Lecture 7: Bacterial Genetics Flashcards
(102 cards)
1
Q
Describe the central dogma of bacterial genetics
A
1) From existing DNA to make new DNA (DNA replication)
2) From DNA to make new RNA (transcription)
3) From RNA to make new proteins (translation)
2
Q
List and describe the 3 forms of DNA. Where are they typically found?
A
1) B form: the one typically seen
2) A form: a slightly tighter coil, found in dehydrated specimens
3) Z form: an even tighter coil, left-handed helix; unknown role in cells, but has been found in many animals (mammals, protozoans, plants) and may provide torsional strain relief (supercoiling)
3
Q
What do complementary and antiparallel describe in terms of DNA?
A
1) Complementary: base pairing rules (A&T and C&G)
2) Antiparallel: backbones run in opposite directions
4
Q
Describe DNA replication in microbes
A
1) Semiconservative replication
2) The two strands of the parental double helix unwind, and each specifies a new daughter strand by base-pairing rules.
3) “The daughter cells are born pregnant”; i.e. new DNA is already being formed in daughter cells as soon as they’re replicated.
5
Q
Initiation of DNA synthesis predates what?
A
Any initiation of cell division
6
Q
Name 3 features of DNA that are involved in replication in bacteria
A
1) OriC: origin of replication
2) Replisome: where proteins and nutrients go to aid in replication
3) Ter: site where replication ends
7
Q
What direction is the replication of the E. Coli chromosome?
A
It’s bidirectional
8
Q
Describe 3 ways a bacterial chromosome can be compacted
A
1) Can be circular
2) Negatively supercoiled
3) Negatively supercoiled and mediated by DNA binding proteins (histone-like proteins).
9
Q
DNA binding proteins are what kind of proteins?
A
Histone-like proteins
10
Q
Describe why DNA may be negatively supercoiled and mediated by DNA binding proteins (histone-like proteins).
A
The nucleoid is supercoiled and compacted, and the scaffolding from the DNA binding proteins keeps it compact, but also allows regions of the chromosome to be accessible.
11
Q
More organization of the chromosome allows for what?
A
Faster gene expression
12
Q
Describe the bacterial chromosome shape, replication speed, error rate, and okazaki fragment length
A
1) Chromosome: circular, some linear
2) Replication speed: 1,000bp/s
3) Error rate: 10^-8
4) Okazaki fragment length: 1,500nt
13
Q
Describe bacterial transcription and translation
A
Transcription: Polycistronic & no post transcriptional modification
-Ribosomes can jump around and translate several proteins at once
Translation: 50S, 30S ribosomes / Protein splicing
14
Q
Define polycistronic transcription
A
Ribosomes can jump around and translate several proteins at once
15
Q
Describe the eukaryotic chromosome shape, replication speed, error rate, and okazaki fragment length
A
1) Chromosome: Linear
2) Replication speed: 100bp/s
3) Error rate: 10^-10
4) Okazaki fragment length: 100nt
16
Q
Describe eukaryotic transcription and translation
A
1) Transcription: Monocistronic mRNA & post-transcriptional modification
2) Translation: 60S, 40S ribosomes / Protein splicing
17
Q
Describe the reading frame of transcription and translation
A
1) There’s a coding strand (5’-3’) and template strand (3’-5’) used during transcription; mRNA strand ends up looking the same as the coding strand but with U instead of T.
2) Then translation occurs via ribosomes to produce a polypeptide from the mRNA
18
Q
What are the 3 stop codons?
A
UAA, UAG, and UGA
19
Q
Describe the importance of redundancy in genetic code
A
The redundancy of the genetic code allows for mistakes to be made, since a single nucleotide mutation may still be able to produce the same amino acid as the original
20
Q
Define missense, nonsense, and frameshift mutations and describe what they result in
A
1) Missense mutation: The changing of an entire nucleotide (i.e. T&A) and you are now coding for a different amino acid
-The least detrimental to a cell
2) Nonsense mutation: Results in a premature stop codon
3) Frameshift mutation: A nucleotide is lost and affects all downstream amino acids; usually a very different protein
21
Q
What is the least detrimental mutation to a cell?
A
A missense mutation
22
Q
What are the 3 possible outcomes of genetic mutations?
A
1) No effect (no change in phenotype)
2) Change in phenotype
3) Fatality
23
Q
What are the two types of point mutations?
A
1) Transition: purine > purine or pyrimidine > pyrimidine (staying the same type of nucleotide)
2) Transversion: purine <> pyrimidine (switching type of nucleotide)
24
Q
What is the most common category of mutations?
A
Point mutations
25
What are the two main categories of mutations?
Point mutations and frameshift mutations
26
Define a transversion mutation and list its 3 possible outcomes
1) Transversion: purine <> pyrimidine (switching type of nucleotide)
2) a) None
b) Nonsense: truncated protein
c) Missense: different amino acid; altered protein
27
What are the two types of transversion mutations? What do each of these result in?
1) Nonsense: truncated protein
2) Missense: different amino acid; altered protein
28
List and define the 2 types of frameshift mutations
1) Deletion: deletion of 1 or more nucleotides
2) Insertion: the addition of 1 or more extra nucleotides
29
List 2 things that can cause mutations and give an example of each
1) Chemical mutation
-Ex: N-methyl-N-nitro-N-nitroguanidine can alter guanine into O^8 methylguanine
2) Environmental mutation
-Ex: The alteration of thymine with UV light into a thymine dimer
30
We must have ways to differentiate wild-type vs mutant; name and define what this process is called
Screening: detection system for a mutant phenotype
31
Name 4 types of mutations
1) Morphological mutations
2) Lethal mutations
3) Conditional mutations
4) Biochemical mutations
32
Give 2 examples of biochemical mutations and define them
1) Auxotroph: must obtain nutrient from the environment because it has lost the ability to synthesize it
2) Resistance mutant: resistance to a pathogen, chemical, antibiotic
33
Name 4 ways to screen for mutants
1) Replica plating
2) Mutant libraries
3) Phage-sensitivity
4) Plasmid selection
34
Describe the replica plating process for screening for mutants
Involves creating two replica plates (one with complete medium and one with an incomplete media) and looking for a species that grows on the complete media but not on the incomplete media
35
What does the Ames test do? Where has it previously been successful?
1) The Ames test is an inexpensive method using bacteria as test subjects to determine the potential carcinogenicity of a substance. (i.e. identifies mutagens)
2) Has been successful in identifying only half of animal carcinogens.
36
Describe the Ames test
1) A culture of auxotrophs is plated onto two petri dishes; one with a minimal media with a small amount of histidine, and another with minimal media with a test mutagen and small amount of histidine.
2) Then the first dish may lead to a few spontaneous revertants (some eventually learn how to survive without histidine), and the second dish can lead to many revertants induced by the mutagen (if the mutagen is a mutagen, this dish should have more revertants/ survivors).
37
Define genes, phenotype, and genotype
1) Genes: The basic unit of inheritance
2) Phenotype: features that are expressed (ex: blue eyes, metabolic trait, etc)
3) Genotype: the gene sequence that exists in an organism
38
Describe cis acting elements and name 3 of them
1) Elements that are intrinsic to the DNA itself
2) Promoter, operator, and terminator
39
Define promoter, operator, and terminator. Also, what do these 3 things have in common?
1) Promotor: areas where RNA polymerase binds and transcription starts
2) Operator: area where effectors bind to limit or allow transcription
3) Terminator: region of DNA that tells RNA polymerase to stop transcribing
4) They are all cis acting elements
40
What's the differences between cis and trans acting elements?
Cis acting elements are a part of the genetic code, trans acting elements are not a part of the DNA
41
Name 3 things that help with gene organization
Operons, regulons, and trans acting elements
42
Define operons and regulons
1) Operons: multi-gene organizations; often several genes in tandem, all controlled by the same promoter
2) Regulons: functional groups consisting of several operons; same promoter precedes [same condition (internal or external) activates transcription of multiple operons at the same time]
43
Define constitutive expression and inducible operons
1) Constitutive expression: always expressed at high levels
2) Inducible operons: (+) or (-) control
44
Name 4 types of trans acting elements
Repressors, activators, corepressors, inducers
45
Describe what allows bacteria to build large structures quickly
Bacteria has many transcription factors and proteins because they’re organized in operons, which allows them to make large structures very quickly
46
Name 2 genetic elements
1) Chromosome
2) Plasmid
47
Describe what plasmids consist of
Always has an origin of replication, typically has an antibiotic resistance marker, an enzymatic marker gene, and RE cut sites
48
What are RE cut sites made by?
Made by restriction enzymes that cut DNA.
49
Describe plasmids:
1) Describe their size, and do they replicate independently?
2) Describe their shape and number of base pairs
3) Are plasmids essential to growth?
1) Small genetic elements that replicate independently of the bacterial chromosome
2) Most are circular, double-stranded DNA molecules. Size ranges from 1,500 to 400,000 base pairs
3) Plasmid genetic information may not be essential for growth
50
What does plasmid genetic information often provide?
Selective advantages (such as antibiotic resistance, toxins, virulence determinants, etc)
51
Name 3 selective advantages
1) Antibiotic resistance
2) Toxins
3) Virulence determinants
52
Define horizontal gene transfer
The mechanism by which bacteria exchange/ acquire DNA
53
What 3 methods of transfer can be used in horizontal gene transfer? Describe them.
1) Conjugation: Physical connection between bacteria mediates transfer
2) Transformation: Uptake of naked DNA from environment
3) Transduction: Viral transfer of DNA into bacteria
54
What 3 things is horizontal gene transfer important for?
Evolution, adaptation and pathogenicity
55
Describe the process of horizontal gene transfer; what does donor DNA go through and result in, and what two things can happen to the result?
1) Donor DNA can go through conjugation, transformation, or transduction to result in a partly diploid recipient cell with the DNA.
2) Then the donor DNA can either be integrated into the chromosome or the donor dna can self replicate (plasmid)
56
Define conjugation
Transfer of DNA, often in the form of a plasmid, by direct cell-to-cell contact
57
What does the donor cell in conjugation contain, and what does it use to transfer genetic material?
1) The donor cell contains the F factor
2) It uses a sex (F) pilus to transfer DNA or a plasmid
58
Who discovered conjugation and what did he use? Describe his experiment
1) Conjugation was discovered by Bernard Davis using a U-tube
2) One half of a U-tube had strain A, the other had strain B, and they were separated with a fine filter. The filter was too small for entire bacteria to pass through or to physically touch the other side, but media could flow through. He discovered that if you didn’t allow physical contact, the two strains would not mix, but that if you allow contact, the two strains would mix (i.e. the transfer of genetic information, some of the auxotrophs received genes to allow them to make something they couldn’t)
59
What did Bernard Davis find?
That physical contact is necessary for the transfer of genes
60
What 3 things do conjugative plasmids require?
1) Have to have the pilin protein (to make the sex pilus)
2) Have to have a type IV secretion system
3) Have to have a coupling protein
61
What allows for plasmids to be integrated into a chromosome?
IS elements, which contain inverted repeats
62
What 2 things are involved in F factor mediated conjugation?
Involves a donor and recipient; the donor has the F-plasmid that encodes for the pilus (which allows for conjugation). F+ donor, F- recipient.
63
Describe the process of F factor mediated conjugation
1) Donor can sense when it’s near an F- recipient, so it constructs a pilus which makes physical contact with the F- cell. Sex pilus then shortens to bring the cells close together.
2) Then a type IV secretion system is constructed (makes the needle accessible for the transfer of something), which now joins the two cells
3) The coupling factor initiates contact with the plasmid and couples it with the type IV secretion system to begin feeding it through
4) The relaxosome makes a cut at the origin of transfer and begins to separate one DNA strand. 5) The intact strand is replicated by the rolling-circle mechanism; creates a single strand of DNA to be fed through the type IV secretion system to the other cell.
6) Accessory proteins of the relaxosome are released.
7) The DNA/ relaxase complex is recognized by the coupling factor and transferred to the secretion system
8) The secretion system pumps the DNA/ relaxase complex into the recipient cell
9) As the DNA enters, the F-factor DNA is replicated to become double-stranded. The new cell now has the ability to make a pilus.
64
What is rolling circle replication found in? List its 5 steps.
-F factor mediated conjugation
1) DNA is ‘nicked’
2) 3’ end elongated; 5’ end is displaced
3) 5’ end complemented with Okazaki fragments
4) DNA replication
5) Circularization
65
What makes physical contact with the recipient cell in F factor mediated conjugation?
The sex pilus physical contact with the F- cell. It then shortens to bring the cells close together.
66
What joins the two cells together in F factor mediated conjugation?
A type IV secretion system
67
F factor mediated conjugation: What initiates contact with the plasmid, and what does it couple the plasmid with?
1) The coupling factor initiates contact with the plasmid
2) It couples the plasmid with the type IV secretion system to begin feeding it through
68
F factor mediated conjugation: What makes a cut at the origin of transfer?
The relaxosome makes a cut at the origin of transfer and begins to separate one DNA strand.
69
F factor mediated conjugation: Which strand of DNA is replicated? What is it replicated by?
The intact strand is replicated by the rolling-circle mechanism.
70
1) What does HFR stand for?
2) Define HFR conjugation
1) Stands for high frequency conjugation
2) When the donor cell integrates the F plasmid into its chromosome.
71
Describe HFR conjugation:
1) What initiates conjugation?
2) What do HFR cells transfer?
3) What leads to more transfer?
4) What eventually happens with the genetic material transferred?
1) HFR cell initiates conjugation by constructing a sex pilus
2) HFR cells transfers its unique genes and the plasmid integrated in its chromosome into a F- cell
3) Longer conjugation leads to more transfer
4) Recombination between donor DNA and recipient DNA occurs
72
1) During HFR conjugation, what determines the amount of material transferred?
2) Does the F- recipient cell typically remain F-?
1) Longer conjugation leads to more HFR cell genes being transferred to the F- cell; 100 minutes at most. 2) It’s rare that the entire F factor is transferred, so some is left behind; this is why the recipient typically remains F-.
73
Describe conjugation in gram positive bacteria (3 steps)
1) The recipient cell has the ability to produce a pheromone that attracts and primes a donor cell
2) Donor cell's plasmid interacts with the pheromone and transmits aggregation substance (AS) which migrates to the cell surface
3) AS then binds to a binding substance (BS) on the recipient cell(s), forming a clump of cells within which genetic exchange occur.
74
Define transformation and list its two types
1) The uptake of naked DNA from the environment
2) Can be either transformation of DNA fragments or transformation of a plasmid
75
Transformation with DNA fragments can result in two different outcomes; what are they?
1) Integration by nonreciprocal recombination
2) Degradation of DNA
76
Define competence
The ability of a cell to take in DNA
77
Describe transformation in gram positive bacteria (4 steps)
1) A competent cell binds a double-stranded DNA fragment
2) DNA is cleaved into smaller fragments (5-15kb)
3) One strand is hydrolyzed at the surface, the other strand is imported
4) Homologous recombination leads to a transformed cell
78
Describe the difference in gram positive and gram negative cells regarding their DNA uptake machinery
Gram negative cells have more machinery (particularly a PiiQ) due to having an outer membrane.
79
True or false: Only certain strains and species are transformable
True
80
1) What did Fredrick Griffith’s Streptococcus experiment demonstrate?
2) Describe the experiment
1) He demonstrated that DNA from a dead cell can be taken up by a live cell.
2) This was proven by using an R-strain that had a capsule and an S-strain that did not. He found that the R-strain is innocuous, but that the S-strain kills mice. He then heat-killed the S-strain and found that the dead cells did not kill the mouse. He then mixed dead S-strain bacteria and live R-strain bacteria and found that this combination killed the mice; he concluded that the live R-strain was transformed to the S-strain by taking up their DNA.
81
1) What did Avery, MacLeod, and McCarty's experiment confirm?
2) What was the experiment?
1) That it was the DNA from the heat-killed bacteria that caused pathogenicity.
2) They used different combinations of R cells and S cells (one type R cells only, one type R cells and type S DNA extract, etc)
Found that it was indeed the genes being transferred from the S cells to the R cells that caused pathogenicity
82
What are the two main types of transformation?
Natural and artificial
83
1) Define natural transformation
2) Define artificial transformation
1) Natural transformation: These bacteria have the necessary machinery and will readily acquire and maintain exogenous DNA
2) Artificial transformation: By molecular methods, we make bacteria capable of taking up DNA (genetic engineering). This can be through either chemically-induced competence (heat-shock) or electroporation (electrical shock)
84
What two things can be done to initiate artifical transformation?
Chemically-induced competence (heat-shock) or electroporation (electrical shock)
85
Describe artificial transformation for industry purposes (5 steps)
1) Vector, such as a plasmid, is isolated
2) DNA containing gene of interest from a different species is cleaved by an enzyme into fragments
3) Desired gene is selected and inserted into the plasmid
4) Plasmid is taken up by a cell, such as a bacterium
5) Cells with the gene of interest are cloned with either one of two goals in mind:
a) Create and harvest copies of the gene OR
b) Create and harvest protein products of a gene
86
Define transduction and list its two outcomes
1) The viral delivery of DNA (bacteriophages); a type of horizontal gene transfer
2) Results in:
a) Lytic infection: Replication into large numbers and causing the cell to lyse
b) Lysogenic state: Integration into the host genome without killing the host
87
Define lytic infection and lysogenic state. What are they the two potential outcomes of?
1) Lytic infection: Replicate into large numbers and cause the cell to lyse
2) Lysogenic state: Integrate into the host genome without killing the host
3) These are the two potential outcomes of transduction
88
1) Prophages are a part of what cycle?
2) Define prophages
1) The lysogenic cycle (of transduction)
2) Defined as a phage or viral DNA that is integrated into the host’s chromosome and has the ability to leave it.
89
When do cells switch from the lysogenic cycle to the lytic cycle?
Cells can switch from the lysogenic cycle into the lytic cycle by factors that cause the phage DNA to no longer be integrated with the chromosome (ex: UV light, antibiotic treatment)
90
2) Define generalized transduction.
2) Typically only what type of DNA is transferred?
1) The transfer of DNA from one bacteria cell to another via a bacteriophage (which transfers DNA from one host to another).
2) Typically only bacterial DNA is transferred.
91
List the steps of generalized transduction (5 steps)
1) Phage infects a bacterial cell, which then hydrolyzes the host DNA into pieces, and phage DNA and proteins are made.
2) The phages assemble and occasionally carry a piece of the host cell chromosome.
3) The transducing phage then injects its DNA into a new recipient cell.
4) The transduced DNA is recombined into the chromosome of the recipient cell.
5) The recombinant bacterium has a genotype that is different from the recipient bacterial cell.
92
Describe the steps of specialized transduction (3 steps)
1) Begins with a prophage that’s induced (UV light, stress, etc) and the phage is de-integrated from the chromosome.
2) Replication of the defective virus DNA with incorporated host genes occurs, which causes the assembly and release of transducing phage particles, which leads to infection of the next cell.
3) After the next cell is infected, one of two things happen:
a) Crossover to integrate bacterial genes, which leads to a bacterial chromosome containing only donor DNA.
b) Integration as a prophage leads to a bacterial chromosome that contains both virus and donor DNA.
93
What are the two potential outcomes of specialized transduction?
A) Crossover to integrate bacterial genes occurs, which leads to a bacterial chromosome containing only donor DNA.
b) Integration as a prophage leads to a bacterial chromosome that contains both virus and donor DNA.
94
What can de-integration include?
Sometimes the rare event of de-integration can include some bacterial genes (up to 5 or 10%)
95
1) Define transposition
2) What thing does transposition require?
1) When DNA ‘jumps’ from one location to another
2) Transposons
96
1) Define transposons
2) What can complex transposons do?
3) What do replicative transposons contain?
1) Mobile genetic elements that can transfer DNA within a cell, contain transposase
2) Complex transposons can carry genes providing resistance to antibiotics or virulence genes grouped together in a pathogenicity island.
3) Replicative transposons contain a resolvase gene
97
How does transposition occur? (2 steps)
1) Transposase cuts DNA
2) Ligation of transposon into target site
98
Name 4 effects of transposon insertions
1) Causes mutations, DNA rearrangements
2) Block transcription and translation
3) Can activate genes
4) Move between plasmids, spreading antibiotic resistance cassettes
99
Describe Agrobacterium:
1) Gram positive or negative? Do they have any appendages?
2) Where are they found?
3) What are they the causative agent of?
4) What's unusual about them?
5) How are they useful?
1) Gram negative bacilli with flagella
2) Found in the rhizosphere (root area)
3) Causative agent of crown gall disease and Morgellons disease (maybe, researchers moving away from bacteria as cause of Morgellons)
4) Unusual chromosomal organization
5) A good research tool for tumor induction and transfection
100
Describe the virulence of agrobacterium; how do they infect plant cells and what gives them a selective advantage?
1) Has its own chromosome and a Ti (tumor-inducing) plasmid that contains virulence genes, which is what infects the plant cell and incorporates into its chromosome. This causes the plant to release excess growth hormones which cause tumorogenesis.
2) During tumorogenesis the plant secretes opines, which the bacteria then feed off (other bacteria don’t eat these) and gives them a selective advantage.
101
How do agrobacterium find plants to infect?
Secretion of acetosyringone from wounded plant tissue act as a powerful chemoattractant for Agrobacterium.
102
Describe agrobacterium as a tool for plant genetics
Discs of leaves can be removed and incubated with agrobacteria whose plasmid has been altered to confer positive genes, and the agrobacteria can transfer these positive genes to the leaves and create a new (and better) plant genotype.
