of sonic hedgehog protein
35
what % of births possess a congenital abnormality?
6%
36
what are some causes of congenital abnormalities?
- inherited genetics - chromosomal abnormalities and single gene defects
- environmental factors - e.g. exposure to teratogens (chemical compounds such as agent orange)
- multifactorial - e.g. folate deficiency causes neural tube defects
- the pre-natal environment - nutrition, alcohol, drugs, smoking
37
why is the intrauterine environment fundamental to foetal programming?
- it dictates how genes function
- small changes can alter genes and therefore predispose to certain diseases
38
what are epigenetics?
the processes which govern the evolution of an individual's phenotype from the genome (heritable factors, not genes)
39
what mechanisms are used to chemically modify chromatin structure?
- DNA methylation
- histone acetylation
-miRNA
40
what are the features of arabidopsis thaliana
- it is a small flowering weed
- it possesses a short life cycle and a small genome
- it is hermaphroditic
- it produces 100s of seeds per plant
41
what coding genes are contained in arabidopsis thaliana's genome?
genes coding for:
- regulatory proteins
- cell communication
- signal transduction
- developmental processes (remodelling of the cell wall, and response to light)
42
describe the process of embryonic development in plants
1 - fertilisation
2 - division of the zygote with asymmetry and polarity of the embryo
43
how are asymmetry and polarity expressed in the plant embryo?
- embryo proper contains dense cytoplasm, and the suspensor transports nutrients to the embryo
- the embryo has 2 groups of cells: at the suspensor end (root) and the opposite end (shoot)
44
what are the 3 types of tissue in plant embryos?
- epidermal tissue cells
- ground tissue
- vascular tissue cells
45
what does seed germination cause?
it causes the formation of:
- roots
- the stem
- apex cotyledons
- the epidermis
- ground tissue and vasculature
and causes the elongation of the cell shape
46
what are the features of the meristem tissue?
- they are groups of self renewing stem cells
- they have the capacity to divide
- the cells can differentiate: shoot and root meristems produce all cell types, other cells may retain some meristem potential
47
what maintains meristem growth?
- the Wuschel gene and positive feedback mechanisms
48
what is the main influencing developmental switch in plant growth?
light
49
what are the main classes of plant hormones?
- auxins
- gibberellins
- cytokinins
- abscisic acid
- ethylene
- brassionsteroids
50
what are the properties of plant hormones?
- they are local and long range
- can be used on their own or in combination
- moderated by other regulators, the environment, and nutritional status
- they are able to control cell specialisation
51
describe the 3 phases of plant morphogenesis
1 - meristematic cell division
2 - cell growth: elongation caused by turgor pressure and orientation of cellulose fibrils
3 - cell differentiation
52
what are the environmental factors influencing flower growth?
- day length
expression of constons genes in the leaves
- prolonged cold periods
53
which structures does specialised leaf growth create?
- sepals
- petals
- the stamen
- the carpel
54
what controls the pattern of whorl type?
- homeotic selector genes which code for gene regulatory proteins
55
what are the essential processes used for multicellular organisms to develop?
- cell proliferation
- cell specialisation
- interaction between cells, each other, and the environment
- migration and movement
56
what are tissues and what are they influenced by?
- tissues are composed of cells that have a common embryonic origin
- structures and functions of tissues are influenced by cell-cell connections and the extracellular matrix
57
what cell types are formed by the ectoderm?
- skin cells of the epidermis
- neurons of the brain
- pigment cells
58
what cell types do the germ cells in germ layers form?
- egg
- sperm
59
what cells types does the mesoderm form?
- cardiac muscle
- skeletal muscle cells
- tubule cells of the kidney
- red blood cells
- smooth muscle in the gut
60
what cell types are formed by the endoderm?
- alveolar cells
- thyroid cells
- pancreatic cells
61
what are the general features of epithelial tissue and their membranes?
- they are abundant and widely distributed throughout the body, lining internal surfaces, and covering external surfaces
- arranged in tight continuous sheets and are closely associated via cell junctions
- the cells are polarised (have a top and bottom)
- the apical membranes face the body surface, body cavity, the lumen, or the duct, and may be specialised
- the basal layer will be anchored to a basement membrane, a point of attachment for cell migration and as a further selective barrier
- there is a thin extracellular matrix composed of the basal lamina and the reticular lamina
62
what are the main functions of epithelial tissue?
- protection - waterproofing, minimising environmental influence
- selective barriers - controlled movement of substances in and out of the body
- filtration - e.g. kidney glomeruli
- secretion - products are released onto their apical surface
- excretion - urine formation
63
what cell shapes can epithelial tissue be classified by?
- squamous - e.g. endothelial cells of blood vessels
- cuboidal - e.g. cells of the ovary, and kidney tubules
- columnar - e.g. lining of GIT
64
what cell layer types can epithelial tissue be classified by?
- simple - one layer e.g. squamous
- pseudostratified - one layer but appears like several e.g. ciliated lining of upper respiratory tract
- stratified - 2 layers e.g. the keratinised top layer of the skin, non-keratinised linings of the mouth and vagina
65
what are the general structure and functions of connective tissue?
- not present on body surfaces
- most types of connective tissue are innervated and vascular
- the general functions are to bind, support, strengthen, protect, insulate, and compartmentalise
- comprised of the extracellular matrix (made up of ground substance and protein fibres, including collagen, elastin fibres, and reticular fibres), and widely spaced cells
- variation in the extracellular matrix determines the structure and function of the connective tissue
66
what cell types make up connective tissue?
- it is derived from embryonic mesenchymal cells, and all forms have undifferentiated progenitor cells called blasts:
- loose and dense CT has fibroblasts
- cartilage CT has chondroblasts
-
67
how is connective tissue classified?
1 - embryonic CT - mesenchyme & mucous CT
2 - mature CT can be classified into:
- loose CT (areolar, adipose, reticular)
- dense CT ( dense regular, dense irregular, elastic)
- cartilage (hyaline, fibrocartilage, elastic)
- bone tissue
- liquid CT (blood tissue, lymph)
68
what are the general structures and functions of muscle tissue?
- comprised of elongated muscle fibres (myocytes)
- there are three types - smooth, skeletal, and cardiac
general functions:
- movement
- maintenance of posture
- controlled movement of substances
- thermogenesis
69
what are the general properties of muscle tissue?
general properties:
- electrical excitability
- contractility
- extensibility
- elasticity
70
describe the features of skeletal muscle
- long cylindrical fibres which are striated due to overlapping fibrils of actin and myosin
- they are multinucleated as they develop from the fusion of 100s of myoblasts
- each fibre will be innervated by somatic motor neurons
- fast speed of contraction
- each fibre is closely associated with a capillary
71
what are the features of smooth muscle?
- short fibres which are tapered at each end
- centrally located ovular nucleus
- non striated
- greater stretch and recall capacity
- slower and longer lasting contractions
- myogenic
72
what are the two types of smooth muscle?
- visceral - skin and some tubular structures, several fibres are innervated by one ANS motor neuron
- multiunit - lung airways and walls of large arteries, each fibre has one ANS motor neuron
73
what are the features of cardiac muscle?
- the fibres are branched with 1 central nucleus
- connected to other fibres by intercalated discs via desmosomes
- there are many gap junctions to allow the myocardium to contract as 1 coordinated unit
- it is myogenic
- there are many mitochondria
74
what cells in cardiac muscle are autorhythmic?
- the pacemaker (SAN) - under autonomic regulation
- the conduction system (AVN, bundle of His, bundle branches, purkinje fibres)
75
what are the two cell types which make up nervous tissue?
- neurons
-neuroglia
76
what are the general features of neurons?
- make up the electrical excitability in the brain in order to convert stimuli into action potentials and then transfer nerve impulses
- they connect all regions of the body to the brain and the spinal cord
- they are long cells composed of a cell body, dendrites, and an axon
77
how are neurons classified?
- bipolar - one main dendrite and one axon
- unipolar - dendrites and one axon fused together
- multipolar - one axon and several dendrites
- pyramidal
78
what are the cell types and functions of neuroglia?
- provide supporting roles to neurons but do not generate or conduct nerve impulses
- smaller but more abundant than neurons
cell types:
- astrocytes
- oligodendrocytes
- microglia
- ependymal cells
- Schwann cells
- satellite cells
79
rank the 4 tissue types from highest to lowest capacity for renewal of parenchymal cells
1 - epithelial
2 - connective
3 - muscle
4 - nerve
80
what are the influences in the microenvironment that regulate a stem cell fate?
- physical elements e.g. cell adhesion molecules of surrounding cells and the ECM
- paracrine signals
- endocrine signals
- nervous stimulation
- metabolic products of tissue activity
80
what are the influences in the microenvironment that regulate a stem cell fate?
- physical elements e.g. cell adhesion molecules of surrounding cells and the ECM
- paracrine signals
- endocrine signals
- nervous stimulation
- metabolic products of tissue activity
80
what are the influences in the microenvironment that regulate a stem cell fate?
- physical elements e.g. cell adhesion molecules of surrounding cells and the ECM
- paracrine signals
- endocrine signals
- nervous stimulation
- metabolic products of tissue activity
80
what are the influences in the microenvironment that regulate a stem cell fate?
- physical elements e.g. cell adhesion molecules of surrounding cells and the ECM
- paracrine signals
- endocrine signals
- nervous stimulation
- metabolic products of tissue activity
81
what are the two essential properties of stem cells?
1 - self renewal - proliferate indefinitely without limit
2 - potency - number of differentiated cell types able to be made
82
what are the different types of stem cells?
- totipotent - able to operate all possible differentiated cells e.g. zygotes
- pluripotent - can divide into all cell types e.g. embryonic stem cells
- multipotent - limited range of differentiated cells e.g. hemotopoietic stem cells in the bone marrow
- oligopotent - can differentiate into a few cell types
- unipotent - can only turn into 1 cell type e.g. epidermal skin cells
83
what are the different layers of skin structure?
epidermis
papillary region
dermis - made up of dense CT and loose CT
reticular region
hypodermis - made up of fatty connective tissue
84
what is the epidermis made up of?
- keratinocytes
- pigment cells (melanocytes)
- dendritic cells (langerhans cells)
85
what is loose connective tissue made of?
- collagen fibres
- macrophage
- fibroblast
- mast cell
- endothelial cell (forming capillaries)
86
what is dense connective tissue made up of?
- fibroblast
- elastic fibre
- collagen fibres
87
where are stem cells stored in the skin structure and where do they act?
- stem cells are present in a bulge halfway up the hair shaft and in the right conditions it migrates to:
1 - the dermal papilla and will generate cells to make the proteins to produce hair
2 - the sebaceous gland to generate different epithelial cells
- some stem cells are present in or migrate to the basal layer of the epidermis to generate epithelial cells that will become skin cells
88
in the skin, how do stem cells cause skin shedding and replacement of cells?
1 - division of the stem. cells causes these cells to migrate upwards and to differentiate into the cells in the cell layer (squamous cells)
2 - the cells will gain proteins on its surface and begin to transition up again and lose their organelles to become dead cells
3 - they will then flake off from the surface
89
what is asymmetrical division used for?
- can be environmental asymmetry or divisional asymmetry
- when the stem cells divide, some of the progenitors have differentiated and some have remained stem cells
- used to maintain stem cell numbers
90
describe transit amplifying cells
- cells go through a specific number of divisions before terminal differentiation is achieved
- this amplifies the end product
91
where are keratinocytes with stem cell potential located?
- they are bound to the basal layer using beta1 integrin proteins
- those which aren't bound to the basal layer do not have stem cell potential
92
if there is a mutation that causes a lack of beta1 integrin, what is impaired?
- there is problems with basal membrane assembly
- impaired wound healing
93
how is the pulse chase experiment used to find the locations of stem cells in the skin? what are the findings?
- nucleotide analogues are introduced into the tissue by being incorporated into the newly synthesised DNA and then traced
- the tracers are BrdU or [3H] thymidine
- cells with high concentrations of tracers are the stem cells and are found at the tips of the basal papillae
- 10% of basal cells and the majority of cells in the bulge are stem cells
94
when is the pattern of organ size and growth determined?
- it is laid down during embryonic growth via short range signals
95
what are cell division, cell growth, and cell death controlled by?
- they are tightly controlled by intracellular programmes and extracellular signal molecules
96
what are the 3 main types of extracellular signalling molecules?
1) mitogens - trigger cell division
2) growth factors - increase cell mass through protein synthesis
3) survival factors - suppression of apoptosis
97
what can molecular signals determine for stem cells?
- the rate of stem cell division
- the fate of the daughter cells
- the number of divisions during transit amplification stages
- exit from the basal player
- time from the basal layer to terminal differentiation
(regulation differs for normal tissue maintenance and wound repair)
98
what are the main signalling pathways used for attachment to the basal layer or extracellular matrix, and what does each pathway do?
- epidermal growth factors - stimulates cell growth and differentiation as a response to cells with epidermal growth factor receptors (EGFR)
- fibroblast growth factors - cell proliferation and differentiation
- Wnt - involves a series of proteins passing signals from cell surface receptors to the nucleus (gene expression and cell-cell communication)
99
define vasculogenesis
the formation of the first primary blood vessels from early embryonic endothelial cells
100
define angiogenesis
a network of vasculature being created from primary blood vessels
101
what occurs during angiogenesis?
1) the basal lamina of existing vessels breaks down
2) endothelial cells migrate into the interstitial space
3) endothelial cells proliferate - the tip cells in the new capillary branch has different gene expression which sends out filopodia and responds to environmental signals (vascular endothelial growth factors)
4) the stalk cell divides and the cells become hollowed out to form the lumen so that it develops and matures
5) the vessel is stabilised by pericyte recruitment
102
what signals are involved in triggering angiogenesis?
- hypoxic conditions are caused by cells low on O2 make HIF1 alpha
- hypoxic conditions trigger the transcription of VegF to act on endothelial cells and turn off the system
103
what does interaction with the Notch signalling pathway control?
it controls which cells become tip or stalk cells in angiogenesis
104
what are the potential uses of stem cells in future research?
- keratinocytes can be cultured to grow replacement skin
- hemotopoietic stem cells can be used to treat leukaemia using bone marrow transplants
- neural stem cells - mice show potential for replacement of degenerated neurons
- somatic cell nuclear transfer
105
why are adult stem cells usually favoured for use over embryonic stem cells?
there are ethical issues regarding human embryo use
106
define bacterial genetics
the study of the mechanisms of heritable information in bacteria, their chromosomes, plasmids, transposons, and phages
107
what techniques are usually used to study bacterial genetics?
- culture in defined media
- replica plating
- mutagenesis
- transformation
- conjugation
- transduction
108
why are bacteria good model organisms?
- they are haploid, meaning a mutation in one gene will be seen immediately through its effects
- asexual reproduction - daughter cells are genetically identical
- short generation time
- grow on plates and liquid culture with defined media (not true for all bacteria)
- easy to store stocks
- easy to manipulate genetically
109
define haploid organism
an organism that only carries one copy of its chromosomes
110
describe the bacterial genome
- made up of single, circular, double stranded DNA chromosomes
- there is a lot less space between genes than in eukaryotes
- introns are extremely rare
- functionally related genes are often grouped in operons
- they often carry plasmids
111
define plasmid
- circular extra-chromosomal DNA which replicates independently
112
describe binary fission
- a form of asexual reproduction common in prokaryotes
1 - the cell elongates and all of the contents increases
2 - the DNA is replicated and segregated into 2 identical daughter cells
3 - once the cell is about twice the original length, a septum starts forming in the middle, which grows from both sides of the cell to split it in 2
113
what are the growth requirements for E.coli?
- E.coli is capable of synthesising all cellular components from simple inorganic nutrients and a carbon (energy) source
114
what are the typical minimal medium components for E.coli and why is each component needed?
- K2HPO4 & KH2PO4 for phosphate and pH control (they form a buffer system of the acid and its conjugate base)
- (NH4)2SO4 for a source of nitrogen
- MgSO4 as Mg is needed for proteins
- CaCl2 - Ca has a probable role in sensing and membrane transport
- trace metals for protein function
- glucose as a carbon source
115
define phototroph
when a bacteria needs a carbon source only to function - generally the wild type
116
define auxotroph
a mutation which causes the bacteria to be impaired in some metabolic capacity
117
what types of auxotroph are there?
- biosynthetic auxotrophs require additional nutrients in order to grow
- catabolic auxotrophs have lost the ability to catabolise some carbon sources
118
what are temperature sensitive mutants? why do these occur?
- mutant bacteria which grow only at a permissive temperature and not at a restrictive temperature
- occurs because enough mutant protein folds correctly at the lower temperature due to the lower amount of energy in the system
119
what did Lamarck suggest about evolution?
he suggested that life is not fixed and that if an organisms uses something more it will increase, and that it will shrink if not used, i.e change is directed by the environment
120
explain the proceedings and findings of the Luria-Delbruck experiment
- it was carried out to see that when a toxic agent was added to a bacteria culture, does the toxic agent cause bacteria to become resistant
1 - E.coli cultures of the TonS strain were grown and aliquots were plated on plates containing T1 phage (the toxic agent)
2 - when set up in parallel, there is a big variation in the number of resistant colonies - this means that mutations are already present in cultures and it is selected for by the toxic agent, so bacteria evolve as a result of the mutation
- this matches the classifications of Darwinian evolution
121
what occurred in the Newcombe experiment? (1949)
1 - the TonS strain of E.coli was plated on 2 plates on grown
2 - nothing is changed about plate B, but plate A is re-spread
3 - they are both sprayed with phage and then grown again
- plate A will have more resistant colonies because the colonies are split up in re-spreading and new colonies form
122
describe the process of replica plating
1 - spread out gauze the same size as the petri dish
2 - dip the master dish onto the gauze
3 - take the clean dish and dip it onto the gauze
123
what occurred in the Lederberg and Lederberg experiment? (1952)
- they created replica plates and observed the position of TonR colonies on replica plates - the position is always the same
- this is because the resistance mutation happened before the replica plating and addition of the toxic agent
124
how can bacteria protect against mutagenesis?
- bacteria have enzymes that can repair DNA
- they can produce melanin and other pigments to protect them from radiation damage
125
what is a replication error?
when a wrong base is inserted by DNA polymerase
126
what are tautomeres and how do they cause mutations?
- they are isomers that exist in equilibrium
- they have different hydrogen bonding patterns
- all our bases have tautomeres, when they arise the wrong base will insert
127
what is base pair slipping?
repeat nucleotides in a genetic sequence can lead to fremashift mutations
128
define mutagens
- they are chemical or physical agents which cause damage to DNA
129
describe the structures and effects of intercalating agents
- they are mutagens
- they usually have flat, multiple ring structures that binds between base pairs - this distorts the helix and can also lead to frameshift mutations
- they can be used to be added to DNA gel to detect DNA
130
what are the two types of substitution mutation?
- transition - a change from one pyrimidine to another, or one purine to another
- transversions - change from a pyrimidine to a purine and vice versa
131
where will point mutations be likely to have a consequence, and where will they not?
- non coding regions - will not have any consequence
- promoters - will either have no consequences or can affect transcription (whether it is upregulated or downregulated)
- protein coding region - can affect sequence or regulation of translation
132
what are the consequences of point mutations and where are they likely to happen if a substitution mutation occurs?
- silent mutations have no effect - third base pair substitutions are often but not always silent
- missense mutations cause a different amino acid to be inserted - most first and second base pair substitutions result in missense
- nonsense mutations cause a stop codon to be inserted - some substitutions in any position cause these
- nonsnese and frameshift mutations are usually detrimental but can be tolerated close to the C-terminus
133
what are inversion mutations?
- part of the DNA removes and re-inserts in the wrong orientation (backwards)
134
what are tandem repeats?
- part of the genome is duplicated
- this leads to the overproduction of proteins encoded in the duplicated region
135
what are transposons?
- they are nucleotide sequences that are able to move themselves around
- this can disrupt genes
136
what are reversion mutations?
- a point mutation resulting in the restoration of the original sequence before a past mutation
137
describe suppressor mutations
- a second mutation occurs that results in the original phenotype being restored
- it does not mutate back to the original mutation
138
what occurs in the suppression of frameshift?
- if an addition mutation occurs, and then the downstream removal of a base pair occurs (intragenic suppression), some of the effects of the frameshift mutation may be negated
139
define intragenic suppression
a mutation which is restored in the same gene
140
explain intergenic suppression
a second mutation occurs in a different gene to the one in which the first mutation occurs, and the effect is to suppress the phenotype of the first mutation
141
what occurs in nonsense mutation?
- it is a type of intergenic suppression
- mutations in the mRNA and the tRNA cause the anticodon to recognise the stop codon and continue translation
142
why do nonsense mutations make the strains faulty?
translation tends to occur past the normal stop codons and produce proteins which are way too long
143
how can some mutations be selected for in cultures?
- some phenotypes can be selected for, such as drug resistance and phage resistance
- penicillin can be added as it will kill any growing bacteria, and auxotrophs will not grow
144
what occurs in cross feeding?
- when a metabolic pathway is blocked, metabolites immediately before the block will accumulate and may diffuse into the media, where a certain bacteria with an early part of the pathway blocked can metabolise it
- both mutants can now grow, but are dependant on each other
145
what is the Ames test and how is it carried out?
- it is used to identify chemicals that are mutagenic
- it is a biological assay and uses a His auxotroph of Salmonella typhimunum
- the bacteria are grown and plated with and without the target chemical and are incubated
- if the chemical is mutagenic, there will be growth as reversions have occurred
146
what is the limitation of the Ames test
- the chemical itself mat not be mutagenic but a metabolite may be
147
define an operon
a group of genes under the control of the same promoter
148
define polycistronic mRNA
mRNA which encodes more than 1 protein
149
describe the diauxic growth of the E.coli in glucose and lactose medium
- there will be growth as glucose is used, and then a lag when glucose runs out and the lac operon is switched on, and then growth occurs afain
- the carbon sources are used consecutively not simultaneously
150
what do the genes turned on in the lac operon code for?
- LacY encodes beta galactoside permease
- LacZ encodes beta galactosidase
- LacA encodes galactosidase acetyl-transferase
151
what occurs in the lac operon when lactose is not needed?
1 - RNA polymerase binds to the first promoter region
2 - the repressor is transcribed and binds to the operator region
3 - the genes cannot be transcribed
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what happens in the lac operon when lactose is needed?
1 - allolactose binds to the repressor, changing its configuration and causing it to leave the operator site
2 - this allows the structural genes to be transcribed
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how is allolactose produced?
- lacZ can convert lactose into allolactose
- even in the repressed state some will be made due to 'leaky transcription'
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how does the lack of glucose enhance transcription in the lac operator?
- the catabolic enhancer protein (CAP) enhances transcription
- when CAP has cAMP bound to it, it can bind to the promoter to upregulate transcription
- cAMP is made by adenylate cyclase, which is an enzyme inhibited by glucose, so cAMP will only be made when glucose is not present
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what are the uses of the lac operon in biotechnology?
- other genes can be placed under the control of the operon to produce other products - this was first used to produce insulin
- IPTG can be added as an alternative source to lactose (due to very similar shape), and some lac promoters have been generated to be insensitive to glucose for ease of biotechnological uses
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define competence
the ability of a bacterial cell to take up extracellular DNA from the environment
- some bacteria are naturally competent, for others it can be induced
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how can horizontal gene transfer occur?
- bacterial transformation
- bacterial transduction
- bacterial conjugation
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in what part of a bacterial colony's life does competence usually occur?
it will happen usually just before the stationary phase
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what causes bacteria to become competent in their growth cycle
- it relies on quorum sensing, as cell density increases, pheromone production increases which will bind to the correct receptor and trigger expressional changes that then lead to competence
- nutrient levels can also trigger competence, low nutrient levels will cause cells to take up DNA from the environment to increase survival chances
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does transformation require competent cells?
yes
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what is quorum sensing?
the ability to regulate genes based on population density
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how do bacteria determine which naked DNA to take up?
- some bacteria will only take up naked DNA of its own species - this is done by the recognition of specific DNA sequences
- some bacteria will take up any naked DNA regardless of which species it originated from
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describe the uptake of naked DNA into bacterial cells
1 - DNA binds to the surface protein on the cell
2 - DNA enters the cell
3 - the DNA binds to the competence specific protein so that it is stabilised and not broken down by the cell
4 - RecA mediated integration occurs
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what occurs in generalised bacterial transduction?
1 - the phage infects the bacterial cell
2 - the bacterial DNA is replaced and the host DNA is degraded
3 - the phage virions are packaged, but occasionally parts of the host's chromosomes are packaged by mistake
4 - the cells lyse
5 - virions insert host DNA into the recipient and DNA can be incorporated into the new bacteria's genome
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what occurs in specialised bacterial transduction?
1 - normal infective virions are made
2 - the phage DNA is incorrectly excised (it does not cut at the ends of the prophages), and an adjacent gene is carried instead
3 - all new phages will have this defect - only genes close to the integration site can be transduced
4 - the new gene is transferred to the next recipient, which will then integrate
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what occurred in the Davies experiment and what did it show?
- occurred in 1950
- a glass tube with a U-bend was used, with a semi-permeable filter dividing each half - small entities can pass through but not cells
- cotton was used to block one end, and the other was suctioned in order to mix the media via a filter
- bacteria from both sides are plated snd there is no growth
- the colonies will grow when the filter is removed
- it shows that cell to cell contact is needed for this form of horizontal gene transfer
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what are the features of plasmids?
- they are almost always double stranded DNA
- most are circular but they can also be linear
- they replicate independently of chromosomal DNA
- they do not have an extracellular form like phages
- plasmids have different copy numbers in the cell
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how does compatibility affect plasmids in a bacterial cell?
- plasmids can be incompatible with each other, they have to be compatible to co-exist in a cell
- related plasmids sharing common replication mechanisms can often not co-exist
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what are the roles of plasmids?
- they carry non essential but often highly useful genes
- e.g. antibiotic resistance or virulence factors
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what is curing?
- when a plasmid is lost from the host
- this can happen spontaneously or can be in response to certain chemicals
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what do conjugate plasmids encode for
- they will mostly encode the genes that will allow transfer to other cells
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what are mating pairs connected by?
an F pilus - this is responsible for the unidirectional transfer of DNA from one bacteria to another
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what does the F in F pilus stand for?
fertility factor
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what is the importance of F being an integrated plasmid?
- it can integrate in a number of locations or exist as a free plasmid
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how is contact made between bacteria using an F pilus?
1 - the donor contains the F plasmid and is looking for a mate
2 - contact is made by the F pilus and the cells pull closer together
3 - transfer of plasmids occur via the moving bridge
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are plasmids transferred as single or double stranded DNA?
single stranded DNA
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what occurs on the leading strand of rolling circle replication?
1 - one strand is nicked at the Double Stranded Origin of replication
2 - the 3' end is used as a primer for replicating the new strand
3 - once a full round has been completed, the old strand is released as ssDNA
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what occurs on the lagging strand of rolling circle replication?
1 - the ssDNA is circularised and ligated
2 - replication is initiated at the single stranded origin of replication. An RNA primer initiates DNA polymerase
3 - once a full round has been completed, the new strand is ligated to heal the nick
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what does Hfr strain stand for?
high frequency recombination strain
it is derived from the F+ strain
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is the F plasmid an episome?
yes, it has integrated into the genome through recombination
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why and how does the F+ strain transfer part of its genome?
- the genome is transferred as it does not contain a plasmid to transfer
1 - the integrated F plasmid is nicked
2 - DNA polymerase replicates a new strand from 3'
3 - the replaced strand transfers to the other cell via the mating bridge (not the whole chromosome)
4 - complementary strands are synthesised in the new cell - it cannot circularise as not all of the chromosomes have been transferred
5 - most is degraded, but occasionally recombination takes place
6 - gene transfer stops when the mating pair breaks apart
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define merodiploid
a haploid strain that is diploid only in some genes
183
how can Hfr strains become F+ and F' strains?
- they can excise from the genome to become F plasmid again
- the occasional excision is imprecise and some chromosomal genes end up in the plasmid forming and F' strain
184
why would you want to clone a gene?
to:
- determine its nucleotide sequence
- identify and analyse control sequences
- identify mutations in the gene
- investigate the structure and function of the encoded protein
- produce large amounts of the encoded protein
- make tagged versions of the product to aid in purification
- investigate the intracellular targeting of the gene product
185
generally, how is the insert for cloning made?
1 - the primers are modified to add on a restriction side and some random nucleotides that the endonucleases can cleave
2 - PCR is performed with the primers
186
why do eukaryotic genes have to be modified in order to be inserted into a bacterium? how is this done
- bacteria cannot splice out introns
1 - the introns will be naturally removed through splicing in the eukaryotic cells, so mRNA is used
2 - mRNA is then converted into complementary DNA
3 - a poly(dT) primer is hybridised to the polyA tail in the mRNA
4 - reverse transcriptase is used to make a single stranded DNA copy of the mRNA
5 - mRNA is removed via alkaline digestion, leaving a single strand of cDNA
6 - to make the second strand, oligo(dG) is added to the 3' end of cDNA using terminal transferase
7 - the oligo(dC) primer is hybridised
8 - DNA polymerase is used to make a second DNA strand
187
how is a construct made and why is it needed?
- it prevents degradation
- it is inserted into a plasmid vector by cutting the DNA and ligating the gene into the DNA
188
how is the host DNA protected from restriction enzymes?
- it is modified by methylation
189
describe briefly the 4 major classes of restriction enzymes
type I - they recognise specific sequences and cleave DNA at sites removed from this
type II - they typically cleave within their recognition site, they are single function and independent of methylase activities
type III - they cleave at sites which are short distances from the recognition site
type IV - target modified DNA
190
what are the functions of type II restriction enzymes?
- they hydrolyse a phosphodiester bond in each strand of the DNA
- most type II restriction sites are palindromic with the cleavage sites symmetrically arranged
191
what do cloning vectors possess?
- an origin of replication
- a selectable marker
- a disruptable marker
- no conjugation ability
192
what are pUC plasmids and what are their functions?
- they are small in size and are multi-copy
- it confers resistance to ampicillin
- it is a multiple cloning site inside the lacZ gene
- LacZ is the disruptable marker and it is recombinated with disrupted lacZ
- however, sometimes it reforms with non disrupted lacZ
- E.coli is treated with CaCl2 to take up plasmids, using either heat shock or electroporation
- all blue colonies have not been disrupted so white colonies are selected
- His tagged protein is added which can bind to nickel, so it can be purified
193
what does recombination allow?
- rapid evolution
- auxotroph to phototroph switch by compartmentation of deleted functions
194
define homologous recombination
switching DNA that is similar
195
define non-homologous recombination
repair of double stranded DNA break by joining with another piece of DNA
196
what occurs in homologous recombination?
1 - 2 homologous DNA helices align
2 - one strand is nicked, catalysed by the enzyme RecBCD
3 - the free 3' end invades the homologous helix, catalysed by RecA, and the DNA is stabilised by SSB protein
4 - a second nick is made in the other piece of DNA
5 - branch migration occurs (requiring RuvAB helicase), and heteroduplexes form
6 - Holliday junction - it rotates to cross and uncross the strands (isomerisation)
7 - the DNA can be nicked in 2 orientations to give differing products - RuvC catalyses the nicking
197
what is the function of RecA?
- it is present in all known organisms
- essential for DNA repair
- it binds single stranded DNA and catalyses branch migration
198
what is the function of RecBCD?
- it has nuclease activity and catalyses the initial nick in DNA needed for recombination
- it has helicase activity
199
how is a double stranded DNA break fixed using homologous recombination?
1 - strand invasion occurs
2 - the homologous DNA can be used as a template to synthesise the lost DNA
3 - ligation and branch migration occurs and then it can be restored through the holliday junction
200
what happens in non-homologous recombination?
1 - the circular DNA finds the specific sequence in the linear DNA
2 - it inserts through a double stranded DNA break, and then ligates
201
what is an insertion sequence?
- small pieces of DNA that can hop from one position to another (transposition)
- carry no novel features
- can disrupt genes but high levels of reversion
202
what are transposons?
- have the same features as insertion sequences but carry additional genes
- they sometimes carry resistance genes or tra genes
