Exam 3 Flashcards
What is a promoter?
A DNA sequence that is needed for the initiation of transcription
The role of tRNA is to deliver __________ during protein synthesis.
An amino acid
Which of the following statements about operons are TRUE?
Genes within an operon encode proteins that function together in the same cellular process (ex. Lac operon, trp operon)
The structure of an operon ensures that all structural genes are expressed at the same time and same level
Operons are common in bacteria but not eukaryotes
In an inducible operon, an inducer binds to the ______________, pulling it off the operator and enabling transcription of the structural genes
repressor
In a repressible operon, a(n) _________________ binds to the repressor, which prevents transcription of the structural genes.
Co-repressor
The structural genes in the lac operon are involved in the __________ of lactose. The structural genes in the trp operon are involved in the __________ of tryptophan
Breakdown, synthesis
Which of the following statements about mutations are TRUE
Mutations are changes in the existing genetic information of an organism
Mutations can occur spontaneously when organisms reproduce
Mutation rate can be increased by mutagens.
A gene that moves to various different sites within a bacterial chromosome is likely positioned within a:
Transposon
Horizontal gene transfer refers to DNA that is:
acquired from an external source
Which of the following is necessary for the integration of foreign DNA into a host chromosome?
Recombination
The enzyme SwbP1 recognizes the sequence 5’-TTCGAA-3’ and cuts between the ‘TT’ within the sequence to produce a sticky end. What single-stranded overhang is produced as a result? (hint: DNA is double-stranded)
5’-TCGA-3’
Which enzymes are commonly used by molecular biologists to amplify a gene of interest and insert it into a plasmid for the purposes of genetic engineering/cloning?
DNA polymerase, Restriction enzymes, Ligase
- DNA polymerase synthesizes DNA in PCR
- Restriction enzymes recognize a DNA sequence and cut it
- Ligase seals the DNA backbone
In bacteria: DNA polymerase replicate DNA in bacteria, RE’s cut foreign DNA/threats, ligase stitches okazaki fragments
KNOW HOW EACH ENZYME IS IN BACTERIAL ENGINEERING AND BACTERIA NORMALLY
3 stages in transcription
Initiation, Elongation, Termination
Initiation
RNA polymerase binds to DNA at a promoter (a DNA sequence that directs RNA polymerase to the start of a gene)
Start of transcription is at the promoter
Elongation
RNA polymerase adds RNA nucleotides to the 3’ end of the growing RNA strand
Termination
Release of RNA polymerase and RNA from the DNA
constitutively expressed
Genes that are always expressed
These are ‘housekeeping genes’ that encode proteins that are always needed for a cell to remain alive and functional
Explain why the expression of most genes can be regulated
Allows the cell to produce proteins that are needed in a given environment/situation and not waste resources by expressing proteins that aren’t currently needed
Not all genes are expressed at the same time
Induce
increase expression
Repress
decrease expression
Operon
Group of adjacent genes (back to back to one another) that are transcribed together and controlled by a single promoter
Genes within an operon encode proteins that function together in the same cellular process
Ensures that all genes are expressed at the same time and same level
Common in bacterial chromosomes, not eukaryotes
The genes within an operon are controlled by a _________ and an _________
promoter, operator
Promoter
region upstream of the gene(s) where the RNA polymerase binds
Sigma factor
the part of the RNA polymerase subunit that is responsible for binding to the promoter
Operator
Region upstream of the gene(s) where a repressor binds
By binding the operator, repressors influence the ability of the RNA polymerase (sigma factor) to bind the promoter
Where regulatory protein(s) bind and impact transcription
Regulatory gene
Encodes a regulatory protein (e.g. repressor), LacI
Repressors are DNA-binding proteins that bind to the operator and turn off transcription of the structural genes
Is always expressed (constitutively) → means that the repressor is always being made
Structural genes
Genes that make up operon
Expression is regulated (may or may not be expressed)
In an inducible operon, the default state (of the operon) is ___
Off
The repressor is bound to the operator, preventing transcription of the structural genes
Mutations
Changes to existing (endogenous) genetic information
Are inevitable
An uncorrected ‘error’
May or may not affect the encoded protein
Horizontal gene transfer
acquisition of new (exogenous) genetic information
What two things are largely responsible for the fact that genomes are dynamic – constantly changing through
Mutations and Horizontal gene transfer
Genome Evolution
Genomes are dynamic – constantly changing through
Create the diversity that is the raw material for evolution
Natural selection acts on populations of organisms to ensure the survival of organisms fit for a particular environment
Explains why bacteria evolve (i.e. antibiotic resistance)
Vertical gene transfer
When a cell divides, it passes its DNA onto its offspring
DNA getting passed down from generation to generation
3 mechanisms of HGT
Transformation
Conjugation
Transduction
Transformation
Uptake of DNA from the environment
Usually from cells that had died
Competency
the ability to perform transformation
Natural Competency
some bacteria have specialized machinery that bind to DNA outside cell and bring it inside; naturally perform transformation
Induced Competency
Make bacteria do transformation
Via electrical or chemical means
Electrical (electroporation) means for induced competency
shock bacteria, shock opens holes in membrane so outside DNA can float in
Chemical means for induced competency
put bacteria in chemicals (salt) with preheat shock that opens holes in the membrane like electrical
Transduction
DNA transfer mediated by a bacteriophage (“offshoot” of bacteriophage infection)
A bacteriophage is a virus that infects bacteria
After infecting a bacterial cell, bacteriophage package their DNA into a capsid so they can infect other cells
Sometimes DNA from bacterial chromosome gets packaged into the capsid instead
Results in the transfer of bacterial DNA from the donor to the recipient
Biotechnology
The use of microbes, cells, or cell components to make a product
Convert cells into factories
Involves recombinant DNA technology (genetic engineering)
Modifying an organism’s genetic material
ex. Food (fermented foods), Vaccine, Antibiotics, Vitamins
Why are restriction sites palindromic?
The 2 identical copies bind as a dimer
RE’s function as dimers; one binds at the top, one at the bottom, then they cut
Polymerase Chain Reaction (PCR)
Technique for amplifying DNA
Used to produce millions of copies of a gene for insertion into a vector (plasmid)
Also commonly used as diagnostic tests (genetic and infectious diseases)
Done with thermocycler machine
Endosymbiotic theory
Eukaryotes evolved from a pre-eukaryote that engulfed and ‘domesticated’ a prokaryote
The prokaryote evolved to be an endosymbiont that we now call the mitochondrion
Still today, mitochondria contain their own genome (which resembles a bacterial genome)
Eukaryotic cell kept bacteria with mitochondria around because it generates energy very well
Chloroplasts kept around → harvest energy from light (photosynthesis)
Bioremediation
Use of microbes to detoxify or degrade pollutants (ex. oil spill cleanup)
Evolved naturally
Bioaugmentation
Addition of specific microbes to degrade a pollutant
Purposefully placed by man
Composting
Arranging organic waste to promote microbial degradation by thermophiles (ex. Compost bin; gets hot inside)
Convert plant remains into the equivalent of natural humus
Benefits of fermentation
Preservation: fermentation results in conditions (acid + alcohol production) that very few microbes can tolerate, especially human pathogens
Improve digestibility of the food: microbes break down fibers
Add nutrients (such as vitamins) and flavor (such as esters and sulfur compounds)
Food spoilage
A process in which food becomes unsuitable for consumption
‘Goes bad’
Often due to microbial activity (mold, fungi)
Humans are in competition with microbes for the nutrients in food
Conjugation
Direct transfer of DNA between two bacterial cells
Involves a donor and a recipient
Initiated by a sex pilus protruding from the donor cell
Donor creates a bridge (sex pilus), reaches out and touches the recipient, pulls in close, then transfers DNA
Sex pili are encoded on…
conjugative plasmids
Conjugative plasmids
Mediate their own transfer from the donor cell to a recipient cell
Conjugative plasmid benefits; it stays in the original cell and transfers a copy of itself
HGT occurs when some of the recipient’s DNA is transferred along with the conjugative plasmid
A donor contains a conjugative plasmid where, on it, is a gene that encodes a sex pilus
It’s the conjugative plasmid that makes the decision to perform conjugation
Plasmids
Self-replicating circular pieces of DNA
Maintained within a cell, inherited without the need for chromosomal integration
100x smaller than bacterial chromosomes
DO NOT encode essential genes (main difference plasmid vs chromosome)
Can be lost and the bacterial cell will still survive b/c there are no essential genes on the plasmid, only specialty genes ( Antibiotic resistance, virulence)
Recombination
Exchange of DNA between two DNA molecules
Most efficient when regions are homologous (similar DNA sequence); Allows DNA to integrate into the chromosome
Is NOT a mechanism of horizontal gene transfer
DNA that is acquired through HGT must be maintained by the recipient cell or it will be lost
If the DNA is a plasmid, it will be maintained on its own
If the DNA is not a plasmid, it has to integrate into the chromosome or it will be degraded by the cell before it can be passed down
Explain the process/steps to recombination
- DNA from one cell aligns with DNA in the recipient cell (there is a nick in the donor DNA)
- DNA from the donor aligns with complementary base pairs in the recipient’s chromosome. This can involve thousands of base pairs.
- RecA protein catalyzes the joining of the two strands
- Result is the recipient’s chromosome contains new DNA
Complementary base pairs between the two strands will be resolved by DNA polymerase and ligase. The donor DNA will be destroyed. The recipient may now have one or more new genes.
Restriction enzymes
Enzymes that cut specific DNA sequences
How we go about Recombinant DNA Technology; we need to cut DNA in a convenient matter so that we can put it in a plasmid
Naturally produced by bacteria as a defense mechanism
Used in bacteria as a defense mechanism but in the lab are used to cut DNA in a specific way for biotech
Different RE’s cut at different locations within recognition site; different DNA sequences
Explain how RE’s are naturally produced by bacteria as a defense mechanism
Bacteria cut up bacteriophage DNA or any foreign DNA
Bacterial DNA is protected because it is methylated
RE’s can’t cut methylated DNA → chromosomal DNA is left alone
blunt ends
Some RE’s cut DNA at the same location on both strands
Cuts right across from each other
sticky ends
RE cuts DNA in a staggered manner, producing an overhang
3 Stages to PCR
- Denaturation
- Annealing
- Extension
Denaturation (1st step in PCR)
Separating target DNA into individual strands
High temperature
Annealing (2nd step in PCR)
Primers bind to target DNA in a sequence-specific manner; needed to allow DNA polymerase to start synthesizing
Low temperature
Extension (3rd step in PCR)
DNA polymerase synthesizes DNA
Intermediate temperature
Model Microbes
Common model systems for recombinant DNA technology
Prokaryotes (bacteria) - E. coli
Eukaryotes – S. cerevisiae (budding yeast)
Easily cultured, well-characterized, maintain plasmids
Taxonomy
Classification system (for organisms) based on evolutionary relatedness
We assign scientific name based on taxonomy
Names are based on binomial nomenclature – genus and species
Three Domain System
1978 proposal by Carl Woese based on rRNA sequence (since ribosomes are a requirement of cellular life and differences in RNA sequence can provide information on evolutionary relatedness)
3 domains:
Eukaryotes
Bacteria
Archaea
How methods of taxonomy have changed over time
Carl Linnaeus originally placed all life into 2 kingdoms: plants and animals
The discovery of microbes led to disagreements around their classification
Fungi were placed in their own kingdom in 1959
Prokaryotes were placed in their own kingdom in 1968
Mitochondria and chloroplasts are derived from bacteria; DNA looks like bacteria more than host cell (eukaryote) → evidence of endosymbiotic theory
Why are viruses not included in the three domains of life?
They lack cellular structure (and aren’t considered living/organisms)
Viruses still have genomes so they can be classified in respect to one another
Where did viruses come from in the first place [if they’re not living things]?
We don’t know fully
Possibly arose from independently-replicating strands of nucleic acids (e.g. plasmids)
Possibly developed from degenerative cells that got whittled down to genetic material and capsid
Symbiosis
Close association between two different organisms that is beneficial to one or both of them
Indicator organisms
Used to detect fecal contamination of water
Coliforms
Aerobic or facultatively anaerobic, gram-negative,
non–endospore-forming rods
Ferment lactose with acid and gas within 48hrs at 35°C
Predominantly E. coli
Presence of coliforms determined by Most Probable Number (MPN) method
Botulism
Food-borne disease caused by consumption of the botulism toxin secreted by the bacterium Clostridium botulinum
C. botulinum forms endospores (heat tolerant), toxin is heat stable (boiling doesn’t destroy it), packaging swells due to gas production by C. botulinum
Avoid bulging cans (due to gas → expanding), no need to avoid a dented can
The toxin inhibits fusion of synaptic vesicles → prevents activation of muscle cells → leads to flaccid paralysis (muscles constantly in weakened position)
Botulism toxin makes us sick, not the bacteria
Where do the fermenting microbes come from?
Indigenous flora or Starting cultures
Indigenous flora
Microbes naturally found on the food
Common for traditional fermented foods
E.g. wines and cheeses aged in caves
Starter cultures
Microbes added to the food
Often transferred from a previous fermentation
Strains can be engineered for desirable traits and re-used
Ethanolic Fermentation
Pyruvate –> ethanol + carbon dioxide
Essential part of bread, beer, and wine making
Production of beer and wine involves the same yeast: Saccharomyces cerevisiae (Brewer’s yeast)
Beer [ethanolic fermentation]
Alcoholic fermentation of grain (starting substance)
Wine [ethanolic fermentation]
Alcoholic fermentation of fruit (starting substance, e.g. grapes)
Synonymous mutation
no change to protein sequence (silent)
Frameshift mutation
Shifts the reading frame
All subsequent codons are out of frame
Frameshift and nonsense cause underlying protein to not be…
functional anymore
Codon
3 mRNA nucleotides that code for an amino acid
64 total (61 sense, 3 nonsense)
Replicative transposon
makes a copy of itself, original piece stays, copy gets put elsewhere
Non Replicative transposon
picks up and moves
Insertion sequence (IS)
the simplest version of a transposons composed of only inverted repeats and transposase gene
How are plasmids vectors
They transfer genes between organisms
Able to self-replicate (ori) independently from the chromosome
Ori = origin of replication
Phylogenetic trees
Display evolutionary relatedness
Utilized for taxonomy
Closely-related organisms should have similar names
Originally based on observable characteristics (e.g. cell morphology)
But the same characteristic can evolve independently in different organisms
Sharing a trait does not mean relatedness
All things are related, it’s a matter or how closely
Now based on genetic relatedness (DNA sequence); more accurate
If bacteria don’t have sex, how do they achieve diversity?
By acquiring DNA through HGT
Horizontal Gene Transfer (HGT)
DNA comes into cell from some outside source
Allows traits to be shared between unrelated bacteria; Unrelated cells that aren’t offspring
Ex. Antibiotic resistance can be shared with other bacteria
Transcription
RNA synthesis
Accessing the information stored within DNA and converting to an intermediate form (mRNA) that will soon be translated into protein
On a gene by gene basis
Cell asks do we want to express the gene and at what level
Non-synonymous mutation
changes protein sequence
Missense (type of non-synonymous)
changes an amino acid to another amino acid
Nonsense (type of non-synonymous)
Changes an amino acid to a stop codon
Cause underlying protein to not be functional anymore
