Prokaryotic Genetics (36-43) Flashcards
1
Q
Why are bacteria good model organisms?
A
- Haploid - see effect of mutation immediately
- Asexual reproduction - daughter cells have same properties
- Short generation time
- Easy to store
- Easy to genetically manipulate
2
Q
What is the make-up of the bacterial genome?
A
A single circular, double stranded DNA chromosome
→ introns are rare
→ grouped in operon (related to same function)
→ often carry plasmids
3
Q
How do bacteria reproduce?
A
Binary fission
→ asexual reproduction
→ cell elongates and content increased
→ DNA replicated and segregated
→ produces identical daughter cells
4
Q
What are the growth requirements for E.coli?
A
Capable of synthesising all cellular components from simple inorganic nutrients and a carbon/energy source
5
Q
What are auxotrophs?
A
Mutant organisms impaired in some metabolic capabilities
6
Q
What are biosynthetic auxotrophs?
A
Require additional nutrients in order to grow
→ e.g. His- require histidine in its growth medium
7
Q
What are catabolic auxotrophs?
A
Lost the ability to catabolism some carbon source
→ rarely a problem as glucose is often carbon source of choice
→ Ara- arabinose mutation - unable to grow on the monosaccharide arabinose (pointless using this as carbon source)
8
Q
What type of catabolic auxotroph is often fatal?
A
Glucose catabolic mutants
→ glucose metabolisms is essential
9
Q
When are conditional mutants lethal?
A
In repressive conditions
→ but not lethal in permissive conditions
10
Q
What are temperature sensitive mutants?
A
Only grow at a permissive temperatures (30C for E.coli) and not at restrictive temperatures (37C for E.coli)
11
Q
What did the Luria-Delbruck experiment predict?
A
Bacteria follow Lamarckian evolution
→ unlike ‘higher’ organisms
→ add toxic agent to bacterial culture and the entire culture becomes resistant
12
Q
What was the conclusion from the Luria-Delbruck experiment?
A
Genetic mutations arise in the absence of selection pressure and are selected for by toxic agents
→ bacteria evolve as a result of mutation
13
Q
What experiments determined bacterial evolution?
A
Luria-Delbruck, Newcombe and Lederberg and Ledeberg Experiments
14
Q
How many substitutions does E.coli DNA polymerase make?
A
~once every 10^7 bases
genome is 5.4x10^6
→ after 2 generations 3 substitutions
→ if not recognised + repaired mutation is inherited
15
Q
What are the 3 types of spontaneous mutation?
A
- Replication errors - wrong base pairs inserted by DNA pol
- Tautomers (isomers) - different H-bonding pattern, wrong base
- Base pair slipping - repeat nucleotides can cause frameshift mutations
16
Q
What are mutagens?
A
Chemical or physical agents that cause damage to DNA
→ increase mutation rate
→ e.g. nitrous acid, reactive O species, UV light
17
Q
What are intercalating agents?
A
Insert themselves into DNA between base pairs
→ usually flat multiple ring structures
→ distors helix
→ causes frameshift mutations
18
Q
What are point mutations?
A
A change to one base pair
→ substitutions
→ insertion
→ deletion
19
Q
What are the 2 types of DNA substations?
A
- Transitions (e.g. purine→purine)
- Tranversions (e.g. purine→pyrimidine)
20
Q
What are the consequences of point mutations?
A
Protein coding parts: can affect sequence or regulation of translation
Promoter: can affect transcription
Non-coding: may have no consequence,
21
Q
What types of mutations can substitutions leads to?
A
- Silent → no change
- Missense → change aa
- Nonsense → STOP codon
22
Q
What type of mutation do insertions and deletions cause?
A
Frameshift mutations
23
Q
What happens to proteins after mutations?
A
- Silent → nothin: genotype changes, phenotype doesn’t
- Missense → often nothing: genotype changed, phenotype may change
- Nonsense + frameshift → detrimental: genotype and phenotype changes
24
Q
What are the types of large scale mutations?
A
Deletions
Inversions
Tandem repeats
Transposons
25
What is reversion mutation?
A point mutation resulting in the restoration of the original sequence
26
What is a suppressor mutation?
A second mutation resulting in the original phenotype being restored
→ e.g. salt bridge reversed stabilising protein
27
What is suppression of frame shift?
When a mutation restores the original frameshift
28
What is intergenic suppression?
A second mutation in a different gene to the original, that suppresses the phenotype of the first mutation
→ e.g. nonsense suppression
29
What are selectable phenotypes?
Drug resistance, phase resistance
30
How do you make a histidine auxotroph?
1. Expose to a mutagen
2. Grow in complex media to allow expression of phenotype
3. Grow in minimal medium + penicillin
→ auxotrophs argent killed by penicillin's they're not grown
4.. Plate on minimal medium + histidine and grow
31
What is phenotype lag?
The time between a genetic mutation and its phenotypic expression
→ bacteria phenotypes aren't seen for several generations
32
What is cross feeding?
The exchange of nutrients between organisms
→ metabolic pathway blocked, metabolite accumulates, if it diffuses out another bacteria can take it up
→ both mutants can grow but are dependant on each other
→ phenotype becomes obvious once isolated
33
What is the Ames test?
Used to identify chemicals that are mutagenic (therefore carcinogenic)
→ assumes if a chemical is mutagenic to bacteria its also mutagenic to humans
34
How can you use biological assay in an Ames test?
(using a His auxotroph, doesn't grow regularly)
Plate the same number of bacteria on plates with and without chemical being investigated
→ mutagenic - lots of reversions
→ not mutagenic - only a few reversions
35
What is a limitation of Ames testing?
Chemical itself may not be mutagenic, but a metabolite might be
36
What is an operon?
A group of genes regulated together under control of the same promoter
→ common in prokaryotes
37
What are housekeeping genes?
Genes that are constantly expressed as they are required for basic cellular function
→ e.g. genes involved in replication and transcription always need to be active
38
Why aren't all genes always constitutively expressed?
Making RNA and protein is energy costly so genes are switched on and off according to the needs of the cell
→ e.g. switching to rich medium with aa now available means genes to make them are switched off to save energy
39
Is the lac operon constitutively transcribed?
No
40
What is diauxic growth?
Microbes presented with 2 carbon sources have bi-phasic growth with a lag phase in-between
→ carbon sources used consecutively not simultaneously
41
Why is a lag phase required in diauxic growth?
Time is needed for genes to be expressed and necessary proteins to be made after the initial carbon source (e.g. glucose) has run out
42
What do the lac genes encode?
lacY: β-galactoside-permease
lacZ: β-galactosidase (cleaves lactose disaccharide)
lacA: galactoside acetyl-transferase (transfers acetyl group to galactoside and glucosides)
43
How is the lac operon normally turned off?
A lac protein represses the operator preventing transcription of the upstream operon
→ RNA polymerase blocked
44
What is an inducer?
A molecule that turns genes on, disables the represser
→ for the lac operon allolactose is the inducer (similar to lactose but has 1-6 bond instead of 1-4)
45
How can allolactose formation be catalysed by β-galactosidase (LacZ) if the lac operon is repressed?
LacZ catalyses the splitting of lactose, even when the lac operon is repressed state there is still a small amount of transcription
46
How is the lac operon switched on?
Allolactose (rearranged glucose) binds to the lac repressor and makes it let go of the operator
→ RNA polymerase can now transcribe the operon
47
What is the purpose of catabolite activator proteins (CAPs)?
They bind to a region on DNA with cAMP just before the lac operon promoter to help recruit RNA polymerase
48
When is the lac operon expressed?
When lactase is present and glucose isn't
49
What happens to cAMP at low levels of glucose?
cAMP attaches to CAP allowing it to bind to DNA
→ CAP helps recruit DNA polymerase to the promoter, resulting i high levels of transcription
→ the enzyme used to make cAMP is inhibited by glucose
50
How does glucose suppress the lac operon?
Glucose inhibits adenylate cyclase, so no cAMP is made
→ CAP can't bind so the lac operon is transcribed slowly
51
What is bacterial transformation?
Induction of competence
→ the ability of a bacterial cell to take up extracellular ('naked') DNA from the environment
52
What does bacterial transformation lead to?
Increasing genetic diversity and the spread of:
drug resistance, novel virulence factors, novel metabolic capabilities
53
What are the 3 types of horizontal gene transfer?
1. Bacterial transformation
2. Bacterial transduction
3. Bacterial conjugation
54
Why does transformation occur at the entry to stationary phase?
At the stationary phase they starting to run out of nutrients and are at the risk of dying, so bacterial cells try to do something
→ quorum sensing: the ability to regulate genes based on population density
55
What is the mechanism for competence?
Cells secrete ComX, cell density increases means more ComX
→ ComX binding with ComP leads to changes in gene expression, cells become competent
56
How does bacteria know what DNA to take up and what to reject?
It recognises specific DNA sequences from the same species
57
What is the mechanism of DNA uptake?
1. DNA bind to surface protein on cell
2. Single or double;e stranded DNA enters the cell
3. Bind to competence-specific protein
→ recombination leads to new traits required
58
What is transduction?
Genetic exchange in bacteria mediated by bacteriophages
59
What is the lytic cycle with phages?
1. Attachment
2. DNA inserted into cell
→ replication, transcription and translation, new virions assembled
3. Lysis and release of new virions
60
What is the lysogenic cycle with phages?
1. Attachment
2. DNA inserted into cell and integrated into genome
3. DNA stays there and is transmitted to daughter cells
61
What is conjugation?
The process of moving genetic material (often plasmids) via direct cell-to-cell contact
62
What are plasmids?
Small double stranded DNA section
→ mostly circular, can be linear
→ replicate individual of chromosomal DNA
→ do not have extra cellular form (like phages)
63
What is an episome?
A special type of plaid that can integrate into the host genome
64
What is the role of plasmids?
Carries non-essential but often highly useful genes for:
1. Antibiotic resistance
2. Virulence factors
3. Bacteriocins
65
What are conjugative plasmids?
Plasmids capable of horizontal transmission
→ most plasmids themselves encode the genes that will allow transfer to other cells
→ some transfer only to same species
66
What is a F pilus?
A thin flexible filament on the surface of bacteria that connects a mating pair
→ allows for the unidirectional transfer of DNA from donor to recipient
→ creates contact and pulls cells closer together
67
How are plasmids transferred?
1. The double stranded plasmids is nicked
2. The nicked strand is unrolled and transferred
3. Once transferred plasmid is made circular and other strand synthesised
68
How is a plasmid copied?
Rolling circle replication (RCR)
1. One strand is nicked creating 5' 3' end
2. 3' serves as primer for replication
3. Once a full round is synthesised old strand released and new strand ligated
4. Single strand circularised and replicated
69
What is a high frequency recombination strain (hfr)?
Has f plasmid integrated into genome through recombination
→ they can transfer part of their genome to another cell
70
How is the development of complex multicellular organisms possible?
Due to the capacity of cells to become specialised and communicate
→ functional units need structural integrity and to be able to receive and respond to stimuli
71
What are the 4 essential processes of development determined by selective gene expression?
1. Cell proliferation
2. Cell specialisation
3. Interactions with environment
4. Cell movement and migration
72
What is the shared sequence of cell development events common to all animals?
egg → cleavage (1st division) → gastrulation → germ layers
73
What are the proteins important for multicellular development?
Cell adhesion and signalling transmembrane proteins
74
What are the 2 types of cell adhesion molecules?
1. Cadherins: mediate cell-cell adhesion
2. Integrins: mediate interaction between cytoskeleton and extracellular matrix
75
What are the 2 types of cell-cell anchoring junctions?
1. Adherens junctions: actin filaments via cadherin proteins
2. Desmosome junctions: intermediate filaments via cadherin proteins
→ connect with the intracellular cytoskeleton
76
What are the 2 types of cell-matrix anchoring junctions?
1. Actin-linked cell matrix adhesion: actin filaments via integral proteins
2. Hemidesmosomes - intermediate filaments via integral proteins
77
How do the differences in DNA regulatory proteins (transcription factors) and non-coding DNA (enhancers) lead to variant in body plan/shape/structure?
The same gene regulatory protien (transcription factors) in different organisms bind to different enhancer regions so different down stream protein transcription
→ leading to different environmental influences of cells
78
How do differences in protein expression and cell-cell communication manifest into anatomical changes?
79
What happens as embryonic development proceeds?
The embryo is divided into broad regions which become the future germ layers (mesoderm, ectoderm and endoderm)
→ the cells within these regions are more committed to their fate dependant on their spacial region
80
What are the 2 stages of cell commitment?
1. Specification: in a neutral environment can differentiate according to fate, in a different environment fate changes depending on cell signals
2. Determination: cells can differentiate according to fate even if in a different environment (fully determined no matter what)
81
What is induction?
Where a signal from one group cells influences the developmental fate of another
→ inductive signals have spacial and temporal influence
82
How is cell fate determined by asymmetrical cell division?
Significant molecules (e.g. mRNAs) are differently distributed causing sister cells to be born different
→ changes protein production and function
83
How is cell fate determined after symmetric cell division?
Sister cells become different as a result of influences acting on them after birth
→ extracellular influences determined by location and susceptibility to inductive signals
84
What are HOX proteins?
Transcription factors that active or repress genes
→ determine the type of structures formed in particular segments
85
Where are the animal and vegetal poles?
Animal pole - ectoderm layers
Vegetal pole - endoderm layers
→ contain differing selections of mRNAs providing polarity
→ before fertilisation there is already polarity - maternally derived
86
What does fertilisation trigger in the polarity of cells?
Triggers cortical rotation 30° rotation
→ offsets the animal pole and location of important mRNA molecules
→ leads to asymmetry of mRNA
87
What occurs after cortical rotations completed?
1h later cleavage follows
→ results in many small cells (blastomeres) without significant change in mass
→ first differences in cell fate
→ ectoderm, mesoderm and endoderm
88
What is gastrulation?
early embryo development processes
→ embryo becomes hollow ball of cells (blastula)
→ blastomeres are predetermined to become 3 germ layers
89
Why is a dorsoventral signal gradient created during gastrulation?
To control tissue pattern and coordinate gastrulation movement
90
What happens during cell migration in gastrulation?
Cells are spatially rearranged some undergoing involution
→ cell shape also changes via convergence or elongation
91
What is neurulation?
The initial stages of nervous system development
→ gives rise to the neural plate, neural tube forming the spinal cord and brain
→ as the central body is formed following gastrulation distinct mesoderm become apparent on either side of the body
92
What are somites?
Precursor cell populations that give rise to vertebrae body plan
→ from vertebrates, ribs and muscle
93
What are the chemical modifications in chromatin structure that can alter foetal programming?
1. Methylation of the DNA
2. Acetylation of histones
3. miRNAs
94
What happens after a seed is germinated?
The short emerges - enlargement of non-meristem cells followed by the shoot
→ rapid cell division of the apical meristems
95
What are the 3 stages of plant morphogenesis?
1. Cell division - meristem
2. Cell growth - elongation (turgor pressure and orientation of cellulose fibrils)
3. Cell differentiation - specialisation
96
What is meristem tissue?
Undifferentiated cells capable of cell division
→ can differentiate into all other tissues and organs of the plant
97
What are tissues?
Composed of cells that have a common embryonic origin
→ structure and properties influenced by cell-cell connections and the surrounding extracellular matrix
98
What are the 4 tissue types?
1. Connective tissue
2. Epithelia
3. Muscles
4. Nerve
99
What does a blastocyst consist of?
Zona pellucida: layer of 3 different glycoprotein, species specific
ICM: becomes the embryo
Trophoblast: becomes supporting tissue, placenta
Hypoblast: yolk sac
Epiblast: gastrulation and neurulation
100
What is epithelia tissue structure?
Creates barriers, is abundant and widely distributed throughout the body
→ arranged in tightly packed continuous sheets
→ line internal and cover external surfaces
→ small gaps between cells to control what goes through layers
→ cells are Polaris and closely associated via cell junctions
101
What are the major functions of epithelia tissue?
1. Protection - waterproof skin
2. Selective barriers
3. Filtration - kidney
4. Secretion
5. Absorption
6. Excretion - urine formation
102
What are the 2 types of epithelial tissue?
1. Covering and lining epithelium: controls transport
2. Glandular epithelium: ability to secrete
103
What is the function of epithelium tissue?
Bind, support, strengthen, provide integrity, insulate and compartmentalise
→ not present on body surfaces
104
What is connective tissue composed of?
1. Extracellular matrix: ground substance - water + GAGs and protein fibres - collagen, elastin reticular
→ ECM variation determines structure and function of connective tissue
2. Cells widely spaced
105
What are the types of connective tissue cells?
Loose and dense CT - fibroblasts
Cartilage CT - chondroblasts
Bone - osteoblasts
Liquid CT (blood tissue, lymph)
106
What is muscle tissue comprised of?
Elongated cell muscle fibres - myocytes
→ electrical stability, contractibility, extensibility, elasticity
107
What are the main types of muscles tissue?
1. Skeletal
2. Smooth
3. Cardiac
→ some control automatic, other self contracting
108
What are the functions of muscle cells?
Movement and locomotion
, maintenance of posture, controlled movement fo substances, thermogenesis
109
What is the composition of skeletal muscle?
Long cylindrical fibres
→ striated due to overlapping fibrils of actin and myosin
→ develop from fusion of 100s myoblasts so multinucleated
110
What is the composition of smooth muscles?
Short fibres tapered at each end
→ central oval nucleus
→ no overlap of filaments, non striated (smooth)
→ greater ability to stretch and recoil
111
What are the 2 types of smooth muscle?
1. Visceral: skin, stomach, intestines
→ several fibres innervated by one ANS synapse
→ connected by many gap junctions to spread action potential
2. Multiunit: lung airways, artery walls
→ each fibre has one ANS synapse
→ not as closely associated, fewer gap junctions
112
What is the function of tissue stem cells?
Form the basis for tissue homeostasis and repair
→ general maintenance + magnified cell proliferation the damaged
113
What influences stem cell niche?
Th micro environment
→ change in environment causes change in cell fate
→ cell adhesion, endocrine signals, nervous stimulation, metabolic products
114
What are the 2 essential stem cell properties?
1. Self renewal: proliferate without any limit
2. Potency/plasticity: how many different cell types?
115
What are the types of stem cells?
1. Totipotent - zygote
2. Pluripotent - embryonic
3. Oligiopotent - epidermal
116
What are the fundamental extracellular signal molecules?
1. Mitogens: remove the hand-brake on the cell cycle
2. growth factors: increase cell mass
3.Survival factors: suppression of apoptosis
